1

              U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

                          PUBLIC HEALTH SERVICE

                      FOOD AND DRUG ADMINISTRATION

              CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

                INTERNATIONAL ASSOCIATION FOR BIOLOGICALS

                    NATIONAL INSTITUTE OF ALLERGY AND

                           INFECTIOUS DISEASES

                     NATIONAL VACCINE PROGRAM OFFICE

                        WORLD HEALTH ORGANIZATION

                                + + + + +

             EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES

               ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT

                                + + + + +

                                WORKSHOP

                                + + + + +

                       Thursday, September 9, 1999

                                + + + + +

                      The workshop took place in the Plaza

          Ballroom, Doubletree Hotel, 1750 Rockville Pike,

          Rockville, Maryland, at 8:00 a.m., Naomi Rosenberg,

          Ph.D., Kathryn Zoon, Ph.D., and Keith Peden, Ph.D.,

          Session Chairs, presiding.

                                                                          2

          PRESENT:

                      KATHRYN ZOON, Ph.D., Session Chair

                      NAOMI ROSENBERG, Ph.D., Session Chair

                      KEITH PEDEN, Ph.D., Session Chair

                      JOHANNES LOEWER, M.D., Panel Chair

                      STEPHEN HUGHES, M.D., Panel Chair

                      RON DESROSIERS, Ph.D., Speaker

                      BEN BERKHOUT, Ph.D., Speaker

                      HSING-JIEN KUNG, Ph.D., Speaker

                      MAXINE LINIAL, Ph.D., Speaker

                      JOHN KAPPES, Ph.D., Speaker

                      JOHN PETRICCIANI, M.D., Speaker

                      DONALD BLAIR, Ph.D., Speaker

                      RUTH RUPRECHT, M.D., Ph.D., Speaker

                      EUGENE MAJOR, Ph.D., Speaker

                      HAIG KAZAZIAN, M.D., Speaker

                      BERNARD MEIGNIER, Speaker

                      GIRISH VYAS, Ph.D., Speaker

                      ALEX VAN DER EB, Ph.D., Speaker

                      THOMAS BROKER, Ph.D., Speaker

                      PAUL, SANDSTROM, Ph.D., Speaker

                      JAMES McDOUGALL, Ph.D., Speaker

                      BRIAN VAN TINE, M.D., Ph.D., Student

                         Speaker

                      SANDRA RUSCETTI, Ph.D., Panelist

                                                                          3

          PRESENT (Continued):

                      CLIVE PATIENCE, Ph.D., Panelist

                      LEONARD EVANS, Ph.D., Panelist

                      DAMIAN PURCELL, Ph.D., Panelist

                      ARIFA KHAN, Ph.D., Late Breaker

                                                                          4

                             C-O-N-T-E-N-T-S
          Retroviral Recombination as an Adjunct to Viral

          Propagation and Pathogenesis (HIV Model), Ron
          Desrosiers, Ph.D. . . . . . . . . . . . . . . . . .6

          Reactivation of Replication-Defective (HIV) 
          Retroviruses, Ben Berkhout, Ph.D. . . . . . . . . 18

          Retrovirus Insertion into Herpes Viruses,

          Hsing-Jien Kung, Ph.D.  . . . . . . . . . . . . . 33

          Foamy Virus Replication:  Implications for Transfer
          of Cellular Sequences, Maxine Linial, Ph.D. . . . 57

          Recombination in Lentivirus Vectors, John
          Kappes, Ph.D. . . . . . . . . . . . . . . . . . . 76

          Panel Discussion of Viral-Viral and Viral-
          Cellular Interactions . . . . . . . . . . . . . . 91

          Designer Cell Substrate, Telomerase Activity and 
          Cell Immortalization, James McDougall, M.D. . . .137

          Introduction to Cell DNA Issues, John
          Petricciani, M.D. . . . . . . . . . . . . . . . .151

          Evaluation of the Transforming Activity and
          Tumor Inducing Capacity of Tumor Cell DNA, Donald
          Blair, Ph.D.  . . . . . . . . . . . . . . . . . .166

          Infectivity of Retroviral Provirus DNA,
          Ruth Ruprecht, M.D., Ph.D.  . . . . . . . . . . .185

          Cells and Their DNA Containing Integrated DNA Viral
          Genomes:  Differentiated Phenotypes of the Human
          Central Nervous System, Eugene Major, Ph.D. . . .197

          Mobile Elements in Mammalian Genomes and Their
          Implications for Cell Substrate Safety, Haig
          Kazazian, M.D.  . . . . . . . . . . . . . . . . .217

          Panel Discussion of Cellular DNA as a Potential
          Source of Oncogenic Activity or Infectious
          Agents  . . . . . . . . . . . . . . . . . . . . .234

          Introduction to Evening Session, Keith
          Peden, Ph.D.  . . . . . . . . . . . . . . . . . .289

                                                                          5

                       C-O-N-T-E-N-T-S (Continued)

          Industrial Experience with Life Polio Vaccine 
          Prepared on the VERO Continuous Cell Line,
          Bernard Meignier  . . . . . . . . . . . . . . . .294

          Proteins of Replication-Incompetent Virions
          for HIV Vaccination, Girish Vyas, Ph.D. . . . . .313

          Unusual Response to Apoptin of Diploid Fibroblasts
          From Cancer Prone Syndromes, Alex van der Eb,
          Ph.D. . . . . . . . . . . . . . . . . . . . . . .327

          Human Papillomaviruses:  a Window into Eucaryotic
          Cellular DNA Replication Mechanisms and Regulation,

          Thomas Broker, Ph.D.  . . . . . . . . . . . . . .341

          Facilitated Detection of Adventitious Agents Using
          Genetically Engineered Cell Lines, Paul
          Sandstrom, Ph.D.  . . . . . . . . . . . . . . . .352

          In Situ Transcriptional Analysis of Integrated 

          Viral DNA, Brian Van Tine, M.D. Ph.D./Student . .368

          Late Breakers:

                Arifa Khan, Ph.D. . . . . . . . . . . . . .376
                Damian Purcell, Ph.D. . . . . . . . . . . .387
                Johannes Loewer, J.D. . . . . . . . . . . .395

                                                                          6

     1                    P-R-O-C-E-E-D-I-N-G-S

     2                                             (8:03 a.m.)

     3                CHAIRPERSON ROSENBERG:  Good morning. 

     4    Please take your seats.  We would like to start the

     5    session so that we try to stay on time.

     6                This morning's session is going to

     7    continue.  I think you'll hear many similar issues to

     8    those that we heard addressed yesterday afternoon.  So

     9    we'll continue discussing retroviruses.

    10                However, this morning we'll move more

    11    toward the complex retroviruses with presentations

    12    addressing issues relating to lentiviruses.

    13                We'll start with our first speaker, Ron

    14    Desrosiers.

    15                DR. DESROSIERS:  Thank you very much.

    16                My laboratory -- excuse me -- my

    17    laboratory uses simian immuno-deficiency virus, SIV,

    18    infection of rhesus monkeys as an animal model for

    19    AIDS, and I'm not exactly sure why I'm here, but I

    20    think I'm here to --

    21                (Laughter.)

    22                DR. DESROSIERS:  -- I think I'm here to

    23    give some examples of retroviral recombination from my

    24    experiments or where there may have been opportunities

    25    for retroviral recombination in our SIV system and

                                                                          7

     1    where we have not seen it.

     2                So I only have three slides in the

     3    carousel, so I'll try to give some explanation of each

     4    of these three, and I should finish well ahead of

     5    time.

     6                One of the things we've been doing in this

     7    SIV system is knocking out the so-called auxiliary

     8    genes.  These are genes that are not absolutely

     9    required for the viruses' ability to replicate, but do

    10    contribute to the virus' ability to replicate in

    11    monkeys and to cause disease.

    12                So we've done such experiments with the

    13    nef gene, the vpr gene, the vpx gene, and the vif

    14    gene.

    15                When we introduced a premature stop codon

    16    into the nef reading frame to eliminate the ability of

    17    nef to be expressed and used that nef stop virus to

    18    infect rhesus monkeys, there was very quickly -- by

    19    two weeks after infection of rhesus monkeys, all we

    20    could detect was revertant virus, that is the

    21    premature taa stop codon had universally reverted by

    22    two weeks of monkey infection to a stop codon,

    23    indicating that there was strong selective pressure

    24    for open functional forms of nef in vivo, and that

    25    such revertant viruses, although they might be rare,

                                                                          8

     1    appear rarely as a rare event.  The strong selective

     2    advantage of such a virus can very quickly result in

     3    that virus being the vastly predominant virus.

     4                We've also made forms of virus with a

     5    large, gaping deletion in the nef gene, and infection

     6    of monkeys with such nef deletion virus shows that

     7    this nef deleted virus is clearly an attenuated virus. 

     8    It replicates much less than the parental virus, and

     9    is much less prone to inducing disease.

    10                So one of the things that we've done is to

    11    look very hard to see whether or in which ways a

    12    virus, such nef minus virus, could regain pathogenic

    13    potential.

    14                We've looked both in cultured cells

    15    producing this nef deletion virus and in rhesus

    16    monkeys infected with nef deletion virus for possible

    17    recombinant viruses.

    18                In discussing use of such strains as

    19    experimental vaccines, the question has been raised

    20    whether such viruses could possibly acquire cellular

    21    sequences in an analogous way, the way the Type C

    22    retroviruses can acquire oncogenes or other such genes

    23    to bring back nef function or to provide some

    24    additional oomph to the virus to replace the loss of

    25    nef.

                                                                          9

     1                And despite extensive efforts looking for

     2    such recombinant viruses, where there would be strong

     3    -- so in the absence of nef, there would be selective

     4    pressure for capture of any gene that created a

     5    selective advantage, and we should be able to see it

     6    in vivo because of the strong selective advantage that

     7    could impart.

     8                And despite extensive efforts looking for

     9    such viruses, we've never found such recombinant

    10    viruses that have picked up extraneous sequences.

    11                We've taken animals infected with nef

    12    deletion virus, and we've serially passaged virus in

    13    rhesus monkeys, and we have been able to increase the

    14    pathogenic potential of such nef deleted SIV by serial

    15    passage in monkeys, and the increased pathogenic

    16    potential is not due, at least in the two lineages

    17    we've looked at, is not due in either case to capture

    18    of cellular sequences or extraneous sequences, but is

    19    actually due to compensatory changes in SIV elsewhere

    20    in the genome that allow the virus to make up for the

    21    loss of the nef gene.

    22                So similarly using nef minus virus, vpr

    23    minus virus, vpx minus virus, and vif minus virus,

    24    we've not seen any examples, despite extensive efforts

    25    looking for it; we've not seen a single example where

                                                                         10

     1    the virus has captured an extraneous sequence and made

     2    it increase selective advantage.

     3                Now, a few years ago we published a paper

     4    where we attempted to directly look for -- we designed

     5    an experiment to see if we could directly demonstrate

     6    retroviral recombination in an infected monkey.  So

     7    this is now -- we're not talking about recombination

     8    with cellular sequences, but recombination between two

     9    different SIV strains.

    10                So what we did -- how do I go backwards

    11    here? -- so we used two different SIV strains for this

    12    experiment.  We used one strain with the deletion in

    13    nef, and the second strain was deleted in both vpx and

    14    vpr.  That is about four kilobase pairs away.

    15                So we inoculated the nef deleted virus in

    16    one leg, and we inoculated the vpr/vpx virus in the

    17    other leg.  Both of these are attenuated viruses.

    18                By two weeks after infection, as reported

    19    in that Journal of Virology paper, by two weeks after

    20    infection all we could detect is wild type virus

    21    resulting from a recombination between these two

    22    different strains.

    23                For recombination to occur, one needs

    24    infection of the two different retroviral strains,

    25    infection of the same cell, and one can easily achieve

                                                                         11

     1    that in cell culture, but there had been some question

     2    how likely an event that would be in a whole infected

     3    organism with the massive pool of cells, and obviously

     4    at least initially a very, very low multiplicity of

     5    infection.

     6                For such recombination to occur, as I just

     7    said, you need infection of a single cell with both

     8    strains, and although at least theoretically there are

     9    a number of or several different mechanisms by which

    10    retroviral recombination can occur, the major way such

    11    recombination occurs relates to the fact that virions

    12    contain two RNA molecules, and when you have two RNA

    13    molecules in a single virion of the two different

    14    genotypes, when a cell is newly infected through the

    15    reverse transcription process and the copy choice

    16    mechanism in the reverse transcription process, one

    17    can then get recombinant viruses, in this case

    18    representing a wild type sequence and fully

    19    pathogenic.

    20                So these experiments illustrated two

    21    things.  They illustrated the ease with which

    22    recombination can occur even in vivo in the large pool

    23    of infected cells in the rhesus monkey.

    24                And the second thing that they illustrated

    25    was the enormous power of selective advantage and how

                                                                         12

     1    selective forces can very quickly selected for strains

     2    of virus with a growth advantage.

     3                And the final thing I want to talk about

     4    is a virus deleted in the vif gene.  So although vif

     5    is generally considered an auxiliary, nonessential

     6    gene, which it certainly is with HIV for growth in

     7    some cell types, it turns out that vif in most cell

     8    types for both SIV and HIV is very important for the

     9    virus' ability to replicate.

    10                In fact, for the strain of SIV that we

    11    work with, the only way we've been able to grow a vif

    12    minus virus is in a vif complementing cell line.

    13                So this shows the growth curves of vif

    14    deleted SIV, which has been produced in a vif

    15    complementing cell line.  Growth of this vif minus SIV

    16    in the vif complementing cell line, viv CEMX compared

    17    to growth of the same virus stock, same amount in the

    18    parental line CEMX 174.

    19                Now, the vif complementing cell line

    20    contains the full vif open reading frame, and the vif

    21    minus the deletion in the vif gene in the virus that

    22    we used is within the vif reading frame.  So it is at

    23    least theoretically possible that there could be

    24    recombination in this complementing cell line between

    25    the vif minus viral sequences and the vif gene

                                                                         13

     1    present, integrated in the host cell genome used for

     2    growing the virus.

     3                And obviously if there was a recombination

     4    for vif-plus virus, that virus would have an enormous

     5    selective advantage both in cell culture and in

     6    monkeys, and we could quickly, quickly see it.

     7                So we've made a number of vif-minus virus

     8    stocks and vif complementing cell lines.  We've

     9    infected at least six different monkeys with vif

    10    deleted SIV grown in vif complementing cell lines, and

    11    we've not seen a single example of virus regaining the

    12    vif sequences from the cell line that it was grown in.

    13                That's basically the sum total of my

    14    experiences involving recombination either with viral

    15    sequences or cellular sequences in the SIV system.

    16                So I'll stop there and take questions.

    17                (Applause.)

    18                DR. KUNG:  Hsing-Jien Kung, UC-Davis.

    19                Hi, Ron.

    20                DR. DESROSIERS:  Hi, Hsing-Jien.

    21                DR. KUNG:  So much of the -- in terms of

    22    detection of the recombinants, usually you need a

    23    selection.  So I was asking you in the nif experiment,

    24    did you grow the virus on just wild type non-

    25    complementing virus in an effort to pick up -- to

                                                                         14

     1    actually pinpoint the selection of that virus?

     2                DR. DESROSIERS:  Well, the --

     3                DR. KUNG:  The vif, the last experiment

     4    you were talking about.  You said you didn't see the

     5    pick-up of the host gene.

     6                DR. DESROSIERS:  That's correct.  So vif

     7    minus virus does not grow in CEMX 174 cells or other

     8    cell lines, but the vif plus virus does.  So we've

     9    transfected vif minus SIV DNA into the vif

    10    complementing cell line to make virus stocks.  We've

    11    expanded those virus stocks in the vif complementing

    12    cell line.

    13                If the vif minus virus had picked up a vif

    14    gene, it would now grow in a non-complementing cell

    15    line, and we've repeatedly tested our virus stocks,

    16    undiluted, large amounts.  We've taken undiluted virus

    17    stocks and put it into six different monkeys, two

    18    different stocks prepared independently.  We've never

    19    seen -- there there was clearly the enormous selective

    20    advantage of minute amount of vif plus virus would

    21    quickly grow out, and we've not seen it in the

    22    examples we've done.

    23                MR. BROKER:  Tom Broker, UAB.

    24                In that last experiment, how much flanking

    25    homologous sequence might you have had, and can you

                                                                         15

     1    consider expanding that outward to ask whether you'd

     2    favor recombination with enough flanking homologous

     3    things allowing crossover?

     4                DR. DESROSIERS:  Yeah, I don't remember

     5    off the top of my head exactly how much, but it was

     6    not a lot.  It was maybe 100 base pairs or so flanking

     7    on each side, but you're right.  I think if one wanted

     8    to investigate it, I think that would be a pretty good

     9    system to do it.

    10                MR. BROKER:  It would be great.  

    11                DR. DESROSIERS:  could expand out further

    12    and further and see under what conditions one might

    13    see it.

    14                MR. BROKER:  Okay.

    15                DR. DESROSIERS:  I think in the experiment

    16    when there was recombination between two retroviruses,

    17    I've always kind of hoped that people would have used

    18    that system a little bit more, too.  One could

    19    actually -- we never went to the trouble to try to

    20    look.  One could actually use that system to look at

    21    where the recombination events were occurring by

    22    putting in third base changes.  You could actually map

    23    where the crossover, quote, crossover events occurred,

    24    and we never took the trouble to do that.

    25                But I think anyone who's interested in

                                                                         16

     1    studying recombination in retroviruses, that would be

     2    a pretty nice way to do it, but, no, we've not done

     3    that experiment.

     4                It was a short stretch of sequence.  We

     5    were actually -- for the experiments we wanted to do,

     6    we wanted to avoid getting recombinant virus, and so

     7    we kept the length of overlap short, but if one wanted

     8    to study it, exactly right.  You could just expand the

     9    length and see if you had a much larger length of

    10    flanking sequences, whether you had increased chances

    11    of seeing recombination

    12                PARTICIPANT:  It's really an extension of

    13    the same experiment.  We know that retroviruses will

    14    promiscuously package all sorts of cellular messages. 

    15    Do you know within the virion population that's coming

    16    out of the complementing line whether you actually do

    17    get vif message actually packaged in the virions that

    18    might be a substrate eventually for a strand jump

    19    recombination?

    20                DR. DESROSIERS:  Yeah, that's a -- we

    21    haven't actually looked at it.  My mind is turning in

    22    theoretical terms, what that means.  It's generally

    23    believed that vif minus virus in a noncomplementing

    24    cell line is capable of a single round of infection

    25    events.

                                                                         17

     1                PARTICIPANT:  Sure.

     2                DR. DESROSIERS:  It can get into one

     3    round, but then not pass.    I think there's really

     4    only room in the virion -- some of our retroviral

     5    experts here can correct me if I'm wrong -- but

     6    there's really only room in the virions for two RNAs,

     7    and I think if a virus did package cellular RNA and

     8    that would leave room at most for a single viral RNA. 

     9    I think that would result in a defective particle,

    10    would not be infectious, but still could be propagated

    11    as a defective virus conceivably anyway in the

    12    presence of a helper.

    13                CHAIRPERSON ROSENBERG:  I need to remind

    14    the questioner to give their names.

    15                DR. LINIAL:  Maxine Linial, Hutchinson

    16    Center.

    17                About your second point, we have shown

    18    with ALV that you can package a full length genome and

    19    a cellular RNA and get recombination.  So it can

    20    occur.

    21                But my main question was:  how much

    22    replication did you get with the delta vif virus, and

    23    if you did see any, in what kind of cells was it

    24    replicating in the monkeys?

    25                DR. DESROSIERS:  The vif minus virus?

                                                                         18

     1                DR. LINIAL:  Un-huh.

     2                DR. DESROSIERS:  The only way we were able

     3    to replicate the vif minus SIV is in the vif

     4    complementing cell line.  So the vif minus virus,

     5    we've tested it.  We can clearly generate high titred

     6    stocks in the complementing cell line, and we've used

     7    that to infect a variety of cell lines, primary rhesus

     8    PBMC, primary macrophage cultures, and inoculated into

     9    monkeys.

    10                And in cell culture, the only way -- the

    11    only system we detect any replication is in the vif

    12    complementing cell line.

    13                In monkeys, the vif minus virus is at the

    14    very most -- the very least the most attenuated strain

    15    we've seen.  The only thing we've been able to measure

    16    in the delta vif inoculated monkeys is very, very weak

    17    antibody responses.

    18                CHAIRPERSON ROSENBERG:  Thank you.

    19                The next speaker is Ben Berkhout.

    20                DR. BERKHOUT:  Okay.  I would like to

    21    continue on some of the themes that have been

    22    introduced by the previous speaker, and I guess we can

    23    quickly go over the introduction.

    24                Of course, most of you should be familiar

    25    with the idea of live attenuated viruses.  These

                                                                         19

     1    entities should replicate and elicit supposed immune

     2    response that confers lifelong protection against

     3    challenge with a pathogenic wild type virus.

     4                Of course, the concept has proven to work

     5    in several systems, for instance, vaccinia, polio, and

     6    measles, and for HIV-1 good results have been

     7    described in SIV infection models of macaques by

     8    several labs, including that of the previous speaker,

     9    and it has been proposed recently by some groups that

    10    one should start clinical trials on humans with the

    11    HIV-1 versions of live attenuated viruses with

    12    multiple gene deletions.

    13                But there are still several safety

    14    concerns.  One of them is the induction of a fulminant

    15    infection in immunocompromised hosts, and second, and

    16    that's the one I would like to focus on today, is the

    17    potential reversion of an attenuated vaccine strain to

    18    a virus variant that can replicate fast and that

    19    potentially can cause AIDS.

    20                Now, all of the studies I will present are

    21    done in tissue culture settings.  So we will only look

    22    at replication potential of viruses, and therefore, of

    23    course, we don't have any measurements of

    24    pathogenicity.

    25                Now, let me start out with a purely

                                                                         20

     1    hypothetical graph.  The idea here is to delete genes. 

     2    As you know, HIV-1 has nine genes; simple

     3    retroviruses, only has three.  So the idea is that we

     4    can probably removed a couple of the accessory genes

     5    and thereby we will credibly decrease the replication

     6    potential of such a virus.

     7                Probably that also has a consequence for

     8    the pathogenicity of this virus, and I did draw

     9    parallel curves.  There's no evidence for that, but I

    10    think it's reasonable for this graph at least.

    11                Probably also the immunogenicity of these

    12    viruses will slowly drop off, and the idea is to reach

    13    a level that is below a threshold for the

    14    pathogenicity such that this virus will not cause

    15    AIDS.

    16                However, of course, replication should

    17    still suffice to induce a good immunogenicity, and of

    18    course, these thresholds are, again, hypothetical, and

    19    we don't know how big this window is.

    20                Even if you managed to identify such a

    21    virus that has these properties, one of the key

    22    questions is how stable is this virus and will it

    23    perhaps not be able to regain replication capacity and

    24    thereby going over this threshold.

    25                Now, why should we release genes?  We

                                                                         21

     1    already heard in the previous talk that when you put

     2    stop codons in, for instance, the nef gene, it's

     3    repaired in two weeks.  Even small deletions or

     4    substitutions will be removed by this virus very

     5    rapidly, and that was shown early on in a paper

     6    published in '95 using the SIV system and virus

     7    evolution in macaques.

     8                A four amino acid deletion was introduced

     9    in nef, and that's shown here.  It was still present

    10    two weeks after inoculation.  However, after 17 weeks,

    11    the virus managed to repair this sequence, at least to

    12    fill in this gap, by duplicating the sequences that

    13    are located over here.

    14                Officially at this point in time, you do

    15    not have the wild type sequence back, but credibly

    16    after 25 weeks, we do see a wild type tyrosine appear

    17    at this position.  It's like the wild type, and after

    18    45 weeks, we also have an aspartic acid here, and we

    19    are almost back to the wild type sequence

    20    demonstrating the enormous repair capacity of this

    21    virus.

    22                So small deletions will not suffice and

    23    we, therefore, have to make bigger deletions.

    24                Now, the system we are going to us is a

    25    tissue culture evolution system, and I will use one

                                                                         22

     1    slide to introduce the system.  The method we have

     2    called forced evolution.  Basically we start out with

     3    a molecular clone of HIV-1 in which the deletions in

     4    accessory gene products have been introduced.

     5                We start with a massive transfection.  Ten

     6    to 40 microgram of molecular clone is electroparated

     7    (phonetic) into cells, and then initially the cells

     8    are cultured, but when there is a sign of virus

     9    replication, it's seen in either syncytia or GAG E24

    10    production.  We start to passage the culture

    11    supernatant onto fresh and infected cells.

    12                Initially these large inocula up to 1 mL,

    13    later on credibly reducing the amount of virus that is

    14    transferred.

    15                So in Section A we will be able to pick up

    16    revertants, and I guess it's important to realize that

    17    this reversion or evolution is a two-step process. 

    18    One needs mutations, and those will be introduced

    19    primarily by the first transcriptase enzyme, which is

    20    error prone.  Of course, these mutations will be

    21    introduced in a random manner, and then will get

    22    selection of those virions that are better than the

    23    input virus.

    24                So its evolution, as I guess it must be

    25    proposed by Darwin, although he used different terms. 

                                                                         23

     1    It's variation and survival of the fittest.

     2                Now, because I think a presentation may

     3    not be complete without seeing an RNA stem loop

     4    structure, I actually show you one example of a study

     5    that we performed on a stem loop structure.  That's

     6    the so-called TAR hairpin that is found at the extreme

     7    five prime and three prime end of HIV-1 genomes.  This

     8    is the wild type situation.

     9                This is a mutant that is completely

    10    replication defective.  Here we have open to the lower

    11    left-hand side of the stem, and after about six months

    12    of culturing initially the cells, later on the virus

    13    passage, we ended up with this revertant.  It has one,

    14    two, three, four substitutions.  It doesn't go back to

    15    the wild type sequence, but what this revertant is

    16    telling us is that this hairpin structure is critical.

    17                Sequences are less critical, but one needs

    18    a base pair lower region here, and of course, we also

    19    have some idea of what this hairpin is doing.  It's

    20    critical in transcription regulation of this virus,

    21    but also because it's present in both ends, it plays

    22    a role in the mechanism of reverse transcription.

    23                So we obtained a rather complete set of

    24    deleted HIV-1 genomes from Ron Desrosiers, and I will

    25    today only focus on one of these deletion virions, and

                                                                         24

     1    that's the so-called Delta 3 virions with three

     2    deletions.

     3                One deletion is in the accessory vpr

     4    protein, and this five prime half of the genome was

     5    combined with this one, which has a deletion in the

     6    nef gene.  Then some of the sequences here at the

     7    border of the three prime LTR are still present

     8    because they are important for replication.

     9                And then there is a second deletion in the

    10    three prime LTR.

    11                Now, it's important to realize that this

    12    deletion in the LTR will be inherited in the progeny,

    13    also in the five prime LTR, and the five prime LTR, as

    14    you probably know, is the transcription promoter at an

    15    untelldish (phonetic) virus.

    16                Over here I've indicated some primers that

    17    will be used in subsequent PCR analysis to actually

    18    check whether these introduced solutions are still

    19    present.

    20                One more thing.  We tried to do efflution

    21    (phonetic) in tissue culture in primary cells.  That's

    22    indicated over here, but in particular, for this Delta

    23    3 variant we found that the replication, while that

    24    much delayed, that it probably will take years to

    25    really obtain the fertent (phonetic) viruses. 

                                                                         25

     1                So to solve that problem, we are going to

     2    use a transformed T cell line, in this case Sup. T1 T

     3    cell line, because there the replication defects of

     4    this kind of mutants are less feared (phonetic). 

     5    There certainly is a replication, delay in

     6    replication, but it's much less feared than in primary

     7    cells.

     8                So the virus with three deletion was

     9    passaged for up to four month in the Sup. T1 T cell

    10    line.  Samples were taken at day 14, 53, 83, 125, and

    11    we analyzed the samples with similar input virus

    12    amounts in a parallel infection, and as you can see

    13    over here, the initial virus or the virus present

    14    after two weeks has a hard time replicating, and then

    15    you see a gradual increase in replication capacity and

    16    a big jump actually is seen already at day 53.

    17                So we were interested to find out what has

    18    happened with this virus.  So we performed PCR essays,

    19    PCR analysis.  PCR analysis across vpr gene didn't

    20    show any insertion or deletion of sequences, and in

    21    fact, the sequenced region, nothing has happened.

    22                This is a PCR across the nef and LTR

    23    deletion, and as you can see over here, something

    24    happens around day 55.  We see the appearance of a

    25    variant that is about 14 base pairs longer, and

                                                                         26

     1    apparently this variant is much more fit than the

     2    input virus because it out competes the input virus

     3    within a couple of weeks.

     4                So at this point we got excited.  We

     5    thought, well, perhaps there is an insertion of

     6    perhaps cellular sequences in these deleted virus, and

     7    of course, we sequenced this region.  

     8                It turned out that the two introduced

     9    deletions were maintained.  The nef deletion was there

    10    and also the deletion in the U3 region was perfectly

    11    maintained.

    12                There also was no insert of cellular

    13    sequences, but what has happened is that in the core

    14    promoter region, and as indicated here this is a

    15    transcription start (phonetic) side, three binding

    16    sites for the constitutive transcription factor, SP-1,

    17    and a tandem repeat for nf kappa B binding.  The

    18    deletion that was introduced is indicated over here. 

    19    Nothing happened over there.

    20                What we did see is a gross duplication of

    21    all the three SP-1 sites.  In addition, there are

    22    seven nucleotides of unknown origin, and we have seen

    23    an onser (phonetic) duplications.  

    24                Yesterday in one of the talks, and I must

    25    say that this apparently is a very popular theme in

                                                                         27

     1    mutant retroviruses, they easily change the number of

     2    repeat elements in their genome, and in fact, we

     3    probably know why that is so, because this duplication

     4    is probably introduced during the first inscription by

     5    a mechanism called slippage realignment.

     6                So RT enzyme has probably copied the

     7    sequences over here.  Then RT and the cDNA is

     8    partially removed and it reanneals back to the

     9    original sequences, and in one step one gets a

    10    duplication of three SP-1 sites.

    11                So we end up with this promoter.  Does

    12    this make a better transcriptional promoter? 

    13    (Unintelligible) in transient LTR reporter assays, and

    14    what we compare is the wild type LTR, the one with the

    15    U-3 deletion, and then the one that's the duplication

    16    of SP-1 site.  So as you compare the last two bars, we

    17    tested that in the absence of the transcriptional

    18    activate of protein TAT and also in the presence and

    19    then in cells that were not more activated by PMA and

    20    PHA.

    21                In the absence of TAT protein, we, in

    22    fact, see that the LTR promoter does not gain function

    23    by duplicating of the SP-1 site.  In fact, it gets a

    24    little worse.  

    25                However, in the presence of TAT protein

                                                                         28

     1    and, of course, in virus replication in this system,

     2    the TAT protein will be around.  We do see a partial

     3    restoration of the LTR function, and in this case

     4    actually it's becoming a little better than the wild

     5    type.

     6                So the transcription promoter is, indeed,

     7    improved in the presence of TAT protein.  

     8                Does this single mutation also improve the

     9    replication of the delta 3 virus?  Well, indeed, it

    10    does, and that is shown here.  So we reconstructed a

    11    molecular clone with three deletions, and then in

    12    addition the six SP-1 sites.  So here is the wild type

    13    virus.

    14                If you only delete vpr there is a small

    15    replication.  So this is the input virus with three

    16    deletions, and if you in this virus then introduce the

    17    six SP-1 sites, there is a rather dramatic increase in

    18    replication capacity.  In fact, this revertant virus

    19    replicates much faster than the virus with a single

    20    vpr evolution.

    21                Now, we thought it was also of interest to

    22    test the replication of this virus not only in the

    23    Sup. T1 T cell line, in which it's evolved, but also

    24    in primary cells, and that's shown here, and there we

    25    do see a different picture.

                                                                         29

     1                This is the wild type virus replication in

     2    primary cells, and over here we see that there hardly

     3    is a difference in the replication of the original

     4    delta 3 virus and the one with six SP-1 sites.

     5                So it seems that the SP-1 site duplication

     6    is beneficial only in the Sup. T1 T cell line that was

     7    used for the evolution experiments.  That may have to

     8    do with the pool of transcription factors that are

     9    present in that particular T cell line.

    10                And, for instance, it has been reported

    11    that Sup. T1 cells have an extremely low amount of nf

    12    kappa B, and one can imagine that perhaps, therefore,

    13    SP-1 is more important for this virus.

    14                So just to sum up, we show that this delta

    15    3 strain is genetically unstable.  It retains

    16    replication capacity in tissue culture, and we have

    17    seen duplication of SP-1 sites.

    18                Now, this is all tissue culture, but I

    19    think there are some additional evidence to suggest

    20    that these viruses are unstable.  The Ruprecht

    21    laboratory has recently demonstrated that some of the

    22    viruses, the delta 3 viruses, do eventually cause AIDS

    23    in infected monkeys, and there's also some evidence

    24    from cohort studies in humans where people infected

    25    with a nef deleted and, therefore, attenuated virus

                                                                         30

     1    strain do over time show a decline in CD-4 T cell

     2    numbers and, therefore, perhaps they are at risk for

     3    going on to AIDS.

     4                So the conclusion will be that these

     5    deleted viruses are safe.

     6                Now, in the context of this meeting, I

     7    should say that the one example I showed you today we

     8    did not find any incorporation of cellular sequences

     9    in these -- in these deletion virions, and like the

    10    previous speaker, in all of the evolution experiments

    11    that we have done so far, we have not come across any

    12    insertion of cellular sequences in these virus

    13    strains.

    14                Now, finally, I will discuss a putative

    15    route to perhaps a more safe vaccine.  What I showed

    16    is that this deleted and attenuated virus is able to

    17    regain replication capacity, and that's indicated by

    18    this arrow.

    19                Now, perhaps this virus may be a nice

    20    starting point to remove additional functions, thereby

    21    again going down on the replication letter.  Perhaps

    22    then again we can try to evolve a faster replicating

    23    variant, and by repeated cycles of gene deletion and

    24    subsequent evolution, we perhaps may end up with a

    25    virus with only three to five genes that is able to

                                                                         31

     1    replicate efficiently, and that perhaps will be more

     2    stable in genetic terms.

     3                And with that I would like to stop.

     4                (Applause.)

     5                CHAIRPERSON ROSENBERG:  Is there a

     6    question?

     7                MR. FALLEAUX:  Hi.  Frits Falleaux.

     8                This may be a silly question for

     9    someone --

    10                CHAIRPERSON ROSENBERG:  Be sure to

    11    identify yourself clearly.

    12                MR. FALLEAUX:  Yeah, Frits Falleaux,

    13    right.

    14                This may be a silly question for someone

    15    from working with adenoviruses and also HIV, but did

    16    someone think of trying to make an attenuated to HIV

    17    which is, in part, replication efficient and in

    18    combination with, for example, a useful promoter that

    19    dries (phonetic) nef so that you have partial

    20    replication in the presence of the inducer, which you

    21    take away after the introduction of protection?

    22                DR. BERKHOUT:  People have worked along

    23    these lines.  For instance, one has introduced the

    24    constitutive CMP promoter in the context of a

    25    replicating HIV-1 virus.

                                                                         32

     1                One of the problems with introducing

     2    exogenous sequences in this final genome is that in

     3    most cases the virus doesn't accept them, and they are

     4    over time kicked out of the genome.

     5                So the only way to safely introduce

     6    exogenous sequences in this final genome will be to

     7    introduce elements that are absolutely required for

     8    replication, and then I think, indeed, it would be of

     9    interest to study better and inducible promoters, but

    10    that hasn't been tested so far.

    11                DR. EVANS:  Leonard Evans, Rocky Mountain

    12    labs.

    13                Did you try to put that promoter, the

    14    duplicate promoter back into the wild type?

    15                DR. BERKHOUT:  We did, and in

    16    straightforward replication curves, there is no

    17    difference in replication.  If you do fairly sensitive

    18    competition assays so the wild type virus, and the

    19    wild type has six SP-1 sites, you do see that in the

    20    wild type context, in fact, the fitness goes down a

    21    little bit.  So --

    22                DR. EVANS:  It goes down?

    23                DR. BERKHOUT:  Yes, it does down a little

    24    bit.

    25                DR. EVANS:  Okay.

                                                                         33

     1                DR. BERKHOUT:  But that difference is

     2    marginal.  So you have to do very sensitive

     3    competition assays with the two viruses to find out. 

     4    So six SP-1 sites are clearly beneficial in the

     5    context of this deletion variant, but they don't make

     6    wild type virus much better.

     7                DR. EVANS:  Okay.  Thanks.

     8                CHAIRPERSON ROSENBERG:  Thank you.

     9                The next speaker is Hsing-Jien Kung.

    10                DR. KUNG:  Thank you.

    11                So in today's talks and yesterday's, you

    12    have heard that retrovirus have significant potential

    13    to recombine with another retrovirus or recombine with

    14    hos genes under selective pressure.  Today I'd like to

    15    discuss with you about experiments that I would

    16    summarize as experiments regarding recombination

    17    between retroviruses and the herpes viruses.

    18                If I can have the first slide.

    19                Let's consider the following scenario.  In

    20    co-infected cells with the retrovirus and the herpes

    21    virus, retrovirus copying to RNA, then into DNA, RNA

    22    copying to DNA, and has a choice of going into the

    23    cell or the genome or has the choice to go into the

    24    herpes viral genome if in the co-infecting cells.

    25                We can argue what is the probability. 

                                                                         34

     1    What is the chance?  Well, based on the most

     2    simplistic view, the simple calculation, simply based

     3    on the mass ratio, you would argue that if the herpes

     4    virus -- and let's take the worst case scenario.  It's

     5    only present as one to ten copies -- one copy, let's

     6    say, in the latent state.  Based on mass ratio, you

     7    would calculate that in every 10,000 integration into

     8    the host genome, you would have one into this size of

     9    herpes virus.

    10                Now herpes virus sometimes will also

    11    replicate.  So it can reach -- so okay.  Let's take

    12    that calculation and say you'll have massive infected

    13    cells, about a million cells, in fact, with

    14    retrovirus.  Then there should be 100 integrants if

    15    herpes virus is in latent state.

    16                Now, herpes virus, of course, also can

    17    replicate, let's say, 2,000 copies of 10,000 copies. 

    18    If it were 10,000 copies, then every integration into

    19    the host chromosome, there's a chance into the herpes

    20    virus genome.

    21                So if you calculate, you say this is not

    22    too rare, but, on the other hand, you can make the

    23    argument that herpes viral replication usually are

    24    combined in certain subnuclear structure, and they may

    25    not be as successful to the retrovirus integrative

                                                                         35

     1    complex, and they may be encapsulated very quickly,

     2    and so all of these theoretical considerations may

     3    also argue against genotypic mixing or recombination

     4    between these two.

     5                Okay.  So I think only experiments will

     6    tell.

     7                Next slide, please.

     8                So today I'm going to talk about

     9    retrovirus integration into herpes virus.  The system

    10    under study is avian retrovirus using reticular

    11    endothelial virus as a model system, REV.  It's a

    12    typical nonacute Type C retrovirus that cause T cell

    13    lymphoma and the B lymphoma in chickens, and we show

    14    before that the mechanism of oncogenesis is through

    15    insertion or activation of C mixing.

    16                Avian herpes virus, Marek's disease virus,

    17    is very, very prevalent.  Until 1970 that was the most

    18    important economic loss in the poultry industry, until

    19    the live attenuated vaccine was developed.

    20                Now, both viruses infect T cells, infect

    21    the same T cell types.  The tumors are derived from 

    22    very similar T cell types.  So co-infection did exist

    23    and has been observed and reported in many papers, and

    24    in fact, many of the retroviruses were isolated from

    25    Marek's diseased chickens, including the chicken

                                                                         36

     1    syncytia virus strain of the REV and avian

     2    myeloblastosis virus, some of you probably know, that

     3    carries the mip gene.

     4                So there is a preponderance of evidence

     5    that these two viruses coexist in chickens.  

     6                So the implication of the study -- I will

     7    show you some experimental evidence, but the

     8    implication of the study is, of course, that you can

     9    generate a hybrid virus with altered gene expression

    10    patterns, which I will show you, and the phenotypes,

    11    emergings of new pathogens, if you will, and can

    12    certainly sometimes broaden the host range because

    13    some of the herpes virus, indeed, can carry retroviral

    14    information.

    15                So with that in mind, let's first tell you

    16    a little bit more about the herpes virus, this Marek's

    17    disease virus.  This virus, although it's

    18    lymphotropic, really the structure is very much

    19    similar to alpha herpes virus with the repeating

    20    sequence flanking the unique, long reaching, and

    21    repeating sequence flanking unique short.

    22                And just in a typical alpha herpes virus,

    23    you are reaching and encode mostly structural and

    24    replication enzymes.  In fact, this virus, now Lucy Li

    25    has entire sequence and, indeed, show a strong

                                                                         37

     1    correlation with herpes simplex virus.

     2                Unique small region also encodes some of

     3    the genes, however.  They are not important in the in

     4    vitro replication.

     5                Now, the interesting part are usually

     6    confined in the more, but divergent region, in the

     7    repeat region, and we now know that in the Marek's

     8    disease virus that we have worked in the past few

     9    years, that it encodes a protein that we can call it

    10    oncogene, called the mac (phonetic), which is in the

    11    Joan Foss family of loosing zipper protein, and if you

    12    remove this, the virus becomes nononcogenic.

    13                Okay.  So in this sense the virus is

    14    similar to herpes simplex virus, but it's not, due to

    15    some of the coding sequences in the repeat region.  

    16                Okay.  But for our purpose, I'd like to

    17    point out this virus is extremely oncogenic, probably

    18    most potent oncogenic herpes virus.  It causes tumors

    19    within four to six weeks in experimental animals, and

    20    these tumors are of polyclonal origin.  Okay.  So it

    21    smells like this oncogene does do something in a

    22    direct way.

    23                But today we'd like to talk about how this

    24    virus can be used as a template for retroviral

    25    insertion and what happens after that.

                                                                         38

     1                Just to give you a sense, this virus is

     2    probably, again, the most successful live attenuated

     3    virus vaccine against oncogenic virus.  I'll tell you

     4    a little bit about the vaccine.

     5                The serotype 1 is oncogenic strand, the JM

     6    MD11 and GA stand, but serotype 2 and 3 are vaccine

     7    strands.  They share about 70 percent homology with

     8    serotype 1.  However, they are not oncogenic in

     9    chickens.

    10                Now, there's another way of making this

    11    virus attenuated virus, by taking serotype 1, okay,

    12    and simply passage.  It's a mysterious way, but it

    13    worked every time.  You passage for a long time.  It

    14    could be five to ten years experiments, but you would

    15    get attenuated virus.

    16                People still do not know exactly what

    17    happened, but there is a correlation of expansion with

    18    certain repeat sequence in the region.  We now know

    19    these attenuated viruses actually still maintain the

    20    mac oncogene.  So what happens most likely is that

    21    these viruses do not replicate very well.  Therefore,

    22    they cannot induce T cell lymphoma in chickens.

    23                In vitro, however, after long passage

    24    viruses tend to replicate quite well.

    25                Okay.  So we said that moving to the real

                                                                         39

     1    experiment.  Now, our story began with this particular

     2    experiment.  They were intended to study our REV -- at

     3    the time it was still retrovirologist -- REV insertion

     4    of the T cell lymphoma.

     5                So Bob Isford took some of the T cell

     6    lymphoma generated by Marek's disease virus and looked

     7    for whether REV virus is present or not.

     8                It turned out REV viruses are not present,

     9    but there was some surprise in that in the low passage

    10    of serotype 1 virus, not serotype 2 or 3, that he

    11    actually could detect hybridization.  This

    12    hybridization was under 30 percent mismatched, but he

    13    could detect hybridization against REV or TR.

    14                And this suggests to us there are some

    15    sequences related to REV, and we call it ALTR remnant

    16    present in the present day serotype 1 viruses.  So

    17    this virus has never seen REV recently, but you can

    18    see that they do have some sequence.

    19                The fact that they show specific bands and

    20    they can map in the band D and band F region, which is

    21    close to the repeat sequence, convinced Bob Isford at

    22    that time that these are not a fluke.  These are

    23    probably real homologous sequence even though it has

    24    diverged significantly.

    25                There are the high passage one now,

                                                                         40

     1    attenuated one.  You can see that the sequence begin

     2    to diverge because of expansion of the repeat

     3    sequence.

     4                However, in this, this is something else. 

     5    I will come back to this.  This GM high virus has

     6    multiple LTR related sequence.  I'll come back to

     7    this.

     8                So then Bob Isford began to sequence of

     9    them.  So just to give you summary, indeed, there are

    10    several patch homologies ranging from 70 to 81

    11    percent, with some nucleotides ranging from 22 to 33

    12    nucleotides.

    13                Now, if there's only one site, you can say

    14    this is very skeptical, but with all of these sites

    15    clustered together in the right order and many of them

    16    diverging at the junction of the retroviral genome,

    17    and that suggests to us this probably is real.

    18                So we took that as LTR remnants.  There

    19    are also some  sequence related to the retrovirus, but

    20    the LTR sequence was most, most prevalent.

    21                And this stretch of sequence is most

    22    interesting.  This stretch of sequence turned out to

    23    be that it's in the enhanced region of the REV LTR. 

    24    This sequence now is present as in the enhancer region

    25    of the herpes virus alpha tif or the VP-16 equivalent

                                                                         41

     1    of the Marek's disease virus, and it's a T cell tropic

     2    enhancer.

     3                So with that we figured that in nature

     4    this had happened, and let's see what happens, whether

     5    in recent -- this is what we call the ancestral

     6    insertion.  We like to see whether in recent time

     7    whether there was any evidence of recent insertion,

     8    and we recall this one, the same picture.

     9                This one has a multiple one, and now

    10    again, this is a virus derived from low passage,

    11    simply by culture them for five years.  Okay?  This is

    12    a passage of 211, and it's completely attenuated, and

    13    they can be potentially used vaccine, but this is a

    14    vaccine experiment that went exactly as intended.

    15                So what happens once we discover there's

    16    some retroviral insertion, and these LTR hybrids under

    17    stringent condition and still stick to the filter

    18    paper.  So we know these are more recent insertion.

    19                And Dick Witter, our collaborator,

    20    collected all these viruses.  So he then did the

    21    following smear by cloning this separately.  So with

    22    that he actually was able to separate these clones and

    23    these LTR sequences of segregate, indicating indeed

    24    they are part of the viral genome, and they are

    25    genetically stable because they have been passaged for

                                                                         42

     1    about 200, 200 passages.

     2                And so what happened is that during this

     3    long term propagation you'll feed DEF, duck embryo

     4    fibroblasts, the primary cells.  We talked about it

     5    two nights ago, that DEF, and it turns out in passage

     6    about 87, that DEF they used for fuller infection by

     7    Marek's disease virus turned out to contain reticular

     8    endotheliosovirus.  So during this evident of clone

     9    mixing fraction, that retrovirus integrate that, but

    10    now they become very stable.

    11                Now, you would argue why do they persist

    12    for so long.  Well, when Dick Witter compared the

    13    replication rate with the wild type, they all in vitro

    14    replicate much better.  We do not know whether that's

    15    due to LTR insertion.  However, the LTRs seem to be a

    16    persister, and the LTR integrants seem to dominate the

    17    culture at passage 211.

    18                So this was a five year experiment ten

    19    years before we began the study.  We could not

    20    control.  We could not add any control.  So Dick

    21    Witter and I think that if this happened nature, on an

    22    evolution or scale; if it had happened in the ten

    23    years prior to our experiments, can we do a more

    24    controlled experiments by doing a co-infraction

    25    (phonetic), and can we make this work in five weeks or

                                                                         43

     1    five days?

     2                So the experimental protocol turned out to

     3    be very straightforward, that you mix the MDV,

     4    infected DEF with REV.  MDV is cell associated virus. 

     5    So it's actually a little bit difficult to do

     6    experiment, but if you mix them together, you passage

     7    them every week, and then by feeding fresh DEF, okay,

     8    and then we took individual passage mass culture,

     9    isolate MDV, and in fact just isolate the cell and

    10    look for MDV mini chromosome imposed fiogel

    11    (phonetic), and the free retroviral DNA should run out

    12    of the gel, and then you probe with REV LTR to monitor

    13    the kinetics of integration.

    14                It turned out it's not difficult at all. 

    15    Okay.  We have repeated this several times now. 

    16    Basically after, in fact, five weeks you can see a

    17    little bit.  This is a southern blot.  So it's not a

    18    most sensitive method, but you can see that retroviral

    19    LTR integration increase, okay, after passage 16. 

    20    It's a huge amount radioactively.

    21                This is just a load to show you that MDV

    22    DNA were loaded at about the same amount.

    23                Now, if you do TRP CPCR, you could

    24    actually detect within one to two passage.  Okay.  So

    25    the integration certainly can be very efficient,

                                                                         44

     1    although as I said, this is radioactive.  You really

     2    do not know what is the population of the integrants

     3    versus no integrated one.

     4                Now, this increasing intensity, of course,

     5    are due to two reasons.  One is that REV is still

     6    present.  It can infect more herpes virus, but,

     7    secondly, of course, it may be the herpes virus with

     8    LTR, in fact, replicated better in some fashion. 

     9    There is no selective pressure except in vitro

    10    replication.  So this may actually contain the LTR

    11    integrate certain places that can enhance the

    12    replication.

    13                So what Dan Jones did and Rhonda Koss did

    14    was -- were to actually look at the insertion sites,

    15    and then something rather interesting was revealed. 

    16    So by looking at -- this is 17 kilobase genome, but

    17    they looked at the integration sites.  Integration

    18    sites are tightly clustered with two insertions in the

    19    GD region, but others are tightly clustered around the

    20    -- close to the boundary between repeat and the unique

    21    sequence.

    22                Now, at Alsets (phonetic), if you look at

    23    the sequence, okay, just very briefly, you found

    24    actually these are the individual integration sites. 

    25    I do not mean to have you look at the sequence, but

                                                                         45

     1    look at the arrow indicating that there's no sequence

     2    facility.  It's a regional facility.

     3                Okay.  Now, at the outset we knew the

     4    integration should not be totally random because we

     5    were selecting viruses, replication virus.  So any

     6    insertion in the essential genes would disrupt its

     7    ability and we may not be able to pick up.  So, again,

     8    this underscored the importance that if you want to

     9    study some recombinant, that the selection pressure

    10    turned out to be an important one.

    11                So we figured that maybe these are the

    12    sequence, the regions.  There are no coding sequence. 

    13    Therefore, it integrates better, but that still cannot

    14    fully account for this tight cluster because the U.S.

    15    region shown by Robby Morgan and Marc Purcell can be

    16    completely deleted, yet in vitro replication, and

    17    still very viable.

    18                So we then began to think this actually

    19    also looked very much like insertion of mutagenicity

    20    oncogene that we studied before, especially the RB

    21    oncogene in avian erythroleukemia in terms of the

    22    tight clustering.

    23                So we began to entertain the hypothesis

    24    maybe this can activate some of the genes near the

    25    boundary.

                                                                         46

     1                But before I say that, before I show you

     2    the data that I have, we have to isolate the virus in

     3    order to do the experiment.  This is a simple whole

     4    cell PCR mapping insertion sites.  We did not have the

     5    virus yet.

     6                So in the past few years Dick Witter was

     7    able to isolate the virus.  This was a heroic effort

     8    because the retrovirus that we used had no markers, no

     9    selection marker.  It's a wild type retrovirus.  So it

    10    depends on how prevalent these integrations are in the

    11    whole mass population.

    12                So Dick was able to isolate several, and

    13    I'm just going to talk about one, quote, RM-1.  For

    14    the first retrovirus, the REV MDV hybrid virus number

    15    one, and this is very interesting because it contains

    16    a solo LTR.  No other retrovirus sequence are present,

    17    and it integrates at a hot spot, the tight cluster

    18    area.

    19                And this shows the retrovirus duplication

    20    of the MDV genome.  So it's authentic integration.

    21                It has a solo LTR, our first in this

    22    region, and then it homogenized to the other region as

    23    well.

    24                Now, the most interesting is the phenotype

    25    of this virus.  This virus, okay, RM-1 -- these other

                                                                         47

     1    two are the clones -- had everything.  Everything else

     2    is very similar to the wild type in that it can

     3    replicate very well inside bursa, inside T cells in

     4    the chicken.

     5                The only thing different is it has no

     6    oncogenicity.  Okay?  So this is the viral clone that

     7    did the test, in vivo test, and this is the wild type,

     8    okay, the oncogenicity seven out of eight, five out of

     9    eight, eight out of the eight, and this is the

    10    attenuated one.  Of course, after long passage it also

    11    has no oncogenicity.

    12                But this virus N and attenuated virus

    13    differ significantly.  This virus can replicate very

    14    well, whereas the attenuated virus does not, do not

    15    replicate very well in vivo.

    16                Okay, and as a result, when Dick Witter

    17    did the challenge protection experiment, so in fact,

    18    it was RM-1, then challenged with the virulent, very

    19    virulent MDV, it turned out this has much better

    20    protectivity than the other attenuated strand.

    21                And this is showing here -- maybe we could

    22    just locate the protections.  So this was done by

    23    taking the RM clone, infect the first as a vaccine. 

    24    Then you challenge it with the very virulent virus,

    25    and it turned out the protection is 100 percent,

                                                                         48

     1    whereas the current vaccine virus is 34 percent, which

     2    this is the Basta (phonetic) vaccine virus, about 78

     3    percent, and so on and so forth.

     4                And the reason is that it replicate very

     5    well inside the chicken, the only things that cannot

     6    cause oncogenesis, and I'd be happy to speculate to

     7    the reason why that's the case, but also because it

     8    can spread very well.  So it serves as very good

     9    protection.

    10                Okay.  Now, I don't mean to say this is

    11    the vaccine virus because it causes other associated

    12    diseases.  Other than oncogenicity, it does cause

    13    thymic atrophy, like the wild type.  So it cannot be

    14    a vaccine yet, but this certainly gives us some clue

    15    as how perhaps to manufacture better vaccine for MDV.

    16                But in the context of this discussion,

    17    this shows that LTR insertion can change the phenotype

    18    of the virus.  We look at other regions, whether

    19    there's any gross change.  There's no gross change,

    20    but we cannot rule out point mutations.

    21                So now, at the molecular level, we'd like

    22    to see whether it activates anything.  Indeed, this

    23    RM-1 has a solo LTR integrate in there, and these are

    24    the northern blot to show, indeed, there is LTR

    25    insertion in here, and there is a transcript with a

                                                                         49

     1    link to LTR from here to here.  That would transcribe

     2    polysystronic message carrying soft 2 open reading for

     3    US-1 and US-10. 

     4                Since this is the closest to the five

     5    prime end, we think this product may be relevant.  We

     6    don't have evidence to show that this is relevant. 

     7    We're just beginning to do that, but I can tell you a

     8    little bit about it.  This is a novel sequence, unique

     9    to the oncogenic herpes MDV strain virus.  The

    10    sequence is novel.  So we do not know the function,

    11    but it does share some homology with US-22 gene family

    12    of this cytomegalovirus as well as HHV-6.

    13                In the case of HHV-6, this particular

    14    protein, it's not the same protein, but it shows some

    15    similarity.  It has been implicated in the

    16    transactivation of HIV.

    17                So we think it may be a co-factor of the

    18    transcriptional factor.  Indeed, recent experiments

    19    show that by Hous Chan's lab that this is a

    20    nucleoprotein.

    21                I cannot tell you more about this simply

    22    because we don't have much data about this protein

    23    yet.  So let me give you a conclusion.

    24                So what I have shown you is that we have

    25    shown retroviral insertion of herpes virus at least in

                                                                         50

     1    this system at several levels.  We show there's

     2    ancestral insertion; that avian retrovirus REV LTR

     3    remnants are present in the serotype 1 MDV, indicating

     4    that infection into the herpes virus in nature

     5    probably happened after the divergence between the

     6    vaccine virus and this virus.

     7                I also show you in the acute co-infection

     8    experiments REV insertion into MDV detectable as early

     9    as second passage.  REV insertion sites are non-

    10    randomly distributed.  REV LTR insertion of mutagen of

    11    MDV genes demonstrated MDV with altered pathogenicity

    12    in the phenotypes also generated.

    13                I'd like to take this in a broader context

    14    and tell you about what happened since our original

    15    discovery.  Now this has been repeated by several

    16    laboratories, and that demonstrate that herpes

    17    retrovirus insertion to herpes virus.

    18                First, that our own actor (phonetic) has

    19    extended REV insertion into the oncogenic strand

    20    experimentally to the vaccine strand, herpes virus of

    21    turkey, and we can see its insertion with no problem,

    22    again within one to two passages.

    23                But here there's a very interesting clone. 

    24    One of the clones -- I'm sorry.  So this you saw

    25    already, but this is REV insertion into HVT.

                                                                         51

     1                There's one clone here that actually

     2    carries the full length of REV.  In fact, we were able

     3    to show this can produce virus after transfection.  So

     4    this is not anything specific for the oncogenic strand

     5    of MDV.

     6                We also showed that rav 1, rouse

     7    (phonetic) associated virus, can also integrate in the

     8    MDV or HVT.  Okay.  So, again, there is nothing

     9    special about REV.

    10                This is probably very significant.  From

    11    Japan, Hiria's group about two years ago showed that

    12    this MDV virus isolate from chicken actually carry

    13    endogenous REV 0.  Now, this experiment did not see in

    14    vitro culturing at all.  So it was an in vivo isolate,

    15    and REV 0 very nicely integrated MDV, again, at a hot

    16    spot that we show for REV.

    17                Furthermore, there are two more

    18    laboratories that I actually did not update this

    19    slide, show last year that the avian erythroblastosis

    20    virus LTR also integrates into MDV and another REV 0-

    21    like sequence integrated into MDV.  So this has been

    22    repeated in the four laboratories independently.

    23                Recently Eric Davidson in Israel did in

    24    vivo experiments to see whether in vivo recombinant

    25    can be detected or not, and they used PCR knowing that

                                                                         52

     1    the cluster region -- so they designed PCR primary

     2    that can easily detect recombinant, and they were able

     3    to detect about 20 of them and were helping them

     4    analyze it.  

     5                Now, a year ago this paper is rather

     6    profound to us.  It's not retrovirus insertion of

     7    herpes virus, but this group found that REV can

     8    integrate into fowlpox virus, and the pox virus is a

     9    vaccinia virus group.

    10                This is profoundly significant to us

    11    because this shows retroviruses can integrate into a

    12    virus that only has a cytoplasmic life cycle.  They

    13    may not need the DNA pk.  You may not other things in

    14    the nucleus to do the job, but you can.

    15                And again, I emphasize this was an in vivo

    16    isolate.  The virus never sees in vitro culture.  So

    17    its recombination in vivo.

    18                Then finally, George Miller's lab found in

    19    EBV infected cells there is a fusion of cDNA.  They

    20    did an isolated virus.  So we do not know the fate of

    21    the virus.  They do find a junction that carried both

    22    retroviral LTR and EBV sequences.

    23                So this may happen in other systems as

    24    well, and I do not -- I have no reason to believe it's

    25    a special for chicken, but I think, again, selection

                                                                         53

     1    is very important.  If you do not have selection, this

     2    kind of integration come and go.

     3                I think I will stop here.  Thank you very

     4    much.

     5                (Applause.)

     6                PARTICIPANT:  I have a comment and a

     7    question.  I think for the sake of the audience it's

     8    also worth noting that there is evidence for ancestral

     9    capture in a number of herpes viruses, ancestral

    10    capture of cellular genes by a process involving

    11    retroviruses.

    12                So, for example, if you look at the

    13    members of the gamma-2 herpes virus group, they --

    14    from new world primates, old world primates, and

    15    humans -- those herpes viruses have genes for

    16    dihydrofolate reductase, cyclin D, and a few other

    17    genes, in all cases lacking introns (phonetic), the

    18    lack of introns suggesting it's acquired by a process

    19    involving reverse transcription, and that's likely to

    20    have been acquired by a process involving co-infection

    21    of cells with a retrovirus.

    22                My question has to do with why do you

    23    think -- it looked to me like that many of your

    24    examples of capture were pieces of LTR and not whole

    25    LTRs.  You showed one example of a single LTR.  Why do

                                                                         54

     1    you think there are so many -- I mean, how does that

     2    happen, and why does it just have small pieces or a

     3    single LTR, and is there any evidence in any cases

     4    that these LTRs or LTR pieces are actually driving

     5    expression of some viral gene?

     6                DR. KUNG:  Okay.  First, I did snow in RM-

     7    1.  I just went through so quickly.  I'm sorry.  The

     8    LTR is promoting insertion activate transcript.  Okay?

     9                Secondly, in terms of why it's LTR, I

    10    think that what happens, LTI/LTR direct recombination

    11    which especially in herpes virus, that you have the

    12    flip-flop of the R region.  So the recombination is

    13    very, very acute.  You have direct recombination

    14    between LTI/LTR you would delete the sequence of.  In

    15    fact, the full length sequencing, the herpes virus

    16    genome, it's not very stable.  It's about ten

    17    kilobase.  So the virus has a tendency in my mind to

    18    spit out the extra sequence, and LTR seems to be

    19    harmless at least.  Yeah.

    20                MR. MINOR:  Philip Minor from NIBSC.

    21                I'm completely ignorant about

    22    retroviruses.  The integrations that you described

    23    were all with co-infections, I guess.  Is it possible

    24    that you could get an integration if you were just

    25    looking at an endogenous retroviral sequence that was

                                                                         55

     1    maybe being transcribed?  Can you pick it up in that

     2    way?

     3                DR. KUNG:  Yeah.  Thank you for asking the

     4    question.

     5                Again, I went so quickly.  There was an

     6    endogenous virus REV 0 integration into the herpes

     7    virus shown by the Japanese group, but also more

     8    importantly is that the retrotransposon, popping -- I

     9    guess later the speakers will be talking about that --

    10    it's very frequent.

    11                I give you one example, not related to

    12    herpes virus, but you probably know the bacula virus,

    13    the autografa californa nuclear polyhedral viruses,

    14    that the Freezen (phonetic) and Miller and their

    15    colleagues did a beautiful study, show that the TET,

    16    it's a transposal element, number four, integrating to

    17    the bacula virus genome transcribed the gene and

    18    contained the entire sequence.

    19                So, yes, my feeling is it's -- all we're

    20    talking about is selection.  If you have a selection,

    21    I think you will be able to detect those endogenous

    22    transposable -- retrotransposable like, and I think

    23    that's also related to Ron's comment.

    24                Many of the herpes viruses captured,

    25    especially like the KSS-3, Kaposi's sarcoma, capture

                                                                         56

     1    a lot of cellular homologs, and many of them are

     2    internalist (phonetic) even though herpes virus are

     3    known to be able to splice out intron.

     4                So my feeling is that some of them may not

     5    be entirely due to retrovirus, but could be due to

     6    retrotransposable.

     7                DR. PEDEN:  Keith Peden, CBER.

     8                You may have said, but does the

     9    integration site have the hallmarks of genuine

    10    retroviral integration, the duplications at the ends?

    11                DR. KUNG:  Oh, yes, yes.

    12                DR. PEDEN:  It does?

    13                DR. KUNG:  Duplication at the end, yeah.

    14                MR. COFFIN:  John Coffin, Tufts.

    15                Just to clarify a little bit your answer

    16    to the previous question, I think it was asked whether

    17    the endogenous viruses could actually be moving

    18    without an infection cycle, and the answer probably 

    19    is no, for all that we know about endogenous

    20    retroviruses.  You still need to get -- in order to

    21    move them from one place to another, you still need to

    22    -- they still need to undergo a complete replication

    23    cycle.

    24                DR. KUNG:  Yes. 

    25                CHAIRPERSON ROSENBERG:  I think we need to

                                                                         57

     1    move on because we are somewhat behind, although I

     2    don't want to cut this short.

     3                The next speaker is Maxine Linial.

     4                DR. LINIAL:  Okay.  Today I'm going to

     5    talk mostly about foamy viruses, but I wanted to start

     6    off raising a couple of issues, some work in my lab

     7    about packaging of avian retroviruses.

     8                Okay.  So for many years my lab has been

     9    interested in defining the minimal packaging region of

    10    the avian retroviruses, and this is a schematic of

    11    what from the literature appears to be defined as the

    12    minimal packaging regions of NLV HIV and the ALV

    13    viruses, and we had previously defined a packaging

    14    region of about 160 nucleotides from the five prime

    15    end of the genome, which are sufficient for packaging. 

    16    That is, you can place this region on any heterologous

    17    RNA, and that RNA will be packaged with a high

    18    efficiency into retroviral particles.

    19                And in fact, we find that such a small

    20    region on a neo or a hygro RNA is packaged only about

    21    2.7 times worse than the intact ALV genome.  So we

    22    believe this is a sufficient packaging region.

    23                More recently we've done a series of

    24    experiments on this 160 nucleotide region and have, in

    25    fact, found that we can delete off the entire three

                                                                         58

     1    prime end, leaving an 82 nucleotide region with this

     2    set computer predicted secondary structure that has

     3    several stem loop regions, and by mutagenesis RNAase

     4    protection and looking at a variety of viruses, we

     5    know that there is one, two, three stems that are

     6    important, and that possibly the region that's

     7    involved in protein binding and recognition of this

     8    RNA may be only a four nucleotide loop.

     9                So we're getting very close to

    10    understanding what the structure of the packaging

    11    region looks like for this virus.

    12                And one other point I want to make is that

    13    retroviral packaging seems to be a hierarchy of

    14    sequences.  The retrovirus most avidly packages

    15    sequences with its own psi regions, but then, of

    16    course, we know that vector RNAs containing psi can be

    17    packaged with very high efficiency, and probably after

    18    that the retrovirus is packaged cellular RNAs or other

    19    elemental RNAs containing psi-like sequences, as we

    20    heard for MLV.  VL-30 sequences have psi-like

    21    sequences and are avidly packaged, but they also

    22    package random cellular RNAs.

    23                And work from my lab and a variety of

    24    other labs has shown that such RNAs can be a player in

    25    packaging cell lines, and we did a lot of work on a

                                                                         59

     1    quail packaging cell line which contains a single

     2    integrated RSC lacking psi, and the unique thing about

     3    this packaging cell line is that the provirus is

     4    exceedingly active and produces about 100 times more

     5    virus than any of the other packaging cell lines that

     6    we looked at, and because it made so many particles,

     7    we could so that what was packaged was random cellular

     8    RNAs, and that we could also show that these cellular

     9    RNAs could be reverse transcribed in new cells and

    10    integrated into the genome with detectable frequency.

    11                And we could find about 100 using neo as

    12    a cellular RNA.  We could find about 100 transductions

    13    of the neo gene in an infection with this virus, and

    14    we propose that this kind of event can occur with any

    15    packaging system if it's looked for, but this kind of

    16    a looking for integrations in the new cell is not

    17    generally assayed in any of these packaging systems.

    18                So now I'd like to turn to foamy viruses. 

    19    This is one of the seven genera of retroviruses, the

    20    spuma retroviruses.  They're a tightly knit family

    21    that's fairly divergent from all the other groups of

    22    retroviruses.

    23                One aspect of the virus that's very

    24    interesting is that unlike, for instance, the gamma

    25    retroviruses which are know -- can be pathogenic in

                                                                         60

     1    their native host with long latency, the gamma

     2    retroviruses or the alpha retroviruses are unlike the

     3    lentiviruses which are known to be pathogenic and

     4    accidently infected hosts but generally not in their

     5    natural host.

     6                The spuma viruses or foamy viruses are not

     7    pathogenic in any host but spar.  So that there is a

     8    whole variety of these viruses, and their life long

     9    infections in their host, and there's no

    10    pathogenicity, and when there's an accidental

    11    infection, for instance, several of these simian

    12    viruses are known to infect people.

    13                Again, there appears to be no

    14    pathogenicity in those hosts, and this is a very

    15    interesting question, is why the life style of this

    16    virus is so different from those of its other

    17    retroviral cousins.

    18                The genome of the foamy virus is very

    19    similar structurally to other retroviruses, has gag,

    20    pol, and env genes.  It also has several open reading

    21    frames and two known products, the transactivator

    22    protein called tas, which is absolutely required for

    23    transcription from the LTR promoter, and uniquely to

    24    this group of viruses there's a second promoter in the

    25    envelope gene, the internal promoter, which seems to

                                                                         61

     1    be responsible for transcription of the accessory

     2    genes, tas, self, and a second gene which is a spliced

     3    variant from tas and bel 2 called bet (phonetic).  Bet

     4    is a major product of this virus.  It's completely

     5    dispensable in tissue culture, but is believed to play

     6    some important role in vivo, although we have no idea

     7    what the role of this protein is.

     8                Some features of this virus that make it

     9    very different than other retroviruses is, first, the

    10    pol protein is not made as a gag-pol fusion.  It's

    11    made from its own splice pol message.  This makes it

    12    more similar to the hepadenoviruses that goes to the

    13    retroviruses.  Again, it has an internal promoter

    14    unlike all the other retroviruses.

    15                And a third feature is that you cannot get

    16    particle egress from the cell with gag alone.  You

    17    must have the envelope protein.  Again, we're similar

    18    to the hepadenoviruses like HBV than to the

    19    retroviruses.

    20                Foamy viruses have been isolated from a

    21    variety of species.  It's extremely prevalent in cats,

    22    both domestic and wild cats.  Recent studies say about

    23    70 percent of individuals cats are infected.  It's

    24    highly prevalent in many bovine flocks.

    25                Recently a virus has been isolated from

                                                                         62

     1    horses, although the prevalence of this virus is not

     2    clear at the moment, and from a variety of primate

     3    species, in fact, every primate species that's been

     4    studied does have a foamy virus.

     5                And in some groups of primate in primate

     6    centers, essentially all of the individuals infected

     7    -- Jonathan Allen at Southwestern, I think, has shown

     8    that all of the baboons there do have foamy virus.  So

     9    although there aren't so many studies in the wild,

    10    these viruses have been isolated from wild animals, as

    11    well.

    12                There have been several isolates from

    13    human, and I'd like to speak briefly about that.  The

    14    type species is called HFV, human foamy virus.  This

    15    was isolated from a nasopharyngeal carcinoma cell

    16    culture from a patient from Kenya many years ago.

    17                Recent studies, however, from the group at

    18    CDC have clearly shown that this human virus is

    19    basically a chimpanzee virus, and interestingly, it

    20    clusters very closely with isolates from

    21    Schweinfurthiae chimpanzees, which is the only

    22    chimpanzee that's present in East Africa where the HFV

    23    isolate came from, which clearly suggests that HFV was

    24    in this culture because the patient might have had

    25    contact with the chimp and acquired the virus through

                                                                         63

     1    a zoonotic infection.

     2                And all the other isolates from people are

     3    clearly linked to having been bitten by a monkey or a

     4    chimpanzee.

     5                So one interesting thing about HFV is

     6    despite the lack of pathology in vivo, as far as we

     7    know, in culture you get two types of infection.  The

     8    first and most dramatic is a cytopathic infection of

     9    fibroblast, and this was what gives the foamy virus

    10    their name because these cells become highly

    11    multinucleic and syncytia, multi-syncytia form.

    12                But there's also a second type of

    13    infection, a long term, persistent infection, and this

    14    is seen and our lab has found it in a variety of human

    15    cell lines, T cells, erythroid cells, monocytic cells,

    16    et cetera, and in these long term, persistent

    17    infections you see absolutely no CPE.  The cells

    18    become infected.  They grow perfectly normally, and

    19    you would not know that they were foamy virus infected

    20    without doing PCR or assaying the virus.

    21                So this is clearly a problem when one

    22    deals with material from primates.  One needs to

    23    assume that the primates are probably infected with

    24    foamy virus, and although many isolations of cell

    25    lines from primates lead to cytopathicity and clearly

                                                                         64

     1    show their foamy virus, since we know very little

     2    about the cells in vivo that are actually infected,

     3    it's very possible that you could have cell lines from

     4    primate cultures that are infected with foamy virus

     5    without CPEs.

     6                This is an example.  This is an indicator

     7    cell line where we have LTR driving betagal in a

     8    hamster fibroblast line, and when you infect these

     9    cells with foamy virus, you can see that that turns on

    10    the betagal expression from the tas transactivator. 

    11    You get highly cytopathic cultures.

    12                And here's an example of a really giant

    13    multi-nucleus syncytia that can occur at high

    14    multiplicity.  On the other hand, when we infect a

    15    variety of human cell lines, these cell lines grow for

    16    years.  This only goes up to 40 weeks, but we've grown

    17    these cells.  They continually produce virus, but they

    18    are never cured of the infection, and there's no CPE.

    19                Here we show that in this experiment we

    20    could not infect a V cell, but this is not a problem

    21    of infectivity.  This is a problem of viral

    22    replication.  In fact, now that we have vectors marked

    23    with gfp and work from other laboratories as well

    24    shows that there are basically no vertebrate cells

    25    that are immune to foamy virus infection.  Everything

                                                                         65

     1    from fish on upwards, and in fact, in humans no cell

     2    types to be seen to be immune to foamy virus.  So

     3    whatever the receptor is, it's extremely widespread.

     4                So the life cycle of foamy virus is very

     5    similar to that of other retroviruses with a couple of

     6    striking exceptions.  As I mentioned or I will mention

     7    again, the virus, most of the viral budding is through

     8    the endoplasmic reticulum, and although some virus

     9    does bud from the plasma membrane, again, in order for

    10    the virus to bud, there must be glycoprotein.

    11                If you have an M minus mutant, virus does

    12    not bud from the cell, and this tends to be a

    13    cytopathic event.  If there is no envelope, the cells

    14    die very rapidly.

    15                The other important thing to mention is

    16    that work from our lab, as well as another lab,

    17    strongly suggests that reverse transcription in this

    18    virus is a late step in infection, and that means that

    19    the functional genome in foamy viruses is really DNA

    20    rather than RNA.

    21                And this is done using AZT as an inhibitor

    22    and show that the stuff that is sensitive to AZT is a

    23    late step rather than an early step in the life cycle

    24    of other retroviruses.

    25                Another point to be made is that there are

                                                                         66

     1    huge numbers of intracellular particles.  In fact,

     2    most foamy virus is intracellular.  Only about one to

     3    five percent of the virus buds from the cell.  The

     4    rest of it is cell associated, and this has suggested

     5    that perhaps like hepadenoviruses, there could be some

     6    type of recycling step where some of these

     7    intracellular particles get back into the nucleus, and

     8    I'll get to that in a moment.

     9                So in terms of the HFV genome, by doing

    10    very sensitive PCR and RT PCR, we found that about 25

    11    percent of the particles released from cells contain

    12    apparently full length, double stranded DNA.  The AZT

    13    experiment strongly suggests that the functional

    14    genome is DNA so that even though there are a large

    15    number of RNA particles, we don't believe that these

    16    are infectious, and they're probably remnants of

    17    abortive reverse transcription events.

    18                And we've also been able to show that if

    19    you extract DNA from extracellular particles, it is

    20    infectious if you put it back into cells with

    21    lipofectamine.

    22                Despite the fact that the foamy virus

    23    functional genome is DNA, what is packaged is RNA, and

    24    this is RNA's protection experiment using wild type

    25    human foamy virus or a deletion mutant in the pol

                                                                         67

     1    gene, looking at RNA's protection.

     2                And I haven't shown you the controls here

     3    for particle numbers, but when you look, and this is

     4    RNA's protection that really only just looks at RNA in

     5    the particles and not DNA.  If we RNA, we don't see

     6    any nucleic acid in the particle, and the same is true

     7    for the pol mutant.

     8                And what we found from this study when we

     9    compared the amount of nucleic acid packaged in the

    10    pol mutant and wild type, it's exactly the same when

    11    it's normalized to the number of particles.

    12                So you don't need polymerase to get genome

    13    into the particle.  Of course, this is completely

    14    dead.  So what's packaged is RNA, and DNA just occurs

    15    sometime during assembly or egress from the cell.

    16                We also believe there are large numbers of

    17    particles in the intracellular particles that contain

    18    DNA.  So when you look at a copy number of foamy virus

    19    persistently infected cells or acutely infected cells,

    20    you can find hundreds or thousands of copies of DNA

    21    per cell, and this has made it very difficult to look

    22    at integrated genomes in these cells.

    23                This is a schematic of what we think the

    24    foamy virus looks like.  Instead of having an RNA

    25    genome, it has a DNA genome.  

                                                                         68

     1                Another feature of the foamy virus which

     2    I didn't mention, but which is also very striking is

     3    that the gag polyprotein is not cleaved except for

     4    four KD at the C terminus.  So you never get

     5    maturation to caps at matrix and nuclear caps as in

     6    other retroviruses.  Basically we believe that this

     7    gag protein is multi-functional, that the carboxy end,

     8    which has many basic residues, probably behaves like

     9    the core protein of hepadenovirus and interacts with

    10    the DNA genome in the particle, and that probably the

    11    amino terminus behaves somewhat like matrix and part

    12    of this protein also behaves like tapsin (phonetic).

    13                So we were interested in looking at

    14    integration, and to do this we used a chronically or

    15    persistently infected H-92 cell line, and we cloned

    16    out single cell clones of this virus, and in these

    17    experiments, these southern blot experiments, we cut

    18    with NAG-1, which cuts ounces in the genome, and use

    19    the bet probe.

    20                And if you do such an experiment, you can

    21    see a large number of -- a large amount of viral DNA. 

    22    This is the cut DNA here.  This is the small amount of

    23    DNA that was not cut.

    24                And in many experiments this obscures the

    25    background of integration.  So what we do is we treat

                                                                         69

     1    the cells for several weeks with AZT, and this gets

     2    rids of all the intracellular DNA in the particles,

     3    and then you can easily see the integrated copy number

     4    in these single cell clones, and what's very striking

     5    is that there is a large number of integrated DNAs.

     6                And we've counted upwards of 20 bands per

     7    cell of, as I said, single cell clones of foamy virus

     8    infected, which were leukemia cells.  We've confirmed

     9    that these are single integrations by cloning out the

    10    junction fragments and showing that they're not

    11    duplications or repeats.

    12                These are bona fide new integrations. 

    13    We've also done FSH analysis.  This isn't a very good

    14    slide, and the arrows are not pointing to all of the

    15    integrated copies.  You'll just have to take my word

    16    for it.  There's a rough correlation between the

    17    integration number that we see by a southern blot and

    18    by FSH analysis, and we do see up to about 20

    19    integrated copies per cell genome of this virus as

    20    well.

    21                And we have been very interested in the

    22    mechanism of these multiply integrated pro viruses. 

    23    I'm not showing you the data.  There is, in fact, a

    24    NLS sequence within the gag genome foamy virus which

    25    Axel Rethwelm's lab shows behave as an NLS.  So after

                                                                         70

     1    infection, much of the newly synthesized gag protein

     2    goes back into the nucleus.

     3                And by using a mutant in the NLS, so that

     4    if you delete the NLS the virus can grow fine in

     5    tissue culture, it does have a slightly lower titre,

     6    and we've shown that by deleting the NLS we prevent

     7    accumulation of all of those multiple integrated

     8    copies, and we now have clones of cells that only have

     9    one, two, or three copies per cell instead of ten or

    10    20 copies.

    11                So this implicates that there could be

    12    some kind of intracellular recycling mechanism that's

    13    responsible for the high copy number.  However, what

    14    complicates this is that we've recently found that the

    15    foamy viruses also have another unexpected feature,

    16    and that is we have made a vector in which we have

    17    replaced the bet gene with GFP, and in this case we

    18    also put in the RSV strong promoter.

    19                And when we use a vector to infect either

    20    BHK cells or these erythroleukemia cells, we get a

    21    high infectivity.  This is shown with the GFP

    22    fluorescent, but surprisingly if we compare

    23    infectivity of either these uninfected H-92 cells or

    24    this single cell clone that has about 20 integrated

    25    copies, we find there's basically no difference in the

                                                                         71

     1    ability to reinfect the uninfected  -- the infected

     2    cells versus the uninfected cells.

     3                So at least in these persistently infected

     4    cells that have huge copy numbers, they're not immune

     5    to super infection, and this is very surprising for a

     6    retrovirus.

     7                So, therefore, we can't say whether we're

     8    getting accumulation of all of these integrated copies

     9    by an intracellular pathway or an extracellular

    10    pathway, and this also suggests that even if a cell is

    11    infected by foamy virus, it would not be immune to

    12    superinfection by another foamy virus or more foamy

    13    virus.

    14                The other point about foamy -- foamy

    15    viruses are not very well studied.  There's very

    16    little information about the packaging sequences of

    17    these viruses.  Several groups have tried to make

    18    viral vectors, and so far this isn't an attractive

    19    genome for viral vectors.  

    20                For one thing, the virus, as I said, is

    21    probably not pathogenic in either accidental or

    22    natural hosts.  It has a very large genome, greater

    23    than 11 KV.  It has at least one gene that we believe

    24    we can delete without -- at least in vitro -- without

    25    any untoward effects on the virus.  It has two

                                                                         72

     1    promoters, so it's a very flexible genome.

     2                And another point is work from Germany has

     3    shown that in infected monkeys you can find foamy

     4    virus DNA in every organ in the body, including the

     5    brain, although there's very low viral replication in

     6    vivo.

     7                So it might be a good gene delivery target

     8    to evoke a wide variety of organs.  So in vector

     9    development several groups have found that, in fact,

    10    there are probably at least two packaging or two sys

    11    acting regions in the RNA, and one is at the five

    12    prime end of the genome where you would expect a psi

    13    sequence to be, but surprisingly there's also a region

    14    in the pol gene that's required for transfer of vector

    15    sequences.

    16                Whether this is another packaging sequence

    17    or has another sys acting RNA function is not known. 

    18    For instance, since pol is not made as a gag-pol

    19    fusion protein and the method of incorporating pol

    20    into the particles does seem to require a gag-pol

    21    interaction, but we can't rule out that perhaps it

    22    also needs to bind to the RNA, and so this region of

    23    pol could be a pol binding sequence.  We don't really

    24    know.

    25                So the packaging regions of this virus are

                                                                         73

     1    not at all well understood at all.

     2                So in summary, all retroviruses have

     3    packaging signals.  Unfortunately those of the foamy

     4    viruses have not yet been delineated.  Foamy viruses

     5    package RNA, although the functional genome appears to

     6    be DNA.

     7                In some cells, at least in tissue culture,

     8    foamy virus infection leads to multiple integration. 

     9    This is an interesting point because one would think

    10    that if such a thing occurred in vivo, and we have

    11    absolutely no evidence for it, that foamy viruses

    12    would be all set up for promoter insertions and

    13    inductions of tumors in infected animals.  Yet this

    14    has never ever been seen.

    15                So it's possible that the replication in

    16    vivo is so meager that the virus never really does

    17    multiply integrate, but nobody has ever been able to

    18    or nobody has ever looked in vivo for foamy virus

    19    integration sites.

    20                Nothing is known about the recombination

    21    between foamy virus and other retroviruses.  Clearly,

    22    experiments that are done in monkeys, infecting them

    23    with other viruses such as SIV or viral vectors, there

    24    is probably foamy virus in all of those animals.  So

    25    it would be very interesting to know something more

                                                                         74

     1    about how foamy virus interacts with other types of

     2    retroviruses.

     3                And also any packaging cells derived from

     4    primates, bovine or feline species need to assess the

     5    effect of foamy virus as well.

     6                And I think that's all I have to say.

     7                (Applause.)

     8                CHAIRPERSON ROSENBERG:  We need to keep

     9    the questions, I'm afraid, brief because we are

    10    seriously behind.  So I believe we're supposed to be

    11    at the break, but we still have one more speaker.

    12                PARTICIPANT:  Maxine, does anybody know if

    13    vaccines have been checked for foamy virus

    14    contamination?

    15                DR. LINIAL:  As far as I know, no.

    16                PARTICIPANT:  You mean nobody has looked

    17    or as far as you know?

    18                DR. LINIAL:  I don't know.  There are very

    19    few reagents.  I mean, there are reagents for the so-

    20    called human or chimp foamy virus, but as far as I

    21    know, there are no good antibody reagents.

    22                PARTICIPANT:  There are.

    23                DR. LINIAL:  There are?

    24                PARTICIPANT:  There are?  They are

    25    checked.  Okay.

                                                                         75

     1                DR. LINIAL:  Are they checked for all of

     2    the simian foamy viruses?

     3                PARTICIPANT:  No.

     4                PARTICIPANT:  By PCR?  Is that --

     5                PARTICIPANT:  (Inaudible.)

     6                CHAIRPERSON ROSENBERG:  Could someone

     7    repeat this so that --

     8                PARTICIPANT:  I think it's by a

     9    combination of tests, including PCR and infectivity

    10    tests; is that right?  Okay.

    11                DR. LINIAL:  One problem is that these

    12    monkeys do get cross-infected with other foamy

    13    viruses.  So, you know, I don't know how many you're

    14    looking at.

    15                PARTICIPANT:  Since foamy viruses seem to

    16    be breaking all of the rules, I wonder if it's worth

    17    asking or is it known whether integration is required

    18    for replication, whether there's significant -- since

    19    there are so many copies of DNA, et cetera, whether

    20    there can be significant expression and replication in

    21    the absence of integration.

    22                DR. LINIAL:  My lab, as well as a lab in

    23    Germany, have made a DD35E integrate mutants, and at

    24    least in tissue culture it's completely dead.  So we

    25    believe integration is required.

                                                                         76

     1                CHAIRPERSON ROSENBERG:  I think we need to

     2    move on.  The last talk in this session is by John

     3    Kappes.

     4                DR. KAPPES:  Just a momentary delay. 

     5    Technology, MacIntosh, a little slower in booting. 

     6    Perhaps I'll begin a short introduction.

     7                The focus of my work -- there we go -- has

     8    been on the possible use of lentiviral vectors for

     9    gene therapy.  Off to a bad start.  That is the second

    10    slide.

    11                The principal concern for using lentiviral

    12    vectors for gene therapy is that they may recombine to

    13    produce replication competent retrovirus, and

    14    underlying the concerns for replication competent

    15    retrovirus is genetic recombination.

    16                There have been a number of different

    17    types of -- and I'm going to focus really just on HIV-

    18    based vectors, although I'll probably refer to them

    19    many times as lenti -- but there have been a number of

    20    different HIV-based lentiviral vectors produced to

    21    minimize the pathogenic properties of any RCR that

    22    could emerge, and those include deletions of most of

    23    the accessory genes, deletions of even the TAT and REV

    24    regulatory genes, the lesions in U3, and while I won't

    25    focus on a lot of these details, I will focus on,

                                                                         77

     1    again, what I think is fundamental to understanding

     2    the risks associated with generating replication

     3    competent retrovirus, and that is genetic

     4    recombination.

     5                The assays which have been used thus far

     6    include, that is, to measure recombination or, if you

     7    will, really more because of the way they've been

     8    applied measurements of RCR, include gag transfer --

     9    oh, TAT transfer it should be -- gag transfer, and DNA

    10    mobilization of marker rescue assays.

    11                It's unlikely, and I'm sure they have not

    12    been suggested by the authors who published on these

    13    assays to be adequate indicators of the risk

    14    associated with these viruses or these viral vectors

    15    in vivo.  Especially that's true in the long term.

    16                So today I will present data from an

    17    approach that I devised to understand the risk, if you

    18    will, of using HIV-based vectors through an analysis

    19    that focuses on genetic recombination.

    20                First, I'll present one slide, the unique

    21    difference that I've used to enable the detection of

    22    recombinant viruses and their analysis, and then

    23    several slides I'll show the detection and

    24    characterization of these recombinants, both

    25    biologically and genetically, and finally, in one of

                                                                         78

     1    two slides I'll show how I have further disarmed or

     2    dismantled or split the functions of the lentiviral

     3    based vector to improve safety.

     4                This depicts the three component systems

     5    for HIV-based vectors, not necessarily analogous to

     6    what might be thought of as third generation, but the

     7    important point is that there is a packaging

     8    construct, a vector construct, and an envelope

     9    construct, and just for this one slide, I wanted to

    10    point out in particular the TAT gene because my

    11    recombination assay is based on TAT.

    12                That is, if recombination occurs between

    13    the vector and the packaging construct, and if TAT is

    14    included, I will be able to select for the recombinant

    15    using this approach.

    16                If lentiviral vectors are generated

    17    through transfection of 2-9-3 T cells, which is what

    18    I will show in every case, genetic recombination can

    19    occur during reverse transcription to generate an LTR

    20    TAT containing structure.  It could contain gag; it

    21    could contain the full packaging construction, but

    22    minimally if it contains TAT and TAT is expressed, it

    23    could confer resistance to puromycin in the cell line

    24    that I call Hela-puro.

    25                This cell line was transduced with this

                                                                         79

     1    construct, which confers resistance to puromycin

     2    selection when the cells infected with a virus or a

     3    vector containing and expressing TAT.

     4                A couple of other points worth noting for

     5    this slide because it's really what I will use in

     6    terms of the components that generate the vector and

     7    the data I'll show in every slide, except for a couple

     8    at the end.

     9                The packaging construct is driven by a CMB

    10    promoter.  It contains gag-pol-vif.  Vpr, vpu, mpf

    11    (phonetic) are deleted, and also importantly nef is

    12    deleted, importantly because there is no overlapping

    13    sequence at this end of the genome, which will become

    14    important for reasons which hopefully will be obvious

    15    later.

    16                So to generate the HIV vectors, I

    17    transfect these three constructs into 2-9-3 T cells,

    18    and that's depicted here.

    19                Through transfection bioparticles are

    20    generated, and these bioparticles can be used to

    21    infect the Hela-puro cell line.

    22                If TAT is expressed, the cell should be

    23    resistant, and that's exactly what is shown here. 

    24    These are the colonies stained with crystal violet, I

    25    believe, about nine days after puromycin selection,

                                                                         80

     1    and what's shown is that there is approximately 1,000

     2    colony forming units per ten to the seventh infectious

     3    particles.

     4                Also, importantly, if this infection is

     5    done in the presence of a niverapine, there are no

     6    resistant colonies detected, implying that it's

     7    mediated through the HIV-1 reverse transcriptase, and

     8    in particular, I chose niverapine because it's

     9    specific for HIV-1 RT.

    10                As I suggested, recombination could occur

    11    through any region of the packaging construct.  As

    12    long as TAT was picked up, resistant colonies could be

    13    produced.  So to confirm TAT was actually present, we

    14    designed primers to amplify the first exon, and that's

    15    what's depicted here in what I call the lenti pool.

    16                This is the lenti pool.  After the cells

    17    were expanded, the high molecular DNA was extracted,

    18    and PCR shows detection of a 219 base pair fragment

    19    similar to that detected in proviral DNA.

    20                Because or at least in part because data

    21    had been published that showed there was no TAT

    22    transfer, no gag transfer, no marker rescue using

    23    lentiviral vectors, I wanted to address each of those

    24    points.

    25                And the first series of slides I just

                                                                         81

     1    showed would suggest that there is TAT transfer if the

     2    system is sensitive enough to detect it.

     3                This slide detects a slightly different

     4    approach for looking toward gag transfer.  This is the

     5    path I just described for TAT transfer.  Because

     6    recombination can occur in many ways where an

     7    infectious virus would not be generated, I chose this

     8    pathway for gag transfer because I was really looking

     9    for an open gag reading frame.

    10                So the difference is instead of going at

    11    the heel of puro cells, I infected the virus particles

    12    that were generated by transfection into 2-9-3 T, and

    13    in 2-9-3 T, if a recombinant forms such that you had

    14    an open gag-pol rating frame, you would expect that

    15    particles could be generated, and if those particles

    16    were pseudotyped by transfecting VSVG into the 2-9-3

    17    T cells two days after infection, then they could

    18    infect the Hela-puro cell line and confer resistance,

    19    and that data is shown here.

    20                We detected 540 resistant colonies when

    21    the virus derived by 2-9-3 T cells was used to infect

    22    the Hela-puro cell line, suggesting both DNA

    23    mobilization and gag transfer.

    24                Another way of looking at DNA -- oh, let

    25    me point out that we also confirmed from the

                                                                         82

     1    supernatants of the puromycin resistant cells the

     2    presence of gag protein, both in culture supernatants

     3    and in viral pellets.

     4                Importantly, the amount of gag detected

     5    was increased by about tenfold if TAT was transfected

     6    into those puro resistant cells, suggesting that the

     7    LTR was truly linked, that is, expressed in sys, with

     8    the packaging construct.

     9                Another experiment, which was just a

    10    further extension of what I've already shown related

    11    to the gag transfer, to more specifically and more

    12    clearly differentiate between artifacts and true DNA

    13    mobilization is the depicted here.

    14                The recombinant, which I referred to as

    15    mobilizing gag or gag transfer is depicted here.  We

    16    don't know at this point whether this is the exact

    17    structure, but it should contain an LTR gag-pol

    18    reading frame, and certainly tat and rre.  If this

    19    structure is respent in the Hela-puro line, it's

    20    possible that when it produces particles because the

    21    Hela-puro cell line contains puromycin and a packaging

    22    signal further puro RNA strained, that it, too, could

    23    be packaged into these particles, mobiled to heal a

    24    TAT line in this case, and confer resistance to

    25    puromycin.  And indeed, that's the result shown here.

                                                                         83

     1                So the data indicate by three assays

     2    recombination of the lentiviral vector components, in

     3    particular, recombination between the gene transfer

     4    vector and the packaging construct.  So all of the

     5    data I've shown just far are really just biological

     6    evidence, and now I would like to turn to the genetic

     7    and structure data, that also supports these

     8    conclusions.

     9                If a combination truly occurred between

    10    the packaging construct and the vector, you'd expect

    11    the two to be linked, and if I used primers in the U3

    12    region and at the end of gag, I would expect to

    13    amplify a product from this lengthy viral pool, which

    14    is what it's called.  This was the product of what I

    15    call gag transfer.

    16                I would expect to amplify a PCR fragment

    17    of about 2,000 base pairs, and indeed, that is what's

    18    shown.

    19                If this PCR product was cloned and

    20    sequenced, and if there was recombination into the

    21    packaging construct, what would be expected is this

    22    sequence, that is, the initiation codon for gag and a

    23    sequence depicted here, and in ten out of ten

    24    recombinants, this is what we found.

    25                This sequence, it's also worth pointing

                                                                         84

     1    out, is different than that of the original vector

     2    sequence.  Remember the vector includes part of gag

     3    because it's important for packaging.  There are clear

     4    differences between these nucleotides.  So this is

     5    further evidence that recombination between the vector

     6    and packaging construct occurred.

     7                Looking instead of here on the three-

     8    pronged end of the genome, we used primers that would

     9    span from TAT, shown by this yellow bar, into the U3. 

    10    By PCR we amplified three, maybe four small bands, and

    11    this one here, which corresponds in weight to what we

    12    expected, was cloned and sequenced.

    13                And that analysis is shown here, and I'll

    14    show this in more detail on a couple of later slides,

    15    but the important point is that by sequencing through

    16    this entire region, what we found is that there is

    17    genetic evidence for recombination similar to what we

    18    found on the five prime end on the three prime end,

    19    and in fact, genetic recombination or different

    20    recombinants were found.

    21                There were six recombinants that were of

    22    this type, one of this type, one of this type, and one

    23    of this type, and what are these types?  This is what

    24    I show on the next one of two slides.

    25                Also depicted here is how I suggest this

                                                                         85

     1    recombination is occurring.  Remember reverse

     2    transcription is going to start up here somewhere, and

     3    the growing DNA chain will anneal to the RNA on the

     4    three-prong end, and as it's extended, in one of the

     5    situations I show I would suggest recombination

     6    occurring into the poly A tail of the packaging

     7    construct, in another case, into the poly A tail at a

     8    different position, and in another into the poly A

     9    tail at yet another position.

    10                What these recombinants have in common is

    11    that all of them extended up into 108 base pairs past

    12    the end of U3 or the beginning of U3, depending on

    13    which way you're going.

    14                The other type of recombinant detectable

    15    is one which went in at yet a different position, in

    16    position 45 or it contained 45 As downstream of the

    17    poly A signal, but it only contained 32 base pairs

    18    upstream of the U3 sequence.

    19                This finding was unexpected, and I thought

    20    for a long time about how it might be occurring, and

    21    while I still can't explain it, here's a possible

    22    rational model based on the limited information I

    23    have.

    24                If this is the RNA tail, poly tail, from

    25    the packaging construct, and this is the DNA which is

                                                                         86

     1    growing from reverse transcription, what I noticed in

     2    all cases just prior to where the recombination even

     3    occurred -- and remember eight of the recombinants

     4    jumped from this position into various areas of the

     5    poly A, and one of them jumped from this position into

     6    I think it was number 47A downstream of the poly A

     7    signal.

     8                What they all have in common is a sequence

     9    of Ps, which was suggested could bring those

    10    structures together to allow recombination.  So based

    11    on this data, I think there are several safety

    12    concerns which I could point out.

    13                One, the regeneration of envelope

    14    deficient recombinant lentivirus requires

    15    recombination of only two genetic elements.  That

    16    would be the packaging construct and the vector.

    17                The LTR packages signal, gag-pol LTR

    18    recombinant contains all of the necessary viral

    19    replication machinery for replication minus envelope.

    20                The long term risk of stably integrated

    21    structures that might look something like this is

    22    unknown.  Lentiviral vector recombinants may infect

    23    nondividing cells if envelope is provided in trains,

    24    and I think this might be particularly worthwhile to

    25    consider because unlike what we've experienced with

                                                                         87

     1    Maloney, the lentiviral vectors, if this structure

     2    exists in vivo, and it can be mobilized to nondividing

     3    cells.

     4                I think especially this issue is important

     5    in cells transduced or CD-34 positive stem cells

     6    transduced with lentiviral vectors since it could

     7    represent a reservoir for ongoing gene expression.

     8                Okay.  Quickly just a couple more slides. 

     9    One possible approach to minimize the risks associated

    10    with genetic recombination between the vector and the

    11    packaging component I would depict, I think, in just

    12    two slides, and this is really based on a study that

    13    Wood, et al., published in EMBO in 1997, where instead

    14    of incorporating reverse transcriptase and integrate

    15    as part of the gag-pol precursor protein, these genes

    16    refused to vpr and expressed in trans with an RT

    17    integrate minus provirus, and what was demonstrated

    18    was that the provirus or that the RT integrate was

    19    packaged.  It was processed, including into mature

    20    integrate and RT, P-51 and P-56 components, and they

    21    were fully functional, so provided an opportunity to

    22    further reduce the basic components of the HIV vector

    23    system by deleting RT and integrate from the packaging

    24    construct, and that's what's depicted here.

    25                So now the packaging construct of what I

                                                                         88

     1    call translentiviral vector looks like this.  It's

     2    broken apart into RT integrate infused with vpr,

     3    provided separately from gag-pro.

     4                And one slide to show data, although there

     5    are others that show very similar results.  If the 2-

     6    9-3 T cells are infected with translentiviral vector

     7    derived from transfection of all the vector

     8    components, this structure can produce viral

     9    particles, but upon infection of the Hela-puro cell

    10    line, there are -- no resistant colonies are

    11    generated, and that's due to a lack of reverse

    12    transcription and integration machinery because even

    13    though infection might occur in the absence of reverse

    14    transcription of the genome, no resistance will be

    15    conferred.

    16                So finally, in conclusion, recombination

    17    does occur between the packaging construct and the

    18    gene transfer vector.  This structure is regenerated,

    19    which contains all the basic replication machinery

    20    minus envelope.

    21                LTR containing recombinants integrate into

    22    the chromosomes of the target cells.  The recombinants

    23    express viral proteins, including tat, gag, and the

    24    entire gag-pol precursor protein.

    25                The expression of the integrated gag and

                                                                         89

     1    pol genes produces virus particles which can be

     2    mobilized if envelope is provided in trans.

     3                Recombination within the RNA poly A tail

     4    may represent a mechanism by which genes without

     5    homologous sequence can be mobilized, including

     6    oncogenes, which we discussed at some length

     7    yesterday.

     8                Separating RT and integrate from the

     9    packaging vector controls the regeneration of a

    10    functional gag-pol structure which is absolutely

    11    required for RCR and DNA mobilization.

    12                Thank you.

    13                (Applause.)

    14                CHAIRPERSON ROSENBERG:  I don't think it's

    15    on.  Switch on the bottom maybe.

    16                DR. KUNG:  I just want to comment that, in

    17    fact, in support of your data there are two --

    18                PARTICIPANTS:  Can't hear you.

    19                CHAIRPERSON ROSENBERG:  Why don't you just

    20    repeat his question after or you can come up here?

    21                DR. KUNG:  Okay.  I just want to comment

    22    that in two naturally occurring avian retrovirus that

    23    carry oncogenes; one is avian rhesoblastosis.  One of

    24    the isolate recombination takes place at the poly A

    25    tail.  Okay?  In that case it's 2-lycine.  Is there a

                                                                         90

     1    crossover point?

     2                Okay, and the other is fips (phonetic). 

     3    I think Mike Bishop published that.  Recombination

     4    also takes place at the poly A sequence.

     5                So there are two naturally occurring

     6    oncogene carrying.

     7                DR. KAPPES:  I was unaware of that.  The

     8    data were very surprising.

     9                CHAIRPERSON ROSENBERG:  I think this

    10    should be the last question because we are actually

    11    even beyond the break.

    12                DR. LINIAL:  What kind of titres are you

    13    getting from the last where you break up RT into PR

    14    and RT integrate?  What kind of titres of vector are

    15    you getting from those constructs?

    16                DR. KAPPES:  Let me be sure I understand

    17    the question.  What kind of titres are we getting when

    18    we separate RT and integrate?  Consistently, and maybe

    19    this isn't the best answer, but it's important I

    20    believe, consistently about three to fivefold less

    21    than what we derive from the lenti.  Under our best

    22    conditions without concentration we can get near ten

    23    to the seventh per mL from the lenti.  So under best

    24    conditions, maybe five times ten to the sixth with the

    25    translenti.

                                                                         91

     1                CHAIRPERSON ROSENBERG:  I'd like to thank

     2    all of the speakers.  Hopefully there's time for a

     3    short break before the panel.

     4                (Whereupon, the foregoing matter went off

     5                the record at 10:12 a.m. and went back on

     6                the record at 10:32 a.m.)

     7                DR. HUGHES:  I would ask everyone to

     8    please come and take their seats.

     9                Mindful of the fact that we are what

    10    stands between you and lunch, we're going to try to do

    11    this in a timely way and conclude the session as it

    12    was originally scheduled.

    13                I think in keeping with the spirit that

    14    the conveners of the meeting suggested for us, we need

    15    to consider the issues that are before us in the

    16    context of the proposal to use neoplastic cells as

    17    vehicles for generating vaccine strains.

    18                And I think in that context, it's probably

    19    important to actually give consideration to the

    20    differences in the types of vaccines that people might

    21    wish to generate, for example, the difference between

    22    live vaccine, kill vaccine, or sub-unit vaccine.

    23                And what we've heard in the last couple of

    24    days are a reasonably good description of the manifold

    25    interactions that viruses have with each other and

                                                                         92

     1    with their cellular hosts, and I think we need to give

     2    consideration to that in terms of how we would think

     3    about detecting interactions between viruses and other

     4    viruses or viruses and their hosts so that the

     5    vaccines that would be produced would be as safe as

     6    possible.

     7                And as Dr. Lewis suggested when the

     8    meeting was opened, we need to think in terms of what

     9    we mean by being safe.  We need to think about the

    10    problems of detecting adventitious agents, and we also

    11    need to understand and think about precisely the kinds

    12    of tradeoffs that exist between understanding issues

    13    of safety and issues of the benefits that are provided

    14    by affected vaccines.

    15                And I think with those remarks, I would

    16    open the discussion both to the panel and to the

    17    audience.  Are there comments from the panel?  Does

    18    anyone have any remarks they'd like to make to begin?

    19                No.  The audience, are there questions? 

    20    We may get to lunch early.

    21                (Laughter.)

    22                DR. HUGHES:  Surely the prospects of the

    23    kinds of interactions that we've seen, interactions

    24    not only between endogenous retroviruses and exogenous

    25    elements, but between retroviruses and herpes viruses

                                                                         93

     1    must have provoked some thoughts that are relevant to

     2    this issue of vaccine development.

     3                Please.

     4                MR. KRAUSE:  Just to get things rolling,

     5    I'm Phil Krause from the FDA.

     6                What does the panel see as the likelihood

     7    that these kinds of interactions or risks or potential

     8    risks might be greater with neoplastic cells than with

     9    other types of cells?

    10                And also, as you say, what kinds of viral

    11    vaccines does one have to be more concerned about? 

    12    You know, I can imagine based on what we heard that we

    13    might be more concerned about these kinds of things

    14    happening if one is dealing with larger DNA viral

    15    vaccines or with retroviral vaccines than with, you

    16    know, small RNA virus vaccines, for instance, but I

    17    would be interested in what people have to say on

    18    that.

    19                DR. HUGHES:  Ladies and gentlemen.

    20                DR. EVANS:  Well, I'm not sure that

    21    there's much difference between a transformed cell

    22    line and a regular cell line, especially like a

    23    primary cell line where you've got a lot of

    24    differentiated cells there at least initially, but

    25    like I said, I'm not sure.  I don't know.  I don't

                                                                         94

     1    know if there's elevated copies in certain cell lines

     2    of endogenous sequences that might be packaged and

     3    transferred, or I don't know if there might be change

     4    when you infect that cell line with some other agent

     5    because you could be inducing sequences and promoting

     6    transfergenic information.

     7                In terms of the types of viruses that --

     8    types of vaccines you might make because of the degree

     9    of heterologous pseudotyping between different types

    10    of viruses in terms of retrovirus transfer, I think

    11    you've got to -- anything that has an envelope protein

    12    that directs the tropism of that virus you're going to

    13    have to be concerned about in terms of transferring

    14    the vector.

    15                In terms of that having a deleterious

    16    effect or whether or not you could detect it, you

    17    know, and eliminate it by monitoring is a different

    18    question.

    19                DR. LINIAL:  In terms of foamy viruses, I

    20    would say that the real danger there is using things

    21    that aren't cell lines because presumably you can

    22    easily assay cell lines, and probably most of them

    23    won't make it to cell lines if they have foamy virus,

    24    but you know, using primary explants from monkeys, you

    25    probably run a higher risk of having foamy virus in

                                                                         95

     1    your material.

     2                DR. PATIENCE:  I think with respect to the

     3    endogenous retrovirus sequences, I think basically we

     4    have to consider every potential cell line on an

     5    individual basis.  As far as I'm aware, at least,

     6    there's no correlation between a neoplastic cell type

     7    and an ordinary cell type.  It really has to be looked

     8    at on every cell.

     9                With respect to the types of gene therapy

    10    packaging systems, there's never been any reports of

    11    lentiviral related sequences in the human genome, and

    12    with respect to the leukemia-based virus systems

    13    probably the closest family is the RTV LH system,

    14    which we could see no cross-packaging by MuLV gag.

    15                MR. FRIED:  Mike Fried, ICRF.

    16                From what we talked about yesterday,

    17    what's the chance of the capture of a cellular gene or

    18    an oncogene?  Has anybody done experiments where

    19    there's an activated rash and then take it and try to

    20    transfer it or an antibiotic gene?

    21                I mean is it easy to pick up or not?

    22                DR. HUGHES:  The best data as far as I

    23    know suggest that those events happen so rarely that

    24    they're not seen in cultured cells, if we're talking

    25    about situations in which there's no homology between

                                                                         96

     1    the cellular component and the viral component. 

     2    You're talking about the straight acquisition.

     3                What can be made to happen, depending on

     4    the systems, and perhaps Dr. Kung would comment for

     5    us, at reasonably frequent -- reasonably frequently in

     6    infected animal systems.

     7                So I think there is a substantial

     8    difference in terms of the history that we have for

     9    cells in culture and infected animals.

    10                Hsing-Jien, you might want to speak to

    11    your experiences with respect to the herpes system.

    12                DR. KUNG:  Okay.  I guess, again, all of

    13    this will vary because of selection pressure.  So I'm

    14    talking about still in terms of tumor selection.

    15                In the case of recombination generates so-

    16    called acute oncogenic containing virus, the

    17    efficiency vary tremendously, but one of the, I guess,

    18    champion is the avian erythroblastosis virus that

    19    Harriet Robinson reports about 50 percent; you can get

    20    50 percent of the tumors that are released oncogenic

    21    virus.

    22                And there, again, I think the reason is

    23    because you select for the tumor, and another reason

    24    is that in the case of RB activation, the retrovirus

    25    integration utilized the five prime LTR.  I think

                                                                         97

     1    Naomi talked about it a little bit.

     2                Five prime LTR integration, therefore it's

     3    five prime LTR.  It goes through the packaging signal,

     4    then splice into the RB gene.   In so doing, you do

     5    not need any deletion in between, and I think John

     6    Coffin earlier recalled that this reach through type

     7    of transcriptional activation can be as high as 15

     8    percent.

     9                So the provirus position could very well

    10    to pick up a packaging sequence linked oncogene, and

    11    I think that is the theoretical base why the RB gene

    12    captured by retrovirus had been very frequent, and I

    13    think that's a pretty general phenomenon.

    14                In our lab the frequency of generating

    15    those recombinants are very high.  At Harriet

    16    Robinson's lab, she reports about 40 percent.  So it

    17    is very high, but again, this is selection, you know,

    18    selection tumor.

    19                I guess.  I don't know.  Maxine, you have

    20    done something that's without selection, just look at

    21    packaging and recombination, right?

    22                DR. LINIAL:  Well, we were using selection

    23    for a juggling marker, and you if you select for

    24    anything with retroviruses, you can basically find it

    25    at some frequency.  How this would occur in the

                                                                         98

     1    absence of selection is unknown.

     2                DR. KAZAZIAN:  Haig Kazazian from Penn.

     3                I just wanted to make a comment with

     4    regard to the last question on endogenous sequences

     5    and whether there be a difference in their movement in

     6    differentiated cells versus undifferentiated cells.  

     7                I'll be speaking this afternoon about

     8    retrotransposons, and these sequences are transcribed

     9    pretty much specifically in undifferentiated cells. 

    10    So they're transcribed in tumor cells, and they're not

    11    transcribed in differentiated cells.  So you can't get

    12    retrotransposons to move in differentiated cells, but

    13    you can very easily in undifferentiated cells.  So

    14    that is a difference that these endogenous sequences

    15    have.

    16                MS. HOROWITZ:  I'm Jill Horowitz from

    17    Wyeth-Lederle Vaccines.

    18                And I'd like to know if anyone either on

    19    the panel or in the audience is aware of studies aimed

    20    at determining whether any of the millions of

    21    recipients of life vaccines have currently or have in

    22    the past harbored viruses that have resulted from some

    23    of these live vaccine undergoing dangerous

    24    alterations.

    25                DR. HUGHES:  Would anyone care to comment?

                                                                         99

     1                DR. DESROSIERS:  I'm not aware of any

     2    specific examples, but I'm going to take the

     3    opportunity to express an opinion, and it seconds

     4    something that Maxine just mentioned.

     5                In terms of adventitious agents or

     6    unwanted virus populations, I worry much, much more

     7    about the use of primary African green monkey kidney

     8    cells or any such substrate used for production of

     9    vaccines than I do about a tumor cell line or an

    10    established cell line that may be extensively

    11    characterized in terms of potential adventitious

    12    agents or unwanted agents.

    13                In terms of things like what might come

    14    from African green monkey kidney cells that could be

    15    problematic, I don't worry so much about the agents

    16    that one can test for.  I worry about the agents that

    17    you can't test for, that you don't know about.  So

    18    even if there are no errors and no problems and all

    19    the agents that you know to test for it can be tested

    20    for and excluded from every lot of every vaccine, I

    21    worry about the agents that you don't know about.

    22                And I'll be honest and say that I'm

    23    surprised that primary African green monkey kidney

    24    cells continue to be used, and I'm a little bit

    25    disappointed that FDA and whoever is involved had not

                                                                        100

     1    had a more serious effort to move away from primary

     2    African green monkey kidneys.

     3                We all know that there are a number of

     4    neurodegenerative conditions and other conditions

     5    where viral etiologies have been suspected for years

     6    and no viral agent identified.  Maybe they're caused

     7    by viruses, but maybe they're not.

     8                I think despite the fact that there may

     9    not be any specific examples of viruses having been

    10    introduced, more and more examples are accumulating of

    11    the dangers of cross-species transmission either

    12    naturally or through other practices, and that's

    13    something I think we need to really worry about that

    14    can be minimized by going to established cell lines.

    15                PARTICIPANT:  Walid Heneine (phonetic),

    16    CDC.

    17                In response to your question, we've been

    18    looking at transmission risks of the chicken

    19    retroviruses that now we know now contaminate the

    20    measles, mumps vaccines and the yellow fever vaccines,

    21    and to the two types of viruses that were implicated,

    22    there are the endogenous type avian rhekosis

    23    (phonetic) viruses and the endogenous avian viruses

    24    which you will hear about in later sessions.

    25                So far we have not seen any evidence of

                                                                        101

     1    transmissions to vaccine recipients that we have

     2    studied by molecular and serologic means.

     3                PARTICIPANT:  You know, I think you raised

     4    a number of very good points, Dr. Desrosiers, and I

     5    would like to explore them with you a little bit

     6    further in the sense that suppose one has decided then

     7    that for new vaccines one is not going to use primary

     8    cells, but then that neoplastic cells represent

     9    potentially a reasonable alternative.

    10                If I sort of follow your comments to their

    11    logical conclusion, it sounds to me like you would say

    12    then that cells which come from non-human species,

    13    even if they're neoplastic, might potentially be

    14    slightly more dangerous than those from humans in that

    15    sense, and that one might also have to worry about

    16    unrecognized adventitious agents in those kinds of

    17    cells depending on their history.

    18                Would you comment on that?

    19                DR. DESROSIERS:  What's the question

    20    again?

    21                (Laughter.)

    22                PARTICIPANT:  The implication of what you

    23    just said was that the species from which a cell comes

    24    matters, whether it be primary or nonprimary, primary

    25    or neoplastic as one might consider as an alternative,

                                                                        102

     1    and there's the further implication raised by what you

     2    said that, in general, one tends to worry about in

     3    this context more about unrecognized adventitious

     4    agents than recognized one, since you can test for the

     5    recognized ones.

     6                I just was wondering whether that was a

     7    reasonable sort of conclusion based on what you said.

     8                DR. DESROSIERS:  Yeah.  I wasn't referring

     9    so much to species of origin.  I was referring to the

    10    fact that I think that it's much easier to extensively

    11    characterize and analyze established cell lines for

    12    what's in them and what's not in them than it is a

    13    fresh kidney every time you want to do something. 

    14    That's what I meant.

    15                DR. ENGLER:  Dr. Engler, Walter Reed Army

    16    Medical Center.

    17                I just wanted to make a comment and ask a

    18    broad question to challenge the panel, speaking from

    19    the perspective of a clinician and an educator, trying

    20    to translate the contents of meetings like this and

    21    the recent Thimerosal meeting for regular folks,

    22    whether it's providers or the patients.

    23                And the complexity alone, and when people

    24    say, "I worry about the unknown agents," we have to

    25    worry about foamy virus transmission, lentivirus

                                                                        103

     1    insertion into herpes, and that all translates into,

     2    for the common man, woman, and child, is there a bad

     3    disease with chronic consequences that can result from

     4    immunization.

     5                And the Hepatitis B vaccine, the original

     6    vaccine, which from my understanding and experience

     7    with it was one of the purest vaccines ever made, but

     8    could not really be sold very effectively to lots of

     9    people, including the highest risk folks that should

    10    have gotten it, like surgeons and doctors, and as you

    11    all are thinking about these things and trying to help

    12    CBER and the FDA, the question you have to ask

    13    yourself is:  if I choose to use a cell line or use a

    14    process to make a vaccine, how do I respond to those

    15    concerns?

    16                You all may think it's a minimum concern

    17    compared to jumping out of an airplane, but we're

    18    dealing with a public right now that is extremely

    19    phobic and growingly so about vaccines, and

    20    congressional representatives who have children who

    21    were adversely affected by one of our old vaccines and

    22    truly believe that this is the cause of a problem.

    23                So you know, yesterday the comment was

    24    made, "Well, why don't you do the vaccine process and

    25    spike it with your worst fear and see how it comes out

                                                                        104

     1    at the other end?"

     2                Now, that's something I can explain to a

     3    patient or a nurse or a doctor.  An awful lot of what

     4    you all have discussed is very hard to translate, but

     5    what's left is, "Gee, I'm not sure that this is safe,"

     6    from your mouths, and so I would just caution before

     7    large investments are made in building a vaccine that

     8    you consider the audience of who you have to deliver

     9    it and how do you write the vaccine information sheet.

    10                DR. HUGHES:  I'm certainly not by training

    11    or persuasion clinically oriented.  I'm a basic

    12    researcher, but as I understood the charge that we

    13    were brought was to try and bring out the issues that

    14    have to do with real safety, with the concerns that I

    15    think the public should have, that the vaccines that

    16    are being made are as safe as intelligent humans know

    17    how to make.

    18                And I would like to be able to say to you,

    19    I would like to be able to say to the people in

    20    Congress who in some sense pay my salary, and I'd like

    21    to be able to say to the public that you have no

    22    concerns; that everything that you're going to see in

    23    vaccines and in your life will be completely safe.

    24                However, that would be false, and I think

    25    the best job we can do under the circumstances as they

                                                                        105

     1    exist in the real world is to try to have a frank and

     2    honest discussion of what are, in fact, real concerns

     3    and dangers.

     4                And as Dr. Lewis pointed out, I believe,

     5    in the opening, his opening remarks, we're dealing

     6    with a situation in which there are costs and benefits

     7    and risks and benefits, and I don't believe that's

     8    avoidable.

     9                The average American, as far as I know,

    10    the most dangerous thing the average American does is

    11    to jump into his car and drive to work every morning,

    12    and by contrast, as far as I know, the administration

    13    or taking of any vaccine strain that's now approved in

    14    the United States has minimal risk.

    15                Now, I'm certain that people don't

    16    understand that, but that seems to me to be a problem

    17    that is distinct from the problem that, as I

    18    understood it, we were given, which is to try and

    19    figure out how to make it as safe as possible and to

    20    discuss the potential risks as clearly as possible.

    21                And certainly if there were some way we

    22    could make your job easier of explaining, I would be

    23    happy to try and do that,b ut I think any discussion

    24    that isn't frank and isn't complete and doesn't have

    25    some clear discussion of the potential risks is doing

                                                                        106

     1    a disservice to exactly the people we're trying to

     2    help.

     3                And I'm sorry it's complicated.  I wish it

     4    were not, but I don't think that's an avoidable

     5    problem, and I think that if there is an answer to

     6    your ultimate concern, it's to try and see that there

     7    is some larger and better scientific education in

     8    general in this country, which I think is appallingly

     9    bad, but I don't really think that's our charge here,

    10    although it's a charge that I think we all must in

    11    some sense want to participate in and have as part of

    12    what we do every day.

    13                Would anyone else care to speak to this? 

    14    Is this different or the same?  Are we done here on

    15    this one?

    16                MR. ONIONS:  David Onions, Glasgow.

    17                I just thought really perhaps to echo the

    18    last speaker and really to echo your comments  that I

    19    think that the FDA is to be congratulated because I

    20    think all regulations should be based on good science,

    21    and what we clearly heard in the last two days is some

    22    excellent science.

    23                If I would just summarize where I thought

    24    we were, I think yesterday we put -- I hope we put --

    25    oncogenic DNA to bed.  I think perhaps there are

                                                                        107

     1    better assay systems that could be used as I

     2    articulated, but I really don't think that's a risk.

     3                I think tomorrow we're going to discuss,

     4    which I think is the risk, as Ron Desrosiers said,

     5    adventitious agents.  I think that's still our big

     6    concern in any new cell substrate.

     7                But I think the novel thing that we heard

     8    today, and I'd really like to put this back to the

     9    panel, perhaps the novel thing we heard today was is

    10    there any concern for the genetic stability of our

    11    vaccines given the kinds of work we heard about today,

    12    for instance, retroviral insertions or retrotransposon

    13    insertions that might occur into vaccines, other

    14    possible recombinations between retroviral sequences

    15    and other sequences.

    16                So should we be looking at other

    17    mechanisms in our final lots, for instance, for the

    18    genetic stability of our vaccine?  Is that an issue or

    19    is it not an issue?

    20                That's really all I had to ask.

    21                DR. HUGHES:  Comments from the panel?

    22                DR. PETRICCIANI:  Well, I guess my comment

    23    would be that there is a lot of plasticity for a lot

    24    of the genetic elements that we've been discussing,

    25    particularly retroviral elements, but it's quite clear

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     1    that the active replication of these viruses for the,

     2    you know, reverse transcription is a process that you

     3    want to avoid at all cost because that is the major

     4    contributor to  the plasticity of the genetic element.

     5                So if we can eliminate that from a

     6    vaccine, that would be a major advantage in

     7    stabilizing the genetic content.

     8                PARTICIPANT:  I think just to try to

     9    follow up on what Phil was trying to say, I'm Hanna

    10    Golding (phonetic) with CBER, FDA.

    11                And the issues that we are really

    12    concerned with is the unknown.  We are dealing with

    13    new cell substrate that are  transformed.  We don't

    14    know their history.  We don't know what's the

    15    etiology.  We don't know what's the multi-event that

    16    occurred, and the question whether some of the

    17    processes or mechanisms that you have described

    18    related to retroviruses might have been participating

    19    in the transformation event of these cells that occur

    20    in vivo.

    21                What we are afraid of, that we not dealing

    22    with new cell substrates; that we don't know what

    23    caused their transformation, whether they're viral

    24    related, whether oncogenes related, whether

    25    combination of both, whether some transformation of

                                                                        109

     1    cellular genes into a retroviral type of elements or

     2    not.

     3                The question is:  was the knowledge that

     4    you had from your labs -- do you think at least there

     5    is a way to start and design certain assays that would

     6    allow companies and sponsors, CBER, to try and test

     7    these type of cell lines beyond the current testing

     8    that is currently available?

     9                DR. HUGHES:  Well, one possibility is

    10    rather than to try and deconvolute the rather complex

    11    history, that many cell lines, many permanent cell

    12    lines, transformed cell lines have is to simply create

    13    anew the kind of cell substrates that Dr. Hayflick, I

    14    believe, described in his talk at the beginning of the

    15    meeting, in which while you can't be sure that there

    16    isn't some retrotransposition of that, you certainly

    17    have an understanding of the way in which the cell

    18    became a more whole and if it is transformed,

    19    transformed, because it is something done in the

    20    laboratory, and you can reasonably exclude under those

    21    circumstances that it was a viral agent if a viral

    22    agent is not employed to make the cell permanent.

    23                Now, if the FDA chooses that path, it does

    24    exclude a number of the standard lines that people

    25    routinely use.  I think given the long passage history

                                                                        110

     1    of some of the lines, it will probably be hard to --

     2    you can certainly look at a number of genes in these

     3    cells.  You can look for certain known viruses, but I

     4    believe as you articulated, and in a sense a cell line

     5    reflection of what Dr. Desrosiers said, it may be very

     6    hard to rule out the sort of unknown agent if the

     7    history of the line is not well known.

     8                Anyone else?

     9                DR. DESROSIERS:  Yeah, I'll comment.  In

    10    terms of any cell line that's being considered, in

    11    addition to screening for all known viral agents in

    12    such a cell one, one can use differential display, RDA

    13    type techniques comparing the cell line to equivalent

    14    normal cells to look for the presence of new sequences

    15    or additional sequences not present in normal cells.

    16                For example, that is how the presence of

    17    Kaposi's sarcoma herpes virus DNA was detected in

    18    Kaposi's sarcoma cells.  It was detected as a new DNA

    19    not present in normal cells of the same type.

    20                So one can use those sorts of technologies

    21    to look for the presence of additional unknown DNAs in

    22    the cell line.

    23                DR. HUGHES:  Dr. Hayflick.

    24                DR. HAYFLICK:  Hayflick, UC-SF.

    25                Several speakers have referred

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     1    continuously to neoplastic cell lines, and I have the

     2    feeling that there is a belief that all cell lines are

     3    neoplastic.  I'm sure that most of the audience knows

     4    that that's not true, and we must be very careful

     5    because there are continuously propagable, immortal

     6    cell populations that do not produce neoplasia in any

     7    test that has been conducted.

     8                It seems to me that the decision to use

     9    CHO cells for the production of non-vaccine

    10    biologicals, which was based on the experimental data

    11    that those cells, although they might produce

    12    neoplasia, do not produce malignancies, was a very

    13    wise decision.

    14                But I think it's very important to

    15    understand that there are continuously propagable cell

    16    populations out there that are not neoplastic. 

    17    Several, in particular, I mentioned the other night,

    18    and they are cell populations that have been

    19    transfected with H TERT (phonetic) and are immortal as

    20    fas as we know.  There are well over 400 population

    21    doublings at this time, and do not show any signs of

    22    neoplasia when inoculated into proper experimental

    23    animals, which brings up another final point that I

    24    would like to make.

    25                And that is the belief that immortal cell

                                                                        112

     1    populations have advantages over diploid cell strains,

     2    for example, that have a limited capacity to replicate

     3    might not be important in the sense of the presumed

     4    advantage of immortality, and the reason for that is

     5    that the numbers of population doublings that you can

     6    get from a diploid cell strain, if limited to 50

     7    population doublings only, yields about 20 million

     8    metric tons wet weight of cells, which I think is

     9    ample for the production of most biologicals needed on

    10    this plant for the next decade or two.

    11                And the answer to that is that WI38 still

    12    exists.  It's been used for the production of vaccines

    13    for 37 years now.  There's an ample supply of these

    14    cells.

    15                And furthermore, one must also be aware of

    16    the fact that when immortal cell populations are used

    17    for the production of biologicals, there is a window

    18    of population doubling during which they are permitted

    19    to be used.  So despite the fact that they're

    20    immortal, there is a limited window of opportunity

    21    from the working cell bank to the production of the

    22    final product during which population doublings the

    23    cells are permitted to be used, despite the fact that

    24    they're immortal.

    25                Thank you.

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     1                DR. HUGHES:  Dr. Coffin?

     2                DR. COFFIN:  One point, that one of the

     3    problems with the more limited cell strains is that

     4    not every agent that you necessarily might want to be

     5    growing in a vaccine will grow on these.  If you

     6    wanted to grow HIV, for example, WI38 would not be a

     7    suitable choice, and you really have very little

     8    choice if you want to grow that except to go to either

     9    primary human cells or to some immortalized, almost

    10    certainly tumorigenic cell line.

    11                And so certainly in some instances it will

    12    be essential -- will not be possible to use these

    13    kinds of cells to do what you'd want to do for all

    14    kinds of vaccines one might want to make.

    15                The actual point I stood up to make was to

    16    actually return to the issue that David raised a few

    17    minutes ago which had to do with endogenous elements

    18    and genetic instability of vaccines and the

    19    possibility of endogenous viral contamination of

    20    products.

    21                We now know quite a lot about the

    22    endogenous virus, the endogenous retrovirus

    23    composition of certain species, some birds, mice and

    24    humans, in particular.  And there's a famous

    25    phenomenon that actually hasn't, much to my surprise,

                                                                        114

     1    been mentioned yet, but which I should mention in

     2    endogenous retrovirology, and that is that if you take

     3    a human tumor line and pass it through nude mice, a

     4    very high fraction of the time this tumor line will

     5    come out producing very large amounts of a very good

     6    looking retrovirus.

     7                As chair of IBC, I have had people walk

     8    into my office showing me these Grade EMs and saying,

     9    you know, "Look what we found."

    10                And I say, "Yes, that's a very old

    11    phenomenon that has been described many times," and

    12    it's because many strains of mice contain an

    13    endogenous provirus.  We know which one it was.  It's

    14    a provirus called BXB1 that produces a replication in

    15    competent xenogropic virus and which can readily

    16    infect human cells, particularly under the sort of co-

    17    cultivation circumstances one gets in putting tumors

    18    into nude mice.

    19                It would seem very obvious that cells that

    20    contain this provirus would be very good ones to avoid

    21    for the production of biologicals.  That actually has

    22    not happened.  Most monoclonal antibodies that are

    23    produced are produced in cells that come derived in

    24    some way from BALB-C mice and actually contain this

    25    provirus and are not unlikely to contain noticeable

                                                                        115

     1    amount of xenogropic MLD contamination, which could

     2    easily infect the recipient if not very carefully

     3    removed, or if they were used to grow vaccines, say,

     4    DNA virus vaccines, could, indeed, contribute to the

     5    sorts of genetic instability that Dr. Kung mentioned

     6    earlier.

     7                And in fact, this is at least a

     8    theoretical possibility with virtually all mouse cells

     9    which contain very large numbers of proviruses, many

    10    of which have not been shown to be replication

    11    competent, but which could in theory at least give

    12    rise to some.

    13                So I think there ought to be a lot of

    14    consideration as to the species of origin of the cells

    15    that are used.

    16                On the other hand, for example, human

    17    cells have not been shown ever to give rise to

    18    replication competent endogenous viruses despite

    19    considerable effort to try to find them.  Some cell

    20    lines give rise to particles and so on, but not

    21    through replication competence.

    22                So I think the species and the strain of

    23    origin in the case of mice ought to be given very

    24    serious consideration in deciding which of these cells

    25    to use to avoid some of these issues.

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     1                MR. ONIONS:  I was just going to make --

     2    David Onions -- I was just going to make one point. 

     3    Ron mentioned using RDA.  Representation difference

     4    analysis is one of the ways for looking for perhaps

     5    new viral sequences in cell lines, and I wanted to

     6    come back to that tomorrow in the panel discussion on

     7    adventitious agents.

     8                But just to make a comment on it briefly

     9    now, I think in many ways it's an excellent idea.  I

    10    think one of the problems is that finding the partner

    11    to use as the sort of direct hybridization partner is

    12    a real problem with these kinds of cell lines, and

    13    when you do these kinds of experiments -- and for my

    14    sins I've done them -- you tend to find that, of

    15    course, any rearrangement that's occurred in those

    16    cell lines, and of course, there are rearrangements in

    17    those cell lines, will also come out in the RDA

    18    analysis.

    19                So it makes it a very messy and also very

    20    labor intensive approach, but I think it nevertheless

    21    does have merit, and I'd like to come back to that

    22    tomorrow.

    23                I think one question I would just like to

    24    ask the panel was we touched on pseudotyping in

    25    several context, and usually when a new cell substrate

                                                                        117

     1    is examined, one of the requirements by the FDA and

     2    other regulatory authorities is we look for retroviral

     3    sequences, for instance, by the kinds of analysis that

     4    Joerg will talk about later, the pertile (phonetic)

     5    assay for retrovirus.

     6                Then if it's positive, we look and see

     7    whether those viruses are infectious for human cells,

     8    and that's part of the equation that's used to

     9    evaluate the safety of that cell substrate.

    10                But what is not usually evaluated is is

    11    there any possibility of pseudotyping with the actual

    12    vaccine virus that you're putting in.  We now know, of

    13    course, that you do get pseudotyping, not just

    14    retroviruses to retroviruses, but of course with VSV,

    15    but also with paramixaviruses, with flaviviruses, and

    16    so on and so forth.

    17                So the question I'm putting to you is: 

    18    should that now be part of our safety evaluation, that

    19    we actually look at the potential of pseudotyping

    20    between any retroviral sequence and the substrate and

    21    the viral sequences that we are actually trying to

    22    make a vaccine from?

    23                DR. HUGHES:  Comments from the panel?

    24                DR. PATIENCE:  I think with respect to at

    25    least the gene packaging therapy cell lines which are

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     1    being used, that has already been addressed.  I think

     2    probably your comment was addressed more at other

     3    viruses and other types of vaccine.

     4                DR. HUGHES:  It also brings up, I think an

     5    issue with at least respect to the first part of the

     6    comment that may be one of the more vexing problems,

     7    and that is I think modern technology, particularly

     8    nucleic acid technology is sufficiently robust that

     9    it's relatively easy to determine that any agent we

    10    know and under stand can be found and, if we're

    11    dealing with a cell line, avoided.

    12                I think the greater concern is how do we

    13    find the at least potentially adventitious agents that

    14    we have yet to learn to recognize, and I'd be curious

    15    to hear more about that tomorrow myself.

    16                Please.

    17                PARTICIPANT:  I just wanted to comment on

    18    the experiment proposed yesterday to spike cell

    19    substrates with tumor virus DNA and look for

    20    clearance.  This was recently just referred to as one

    21    of the most understandable parts of this meeting.

    22                I also feel it's one of the things that

    23    brings the greatest concern.  I think asking

    24    manufacturers to spike their materials with a

    25    dangerous tumor virus DNA poses much more risk than

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     1    any potential endogenous things you're worried about

     2    in these cell substrates.

     3                So it's a bad idea from a public safety

     4    risk perspective.

     5                This same experiment, by the way, I think

     6    is in the CBER draft guidelines.  So I think it's very

     7    relevant for discussion.

     8                Besides being a bad idea from a risk

     9    perspective, it's also not necessary from a scientific

    10    perspective.  It's much more likely that a spike --

    11    first of all, you could use a spike for a harmless DNA

    12    sequence instead of a tumor sequence, and also it's

    13    much more relevant to use PCR analysis of endogenous

    14    sequences versus a spike because a spike might clear

    15    differently than the endogenous sequences.

    16                Thank you.

    17                DR. HUGHES:  As a comment, when I heard

    18    the statement made that you're referring to, I thought

    19    the proposal was to add something that was easily

    20    scorable, and I thought it was either plasmid or

    21    lambda that was proposed.

    22                I would also say that it was not clear to

    23    me in the discussion yesterday that that was not a

    24    proposal to show that the process of clearance was

    25    valid, as opposed to a manufacturing process that was

                                                                        120

     1    proposed.

     2                MR. COOK:  I made the comment.  Jim Cook,

     3    University of Illinois.

     4                I think that you've mistaken the point. 

     5    The point was that an experiment could be done to find

     6    out how the clearance process works.  This wasn't

     7    proposing to put this in some kind of vaccine, run it

     8    across into the population and see if polyoma DNA

     9    causes tumors in man, which it doesn't.

    10                (Laughter.)

    11                MR. COOK:  So I think doing experiments to

    12    find out whether some kind of process is safe or not

    13    is perfectly valid.  That doesn't mean that that has

    14    to be put into some kind of production schedule.

    15                I think it's still a good idea, and I'd

    16    like to see the experiment done.

    17                MR. LEWIS:  Andrew Lewis, FDA.

    18                I'd like to sort of take the panel back to

    19    some more generic concepts, and I just think based on

    20    what we heard this morning the question I'd sort of

    21    like to have a poll of the panel would be as

    22    regulatory people do we really need to be worried

    23    about viral-viral and viral-cellular interactions.

    24                It seemed to me from the data that was

    25    presented this morning that there is reason to be

                                                                        121

     1    concerned about these types of thing, but perhaps I'm

     2    wrong.  Perhaps this is something that these are

     3    experimental conditions, and maybe we don't have to

     4    worry about them in the world of generating vaccines

     5    in neoplastic cells or in primary cells.

     6                So I'd like to have a sense of how you all

     7    think about that because this sort of helps us in

     8    deciding how seriously we should go about taking these

     9    concerns.

    10                DR. HUGHES:  Well, I'll begin by saying I

    11    think it would certainly, given the plasticity of

    12    retroviruses, be worth paying attention to.  I would

    13    also say that if you're asking -- if the question

    14    really has to do with neoplastic versus nonneoplastic

    15    cells or permanent cells versus primary cells, since

    16    the cells of many species, whether they're transformed

    17    or not, whether they're permanent or not, have

    18    endogenous retroviruses, and certainly things from

    19    primary culture can have all sorts of exotic exogenous

    20    as well as endogenous viruses; I'm not convinced that

    21    there is an enormous difference that is engendered by

    22    making the cells either permanent or neoplastic, but

    23    that doesn't take away from the larger question of

    24    whether or not that's a real issue.

    25                Shall we just go down the panel?

                                                                        122

     1                Dr. Desrosiers?

     2                DR. DESROSIERS:  Pass.

     3                DR. HUGHES:  Ben?

     4                DR. BERKHOUT:  Well, regarding SIV and

     5    HIV, we haven't seen so far an example where a

     6    defective fire resisity (phonetic) parrots by picking

     7    up cellular sequences.

     8                On the other hand, limited number of

     9    experiments have been done so far.  So I think we

    10    still should do more analysis, in particular, of the

    11    monkeys that were infected with live attenuated SRV

    12    strains to see if this really is impossible.

    13                You know, it may need some more time to

    14    actually say that is impossible.

    15                DR. KUNG:  I guess I have to take a

    16    practical point of view.  I think the theoretical

    17    potential is there.  It's almost impossible to avoid,

    18    and I'm actually thinking we have no choices in the

    19    sense that I think these things will happen, to pick

    20    up DNA from retrovirus, and there are a number of

    21    endogenous viruses, some of the unknown ones we don't

    22    even know.  So it's hard to even detect them.

    23                But you know, in all likelihood, without

    24    any particular selection, those events will come and

    25    go, and so if I take a practical point of view, my

                                                                        123

     1    feeling is that it's not a serious problem.  That

     2    would be my feeling.  There's no other choice.  If you

     3    had better choices, that would be fine.

     4                It's very difficult to even identify those

     5    things, and so I don't know what the practical

     6    solution to that.  It does happen.  I think all on

     7    this panel would say it does happen.

     8                DR. LINIAL:  I guess there are two issues. 

     9    The first, I agree with Hsing-Jien and Steve Hughes,

    10    and that is the problem of retroviral contamination or

    11    contribution to other viral vaccines.  I think clearly

    12    it's a possibility, but it sounds like, you know, all

    13    the common ones are being screened for, and people are

    14    doing the best they can.

    15                The other issue is using retroviral

    16    vectors or retroviruses as vaccines, and I think

    17    that's something that really needs a lot more

    18    discussion.  We've heard a lot of evidence here that

    19    none of these things are really safe, and I have a lot

    20    of concerns about using retroviruses to put into

    21    people, especially possibilities of defective

    22    lentiviruses, which we know can cause disease, or even

    23    in the case of foamy viruses until we know a lot more

    24    about what happens when they're widespread in the

    25    human population, which they aren't now.

                                                                        124

     1                I would be very cautious about using

     2    retroviruses as agents as opposed to worrying about

     3    what retroviruses are doing to other agents.

     4                CHAIRPERSON ROSENBERG:  I guess I would

     5    make two comments.  I agree with what some of the

     6    other panelists have said.  There's nothing that

     7    really convinces me that there's a difference per se

     8    between using a neoplastic or an immortal-like cell

     9    for a substrate.  I think it more reflects the

    10    properties of the particular cell, not whether it's

    11    normal or neoplastic or falls into one of these

    12    intermediate categories.

    13                Another point I think with regard to the

    14    retroviruses.  I think it's very difficult to really

    15    accurately assess the risk, but we've seen examples

    16    presented here, and certainly there are many others

    17    where situations that involve replication of the

    18    viruses or have the risk of replication of the viruses

    19    clearly can lead extremely rapidly -- we've seen

    20    several presentations that have emphasized how rapidly

    21    changes can occur in these populations.

    22                So certainly circumstances that have a

    23    risk of replication, I think, must be avoided because

    24    the circumstances that can follow are quite

    25    unpredictable.  I think that's another message that

                                                                        125

     1    has come through.

     2                DR. KAPPES:  Nava, would you like to say

     3    something?

     4                DR. SARVER:  Sorry, yes.  Nava Sarver from

     5    the NIH.

     6                I'd like to ask the panel and perhaps some

     7    people in the audience.  There has been, I think, the

     8    stringentest (phonetic) case of potential risk with

     9    retroviral replication, has been in the case of gene

    10    therapy where they've been used extensively for

    11    delivering whatever therapeutic genes or replacement

    12    genes, and I'd like to know. 

    13                I mean, I'd like to know -- the FDA and

    14    the audience and the people in the panel that have

    15    been involved in gene therapy -- whether there are any

    16    documented cases where some of the concerns that are

    17    expressed here have been materialized, and from the

    18    limited knowledge that I have, I am not aware of any

    19    identified risk that has occurred in humans, you know,

    20    with the application of gene therapy.

    21                So perhaps this could be taken as a case

    22    study and really exhausted and see what are the risks

    23    and are they meritorious.  I mean, can we really rely

    24    on the concern that is expressed here when we

    25    extrapolate this to gene therapy and learn from that?

                                                                        126

     1                DR. KAPPES:  I think we have no choice. 

     2    All of what we're speaking of is highly complex.  Each

     3    individual question is complex and needs to be

     4    addressed individually.

     5                I think my simple answer to whether we

     6    need to worry about these interactions:  yes, and I

     7    think that's the obvious answer.  No one would

     8    probably disagree. 

     9                Maybe that isn't exactly how the question

    10    was posed.  The implications are tremendous.  I don't

    11    see alternatives.

    12                In the case of HIV vaccines, we have to

    13    continue to look at how the virus is different, for

    14    example, in comparison with what we have learned,

    15    Nava, maybe as you're referring to with Maloney.

    16                One issue I pointed out that's clearly

    17    different with lentiviral vectors compared with

    18    Maloney is lentiviruses and the recombinants

    19    themselves are likely to have the capacity to infect

    20    nondividing cells.  So if you have recombination and

    21    if that recombinant can be pseudotyped by

    22    superinfection or even normal cellular receptor ligand

    23    interactions, it's possible to mobilize that

    24    recombinant to adjacent cells, and that's very

    25    different than what you might see with Maloney.

                                                                        127

     1                It sounds hypothetical, theoretical, and

     2    certainly it is.  So I don't have -- there is no -- I

     3    guess my answer is there aren't easy answers, and this

     4    is the platform by which I think the FDA, in

     5    particular, has established to try to address these

     6    important questions.

     7                DR. PETRICCIANI:  I guess my comment would

     8    be to compare vaccine use with gene therapy, is the

     9    issue with vaccines.  You're involving far more people

    10    probably with the potential risk, and using a greater

    11    number of division of cells, and possibly the benefit

    12    is less for people than gene therapy.  There's less

    13    personal benefit.

    14                So it changes the equation in my mind.

    15                My comment about the use of retroviruses

    16    is that replication is something that is to be avoided

    17    if possible, but you know, if that is present, then

    18    you're definitely going to transmit other genetic

    19    elements.  That's something that will happen

    20    regardless of the cell type.

    21                So the question between neoplastic cells

    22    or permanently dividing cells in my mind becomes less

    23    of an issue rather than the types of genetic elements,

    24    the endogenous elements that will be transmitted.

    25                I think that John Coffin made a good point

                                                                        128

     1    about the species of selection.  If you're going to

     2    administer a retrovirus to a human, perhaps you might

     3    want to think about using a human cell line where

     4    there's no new genetic element coming in from a

     5    different species.  That might be more advantageous

     6    than choosing something completely different, like a

     7    mouse.

     8                DR. SARVER:  I'm not trying to minimize. 

     9    You know, I see the difference.  I mean, it's

    10    definitely a vast difference between gene therapy and

    11    a case where you have the benefit definitely outweighs

    12    the risk, and the naive individuals that are going to

    13    be vaccinated.

    14                What I tried to say is perhaps the gene

    15    therapy field can actually give a quantifiable number

    16    to the risk, to put the probability that something, an

    17    adverse effect can actually take place.

    18                And granted we don't have that many number

    19    of patients in gene therapy, despite all of the hype

    20    that we hear, but nonetheless, it's a defined

    21    population of patients, and it can be used as a

    22    probability study.  What are the chances and can we

    23    actually put a definite number on the probability that

    24    something risky is -- that something bad can take

    25    place?

                                                                        129

     1                This is what I was driving at, whether we

     2    can.  I mean just to work up the numbers and to say

     3    are we talking with infinitesimal probability or is

     4    the probability really a quantifiable number.

     5                DR. EVANS:  One comment about that is

     6    we're going to have to wait for a long time after

     7    those experiments to see if there's any problem.  I

     8    mean this could go on for 40, 50 years and something

     9    could come up.

    10                PARTICIPANT:  I was just going to make a

    11    comment on that comment from the last speaker.  I

    12    really don't think it is a very good object lesson

    13    because, first of all, the numbers are small.

    14                Secondly, a high proportion of the

    15    therapies that are being produced are in terminal

    16    patients.  So there's a high correlation between

    17    having had the retrovirus vector and being dead.

    18                (Laughter.)

    19                PARTICIPANT:  And that's not meant to be

    20    a flippant comment, but it's true.

    21                And I think the third comment is that we

    22    know that a lot of these people have had delivered

    23    BLS-30 sequences.  To my knowledge, there's not been

    24    a particular study that's actually followed up the

    25    consequences of having BLS-30 mainly because these are

                                                                        130

     1    terminal patients with other problems.

     2                DR. PATIENCE:  I've got a few points down

     3    here relating to this question and earlier with

     4    respect for gene therapy and the use of cell lines,

     5    such as WI38, it's not practical.  Basically a lot of

     6    these systems need to go back to single cell clonings,

     7    and by the time you got through your 50 doublings, you

     8    just wouldn't have a useful packaging cell life.

     9                With respect to do we have to be worried,

    10    well, I think we are looking at Maloney or at least

    11    murine leukemia virus, potentially lentiviruses.  A

    12    lot of information is known about these viruses.  So

    13    we can make the systems as safe as we intellectually

    14    possibly can and biologically possibly can.  They're

    15    well understood, which may be reassuring.

    16                Coming back to David Onion's point, with

    17    respect to the patient either dies or receives a gene

    18    therapy vector or another treatment, I think that

    19    really does touch on an important point, which is that

    20    we have to look at individual scenarios with respect

    21    to what species barriers we may be jumping. 

    22    Retroviruses, for instance, their behavior when you

    23    cross the species barrier may be totally different to

    24    what you can see in vitro.  You in very many cases

    25    just cannot predict whether they have any potential

                                                                        131

     1    pathogenic nature or not.

     2                And then another order of magnitude on

     3    from there is, yes, okay, there is a definable risk

     4    with respect to the recipient of a therapy, but does

     5    it not -- at least personally speaking, does it not

     6    become more of a public health issue if there's the

     7    potential for transmission?  I think maybe that takes

     8    it on to a different level of assessment.

     9                DR. RUSCETTI:  Yeah, one slightly

    10    different point I wanted to make at least in

    11    relationship to using mouse packaging cell lines, you

    12    may generate recombinant viruses and there's some

    13    instances where that has happened, but it's not clear

    14    that that actually would be a risk, even if you did

    15    give it inadvertently to patients.

    16                We know from studies of mouse retroviruses

    17    that there are very specific changes.  These viruses

    18    have to have certain sequences that make them

    19    pathogenic, and you may generate lots of different

    20    recombinants that don't have those.  Even to cause a

    21    disease in mice there really is very little evidence

    22    that you can generate pathogenic retroviruses in

    23    vitro.

    24                For these then to cause diseases in humans

    25    I think are even less likely.  These viruses have to

                                                                        132

     1    replicate at very high titres and certain cell types

     2    and perhaps carry certain envoy genes that might have

     3    biological effect in human cells.

     4                So I just wanted to bring up the point

     5    that even though you might generate recombinant

     6    viruses, they may pose no risk at all to humans, but

     7    granted we don't know, and I think everybody will say

     8    here we're not willing to say that there's no risk at

     9    all for using these sorts of lines as substrates for

    10    vaccines.

    11                DR. BROKER:  Tom Broker, UAB.

    12                I wanted to kind of extend one

    13    consideration Nava brought up, and that is there

    14    really is a lot of other experience that I think this

    15    particular concern today can be referred to, and one

    16    of them is simply the blood bank system, the blood

    17    transfusions that are very commonplace, the use of

    18    clotting factors which have been produced in various

    19    ways, and the use of interferons, for example, and a

    20    variety of other proteins that are routinely delivered

    21    to patients.

    22                I think there is a lot of experience here

    23    that could relate to the risk of transfer of agents

    24    either from contemporary blood donors or through the

    25    manufacturing process.

                                                                        133

     1                A second thing I wanted to bring up is

     2    that there is concern about, that the general public

     3    has, as well as the professional communities, about

     4    simply immune reactions to the very product that

     5    you're trying to transfer.  In the case of vaccines,

     6    that's the objective, but in the case of contaminating

     7    components in a vaccine, the question is how many

     8    different things can you inject into a given person

     9    before you start getting negative effects.

    10                Something I've experienced, I helped

    11    monitor an Internet site for laryngeal papillomatosis,

    12    and there is an extraordinary amount of what I call

    13    Internet paranoia that is not to be dismissed, patient

    14    support groups that absolutely go ballistic over the

    15    most elementary rumors warning people not to engage in

    16    a certain therapeutic protocol, for example.  It's out

    17    there, and it's going to be unavoidable.

    18                The fourth thing that I wanted to mention,

    19    which I'll talk a little bit about tonight and

    20    tomorrow, is that a number of cell lines that we have

    21    felt to be relatively stable, in fact, continue to

    22    evolve.  There is a certain degree of genetic

    23    instability in cell lines that, while you may know

    24    what they are today, ten passages from now they may

    25    not be what you thought they were.

                                                                        134

     1                And so rather continuous monitoring of

     2    what you've got is going to probably be a necessity.

     3                Specifically, I believe this is related to

     4    the particular growth environment that the cell line

     5    has experienced in its most recent history.  If the

     6    demands are to divide rapidly and produce large

     7    amounts of material, they will have a certain

     8    phenotype.  If they are put into a more quiescent

     9    stage, it appears that they'll be able to alter their

    10    gene expression at least, if not underlying genetic

    11    changes, to kind of adapt to the demands of how much

    12    growth is expected out of them.

    13                And the final point I wanted to make, it's

    14    a variety of different ones, of course, but that is

    15    the immune status of the recipient is going to affect

    16    the risk of anything that's transferred into a

    17    patient.  For example, there's over 21,000 solid organ

    18    transplants a year, 25,000 bone marrow transplants a

    19    year in AIDS and a variety of other immunosuppressive

    20    disorders, and what works in the general population

    21    may not work for the several hundred thousand people

    22    in the U.S. who have compromised immune status where

    23    reactivation of a contaminating virus, for example,

    24    will be very different in that recipient compared to

    25    an otherwise healthy person.

                                                                        135

     1                So I've raised a lot of issues, but I

     2    think they all are relevant to the concerns of this

     3    session.

     4                Thank you.

     5                DR. HAYFLICK:  Hayflick, UC-SF.

     6                One of the panel members made mention of

     7    the fact that a limitation on the use of WI38 or other

     8    human diploid cell strains is the observation that

     9    they can only undergo about 50 population doublings

    10    which precludes their usefulness when cloning is

    11    necessary in order to produce a particular product.

    12                That was true.  It no longer is because

    13    WI38 and other normal human diploid cell strains can

    14    be immortalized by transfection with H TERT and

    15    undergo hundreds and hundreds of population doublings

    16    with full retention of their normal properties.

    17                Thank you.

    18                DR. HUGHES:  All right.  It is now

    19    slightly past 11:30, and unless someone on the panel

    20    or in the audience has an additional concern that

    21    can't be dealt with after lunch, we'll go to lunch.

    22                Thank you very much.

    23                (Whereupon, at 11:35 a.m., the meeting was

    24    recessed for lunch, to reconvene at 1:00 p.m., the

    25    same day.)

                                                                        136

     1              A-F-T-E-R-N-O-O-N  S-E-S-S-I-O-N

     2                                             (1:07 p.m.)

     3                DR. ZOON:  If we could get started, we

     4    will now enter into our next session, which is

     5    entitled "Cellular DNA as a Potential Source of

     6    Oncogenic Activity or Infectious Agents."

     7                I am obviously not Rob Breiman.  I'm Kathy

     8    Zoon, and I'm the Director of the Center for Biologics

     9    and happy to have an opportunity to chair this very

    10    important session.

    11                I do want to announce that we are adding

    12    one additional speaker this afternoon.  So our break

    13    will be delayed by about 15 to 20 minutes in order to

    14    accommodate an additional talk that was scheduled for

    15    tomorrow, which we are now having this afternoon.  So

    16    I would appreciate your patience.

    17                And also I will ask all the speakers,

    18    because we are having an extra speaker this afternoon,

    19    that please keep to your time so that we give

    20    everybody an opportunity to have their full time to

    21    present their information.

    22                With respect to this topic, this is not a

    23    new topic that either the Public Health Service has

    24    been working with, and particularly the FDA, over a

    25    number of years, whether it's vaccine safety, blood

                                                                        137

     1    safety, or new therapeutic products, including gene

     2    therapy, and obviously it's not without its

     3    controversy over the years.  Levels, acceptable levels

     4    of cellular DNA has been a subject of many

     5    discussions, all of which over the years have

     6    developed into a position in many cases for a number

     7    of the products regulated into scientifically sound,

     8    I believe, estimates of risk involved with DNA, but as

     9    we enter into new areas and looking at new types of

    10    applications of cell substrates, we want to insure

    11    that the very best science underpins our scientific

    12    decision making and regulatory policy.

    13                And we appreciate the opportunity of all

    14    the participants in this conference in contributing to

    15    those efforts.

    16                So I now have the pleasure of introducing

    17    Dr. James McDougall from the Fred Hutchinson Cancer

    18    Research Center, and he's going to be talking to us on

    19    designer cell substrate, telomerase activity and cell

    20    immortalization.

    21                DR. McDOUGALL:  Okay.  First of all, I

    22    should thank the organizers for allowing me to talk

    23    this afternoon.  I have to get back to Seattle.  The

    24    sun's shining there.

    25                (Laughter.)

                                                                        138

     1                PARTICIPANT:  It won't be.

     2                DR. McDOUGALL:  It won't be by the time I

     3    get there, no.

     4                We really got into a real interest in

     5    telomerase and its inductions because we found out

     6    some while ago, maybe a couple of years ago, that one

     7    of the oncogenes that the high risk human papilloma

     8    viruses induces this enzyme in human epithelial cells,

     9    and this first slide just demonstrates the fact that

    10    using a trap assay to see the repeat sequences that

    11    are produced by the telomerase enzyme, that the 16E6

    12    oncogene induces that enzyme, but the other oncogene

    13    of papilloma virus HPV16 or 18 does not do so.  E7

    14    does not induce it.

    15                If we put the two together, we actually

    16    see an enhancement of that telomerase induction, and

    17    if we look at immortalized cell lines that come out of

    18    this sort of experiment, and these are in human

    19    mammary epithelial cells; if we look at a long term

    20    one, in fact, this one is human skin keratinocytes,

    21    passage for 84 times, we see that that telomerase

    22    level, if anything, increases in these cell lines.

    23                One other thing that we were interested in

    24    in this context was does this do anything for the

    25    immortalized cell, for the ability to immortalize

                                                                        139

     1    cells, and does it do anything for maintaining the

     2    telomere length in cells, which is the key point that

     3    one's interested in here.

     4                And if we look at clones of keratinocytes

     5    that are just expressing HPB16 E7, you see that in

     6    early and late passage, there is a loss of telomere

     7    length in each one of these examples.  The average

     8    length is dropping.  So the telomeres, as they do in

     9    any replicating human cell, gradually shorten.

    10                However, if we add E-6 to that mix, we now

    11    see that they maintain their telomeres at a standard

    12    length.  So clearly E-6 induction of telomerase here,

    13    and these are cells pre-immortalization or pre-

    14    transformation, they maintain their telomeres quite

    15    efficiently.

    16                And the third thing about the human

    17    papilloma virus situation is that as with many, if not

    18    most, if not all cancers, they are very -- this is

    19    cervical cancer associated with HBV16 or 18 -- they

    20    are all positive for high levels of telomerase

    21    activity.  So the normal cells from the same patient

    22    are negative, as one would expect.  The tumor is

    23    always positive.

    24                So this was an observation that was of

    25    interest to us in terms of HPV and led us into looking

                                                                        140

     1    more closely at the effects of telomerase itself, and

     2    this is just a cartoon of the ends of the chromosome,

     3    the telomere ends and the telomerase RNA template is

     4    here, and this really should now be called with the

     5    acronym H TERT, H-t-e-r-t, which is the catalytic

     6    subunit of reverse transcriptase that is the active

     7    part of the enzyme here.

     8                So we set out to look at the effect of

     9    this H TERT upon primary epithelial cells, but first

    10    of all repeated the Bodner experiment from Gerund and

    11    their associates, in which they put H TERT into human

    12    fibroblasts, and what you see clearly is that those

    13    become immortalized.

    14                And as Dr. Hayflick pointed out, if you

    15    had 20 tons of WI38, there's no reason why you

    16    shouldn't have 2,000 tons of WI38 if you add H TERT.

    17                One of the characteristics we looked at

    18    was the presence of P16 in these cells, P16 which

    19    always increases with senescence, and we looked at the

    20    profile of P16 in these cells going through extended

    21    life span.

    22                Now, we've taken these up to about 300

    23    population doublings, and what you see is a very

    24    slight increase in the P16 levels from the earlier

    25    picture, but it never reaches anything like the level

                                                                        141

     1    of P16 that we see in human keratinocytes.

     2                By the time they've gone through 16 to 20

     3    passages, they have very high levels of P16, and they

     4    senesce.

     5                Now, the interesting thing that really

     6    came out of this and that we were clued into by the

     7    papilloma virus E6/E7 results was when we looked at

     8    mammary epithelial cells, we found that if you looked

     9    pre-M0, this first point at which mammary cells tend

    10    to senesce, we found that when you added 16E6 to these

    11    and immortalize them, you lose expression of P16 or it

    12    was greatly reduced, which was not the case if you put

    13    E7 in.

    14                And the same picture occurs with the H

    15    TERT.  So H TERT added to HMECs in order for those

    16    cells to become immortalized, you have to reduce the

    17    -- you have to have a reduction in the P16 level, and

    18    if we look at skin keratinocytes, the picture is

    19    exactly the same.

    20                Where E7 is present, together with H TERT,

    21    you do not get a reduction in P16 level, but you don't

    22    need to because E7 is binding the retinoblastoma

    23    protein, and therefore, if you bind the retinoblastoma

    24    protein, you will actually -- sorry.  Here -- you're

    25    actually achieving the same result as if you were

                                                                        142

     1    knocking out P16, but always in the case of H TERT

     2    alone in order for the cells to become immortalized,

     3    you have to down regulate P16, and that holds in a

     4    number of different types of human epithelial cell

     5    lines.

     6                So the situation is that if you put H TERT

     7    into fibroblasts, then clearly they carry on, and

     8    there are no major phenotypic changes observed in

     9    those cells, but if you put H TERT into epithelial

    10    cells, human epithelial cells from any source

    11    whatsoever, they have to lose expression either of P16

    12    or they have to have the RB pathway knocked out in

    13    order for those cells to survive and become

    14    immortalized.

    15                Now, loss of P16, as I'm sure most and

    16    perhaps everybody here knows, is a major feature of

    17    many tumors.  Up to about 50 percent of human tumors

    18    looked at have lost P16 expression in one way or

    19    another, and that's certainly true in a number of cell

    20    types, and I've just put a number of references to

    21    show this effect.

    22                So the P16 locus is part of the N4A locus,

    23    and P16 is actually one of the two transcripts from

    24    this locus.  So there's P16 and P14, and we were

    25    interested in the fact that when we looked at P16 in

                                                                        143

     1    most of the cell lines, P16 was lost, but P14 remained

     2    transcribed and unaffected.

     3                However, in more recent experiments that

     4    we've been looking at in adenoidal epithelial cells we

     5    now find that some of those have lost P14, but still

     6    express P16.  So it looks as though you can use one or

     7    the other of these transcripts to allow this feature

     8    to occur.

     9                So, again, all of the H TERT immortalized

    10    epithelial cells that we look at have lost expression

    11    of one or other of these genes.

    12                And as you know, these genes have very

    13    important roles in the cell cycle pathway, P16 here

    14    and P14 here, with its inhibitory effects on MVM2.  So

    15    these are extremely important genes.

    16                Now, we were interested in how P16 was

    17    being down regulated in these H TERT cells.  One of

    18    the ways we approached it was just to look for loss of

    19    heterozygosity, and obviously we could do that in a

    20    number of ways.  This is just using comparative genome

    21    hybridization where you compare the cell line that

    22    you're interested in with a known normal cell line, a

    23    competitive hybridization onto a normal karyotyp,

    24    and when you do that, you  should always see a line

    25    that goes right down the middle here, showing that

                                                                        144

     1    there's no loss or gain of any sequences in either of

     2    the cell lines that you're looking at.

     3                But what you see on here with a little bit

     4    of noise on some of the chromosomes is a clear loss of

     5    the short chromosome line, and in fact, this cell line

     6    when we look more closely has an isochromosome of the

     7    long arms of nine, but has lost the short arms, and

     8    that's where P16 locus maps.

     9                So clearly loss of heterozygosity is one

    10    of the mechanisms for this.

    11                Now, a second mechanism that goes right

    12    back to some of the talks yesterday is methylation. 

    13    Showing again a number of H TERT immortalized cell

    14    lines from in this cases keratinocytes or some of the

    15    adenoidal epithelial cells, and in every case when we

    16    look at these, we see that at least one and sometimes

    17    both alleles where there's not a lot of heterozygosity

    18    are, in fact, the promoter is heavily methylated over

    19    time.

    20                And so by the time any of these cells

    21    become immortalized, there's clearly methylation of

    22    the promoter of P16.  So clearly two mechanisms are

    23    operating in these H-term immortalized human

    24    epithelial cells to actually down regulate P16.  

    25                We haven't looked at P14, but I suspect

                                                                        145

     1    the same sort of results will show for that.  Sorry. 

     2    I went the wrong way.

     3                Now, one other feature that concerned us

     4    is that, as I said, P16 loss is very common in many

     5    tumors.  What about the phenotype of H TERT

     6    immortalized epithelial cells as opposed to the

     7    fibroblasts where we have not seen phenotypic changes,

     8    and I qualify that by saying "yet."

     9                One of the ways to ask this question is to

    10    look at organotypical RAFT cultures in which we put

    11    the epithelial cells onto a collagen and fibroblast

    12    containing substrate and allow the cells to

    13    differentiate and produce what amounts to essentially

    14    a normal skin.

    15                Since we're putting these genes on with

    16    vectors, we also look to see what the vector, which is

    17    the LXSN retroviral vector that we use in these

    18    systems, and you can see essentially the result is the

    19    same.  Even though this is a slightly smaller RAFT

    20    culture, basically the differentiation pattern is

    21    identical.

    22                And just to show you with HPV, if we add

    23    E7 to this type of RAFT, we begin to see very definite

    24    changes in that there's not a clear difference between

    25    basal cells and surface cells, and there are obviously

                                                                        146

     1    replicating cells high in the differentiating surface,

     2    which is obviously abnormal and what you see in

     3    dysplasia, for example, in cervical carcinoma.

     4                What happens when we look at the H TERT

     5    cells?  Well, again, we can find divisions higher up

     6    in the level, but equally importantly, if we  look at

     7    cytokeratin, in this case Cytokeratin 1, so here's a

     8    hemotoxin and eosin stain of a RAFT, and here it is

     9    with the TERT in it, showing that there clearly is a

    10    change in the phenotype here.

    11                And if we look at the Cytokeratin 1

    12    expression, you can see that it's different in the

    13    TERT RAFT.  It's lost from these basal cells, and it's

    14    clearly very positive in these upper layers, whereas

    15    here it's much more evenly distributed.

    16                And in one other example, if we look at an

    17    integrin, Alpha 6, Beta 4, we see the same sort of

    18    changes, where Alpha 6, Beta 4 is normally expressed

    19    in normal epithelial cells just in these basal and

    20    super basal cells.  In the TERT immortalized

    21    keratinocytes, it's expressed right through the

    22    surface.  So clearly a phenotypic change is going on

    23    here.

    24                And my last example of the fact that there

    25    are clearly phenotypic changes in these cells is that

                                                                        147

     1    if we do a karyotypic analysis of a number of cell

     2    lines, and this is just a subset of the ones we've

     3    looked at, we find some that clearly are quite normal,

     4    and as far as we can see, there is no karyotypic

     5    change in these by any mechanism we've looked at.

     6                But there are some cell lines that clearly

     7    are changing.  Here's the isochromosome 9-1 with the

     8    P16.  This nevertheless has lost P16, but has done so

     9    only by methylation on both alleles, and here is

    10    another H TERT cell line which has really quite

    11    massive karyotypic changes in it, another one here

    12    with an extra chromosome.

    13                So clearly there are phenotypic changes in

    14    terms of cell differentiation, and there are changes

    15    in the keriotypes of these cells.  So H TERT

    16    immortalization is not completely benign.

    17                So conclusions from this:  telomerase,

    18    whether by E6 or H TERT, is not sufficient to

    19    immortalize human epithelial cells unless this pathway

    20    is inactivated.

    21                Secondly, we can get inactivation through

    22    E7 binding of RB by methylation of P16 promoter or by

    23    chromosomal deletion.

    24                Thirdly, we can show that E6 activates H

    25    TERT, but we're not clear about how that works. 

                                                                        148

     1    Although E6 induces myc and myc activates telomerase

     2    in some cells, that doesn't seem to be the pathway for

     3    E6, since we know that E7 induces myc to quite high

     4    levels, but does not result in the activation of

     5    telomerase.  So clearly there are two different

     6    pathways here.

     7                We've found that epithelial cells

     8    immortalized by TERT can exhibit chromosomal

     9    abnormalities, and lastly, that epithelial cells

    10    immortalized by E6, E7 or H TERT with P16 loss have

    11    similar changes from the normal differentiation

    12    program seen in organotypic culture, and these are

    13    remarkably similar to what we see both in experimental

    14    systems and in cervical carcinoma.

    15                Thanks.

    16                (Applause.)

    17                DR. ZOON:  This presentation is now open

    18    for discussion and questions, and if I could ask each

    19    of the participants to identify themselves when they

    20    get to the microphone.

    21                Dr. Fried.

    22                DR. FRIED:  Mike Fried.

    23                What happens in the P16 knockout mice? 

    24    Are the epithelial cells immortal?

    25                DR. McDOUGALL:  I cannot remember.  I

                                                                        149

     1    think they're perfectly normal actually.  I don't

     2    really know the answer to that.  So I won't speculate.

     3                DR. FRIED:  Okay, and what do you think is

     4    the difference between the fibroblasts and the

     5    epithelial cells when you add H TERT?  I mean one goes

     6    and one doesn't.

     7                DR. McDOUGALL:  I do not know.  Again,

     8    that's something that we're looking into and trying to

     9    think up the right experiments for, but we don't know.

    10                DR. HAYFLICK:  Is this working?  Yes, I'd

    11    like to -- this is Hayflick, UC-SF.

    12                I'd like to congratulate you on a series

    13    of very elegant experiments.  I think, however, that

    14    one must emphasize that telomerase expression is

    15    necessary, as far as we know, for immortalization of

    16    cells, but insufficient for production of cancer in

    17    those cells.

    18                Telomerase is an immortalizing enzyme, and

    19    as far as we know not a cancer causing enzyme, and the

    20    evidence for this is as follows.

    21                Virtually all transformed human cell

    22    populations, certainly, have telomerase expression. 

    23    There are some exceptions, of course.

    24                Furthermore, those cell populations have

    25    been transformed, as you so well demonstrated, in many

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     1    cases by oncogenic viruses and also by radiation and

     2    carcinogens, and as a result of that immortalization

     3    telomerase is expressed.

     4                But even more to the point is the fact

     5    that post fertilization of human sperm and egg,

     6    telomerase is expressed for the first three months of

     7    gestation in all cells.  That expression then

     8    diminished and remains constant in all of the stem

     9    cell populations, that is, normal stem cell

    10    populations, during embryonic development and then

    11    during maturation and adulthood.

    12                So that the mere expression of telomerase

    13    is insufficient in itself to produce cancer, and I

    14    think this is very important to recognize.

    15                It's also been shown, as you demonstrated

    16    here, that as the cells undergo many population

    17    doublings after H TERT administration, there are some

    18    -- there is aneuploidy developing, and I was happy to

    19    see that you have also made this same observation.

    20                Thank you.

    21                DR. McDOUGALL:  Yes.  Thank you.

    22                Obviously I agree with everything you

    23    said, except one thing, and that is that I draw a

    24    parallel with what we see with E6, E7, and that

    25    parallel is that E6, E7 also are not sufficient to

                                                                        151

     1    induce cancer.  There have to be other genetic

     2    changes, and my interpretation is that it's highly

     3    likely that H  TERT immortalized cells will eventually

     4    produce those genetic changes, not because of H TERT,

     5    but simply because that is a fact of cells as they

     6    become immortalized and continue to replicate.

     7                So I'm not suggesting H TERT is itself

     8    sufficient, but I think the cells are certainly

     9    capable of progressing.

    10                DR. HAYFLICK:  Yes, I agree.  There are

    11    downstream effects with P53, P16, P21, et cetera, that

    12    are essential for the expression of malignancy or

    13    mutagenicity.

    14                Thanks.

    15                DR. ZOON:  Okay.  Thank you very much. 

    16    Thank you.

    17                Our next speaker I have a great pleasure

    18    to introduce, and a long time colleague, is Dr. John

    19    Petricciani.  He's representing the International

    20    Association for Biologicals, and he will be giving an

    21    introduction to cell DNA issues, a subject which he

    22    has become vastly familiar with over the past 20

    23    years.

    24                So, John, it's a pleasure.

    25                DR. PETRICCIANI:  Thank you very much,

                                                                        152

     1    Kathy.  It's a pleasure to be here.

     2                And as Kathy pointed out in her

     3    introductory comments, this is not a new issue.  In

     4    fact, it's been around for decades, more decades than

     5    some of us would like to remember, in fact.

     6                Because the DNA issue is so ubiquitous,

     7    it's already been discussed almost from the very first

     8    lecture here.  So what I'm going to do is skip through

     9    a number of the introductory slides rather rapidly so

    10    as not to bore you and put you to sleep after lunch,

    11    and then we'll get into some of the other pieces of

    12    this where I think there's room for some discussion.

    13                It hardly needs mentioning again that

    14    there is a rationale for being concerned about DNA,

    15    and it relates simply to the issue of safety of

    16    products that make contain even small amounts of DNA

    17    because of the theoretical possibility that the DNA in

    18    those products, once transferred to the patients

    19    receiving the product, may impose an oncogenic risk or

    20    some other type of pathology.

    21                The risks, just to be a little bit more

    22    specific, could include, for example, if there were a

    23    complete viral genome in the contaminating DNA,

    24    possible at the extreme, possible uptake and

    25    expression of the viral genes to the extent that

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     1    intact virus could be replicated.

     2                Of concern primarily in recent years to

     3    the DNA vaccine and the gene therapy communities has

     4    been the possibility of DNA causing mischief in terms

     5    of insertional mutagenesis, activation or inactivation

     6    of genes, up regulation or down regulations of genes.

     7                So that's not an issue peculiar to cell

     8    substrates and has been primarily an issue, as I just

     9    mentioned, for those two other therapeutic areas.

    10                The real area of concentration of concern

    11    over the past several decades in the context of this

    12    meeting has been on the possibility of tumor induction

    13    with very specific emphasis on the possibility of an

    14    oncogene being transferred from the cell substrate to

    15    the recipients of the product.

    16                In addition, there has been some

    17    discussion of the possibility through residual

    18    cellular DNA of activating proto-oncogenes or

    19    inactivating tumor suppressor genes.  Both of those

    20    possibilities, again, are not unique to this, but of

    21    special interest to the people involved in gene

    22    therapy and DNA vaccines.

    23                Well, there's a lot known about DNA and

    24    the possible mischief that it could cause, and it goes

    25    back many decades.  We know that -- and I'm not going

                                                                        154

     1    to dwell on this because some of these have already

     2    been discussed, but even going back to 30, 40 years

     3    ago, it's been clear that nucleic acid extracted from

     4    phage can be infectious for spiroplasts under the

     5    appropriate conditions.

     6                So we know that viral nucleic acids for

     7    many years can, in fact, be infective.  We also know

     8    that viral DNA can transform cells, and there are two

     9    examples out of many up there on the screen, and as

    10    was mentioned yesterday several times, cellular DNA

    11    was demonstrated to transform 3T3 cells in a classic

    12    experiment in the 1970s.

    13                Two out of 26 human tumors formed foci in

    14    the 3T3 assays, but complicating the interpretation of

    15    those data is also the fact that normal human DNA also

    16    caused foci in that assay system.

    17                It's also well known that viral DNA can be

    18    infective for animals, and we're going to hear a

    19    little bit later about the SIV story.  So I'm not

    20    going to dwell on that.

    21                And finally, in another classic

    22    experiment, it was shown that viral oncogene, a viral

    23    oncogene, v-src, could, in fact, cause tumors in

    24    chickens.

    25                Now, on the other side of the coin to all

                                                                        155

     1    of those positive things associated with nucleic acids

     2    and things to worry about are a bunch of negative

     3    results, and going back to the 3T3 experiment that I

     4    mentioned a moment ago, it's important to remember

     5    that 24 out of 26 of those tumors, in fact, did not

     6    cause foci in the 3T3 assay, and equally important is

     7    the fact that very high molecular weight, human tumor

     8    DNA, was required in order to get a positive, and the

     9    molecular weight was around 30 million, and that 20

    10    micrograms was about a minimum that would give a

    11    positive readout in that system.

    12                There's a recent report within the last

    13    couple of years of up to 250 micrograms of hybridoma

    14    DNA being negative in terms of any evidence of tumor

    15    formation in mice over a two-year period.

    16                Also 100 micrograms of Hela and ten

    17    micrograms of T24 DNA have been negative in an anti-

    18    thymocyte newborn rat assay, and it's here important

    19    to point out that the T24 cell has an activated ras

    20    oncogene associated with it.

    21                And then there's a study which was

    22    initiated actually during the last year that I was

    23    still at CBER, and that was an attempt to do a study

    24    in a non-human primate to try to assess the potential

    25    tumorigenic risk associated with tumor cell DNA, and

                                                                        156

     1    in that study we used T24, which as I just mentioned

     2    has an activated ras oncogene in immunosuppressed

     3    rhesus monkeys, and there were both chronic over a

     4    two-week period, and acute inoculation of the monkeys

     5    intramuscularly, intravenously, and intracerebrally.

     6                And those studies were last reported in

     7    terms of a follow-up of the inoculated animals about

     8    four years ago, and that was after eight years from

     9    the initial inoculation.

    10                It's now 12 years since the inoculations

    11    were done, and we're in the process of trying to

    12    locate the monkeys in their colony down in Louisiana

    13    for another follow-up.

    14                And finally, I just would like to point

    15    out that we do have a normal daily burden in all of

    16    this of about one nanogram of proto-oncogenes that

    17    result from normal cell death in our own bodies.  So

    18    it's not as if there's a zero background that each of

    19    us experiences.  There is a significant background of

    20    DNA, in particular proto-oncogene DNA.

    21                Now, it's been pointed out by us and a

    22    number of other people in recent years, again, with

    23    special emphasis on the DNA vaccine area that foreign

    24    DNA meets significant physiologic obstacles when we

    25    encounter it, and I'm not going to go through these

                                                                        157

     1    because they're well described in more detail in the

     2    literature.

     3                The point here is simply that there are a

     4    number of hurdles that DNA meet ounces it gets into a

     5    person or an animal.

     6                Now, there is a little bit actually of

     7    human experience with what one would consider

     8    potentially dangerous DNA from products.  The first

     9    one, which I'll mention, is a -- actually it was

    10    mentioned during the first day of the meeting by Dr.

    11    Hayflick, and that is there was an experiment done in

    12    the mid-1950s in which adenovirus vaccine was produced

    13    in retrospect in Hela cells, and it was a crude

    14    vaccine with an extremely high probability that Hela

    15    cell DNA was a contaminant of the vaccine.

    16                It was administered to only six people,

    17    and a number of years ago we asked the Army, U.S.

    18    Army, which is where this study was done, to conduct

    19    a follow-up study on those six people.  They were able

    20    to actually find the people, locate them, and do a

    21    medical assessment, and that was 25 years after the

    22    initial inoculation.

    23                And the result of that follow-up was that

    24    all six people were alive and well and without any

    25    evidence of neoplastic disease.  A very small number

                                                                        158

     1    of people, but it's important in talking about

     2    potential risks to try to understand what data are

     3    available to try to put things into perspective.

     4                Another piece of information which, again,

     5    is perhaps not as complete as one might want, but

     6    nevertheless is important to recognize, is a study

     7    that was done in the 1970s in which individuals that

     8    had received blood transfusion from donors who were

     9    later found to develop a neoplastic disease of the

    10    lymphoid system, either lymphoma or leukemia, were

    11    followed up to see whether or not there was an

    12    increased risk in the recipients of blood from those

    13    individuals who later developed neoplastic disease of

    14    the lymphoid system.

    15                And the results of that study showed that

    16    there was, in fact, no increased risk for cancers of

    17    the lymphoid system seven years after the blood

    18    transfusions had been given.

    19                Now, that's important also not just

    20    because of the potential for transmitting potentially

    21    oncogenic cells in the transfusion.  It's important

    22    because recently it's been shown that human blood for

    23    transfusion contains a significant amount of human

    24    DNA, which obviously derives from cell death during

    25    the holding of the blood for transfusion in blood

                                                                        159

     1    banks.

     2                That amount of DNA is substantial.  It can

     3    range from 75 to 450 micrograms of DNA.  So the point

     4    here is that not only were potentially oncogenic cells

     5    transfused, but also the potentially oncogenic DNA in

     6    the blood packs.

     7                Now, as was stated a number of times,

     8    these discussions are not new today, and there have

     9    been a number of previous discussions, and I won't do

    10    anything but highlight these very quickly.

    11                The first significant one to tackle the

    12    DNA issue was 15 years ago in 1984 at an FDA and NIAID

    13    workshop, and at that point there was what's been

    14    attributed to that meeting, a ten picagram per dose

    15    outcome, and let me just show you the actual statement

    16    and then make a comment about it.

    17                The statement of the working group on DNA

    18    stated that procedures for the production of

    19    biologicals must demonstrate that no cellular or other

    20    unwanted DNA molecules will be in the final product at

    21    a level which would have a biological activity, that

    22    is, activities which could induce changes of normal

    23    cellular processes.

    24                I think that is really the point.  It's

    25    not whether it's ten picagrams or 100 milligrams.  The

                                                                        160

     1    point is that what we don't want in the product is DNA

     2    or other unwanted materials that are going to have

     3    biological effect, and that point is often in this

     4    because of the numbers, whether it's 100 picagrams or

     5    whatever it is, but this is really the issue.

     6                A couple of years later, in 1986, WHO had

     7    another working group and reassessing the data in a

     8    more thorough manner than could have been done at the

     9    workshop, and that's where the 100 picagram per dose

    10    target came from.

    11                More recently, Dr. Temin did some

    12    calculations which were published in terms of risk of

    13    DNA in products, particularly emphasizing DNA

    14    vaccines, and came up with a risk of activating a

    15    proto-oncogene of less than one in ten to the minus 16

    16    per DNA molecule.

    17                Now, I've translated that into the risk

    18    associated with 100 picagrams of DNA so that the

    19    numbers here are all normalized to 100 picagrams of

    20    DNA.

    21                A couple of years later Dr. Reinhardt

    22    Kurth did similar calculations in terms of the risk

    23    that might be associated with DNA in terms of the

    24    probability of two independent activating events in a

    25    single cell, and translated into a 100 picagram basis,

                                                                        161

     1    the risk would be one in ten to the 12th, which is

     2    very essentially the same as Dr. Temin's calculation.

     3                Another group from the Netherlands

     4    estimated on a worst case basis for hybridoma DNA that

     5    a risk of an adverse event would be one in five times

     6    ten to the eight.

     7                And then most recently, Dr. Krause and

     8    Lewis estimated in a published paper that if cellular

     9    DNA were to encode an entire viral genome, that there

    10    is a possibility that it could become infectious

    11    during the production of viral vaccines.  They

    12    estimated that if there were 100 genome copies in the

    13    cell substrate and that if product contained 100

    14    micrograms of cell DNA, then there would be a risk of

    15    about one in 400,000 to the recipients of that

    16    vaccine.

    17                However, if you normalize that to 100

    18    picagrams as I've done here, then the risk becomes

    19    about one in four times ten to the ninth.

    20                Now, that brings us up to the present, and

    21    I think the question here is whether there are issues

    22    on which there is still some disagreement and on which

    23    there should be additional discussion.  One of them

    24    you've already heard about, and that is the question

    25    of whether based on phenotypic characteristics of the

                                                                        162

     1    cells there is a relative risk associated with them in

     2    terms of one cell being more tumorigenic than another,

     3    and we've already had some discussion about that.

     4                Another issue or another difference is in

     5    terms of the amount of  DNA that may be in a final

     6    product, and what we haven't heard much about is

     7    purification and downstream processing of various

     8    products, including the live viral vaccines using

     9    contemporary methods and how that may be applied to

    10    reduce the amount of DNA to levels that are not

    11    worrisome.

    12                And then there is the issue of the size of

    13    the DNA, and what are we really worrying about in a

    14    product and is it of sufficient size to even be a

    15    cause for concern?

    16                In my mind the real question is in terms

    17    of the cells themselves, is not how well or how badly

    18    a cell is behaving in a tumorigenicity test, but

    19    really whether the cellular impurities in the product

    20    carry a risk.

    21                For example, I could argue that a highly

    22    malignant cell may have one copy of three activated

    23    oncogenes while a very mildly tumorigenic cell has a

    24    100 copies of a single oncogene, and I don't know

    25    which one is worse in terms of using it as a product.

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     1                And I think the real point is to

     2    understand what it is that's in the product and to

     3    understand what the characteristics of the cells are

     4    in terms of whether you have activated viral oncogenes

     5    or other genetic elements that you can focus on and be

     6    sure that they're not there in the final product or

     7    minimized.

     8                Well, let me summarize here and just again

     9    reiterate.  We do know that viral DNA can infect in

    10    vitro and in vivo.  We do know that viral DNA can

    11    transform cells in vitro, and we do know that viral

    12    oncogenes can cause tumors in vivo.

    13                But at least to my knowledge, we also know

    14    that tumor cell DNA has not caused tumors in any

    15    animal system of which I'm aware, and that makes

    16    people a little nervous because there's a disparity

    17    between all of those positive results and the fact

    18    that try as one might, there's been no evidence that

    19    tumor cell DNA can cause tumors in animals.

    20                Another point to try to remember is that

    21    when we look at probabilities of adverse events, they

    22    are a direct function of the assumptions that you make

    23    that go into the calculations, and you can jiggle

    24    around the assumptions and make things look better or

    25    worse by orders of magnitude, and it's the assumptions

                                                                        164

     1    and the validity of the assumptions that are really

     2    important in making a legitimate probability

     3    assessment of adverse events.

     4                Now, in the literature, when you try to

     5    look at some of the studies that have been done in the

     6    past, it's important to look at the details of those

     7    studies and not just use the numbers that come out of

     8    them, like 20 micrograms of tumor cell DNA causes foci

     9    in the 3T3 assay.  The devil is in the details, and

    10    unless you look at what the things like the 30 million

    11    molecular weight that's required, whether you look at

    12    whether facilitators like DMSO or calcium phosphate

    13    precipitation are needed to get the result, then

    14    you're using things inappropriately and misleading

    15    when you try to use those numbers in doing a

    16    calculation.

    17                Now, there are, I think everyone would

    18    agree, less than all of the data that we might need to

    19    make some really firm conclusions in this area. 

    20    Unfortunately, the lack of data has been with us for

    21    15, 30 years, and I think if there were ever a

    22    justification for a research based regulatory

    23    organization, this is an example of where it is

    24    because there are probably generic pieces of data,

    25    basic ones.

                                                                        165

     1                For example, dose response data could be

     2    very helpful in understanding whether, for example,

     3    there is a threshold below which we really don't have

     4    to worry about DNA, and those data don't exist.  At

     5    least I'm not aware of them.

     6                So there are areas where focused

     7    regulatory research could be very helpful so that we

     8    don't have to come back here 15 years later and go

     9    through the same thing again.

    10                Now, one final comment, and that is I do

    11    think that there is one very special case, and that's

    12    been discussed particularly today, and that is live

    13    viral vaccines produced in tumor cell lines.  It

    14    presents very special problems and needs to be

    15    addressed with the greatest of care.

    16                But I think that that is one element in a

    17    very big array of possible products, and we shouldn't

    18    confuse the very special case with all of the other

    19    ones that are more easily addressed, and we shouldn't

    20    make the whole area very difficult when some of them

    21    can be handled relatively straightforward.

    22                (Applause.)

    23                DR. ZOON:  Thank you for that very nice

    24    summary.

    25                Because it ran over, I would ask unless

                                                                        166

     1    there's a burning question that we'll move on to the

     2    next presentation.

     3                If I could then introduce our next

     4    speaker, Dr. Donald Blair.  Dr. Blair is from the

     5    Frederick Cancer Research and Development Center at

     6    NCI, and he will be discussing evaluation of the

     7    transforming activity and tumor inducing capacity of

     8    tumor cell DNA.

     9                DR. BLAIR:  Okay.  Thank you very much.

    10                The subject of the transforming potential

    11    of DNA from transformed cells has been something that

    12    has come up frequently from the start of this meeting. 

    13    In fact, this morning someone made the comment that

    14    the question had finally been or the danger or hazard

    15    of DNA had finally been put to bed.  If that's true,

    16    then I am going to tell what is probably part of the

    17    bedtime story, which is what I was asked to do was to

    18    review some of the data from a series of experiments,

    19    most of which were done over ten years ago in

    20    evaluating the ability of transformed trell (phonetic)

    21    DNA to induce foci and tumors in systems, in tissue

    22    culture, and in animals.

    23                So what I will try to do is to sort of go

    24    through some of the techniques that were used in some

    25    of these experiments and try to summarize the results

                                                                        167

     1    that we had obtained in some studies that we did, plus

     2    adding some from some others.

     3                Okay.  So what I'll do is review this data 

     4    and some of our results, and the whole question of

     5    this is based on the need to have an effective way to

     6    transfer mammalian DNA, transfer traits from DNA into

     7    mammalian cells.

     8                And attempts have been made ever since the

     9    observations you could do this in bacteria were made

    10    over the years, and in general, most of them were

    11    relatively difficult to reproduce and difficult to

    12    obtain.  It wasn't really until the development of the

    13    technique by Graham and Van der Eb of using calcium

    14    phosphate to transfer DNA that there was really a

    15    reliable way of doing transfer of DNA into mammalian

    16    cells.

    17                Following that observation and using those

    18    techniques, there was a rapid development of and

    19    demonstration that one could transfer viral DNA.  One

    20    could transfer the oncogenes of transforming viruses

    21    and induce transformed cells using purified DNA, but

    22    it was in 1979 that the observations out of Weinberg's

    23    lab and also out of Mike Wigler's lab that it was

    24    possible to transfer the transformed phenotype from

    25    chemically transformed mouse cells to recipient cells

                                                                        168

     1    through transfection and through the transfer of DNA

     2    and chromatin.

     3                And following that, the demonstration,

     4    again, by Weinberg, Cooper, and Wigler's lab that one

     5    could do this from human tumors and that, indeed, the

     6    material that one was transferring was the identified

     7    oncogenes, such as ras.

     8                These observations and this technique led

     9    to a great outburst of attempts to use this technique

    10    to try to evaluate whether all tumors or all DNAs were

    11    capable of doing this and whether one could use this

    12    technique to identify and isolate transforming genes,

    13    and as probably everyone is aware, this went on for

    14    some time with a varying degree of success.

    15                The technique was based on the observation

    16    that if one transferred this DNA onto NIH3T3 mouse

    17    fibroblast, one could see foci of morphologically

    18    transformed cells, some better, some easier to see and

    19    some less easy to see.  As we pointed out,

    20    occasionally normal cells would also transfer this

    21    activity.

    22                But the crucial factor here in

    23    transferring the human DNA was the fact that within

    24    these foci, one could identify the presence of human

    25    genetic material using repeat probes, and furthermore,

                                                                        169

     1    that one could take these initial foci, take the DNA,

     2    transfer this again, and retain a defined set of human

     3    sequences which eventually could lead to the

     4    identification of specific transforming genes

     5    associated with those sequences.

     6                So the initial assay was fairly simple

     7    using the calcium phosphate procedure, transfecting

     8    that onto NIH3T3 cells with various treatments to

     9    enhance that DNA uptake, incubating those cells until

    10    one sees foci of morphological transformation.

    11                The initial results, however, were

    12    relatively -- this process was relatively inefficient,

    13    and so a lot of people set out to try and make this a

    14    little more efficient to try to enhance it, and we and

    15    Wigler's group, as well, devised an assay which was

    16    called the nude mouse tumor assay to try and increase

    17    the sensitivity of this kind of assay, and this simply

    18    summarizes the sort of final version that we and a lot

    19    of other people over the years have used, and that is

    20    to do the transfection of high molecular weight DNA

    21    from tumors or tumorigenic cell lines, in the presence

    22    of a selectible plasmid, DSV2 neo, and to select the

    23    cells which had taken up DNA by selection for neo

    24    resistance to accumulate a population of cells, all of

    25    which contained at least some human DNA.

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     1                Those pools of colonies could then be

     2    trypsinized, injected into mice to generate tumors, or

     3    could be replated and looking for foci.  The advantage

     4    of the tumor assay at the time was that this was a

     5    more biologically significant endpoint.  Furthermore,

     6    it appeared to be a bit more sensitive than looking

     7    for foci.  It avoided a heck of a lot of tissue

     8    culture and time staring at microscopes, and it did

     9    pick up some transforming genes that couldn't be

    10    recognized by morphological transformation since they

    11    induce relatively poor morphological transformation.

    12                Again, the products of these would be

    13    reisolated, checked for the presence of human DNA,

    14    transferred to a second cycle in order to validate

    15    that, indeed, this was a genuine transfer.

    16                The critical features of this that we

    17    found over the years were really, as has been alluded

    18    to, the state of the DNA.  The DNA appeared you have

    19    to have it as relatively high molecular weight. 

    20    Fragmentation of any significant amount destroyed the

    21    majority of the activity in most cases, although with

    22    the appropriate restriction enzymes you could retain

    23    activity with fairly small pieces due to the size of

    24    the genes, as it turns out, you were transferring.

    25                The recipient cells, however, in general

                                                                        171

     1    had to be 3T3s.  They were ideal because they had a

     2    relatively low spontaneous transformation rate.  If

     3    one used seed cultures and controlled the serum and a

     4    variety of other variables, one could retain their

     5    normal state.

     6                However, they could be transfected stably

     7    to high levels, and this appeared to be critical

     8    because we tried a number of other cells, and although

     9    we could in some cases transfer genomic DNA, the

    10    efficiencies were low, and the amount of DNA the cells

    11    retained was generally much lower than what we saw in

    12    3T3.

    13                So in general, this was the cell line of

    14    choice that everybody has tended to use.

    15                Now, in using the -- the problem both in

    16    foci and in tumors was that you had a background, and

    17    this simply sort of summarizes some data that we

    18    accumulated over the years of controls.  Most of these

    19    were transfected either just with DSV2 neo or in many

    20    cases with human placental DNA as a control, and the

    21    percentage of tumors which arose within the first ten

    22    weeks after injection of around three million

    23    transfected cells back here turned out to be around

    24    ten or 11 percent.

    25                The feature of these tumors is we could

                                                                        172

     1    never recover human DNA from these in cases where we

     2    had transfected human DNA, and we were unable to

     3    retransfect this and generate a second round.  So

     4    these appeared to be the kind of spontaneous

     5    transformance that Dr. Rubin talked about earlier in

     6    this meeting.

     7                So with that, however, there were a number

     8    of cell lines clearly, which everyone is well aware,

     9    which contained transforming DNA where one could see

    10    foci and one could get tumors in a tumor assay which

    11    arose relatively early, and one could get secondaries

    12    by taking the primary tumor of these cell lines and

    13    retransferring them.

    14                It's interesting that most of these, with

    15    the exception of MNG HOS are ras.  All of the genes

    16    recovered and the genes apparently responsible for

    17    this transformation were ras.

    18                This was a cell line which we used as a

    19    control.  It's a single copy MOS transformed cell

    20    line, and it allowed us to make an estimate of really

    21    how efficient this was.

    22                We knew if we took 100 micrograms of

    23    genomic DNA we could generate about a single focus,

    24    which gave us an efficiency of around ten to the minus

    25    seventh to ten to the minus eighth.

                                                                        173

     1                If we did the reconstitution experiment

     2    with a clone plasmid DNA and at the maximum

     3    efficiency, we could get the same single focus with

     4    around ten to the minus fourth micrograms, again,

     5    roughly within certainly orders of magnitude, the same

     6    kind of efficiency.

     7                But the efficiency is very low.  It's a

     8    lot of DNA being put on these cells to get very few

     9    transformants, and given that we were pushing this

    10    assay and others were pushing this assay, it became

    11    clear that we could generate transforming genes out of

    12    things that weren't transforming genes in our original

    13    tumors.

    14                In our case we took a cell line called OV

    15    CAR 3, generated an active transforming agent which,

    16    upon analysis and characterization, turned out to be

    17    a fusion between sequences on Chromosome 8 and

    18    sequences in the 9P23-24 on Chromosome 9.  These

    19    sequences were formed during the transfection.  They

    20    could be continuously transferred, but they were not,

    21    indeed, transforming sequences in the original tumor.

    22                And as more and more people pushed this

    23    assay to many of the genes, which were recovered from

    24    this kind of transfections, turned out to be fusions

    25    or transfection generated fusions or activations,

                                                                        174

     1    deletions of control elements, other aspects.

     2                It's interesting that at least in our

     3    hands we only saw these kinds of things with DNA from

     4    tumors, not with human placental DNA, which we ran

     5    extensively as controls.  Whether that has something

     6    to do with the methylation state, which was referred

     7    to earlier, I don't know, but the generation of sort

     8    of spontaneous activation seemed to be more prevalent

     9    in tumors.

    10                However, in terms of what was negative,

    11    which was part of why I was originally asked to do

    12    this, because we had a lot of negative, a number of

    13    cell lines pretty capable of forming tumors were

    14    repeatedly negative in our assays, in addition to what

    15    we expected the human fetal lung, human placenta, and

    16    others have shown that cell lines like VERO and MRC5s

    17    are also negative or have been negative when pushed

    18    into these assays and assayed in this way.

    19                However, if we summarize all of the

    20    analysis that we did, we looked at about 27 cell lines

    21    and 52 primary tumors and recovered a total of around

    22    nine positive transfections when all was said and

    23    done.  Most of those are ras, five of the cell lines,

    24    both of the tumors.  One of them was nep and one ovc,

    25    a gene which we called ovc.

                                                                        175

     1                I also added, because this recently

     2    appeared in National Journal of Cancer a summary from

     3    Dr. Janssen, where he had summarized a similar set of

     4    experiments using a relatively similar assay protocol,

     5    129 tumors and cell lines, most of which turned out to

     6    be ras.  A number of others were genes that he also

     7    identified.  A large percentage of these were

     8    transfection generated recombinants and activations.

     9                So the conclusion, I think, from our data,

    10    from the data of others over the years was that this

    11    was not an easy thing to do, to transfer 3T3 cells

    12    with genomic DNA, and while it does detect oncogenes,

    13    it does so at relatively low efficiency and most of

    14    them are ras.

    15                Now, as we look back now, from what we

    16    know about the situation, it is perhaps not surprising

    17    that that's the case because as we all know and as

    18    we've heard, it takes a number of changes to make a

    19    cell become a transformed cell, and this is simply

    20    taken from the recent paper in Nature by Weinberg in

    21    which he essentially took a normal human fibroblast to

    22    a transformed cell by making the addition of

    23    telomerase, large T antigen, and ras in order to make

    24    the escape from senescence to get through crisis to an

    25    activated series of tumor suppressors, and finally to

                                                                        176

     1    activate the mitogenic pathways.

     2                The cell line that we used for most of

     3    these studies, NIH3T3, appears to have a P16

     4    inactivated, and certainly that might tend to bias

     5    what genes we were able to detect, but in any case,

     6    the detection of transforming genes and the ability of

     7    DNA to transform 3T3 cells was relatively low even in

     8    the situation where we were talking about optimizing

     9    our conditions and optimizing the system in order to

    10    detect transforming DNA.

    11                So certainly a fraction of tumorigenic

    12    cell lines were able to transform 3G3 cells.  A large

    13    number of them, however, a large percentage, probably

    14    70 to 80 percent were not.

    15                Rearrangements during transfections,

    16    however, could also generate transforming genes, and

    17    they've contributed to this, but the frequency even

    18    there, again, was fairly low.

    19                So even under optimal conditions for

    20    transfer, for getting DNA into cells, the ability to

    21    transfer this DNA into transformed cells is very low,

    22    and since there's multiple genetic alterations in

    23    rodents and human cells in order to generate these

    24    cells, that's probably responsible in part, despite

    25    the fact that we were getting as much uptake as we

                                                                        177

     1    could, why there's a relatively low frequency of

     2    transformation.

     3                And so the basic conclusion seemed to be

     4    that, yes, the DNA was transforming, but, no, it's a

     5    difficult thing to do even when you try and bias the

     6    system in any way, and when you go to primary cells

     7    and go to reduced levels of DNA, reduced levels of

     8    uptake, the frequency became at least in our hands

     9    and, I think, everyone else's as relatively

    10    undetectable.

    11                So I will stop at that point.

    12                (Applause.)

    13                DR. ZOON:  Thank you very much for that

    14    excellent overview.

    15                Please identify yourself.

    16                DR. FRIED:  Mike Fried.

    17                It's interesting.  We note today that

    18    NIH3T3 cells are not only missing P16, but they're

    19    missing this P14 arf, which Jim McDougall referred to,

    20    and arf is a very interesting protein because it's

    21    upstream of P53, and it's actually only induced by

    22    oncogenes, by myc, by ras, E1A, and others, it then

    23    turns on P53.

    24                So if the arf NIH3T3 cells are arf

    25    negatives, and as most cell lines partially have a lot

                                                                        178

     1    of changes, but without arf, it allows oncogenes to be

     2    expressed.

     3                Well, in normal cells if these oncogenes

     4    came in, P53 would be turned on.  The cell would go

     5    through apoptosis and be taken out as a defense

     6    mechanism.  So I think that explains why NIH3T3 were

     7    so sensitive for those detecting oncogenes, and most

     8    of the cell types aren't.

     9                DR. BLAIR:  No, I think that it's clear

    10    that each cell with the pattern of inactivations and

    11    3T3 having the particular set that it did probably

    12    made it possible to do this, although I think the

    13    point that they do incorporate a large amount of

    14    incoming DNA and then take some up also contributes to

    15    the fact that you were able to do these experiments.

    16                DR. FRIED:  Sure, but I'm just saying that

    17    most cells picking up oncogenes would turn on P53 and

    18    be taken out of the population.

    19                DR. KRAUSE:  Phil Krause, FDA.

    20                Don, have you done experiments in which

    21    you directly injected DNA from tumors into animals?

    22                DR. BLAIR:  We didn't try to directly

    23    inject the DNA.  We did some experiments directly

    24    injecting cloned DNA, and that in our hands was

    25    relatively clone V MOS DNA at the time, and those

                                                                        179

     1    experiments were not terribly efficient, and so we

     2    never really tried with genomic using tumor DNA

     3    directly into mice, no.

     4                DR. KRAUSE:  And I think I and a lot of

     5    people are intrigued by the Weinberg experiment that

     6    you summarized.  Would you care to speculate?  What do

     7    you think would happen if you tried to inject those

     8    three DNAs directly into an animal?

     9                DR. BLAIR:  I mean, I guess my feeling

    10    would be that the probability of getting all three

    11    into the same cell might be probably fairly low, and

    12    whether you could see the responses that he sees in

    13    cell culture, I would think it would be unlikely, but

    14    you'd have to do it.

    15                DR. KUNG:  I'd like to make a -- oh, go

    16    ahead.

    17                DR. ZOON:  Please identify yourself

    18    though.

    19                DR. KUNG:  Oh, sorry.  Hsing-Jien Kung

    20    from UC-Davis.

    21                I'd like to make a comment because from

    22    Dr. Petricciani's talk, he mentioned the injection of

    23    V-SARC DNA can cause tumor, and in fact, I did that

    24    experiment, and had I known that I would be invited to

    25    this FDA meeting 18 years later, I would have kept

                                                                        180

     1    that chicken longer, but I didn't.

     2                (Laughter.)

     3                DR. KUNG:  But the one point I'd like to

     4    make is that that experiment in that paper, we also

     5    said showed that the tumor actually regressed.  All

     6    the tumors inject with V-SARC DNA, tumor regressed,

     7    and we felt at that time was due to immune response

     8    because we did not have nude chicken.  We had the

     9    immunocompetent chickens there.

    10                And so it's really not a cancer in that

    11    sense.  All the tumors regress.  Whereas if we

    12    injected the entire rsv DNA, okay, doing direct

    13    injection, then the tumor will, in fact, cause

    14    mortality of the chickens.  Okay?  So there is a

    15    difference.

    16                And I'll finish the story.  We never

    17    published because it was a negative result.  So we did

    18    inject C-SARC, and C-SARC never caused tumor.  The

    19    assay was very sensitive in the sense that if you have

    20    a point mutation 527, the C-SARC point mutants, that

    21    can cause tumors.

    22                And so the assay was very sensitive, but

    23    it would pick up something that I would not call it

    24    cancer.  Okay?

    25                DR. ZOON:  Thank you for that comment.

                                                                        181

     1                The last comment/question.

     2                DR. BERKOWER:  Yes, Ira Berkower.

     3                One of the problems is I see a contrast

     4    between what you said and what Dr. Petricciani said,

     5    and that is the relative risk between tumor DNA and

     6    normal DNA.  You had no transforming events with

     7    normal DNA.  So can you put an upper limit on the

     8    relative risk due to the tumor DNA versus normal or

     9    lower limit maybe?

    10                DR. ZOON:  Could we have the mic, please?

    11                DR. BLAIR:  I think it works now.

    12                (Laughter.)

    13                DR. BLAIR:  I think, you know, what we saw

    14    was that when we did normal DNA, placental DNA and at

    15    least one or two human normal cell populations, we

    16    didn't get any tumors or foci that contained DNA that

    17    we could transfer.  We were doing on the order of 300

    18    micrograms of DNA at a shot.  We probably did the

    19    placental DNA ten or 20 -- maybe ten times.

    20                So in that sense, we never saw anything at

    21    that level that we could verify that there was a focus

    22    or a tumor that was due to the transfer of the DNA. 

    23    So that's the only limit I could put on it.

    24                DR. BERKOWER:  But would you say tumor DNA

    25    is at least one log more tumorigenic?  It seems to me

                                                                        182

     1    that it is just from what I've seen in here.

     2                DR. BLAIR:  Well, if you talk about the

     3    active ones, yes, but if you talk about the other ones

     4    that are not active in our hands, no, they were no

     5    more active than -- we didn't see anything.  So --

     6                DR. BERKOWER:  And, secondly, you know,

     7    yesterday we were trying to get at this question

     8    whether some tumors are more worrisome than others. 

     9    It would also seem from your data, as opposed to what

    10    was being said on the podium yesterday that some

    11    tumors are much more worrisome than others.  Others

    12    are almost not worrisome.

    13                This would be towards getting a molecular

    14    discriminator of the risk.

    15                DR. BLAIR:  Well, I think, you know, this

    16    assay clearly is biased, and so one could argue that

    17    a tumor that has an activated ras gene would be more

    18    likely to be detected in this assay.  In that sense,

    19    tumors that had activated ras genes were more readily

    20    detectable.

    21                In some of those there were amplifications

    22    that may have helped to increase the sensitivity of

    23    the assay, but this is, I think -- you know, this is

    24    within a very specific set of conditions designed to

    25    optimize the detection of, you know, morphological

                                                                        183

     1    transformation of NIH3T3 cells.

     2                DR. BERKOWER:  And lastly, one thing I

     3    used to take comfort in was this requirement for very

     4    high integrity, high molecular weight DNA, but in

     5    fact, that may be entirely spurious.  That may be

     6    almost an artifact, again, of the method because if

     7    there is more than one step needed, all you're really

     8    measuring there is how far those two genes are apart

     9    from each other.  You just want to get them into the

    10    same cell.  You're not actually measuring anything

    11    about the DNA.  You're measuring something about the

    12    genes, that they're far apart.  So they need to be on

    13    the same Ps or something like that.

    14                DR. BLAIR:  Well, the 20 kilobase size, 20

    15    to 30 kilobase size, is pretty small on the genomic

    16    scale.  So they're pretty close together.  I don't

    17    think the linkage is probably something that you would

    18    -- that would be a problem in this particular kind of

    19    experiment.

    20                DR. ZOON:  Okay.  If we could have one

    21    last comment, and then we need to move on.

    22                DR. HUGHES:  Yes.  Let me respond by

    23    saying that you need to take into account that even

    24    though the DNA comes from an abnormal cell, if the

    25    transforming principle you pick up is not present in

                                                                        184

     1    the cell DNA that you began with but was created by

     2    transfection, those are events that will happen with

     3    normal DNA, and the reason that those are seen with

     4    DNA from tumor lines or cell lots and not with normal

     5    DNA represents the very large additional number of

     6    experiments that occurred.

     7                But those are certainly events that will

     8    take place with normal DNA if you do enough of it.

     9                The other important point is in trying to

    10    put some sort of form or assessment of risk, what

    11    you're really measuring in these assays is the risk to

    12    3T3 cells, which is not the same thing as the risk to

    13    cells that are not immortalized and don't have lesions

    14    in them which do not make them necessarily oncogenic,

    15    but which certainly make them immortal and, as a

    16    consequence, easier to get into a state in which you

    17    can measure tumorigenicity.

    18                So I don't think you can reasonably use

    19    this as a measure of either the safety of normal DNA

    20    or the danger of DNA from a transformed cell.

    21                DR. ZOON:  Thank you very much for that

    22    comment.

    23                And thank you, Don.

    24                Our next speaker I'm very pleased to

    25    introduce.  We've introduced her many times at

                                                                        185

     1    workshops, is Dr. Ruth Ruprecht, who is from the Dana-

     2    Farber Cancer Institute, and will speak to us this

     3    afternoon on infectivity of retroviral provirus DNA.

     4                Thank you for coming.

     5                DR. RUPRECHT:  Thank you for inviting me.

     6                DR. ZOON:  The lights, please.

     7                DR. RUPRECHT:  I'm speaking about the

     8    infectivity for retroviral DNA.  My laboratory came

     9    upon this issue in a somewhat surreptitious way.

    10                PARTICIPANTS:  Microphone.

    11                DR. ZOON:  Switch it on.

    12                DR. RUPRECHT:  Is that better?

    13                Our experiments started with a mutant of

    14    SIV made by Ron Desrosiers' laboratory called SIV

    15    Delta 3.  This mutant is built on the backbone of SIV

    16    MAC 239 and has three big deletion, one in the vpr

    17    gene, and two in the nef gene.  The third deletion

    18    also overlaps with the LTR, the negative regulatory

    19    element in it.

    20                When Ron's group used this virus in

    21    juvenile and adult rhesus monkeys, it was attenuated,

    22    and it turned out to be protective in about 50 percent

    23    of the recipients upon challenge with wild type nef-

    24    plus virus.

    25                We wanted to develop a neonatal macaque

                                                                        186

     1    monkey model, and we gave these neonatal animals the

     2    live SIV Delta 3 orally.  As you know, we have a

     3    rather surprising result which we reported in 1995. 

     4    These animals developed AIDS that looked for all

     5    intents and purposes exactly like the disease induced

     6    by the wild type virus.

     7                Follow-up data then showed that the SIV

     8    Delta 3 was 100 percent pathogenic in the initial

     9    cohort.  We were obviously concerned that we may have

    10    had a contamination reason, immunosuppressive virus,

    11    and we ruled out any known immunosuppressive virus in

    12    this initial series of animals.  Therefore, we knew

    13    that there was no wild type SIV, and we also knew that

    14    there was no simian retrovirus Type D present in the

    15    animals to explain this result.

    16                But we couldn't test for an adventitious

    17    agent that may have been present in the initial CMX-

    18    174 cells, and we also could not test for an

    19    adventitious agent that may have been present when we

    20    grew up the virus in rhesus macaque PBMC.

    21                Therefore, we decided to go to pure DNA,

    22    and we felt that the truth would be in the DNA.

    23                The initial four animals are shown here. 

    24    The first three got cell free virus.  The fourth one

    25    got infected blood.

                                                                        187

     1                We decided to directly clone the virus out

     2    of animal 94-1 for 80 weeks post inoculation.  At this

     3    time this infant already had immunodeficiency.  We

     4    were able to clone virus out without any intervening

     5    amplification in vitro.  

     6                So what we did was we took PBMC and cloned

     7    the provirus genome in two halves, the five prime half

     8    and the three prime half, and then reconstructed a

     9    replication competent virus.

    10                In doing so -- next slide, please -- we

    11    realized that 90 percent of the proviral halves that

    12    we cloned out were actually defective, and only a

    13    small minority of the seemingly clones of right sides

    14    were actually giving rise to replication competent

    15    viruses.

    16                Now, in order to continue studies with

    17    proviral DNA, we needed to first conduct a positive

    18    control experiment.  We needed to make sure that wild

    19    type SIV MAC 239 would yield similar virus replication

    20    kinetics and disease in rhesus macaques.

    21                Afterwards our plan was to then take

    22    proviral DNA of SIV Delta 3 of the virus that we

    23    cloned out of the animal and compare the

    24    pathogenicity.

    25                Earlier this year we published the results

                                                                        188

     1    that we obtained with the parental SIV MAC 239.  I

     2    will go through these results really quickly.

     3                Intramuscular inoculation of 400

     4    micrograms of DNA in plasmic form into one infant, and

     5    500 micrograms of DNA into two adults resulted in

     6    systemic infection, persistent viremia, and death

     7    eventually in all three animals.

     8                The viral loads in the infant and in the

     9    adult were actually reasonably similar to what you're

    10    seeing when infections are conducted with cell free

    11    virus.

    12                The same goes for cell associated proviral

    13    DNA load.  The kinetics are similar to what has been

    14    published by other groups with cell free virus, and

    15    the same statement also holds for infectious cells in

    16    the PBMS.

    17                So intramuscular inoculation of super

    18    coiled plasmid DNA and coding full length SIV MAC 239

    19    is, first of all, reproducible.  We were able to

    20    maintain the provirus stably in a single plasmid.

    21                The three animals became viremic and they

    22    all succumbed to AIDS after longer term follow-up, and

    23    after this series of experiments, we felt that

    24    intramuscular inoculation of cloned proviral plasmid

    25    DNA is reproducible and would be a wonderful tool to

                                                                        189

     1    study viral pathogenesis because if you rely on pure

     2    proviruses in plasmid form, it can easily quantify

     3    your inoculum, and if you're looking at viral mutants,

     4    you can eliminate any parameters that may affect the

     5    infectivity of your inoculum.  Standardization, in

     6    other words, is very easily achieved with super coiled

     7    plasmid DNA.

     8                So after this series of controlled

     9    experiments were finished, we then took the provirus

    10    that we got out of infant 94-1.  This provirus is

    11    called SIV Delta 3-plus.  We inoculated a series of

    12    newborn rhesus macaques and adult macaques with this

    13    SIV Delta 3-plus in DNA form and compared it with SIV

    14    Delta 3, the parental vaccine strain.

    15                First of all, infant VR-6 had high virus

    16    load and died of AIDS.  So the proviral clone, SIV

    17    Delta 3-plus, is not only infectious and replication

    18    competent in vivo, but it is also pathogenic, and it

    19    shows very clearly that the progeny virus that was

    20    isolated from the vaccine recipient is a pathogenic

    21    virus.  There was no other adventitious agent present,

    22    and even with very sensitive PCR analysis, we've made

    23    sure that there was no full length nef gene present.

    24                Another interesting set of data when it

    25    compared virus loads in adults inoculated with SIV

                                                                        190

     1    Delta 3-plus proviral DNA in comparison with adults

     2    given the parental SIV Delta 3 DNA.  It's actually

     3    somewhat of a shocker.

     4                The adult animals given the parental

     5    vaccine virus DNA are shown in the dashed line.  The

     6    peak viral RNA levels were approximately 10,000 copies

     7    per milliliter.  The three animals then were able to

     8    control the virus and were not viremic in long term

     9    follow-up.

    10                The picture in the adults given the

    11    progeny virus that evolved in that long infant

    12    (phonetic) is quite different.  First of all, peak

    13    YLRNA levels are two logs higher, and secondly, the

    14    animals are persistently viremic a short time later.

    15                This shows very clearly that the virus has

    16    become much more aggressive as it adapted in the

    17    infant, in the initial infant, and that what has been

    18    happening in vivo was selection of the fittest.  Even

    19    though virus isolation in adult vaccine recipients of

    20    SIV Delta 3 is negative, we have to know that the

    21    vaccine strength persists in the lymphoid tissues. 

    22    The virus actually continues to replicate at all times

    23    and uses the faulty reverse transcriptase to do so,

    24    and as John Coffin has published and other groups have

    25    published as well, lentiviral reverse transcriptase is

                                                                        191

     1    a lousy, error prone enzyme that generates a number of

     2    mutants.

     3                The virus is then selected for optimal

     4    replicative capacity, and over the years a more

     5    aggressive virus emerges, and then natural selection

     6    in the host environment eventually will compensate for

     7    the initial attenuation and replicative capacity that

     8    was engineered into the vaccine strain.

     9                I should say that the peak viremia that we

    10    saw in these three adults is actually in the same

    11    order of magnitude as the peak viremia seen with SIV

    12    MAC 239.

    13                Now, in terms of infectivity of DNA, a

    14    number of questions should be addressed for which we

    15    don't have experimental answers.  First of all, what

    16    is the minimal infectious dose for intramuscular

    17    inoculation of cloned SIV MAC 239 plasmic DNA?

    18                Second, which route of inoculation is the

    19    most infectious?

    20                And, third, what is the infectivity of

    21    genomic DNA isolated from an SIV MAC 239 infected

    22    animal?

    23                Now, there are some indirect answers to

    24    some of the questions regarding the infectivity and

    25    the amount of DNA received to achieve systemic

                                                                        192

     1    infection.  In 1991, Norman Leitwin and his colleagues

     2    have published a series of an experiment that showed

     3    the infectivity of a lambda clone containing the

     4    provirus of SIV MAC 239. 

     5                Four cynomolgus macaques were inoculated

     6    intramuscularly with 200 micrograms of this lambda

     7    clone.  Three out of the four animals were infected,

     8    but actually long term follow-up data were not

     9    provided, and we don't know whether these animals went

    10    on to develop disease.

    11                Now, 200 micrograms of the lambda clone is

    12    equivalent to about 50 micrograms of our plasmid DNA. 

    13    So we were approximately ten times above the amount of

    14    provirus that's used by Norman Leitwin in our own

    15    experiments, which were conducted with 500 micrograms

    16    of proviral DNA plasmid.

    17                A number of publications have appeared

    18    over the years showing the infectivity of DNA in

    19    animals, and I've just summarized some of them for you

    20    here.  The references are in the Liske, et al., "AIDS

    21    Research on Human Retrovirus," a paper that appeared

    22    earlier this year.

    23                I would like to focus on the SIV

    24    experiments by Elizabeth Sparter because there is some

    25    more quantitative information available in that paper.

                                                                        193

     1                But, first, polyoma virus.  There is some

     2    information about infectivity of plasmid DNA as

     3    compared to cell free virus, and the infectivity of

     4    the super coiled polyoma plasmid DNA is about four to

     5    five logs lower than that of cell free virions for a

     6    given route of inoculation.

     7                For SIV Elizabeth  Sparter and colleagues

     8    have looked at intramuscular and intradermal

     9    inoculation at various doses.  The lower dose of 30

    10    micrograms per animal was no longer fully infectious,

    11    but we don't really have a threshold from these

    12    experiments either.

    13                To conclude, there is actually a positive

    14    note also to the experiments I've described.  If there

    15    ever is a nonpathogenic, avirulent, live attenuated

    16    AIDS vaccine, the way to go would be through

    17    infectious proviral plasmid DNA because, first of our,

    18    our experiments have shown that cell free virus can be

    19    replaced by infectious plasmid DNA.  This would bypass

    20    the need for preparing virus stock in human neoplastic

    21    T cell lines.  Standardization is easy and is very

    22    reproducible.

    23                Systemic infection can be induced very

    24    reproducibly.  I can tell you that with 500 micrograms

    25    in rhesus monkeys, given intramuscularly, 18 out of 18

                                                                        194

     1    animals became systemically infected.  So this is

     2    highly reproducible.

     3                And lastly, infectious proviral plasmid

     4    DNA is stable and can be stored very easily.

     5                (Applause.)

     6                DR. ZOON:  Thank you, Ruth.

     7                This presentation is now open for

     8    questions, discussion.

     9                Yes.

    10                DR. MURPHY:  I'm Brian Murphy, NIAID.

    11                Have you actually taken SIV that's

    12    integrated into genomic RNA and into genomic DNA,

    13    taken the genomic DNA itself and not taken the

    14    provirus out, clone it up, put it into lambda, put it

    15    into provirus, and seeing if you can initiate an

    16    infection with genomic DNA containing a complete copy

    17    of SIV?

    18                DR. RUPRECHT:  No, we have not done this

    19    yet, and to my knowledge, there are no reports of it.

    20                DR. HUGHES:  A brief comment to clarify a

    21    misconception.

    22                DR. ZOON:  Please identify yourself.

    23                DR. HUGHES:  I'm sorry.  I'm Steve Hughes

    24    from ABL.

    25                And that is the role of RT in engendering

                                                                        195

     1    mutations in any retrovirual life cycle is undefined. 

     2    It is unlikely that -- there are three enzymes that

     3    are involved in the replication of the genome:  RT,

     4    the host DNA dependent polymerase, and the host DNA

     5    dependent RNA polymerase.

     6                While it's unlikely that the DNA dependent

     7    DNA polymerase makes a significant contribution, the

     8    relative contributions of RT and the host DNA

     9    dependent RNA polymerase is completely unclear, and

    10    although it doesn't really matter here, it's led to

    11    some important misconceptions in terms of the drug

    12    treatment for HIV.

    13                DR. RUPRECHT:  Well, what is known though

    14    is that the mutation rate of lentivirus is orders of

    15    magnitude higher than the mutation rate of DNA

    16    polymerases.  So that gives the virus a chance to

    17    adapt, and as you have seen, it adapts rather quickly

    18    over 80 weeks.

    19                DR. HUGHES:  The question is whether or

    20    not it is RT that is responsible for the errors, and

    21    it's entirely possible it's a cellular  RNA polymerase

    22    that's responsible for many of those errors.

    23                DR. RUPRECHT:  I think that's unlikely,

    24    but that's my personal view.

    25                DR. LINIAL:  Maxine Linial.

                                                                        196

     1                Did you compare the sequence of the Delta

     2    3-plus the whole cDNA and the Delta 3?  And if so, how

     3    many different changes were there in the genomes?

     4                DR. RUPRECHT:  We have sequenced the whole

     5    Delta 3-plus, the entire genome, and we found some

     6    very intriguing mutations, but right now without

     7    sequencing more progeny viruses, you can't really see

     8    yet which of the many mutations that we have seen are

     9    the important ones, but that's what we're studying

    10    right now.  Stay tuned.

    11                DR. PURCELL:  Damian Purcell from

    12    Macfarlane Burnet Center, Australia.

    13                We did very similar experiments which I'm

    14    going to present tonight, and I was curious with your

    15    Delta 3 mutation, the U3 deletion particularly, did

    16    you build that into the five prime end of the

    17    construct?

    18                Because when we failed to do that, we find

    19    reversion of the virus very quickly, presumably a

    20    contribution from the presence of high level of

    21    plasmid DNA containing this element.

    22                DR. RUPRECHT:  Well, during the in vivo

    23    replication, the deletions in the three prime end were

    24    followed with PCR in all animals, and what we noticed

    25    in every animal that went on to progress to disease

                                                                        197

     1    was an increasingly large deletion in the area of the

     2    delta nef and delta nre.  So, in other words, the

     3    virus actually lost additional sequences.  

     4                Now, through the process of retroviral

     5    replication and the mechanisms of the reverse

     6    transcriptase, any deletions in the three prime LTR U3

     7    region will automatically be copied into the upstream

     8    element.  So they will be present also in the five

     9    prime LTR in the next round of replication.

    10                So overall the Delta 3-plus virus lost 180

    11    base pairs in the LTR, which then will also obviously

    12    be lost in the five prime.

    13                DR. ZOON:  Thank you very much.  We very

    14    much appreciate it.

    15                We'll now go on to the next paper by Dr.

    16    Eugene Major of the National Institute of Neurological

    17    Disorders and Stroke, and the title of his paper is

    18    "Cells and Their DNA Containing Integrated DNA Viral

    19    Genomes:  Differentiated Phenotypes of the Human

    20    Central Nervous System."

    21                DR. MAJOR:  Thank you.

    22                I feel a little bit like Monty Python, I

    23    think, standing here because I'm about to say, "And

    24    now for something maybe not completely, but just a

    25    little bit different."

                                                                        198

     1                I've been sitting here since Tuesday

     2    night, and what I didn't want to do is to be too

     3    redundant about talking about virus systems that I

     4    think we're somewhat familiar with, but I would like

     5    to talk to you a little bit about the human polyoma

     6    viruses and the nature of the integrated DNA that we

     7    can find in sequence in cells that they transform, and

     8    so that would be a new virus we haven't talked about

     9    yet.

    10                And also then talk about -- and if we

    11    could have the first slide, which is what it says on

    12    this slide, and talk about cells we haven't talked

    13    about yet, which are cells from the human central

    14    nervous system, and a particularly unique cell line. 

    15    At least we think it's unique, that probably would fit

    16    into that category of a design cell substrate, whether

    17    it's useful for vaccine development or not I'm not

    18    sure, but certainly it's one that has unique

    19    properties, which was established by the introduction

    20    of a viral immortalizing gene.

    21                So we'll talk a little bit about copy

    22    number of the human polyoma viruses in cells that it

    23    transforms, and the ability to rescue the integrated

    24    viral DNA from these cells.  We won't talk about

    25    integrated lentivirus in the human central nervous

                                                                        199

     1    system.  We won't have time for that, and then we'll

     2    go on to talk about a specific what we think is an

     3    interesting cell line which we made a number of years

     4    ago from the human fetal brain.

     5                Just a few comments then.  These

     6    particular bullets here can describe SV40 DNA

     7    integrated in human immortalized cells, and it's

     8    relatively true, generally true for human cells

     9    whether they are myoblasts, muscle cells, epithelial

    10    cells, fibroblasts, central nervous system cells.

    11                Generally speaking, and it's also true for

    12    BK and for JC, the human polyoma viruses in cells that

    13    it transforms, not human cells, but rodent cells.

    14                And I should say just parenthetically here

    15    that the human polyoma viruses are pathogenic agents

    16    in the human population.  DK itself is associated with

    17    kidney infections.  JC is associated with a

    18    demyelinating disease.  Both of diseases or both of

    19    those viruses are associated with infections in

    20    reasonably severely immune compromised individuals.

    21                JC, for example, causes neurological

    22    complications in approximately ten percent of all AIDS

    23    patients.  So it's a rather substantial infection.

    24                In most of these cases, there is a low

    25    copy number of the viral DNA which is integrated, and

                                                                        200

     1    frequently what we find in human cells, that it's

     2    integrated in tandem copies, either head to head or

     3    head to tail, and one to two viral copies.

     4                It's usually located at a unique

     5    chromosome.  There aren't a lot of duplications there,

     6    and it's been reported in the literature that SV40

     7    DNA, for example, will integrate in human Chromosome

     8    17, 12Q23.  I'll show you evidence of 2Q35.  Usually

     9    it's a single integration site that takes place.

    10                Integration -- this actually should say

    11    does not select for a cell phenotype, and what I mean

    12    here is that integration in a particular chromosome is

    13    not pathomnemonic for a particular type of cell.  In

    14    other words, you won't get integration of these

    15    particular DNAs.  If it's a fibroblast in a particular

    16    chromosome; if it's a myoblast it will be another

    17    chromosome.  That's not what we mean here.  

    18                The integration site, however, during the

    19    life span of the cell line is generally stable.  We

    20    have not seem translocations, for example, of

    21    chromosomal content from one chromosome to the other. 

    22    From very early passages to much later passage, the

    23    site of integration of the chromosome remains the

    24    same.  

    25                In all of these cases, if it's SV40 or if

                                                                        201

     1    it's JC or VK, the T protein is synthesized, and it

     2    binds P53, the retinal blastoma gene products, and of

     3    course, it interacts with the cell cyclins, and this

     4    has been discussed quite a bit at the meeting already.

     5                SV40 immortalized human cells, as Leonard

     6    Hayflick has reminded us a number of times here, is

     7    nontumorigenic in the conventional ways in which it

     8    has been tested, in introduction into immunodeficient

     9    rodent models.

    10                Now, BK NJC, the human polyoma viruses,

    11    will induce tumors in rodents, hamsters particularly,

    12    rats and mice.  JC itself, if inoculated

    13    intracranially, will induce gliomas, Grade 4 

    14    glioblastoma multiform.  In New World monkeys it's

    15    perhaps the only good primate model for gliomas that

    16    really exists.

    17                But SV40 immortalized human cells is

    18    generally nontumorigenic, and personally I like to

    19    make the distinction as we discussed here in the

    20    terminology between neoplastic, which is cancer

    21    causing, and immortalized, which is a term that at

    22    least we use to simply indicate that it has an

    23    unlimited life span.

    24                this is an archive slide.  I had to dig

    25    this out from the archives in my own collection here,

                                                                        202

     1    SV40 T protein and its immortalized human cells.  All

     2    of the cells contain T protein, and these can be

     3    cloned out.  As you can tell, this is a pretty old

     4    slide because these are immunoprecipitation

     5    experiments, simply demonstrating that the SV40 T

     6    protein or JC or BK will immunoprecipitate along with

     7    P53.

     8                Now, let's turn our attention to, for

     9    example, the way that we can look at the nature of the

    10    integrated DNAs which are present in, let's say, BK

    11    virus, the human polyoma virus, transformed hamster

    12    tumor cells.

    13                Now, before the advent of gene

    14    amplification techniques and PCR, what we used to do

    15    to be able to get these DNAs out of the transformed

    16    cells is do cell fusions, and this is a technique that

    17    we used to use a decade ago or so.

    18                So if we wanted to take a look or even

    19    grow BK virus, for example, what we would do is do an

    20    experiment in which we would take BK virus transformed

    21    hamster cells, and we'd physically fuse it in co-

    22    cultures with a cell that BK is very permissive to,

    23    which is human embryonic kidney.

    24                And when we do that, you form

    25    Heterokaryons and synkaryons, and you'd be able to

                                                                        203

     1    rescue the viral genome by excision, DNA replication,

     2    and then the virus would grow, and in many cases a

     3    long time ago, this is the way we used to propagate

     4    these viruses.

     5                When the fusions begin to form, for those

     6    of you who may not be too familiar with that, you'll

     7    get a heterokaryon that's formed, which is a single

     8    cytoplasm which contains two nuclei, one of the

     9    transformed cells, for example, and one of the

    10    permissive cell.

    11                Then as those two nuclei begin to fuse,

    12    what happens is that the viral DNA which is integrated

    13    in a particular single chromosomal location site is 

    14    excised.  We don't know what the mechanism of that is,

    15    but here you see actually a synkaryon, which is a

    16    fused cell in which you have the nuclear content, the

    17    chromaton from two of the different cells, and viral

    18    DNA begins to replicate after it's been excised.

    19                Now, we measure replication or we detect

    20    replication here by using biotinylated DNA probes

    21    that's specific to the viral DNA, and the chromogen

    22    here is diaminobenzidine, and in the presence of strep

    23    avid and horseradish peroxidase at areas where the DNA

    24    is replicating, which is, of course, in the nucleus,

    25    then you'll get a brown precipitate from the oxidation

                                                                        204

     1    of the chromogen, which is diaminobenzidine.

     2                We've quantitated this technique so that

     3    we can tell when we look at these particular kinds of

     4    nuclei that we have several thousand, if not more,

     5    copies of the viral DNA  which is present in the

     6    nucleus of these cells, and it's a technique we've

     7    used quite a bit for diagnosis of viral infections in

     8    the human brain.

     9                Now, what happens with, for example, if we

    10    do this experiment now with BK, but let's say JC, and

    11    JC grows very much more slowly even in a fused

    12    culture, that actually the nuclei in which the DNA has

    13    now been excised will replicate and begin to divide. 

    14    The cell begins to divide even before virions are

    15    made, and that's what you see here, a very high copy

    16    number.

    17                These fused cultures will undergo cell

    18    lysis with time, and we can go into these cultures and

    19    we can go ahead and look at the viral DNA which is

    20    present there, whether it's BK, for example, or

    21    whether it's JC.  These are simply the genetic maps of

    22    all the primate polyoma viruses that are before you

    23    with JC.

    24                And interestingly enough, at least for us

    25    in looking for BK and JC which has now been excised

                                                                        205

     1    from the transformed cells, that the DNA that we

     2    sequence is perfectly excised from whatever region of

     3    the genome that it was integrated, and integration I

     4    should say within the viral genome is random also.

     5                Now, in order for transformation to exist,

     6    to take place, the integration usually takes place in

     7    sequences obviously that are not involved with the T

     8    protein coating region, the early region of these

     9    viral genomes.  So they'll be in the late region, in

    10    the capsid coating regions here or, for example, in

    11    here for JC or BK.

    12                However, and they're linear obviously if

    13    they're integrated within the chromosome.  However,

    14    upon excision, replication and virion production with

    15    these DNAs, what the DNAs that we get out in the

    16    nucleotide sequences are exactly the ones that we put

    17    in.  We don't really find any cellular chromosomal

    18    DNA.

    19                Now, we usually take extracted virion

    20    populations so there could be a selection for

    21    encapsidation of these human viral DNAs that come out,

    22    and it's possible that there could be free viral DNAs

    23    present in these particular kinds of fusion cultures

    24    that have chromosomal DNA, which we wouldn't see by

    25    the particular kinds of assays.

                                                                        206

     1                If, in fact, however, we take these

     2    viruses and we grow them in permissive cells in

     3    culture, there are a lot of rearrangements that take

     4    place in the genome, and in nature this takes place as

     5    well.  There's a lot of genetic changes that take

     6    place in BK virus as it grows in the human kidney or

     7    in JC virus as it grows in the kidney or grows in the

     8    brain.

     9                But if the DNA remains integrated in one

    10    of its transformed cells, there doesn't appear to be

    11    any rearrangements that take place.

    12                And that's also true if we take plasmids,

    13    conventional colicin derived E. coli plasmids and we

    14    transfect these DNAs into cells that are very

    15    permissive, JC, into cells from the human brain, BK,

    16    and to kidney cells.

    17                Without excising the DNA before we do the

    18    experiment, if we simply transfect these DNAs  that

    19    are infectious, even if the plasmid DNA is still

    20    present, we just take the whole nine KB or ten KB

    21    vector that we made, retransfect it into cultures.  We

    22    allow lytic infection to take place, for example, BK

    23    and HEK cells, and then we sequence the DNA when it

    24    comes out, and whatever that mechanism is for

    25    excision, there are no plasmid nucleotides left as

                                                                        207

     1    well.  So it's a very specific kind of mechanism.

     2                So we're certain, of course, that the DNA

     3    that is integrated into these particular transformed

     4    cells is wild type DNA, and we can determine what

     5    those nucleotide sequences are.

     6                Now, I'd like to turn your attention now

     7    to a particular kind of cell line that we made because

     8    we needed to have in the laboratory a consistent

     9    source of cells that we wanted to work with that would

    10    be a cell line or an immortalized cell line, but

    11    wouldn't have the probability of being able to excise

    12    its DNA.

    13                And so we were able to get a hold of one

    14    of the origin infected mutants of SV40, which Jasha

    15    Gluzman had used and he had created some years ago in

    16    the creation of a series of cell lines called COS

    17    cells.

    18                And that I'll describe to you is really

    19    the human fetal brain counterpart of the COS cell, and

    20    the advantage that we thought we would have at the

    21    particular time is to make an immortalized line of a

    22    reasonably difficult cell to work with in the

    23    laboratory derived from human fetal brain, that we

    24    would not have to worry about viral DNAs excising and

    25    replicating.

                                                                        208

     1                So we used an origin infected mutant of

     2    SV40.  It cannot replicate.  The immortalizing gene is

     3    SV40 T protein.  The cell lines were established from

     4    the CNS, and I'll talk to you about the differentiated

     5    phenotypic properties.

     6                I'll spend just a couple of slides here to

     7    show you the way we handle these kinds of cultures,a

     8    nd we have been culturing cells derived from human

     9    fetal brain for quite a long period of time now as

    10    targets for viral infection for human CNS diseases.

    11                What you see here is a heterogeneous

    12    population of cells from an eight week gestational

    13    aged human brain.  There are cells here which

    14    areneuronal in definition, in glial, as well as

    15    progenitor cells.

    16                We have ways to be able to physically

    17    separate out the different population of these cells

    18    so we can grow glial cells which are predominantly

    19    astrocytes and progenitors for the  oligodendrocyte

    20    (phonetic), which is the myelinating producing cell 

    21    with CNS.  The astrocyte performs a variety of

    22    functions in the human brain.  It performs part of the

    23    blood-brain barrier.  It biochemically modulates the

    24    number of neurotransmitters.  It produces chemokines,

    25    cytokines.  It's a real work horse.

                                                                        209

     1                And actually the human brain consists

     2    predominantly of the astrocyte.  Fifty-five percent of

     3    all of the cells in the human brain is the astrocyte. 

     4    About 35 percent are neurons.  These are a neuronal

     5    population.

     6                This is just an Omarski optics of a phased

     7    contrast, viable cells of the human brain.  Here are

     8    the cells that have attached to plastic, at the glial

     9    cells, the cells here that have the round body and the

    10    process bearing cells are progenitor neurons.

    11                And viruses, different kinds of viruses

    12    will go ahead and infect different cell types, what's

    13    here.  This is just another view of the same kind of

    14    culture, and we phenotypically characterize these

    15    cells as either being fliel or neuronal by different

    16    markers.

    17                And what you see here is a digitized image

    18    of phenotypic markers in these cells that we grow. 

    19    The astrocyte, for example, in glial cells will

    20    express an intermediate filament called glial

    21    fibrillary eosinic protein, and that's what's marked

    22    in the green here.  The red actually in these cells is

    23    marked by an antibody which we recently made to

    24    another intermediate filament called nestin, which is

    25    a marker for progenitor stages of differentiation. 

                                                                        210

     1    It's not unique to the brain, although the nestin that

     2    we used as an antigen to make this antibody we closed

     3    from the sequences from the human developing train.

     4                We can actually identify cell types here

     5    that have both markers, have both nestin, for example,

     6    and FGAP, and we know that these cells are in a

     7    differentiated pathway to either go to a glial cell or

     8    in other cases to go to a neuron, and so we

     9    characterize these cells this way.

    10                So we have this availability in the

    11    laboratory.  We were fortunate enough to be able to

    12    get one of the origin defective mutants from Jasha

    13    Gluzman, and for those of you who may not remember,

    14    but what was done is that there was a restriction at

    15    the nucleus site in the replication origin of the SP40

    16    DNA.  This is just in comparison to the human polyoma

    17    viruses and the deletions made in that origin.

    18                The one we used to introduce into human

    19    fetal brain cultures had an 11 base pair deletion in

    20    the origin here.  So the virus was dead.  It could not

    21    replicate its DNA.  It would never make proteins.

    22                So we took that construct.  We inserted it

    23    into a 12 week gestational age, mixed culture of human

    24    brain, and this is what we got.  This simply goes

    25    ahead and gives you something of an idea of the time

                                                                        211

     1    frame within which we did this from the initial time

     2    of the transfection that was taking place.  We did not

     3    clone these cells.  We simply collected those cells

     4    which survived as a matter of growth.

     5                And after a period of about five passages

     6    or so in culture, they passed through, I guess, what

     7    would be called the replicated senescence period or

     8    M0, perhaps M1.  All of the cells became T protein

     9    positive.  Those were the cells that grew out.  They

    10    did enter a crisis phase later on in passages 31 or

    11    38.  This would represent perhaps 80 cell doublings in

    12    tissue culture, and then from then on, developed into

    13    a permanent or immortalized cell line.

    14                And these are just phenotypically what the

    15    cells looked like.  They have some interesting

    16    biological characteristics, however.

    17                All of these cells, regardless of whether

    18    they're at this, the early stage, or the post crisis

    19    stage are anchorage dependent for growth.  They're

    20    absolutely non-tumorigenic in rodents or in non-human

    21    primates at least under the systems in which we have

    22    inoculated them.  They're contact inhibited for

    23    growth.  They're somewhat serum dependent for growth.

    24                For our particular purposes in the

    25    neurobiology of some of these cells, they are quite

                                                                        212

     1    interesting because they produced nerve growth factor,

     2    brain derived neurotropic factor.  They produce a

     3    great amount of the vascular endothelial cell growth

     4    factor.  They're Class 2 positive, Class 1 positive,

     5    CE-4 negative.

     6                But when you look at the susceptibility to

     7    virus infections, these particular cells are

     8    susceptible to JC virus and to BK.  They're also

     9    susceptible to HIV-1, HTLV-1 as well, to all of the

    10    herpes viruses.  Well, we haven't tested EBB, but

    11    certainly CMV, herpes Type 1 and Type 2, varicella

    12    zoster virus, which isn't on this list, as well as

    13    vesicular stomatitis virus, which can be pseudotyped

    14    into HIV lentiviral vectors.  They're actually

    15    exquisitely sensitive to those.

    16                So all of these cell types or these cell

    17    types are sensitive to a variety of different kinds of

    18    viruses.

    19                Now, we cloned out -- the fact that it was

    20    non-tumorigenic, there was nothing we could do with

    21    these cells to make them tumorigenic.  So we thought,

    22    well, there might have been mutations in the T protein

    23    coating region of the DNA that was inserted into the

    24    chromosome of these particular cells.  So we cloned

    25    out just the T protein coating sequences, and that's

                                                                        213

     1    what you see here in this particular slide.

     2                And we found actually there were five

     3    nucleotide changes in the DNA in these cells compared

     4    to the DNA that we initially put into the transfection

     5    experiment, and some of these nucleotide changes would

     6    predict differences in amino acid calling for the

     7    protein, but we haven't sequenced the protein, so

     8    we're not sure.

     9                But they don't really fall in locations

    10    which could tell us much about any differences in the

    11    biology of what T protein may be doing in these cells,

    12    but at least we know that, and we do know that in FSH

    13    staining, which was done by Tom Glover at Ann Arbor --

    14    you probably can't see that very clearly here -- that

    15    there is a single integration site of the SV40 DNA in

    16    Chromosome 2Q35 location, here, and by sequencing

    17    analysis, we know the location along the SV40 DNA,

    18    which that integration had taken place, and we also

    19    know that it's in a direct tandem type of copy.

    20                Now, we began to be interested in this

    21    cell line for a whole variety of other purposes here,

    22    and in collaboration with many other laboratories that

    23    actually have this cell line, because we made this

    24    cell line back in 1985.

    25                I will go through this relatively quickly

                                                                        214

     1    here.

     2                If we continued to grow the SV40 T protein

     3    immortalized human fetal brain cell line, which we

     4    call SVG, in standard laboratory medium E-MEM in fetal

     5    bovine serum, it's phenotypic characteristics are that

     6    of a glial cell, and these are simply phenotypic

     7    markers that we use by flow cytometry or staining in

     8    order to be able to define the type of cell that it

     9    is.

    10                They have fragments of cholera toxin,

    11    cholera toxin and tetanus toxin, another surface

    12    marker, which is gliactocil sulfatide called A2B5 on

    13    their surface, and this really defines a type of cell

    14    which is either terminally glial or it's a progenitor

    15    to a glial cell.

    16                However, if we treat this cell with

    17    molecules that induce cyclic  EMP, for example, like

    18    forscalin (phonetic), and on the next slide I'll show

    19    you that, or if we take these cells off of serum and

    20    put them in medium which we use in the laboratory as

    21    well as other laboratories use that are selected for

    22    neurons, that we get a shift in phenotype.

    23                And what happens is that we lose the A2B5

    24    population.  We increase the cholera toxin population,

    25    and we also increase the number of cells which are

                                                                        215

     1    cholera toxin/tetanus toxin positive in the neuronal

     2    phenotype of these cells.

     3                Cholera toxin/tetanus toxin denotes a cell

     4    which is in a neuronal lineage, and this was something

     5    of a surprise to us because we had thought that after

     6    so many passages in culture, that in fact this would

     7    be a stable phenotype of these cells, and the presence

     8    of the SV40 T protein would lock those cells into a

     9    certain time in which they were immortalized.

    10                DR. ZOON:  Could you please summarize?

    11                DR. MAJOR:  Okay.  I'll skip that then, if

    12    I have to summarize this, and I'll say, for example,

    13    these are simply cells that we can treat to force them

    14    into a neuronal phenotype.  This is what the cells

    15    look like, for example, if we put them on neural basal

    16    medium.  These are the cells as they look like if

    17    they're in glial medium.  In the presence of serum, of

    18    course, they will go ahead and divide.

    19                If we infect these cultures with JC, for

    20    example, you can't see that, but JC will infect these

    21    cells into glial phenotype because JC is tropic for

    22    glial cells, and it's at a molecular level.

    23                However, if we take these cells and we

    24    induce the neuronal phenotype, this is the same parent

    25    cell; then infect those, then JC will not infect that,

                                                                        216

     1    and it's not a matter of attachment or entry because

     2    we've already shown that, in fact, susceptibility to

     3    JC infection of these particular cells is at the level

     4    of transcriptional control, a very much different type

     5    of cell.

     6                So let me then summarize by saying at

     7    least that here what is surprising to us is that after

     8    many passages in culture, 50, 60, 70 -- some other

     9    laboratories have passed this out 150 times -- what we

    10    seem to have been able to do is to immortalize a

    11    population of human central nervous system progenitor

    12    cells.

    13                That these cells still are multi-potential

    14    for the different phenotypes of the cells that we can

    15    grow either in a glial lineage or in a neuronal

    16    lineage, and we use these now back and forth in this

    17    particular lineage patterns to ask questions of

    18    neurobiology, to ask questions of cell susceptibility

    19    to the human viruses, and for a variety of other kinds

    20    of questions that can be asked.

    21                And, again, the surprising thing is that

    22    even in the presence of SV40 T protein, which is

    23    continually made in these cells, there's still multi-

    24    potential for differentiation, and they respond to

    25    signals that the human brain responds to also in

                                                                        217

     1    lineage pathway commitments as it develops.

     2                And so this is actually from page 19 of

     3    the information that Andy Lewis had sent out to all of

     4    us, talking about perhaps getting to know a little bit

     5    more about the progenitor nature of the cells and

     6    designing specific kinds of cell lines that may or may

     7    not be useful for certainly vaccine development, but

     8    certainly cell lines that are extremely helpful for us

     9    in understanding other kinds of questions at least in

    10    neurobiology.

    11                Thank you.

    12                (Applause.)

    13                DR. ZOON:  Thank you.  Thank you very

    14    much.

    15                If we have time for one quick question,

    16    comment?  No.  Thank you very much.  I appreciate it.

    17                Our last presentation in this session is

    18    by Dr. Haig Kazazian of the University of Pennsylvania

    19    School of Medicine, who will speak to us on mobile

    20    elements in mammalian genomes and their implications

    21    for cell substrate safety.

    22                Thank you for coming.

    23                DR. KAZAZIAN:  Thank you.

    24                Well, I'm going to say some things about

    25    endogenous DNA.  I thought I'd start off by telling

                                                                        218

     1    you a little bit about retroelements and L1 biology,

     2    and then I'll tell you a little bit about an assay for

     3    retrotransposons that we use, and something about the

     4    number of active human retrotransposons, and I'll try

     5    to give you an estimate of the insertion frequency in

     6    germ lines and tell you what I think we know about the

     7    potential for insertion frequency in somatic cells.

     8                So let me go ahead and start.

     9                DR. ZOON:  Do you want me to get this

    10    slide?

    11                DR. KAZAZIAN:  There we go.  Retroelements

    12    are sequences in the DNA that have been put back into

    13    the DNA by reverse transcription, and there are a

    14    number of different types of retroelements, but

    15    retroelements probably make up something on the order

    16    of a third of human DNA.  So a large fraction of human

    17    DNA.

    18                There are non-LTR, non-long terminal

    19    repeat, or poly-A retrotransposons.  They have no long

    20    terminal repeats.  They have a three prime poly-A. 

    21    They usually have two open reading frame, and they're

    22    reverse transcribed by an unusual mechanism, target

    23    prime reverse transcription or nick and prime

    24    mechanism.

    25                And an example of these is mammalian L1 or

                                                                        219

     1    line elements, and we'll be talking about those.

     2                Long terminal repeat retrotransposons, we

     3    don't have any of these in humans that we know of. 

     4    They have long term repeats.  They have two open

     5    reading frames.  They have reverse transcription prime

     6    by a TRNA, so very different from this group of

     7    retrotransposons as far as their reverse transcription

     8    goes.

     9                An example of this group is TY1 of yeast.

    10                Retroviruses, as you've been hearing a lot

    11    about, have long term repeats.  They usually have

    12    three open reading frames.  So they have an envelop

    13    gene that gets them to be infectious.  There's no

    14    envelope gene in these retrotransposons.

    15                Reverse transcription in retroviruses is,

    16    again, primed by a TRNA, like that of LTR

    17    retrotransposons.  A good example is HIV-1 of human.

    18                There are non-autonomous retroelements in

    19    humans.  These are transcribed, but contain no

    20    functions for retrotransposition.  Examples of these

    21    are alu elements, small nuclear RNA pseudogenes and

    22    process pseudogenes, and so they need reverse

    23    transcription and help by other elements, perhaps non-

    24    LTR retrotransposons.

    25                So what we're talking about is

                                                                        220

     1    retrotransposition which is an element being

     2    transcribed into an RNA, reverse transcribed, and

     3    integrated at a new site in the genome.  So this is a

     4    copy and paste mechanism, duplicative mechanism for

     5    actually expanding the genome.

     6                And the major retroelement, the major

     7    player in the human genome is the so-called L1 or line

     8    element, and when I made up this slide, I said

     9    approximately  100,000.  There are new estimates that

    10    say there are up to 400,000 or so of these line

    11    elements in the human genome.  These are database

    12    estimates, which are probably more accurate now, now

    13    that we have something like 12 to 13 percent of the

    14    human genome in the databases.

    15                The majority of L1s are truncated at their

    16    five prime end.  That means they're short, and they

    17    have mostly three prime end sequences and not five

    18    prime end sequences, but about 3,000 L1s are full

    19    length, and some of these can retrotranspose.

    20                So what does a full length L1 look like? 

    21    Here's the structure of that element.  It's six

    22    kilobases in size.  It's got at the five prime end an

    23    internal promoter sequence which is relatively poorly

    24    defined at the moment, but is mostly present in the

    25    first 100 base pairs.

                                                                        221

     1                It has a first open reading frame which

     2    makes a protein which is likely to be an RNA binding

     3    protein.  It has a second open reading frame, which is

     4    quite long, most of the element, and within this

     5    second open reading frame is an endonuclease domain,

     6    a sequence which encodes an endonuclease; a reverse

     7    transcriptase domain which encodes a reverse

     8    transcriptase, and a conserve domain at the C terminal

     9    end, which may be a zinc knuckle sequence.

    10                It's got a very short, three prime

    11    untranslated region, and then it ends in a poly-A

    12    tail, and it's usually flanked by target site

    13    duplications.

    14                So in the early 1990s, we were able to

    15    isolate the precursors for a couple of insertions that

    16    occurred in humans, and these full length precursors

    17    were good candidates for active retrotransposable

    18    elements.

    19                So in the mid '90s, John Moran came into

    20    the lab, and he devised this assay for L1

    21    retrotransposition.  So in this assay then a full

    22    length element has in its three prime UTR inserted a

    23    marker cassette or a retrotransposition cassette which

    24    will tell us when a retrotransposition event has

    25    occurred.

                                                                        222

     1                And this particular cassette has a

     2    backwards neomycin gene which is split by a forward

     3    intron.  So here's the spliced donor site and the

     4    spliced acceptor site of that intron, which is in this

     5    transcriptional direction.

     6                A cassette is also flanked by a promoter

     7    for the neomycin gene and a poly-A signal for the

     8    neomycin gene.  

     9                So when transcription occurs from this

    10    promotor, we go through this intron in this direction. 

    11    We can then splice it out, and then with reverse

    12    transcription, we can have this neogene now

    13    transcribed and put back into the genome in the right

    14    orientation now with no intron disrupting it and

    15    driven by its own promoter, and having its own poly-A

    16    signal, we can get G4 and 8 resistant cells.

    17                So this, as it turns out, is the only way

    18    you can get G4 and 8 resistant cells, is with a

    19    retrotransposition event using this reverse

    20    transcriptase.

    21                So we put this receptor into a piece of

    22    plasmid and a nice receptor zone goes into the cell

    23    and into the nucleus and has about 20 or so copies,

    24    and we infect Hela cells with the constructs by

    25    lipofection, and we have on this P-sep (phonetic)

                                                                        223

     1    element a hygromycin resistant gene.  So we hit the

     2    cells with hygromycin.

     3                And so we end up with cells that are

     4    expressing the marked L1 that have been transfected,

     5    and we outgrow and plate dilutions in medium

     6    containing G41A, and the G41A resistant cells then

     7    contain an L1 retrotransposition event.

     8                And here is an example of the kind of

     9    assay that we get when we put in an active element. 

    10    It turns out this was a precursor of an insertion into

    11    a Factor A gene causing hemophilia in a child.

    12                So you can see that this is with plating

    13    out something like a million transfected cells and

    14    getting something on the order of 1,000 positive

    15    retrotransposition events.  Each one of these blue

    16    specks is a colony of cells which contains an

    17    individual retrotransposition event, and that was

    18    proven by cloning a number of these events and showing

    19    that they look like the retrotransposition events that

    20    are seen in an endogenous human situation.

    21                Here is a construct that has a big

    22    deletion in the element, and here is a construct that

    23    simply has a point mutation in the reverse

    24    transcriptase domain of the element.

    25                I should say that we now have on the order

                                                                        224

     1    of nine of these active human elements in the

     2    laboratory, and the extent of retrotransposition

     3    varies considerably.  So with some of those elements

     4    we get much less retrotransposition than is seen here,

     5    and with others we get much more.

     6                So we have one element in the lab now that

     7    will retrotranspose at a frequency of about one in

     8    every ten transfected cells, a very high frequency.

     9                I should also say that in other

    10    experiments we've shown that the site of

    11    retrotransposition is relatively unbiased.  That is,

    12    the insertions into introns and genes is about the

    13    frequency that one would expect for the size of

    14    intronic sequences or the proportion of intronic

    15    sequences within the genome.

    16                So it turns out that there have been on

    17    the order of 13 natural L1 insertions in humans

    18    causing disease, and it turns out that 12 of those 13

    19    have come from a very small subset of L1 elements

    20    within the genome.

    21                And so we decided to see if we could make

    22    an estimate of the number of active human L1 elements

    23    in the genome, and we got Gary Swyrbl to do a count of

    24    the number of full length elements of this subset in

    25    the haploid genome.  He came out with a number of

                                                                        225

     1    about 80.

     2                We isolated a number of these subset

     3    elements specifically from the genome and found out

     4    that roughly a quarter of them were capable of

     5    retrotransposition.

     6                So from that we could make an estimate

     7    that the number of active human L1 elements in the

     8    genome was on the order of 40 per diploid genome, and

     9    so you could make a little range on that estimate and

    10    say perhaps 30 to 60 active elements in the genome,

    11    and yet now we're sitting with hundreds of thousands

    12    of copies that have built up over evolutionary time.

    13                So I just wanted to give you an idea of

    14    the number of recent retrotransposition insertions in

    15    humans and mouse.  I mentioned that L1 insertions, the

    16    number here was 12.  I know of another one now.  So

    17    let's say it's 13, and there have been a number of

    18    non-L1 insertions, particularly alu insertions, in

    19    humans, and we think that those are driven by L1s,

    20    reverse transcriptase, but there's no direct evidence

    21    for that yet.

    22                But in any case, if we add those up, we

    23    get on the order of 30 or so human insertions that

    24    have been seen that cause disease.  That's the only

    25    way we would know of them, and as you'll see on the

                                                                        226

     1    next slide, that makes up something on the order of

     2    one in every 600 mutations.

     3                In the mouse it's very different. 

     4    Spontaneous diseases that have been characterized in

     5    the mouse are on the order of only 200 or so, and so

     6    the number of insertions or the frequency of

     7    insertions causing mutations in the mouse is on the

     8    order of ten percent, very much higher than it is in

     9    humans.  So I guess we're lucky in that regard.

    10                So when does L1 retrotransposition occur? 

    11    Well, L1 RNA and the orf-1 protein have been found in

    12    primary spermatocytes in leptotene and zygotene

    13    stages.  This is miotic prophase, and this is mouse

    14    work.  This is not human work.  

    15                Timing of retrotransposition events is

    16    really unknown, but it's thought to be early or during

    17    germ line production.  

    18                So we can make an estimate of the

    19    frequency of retrotranspotion insertions in the human

    20    genome.  As I said, the number of known insertions is

    21    on the order of 28.  This was back in March I made

    22    this estimate.  It's now 31 that I know of, but the

    23    number of mutations in the database is higher.

    24                So the number of mutations in the database

    25    when I made this calculation back in March was 16,650. 

                                                                        227

     1    Okay?  That was from the human gene mutation database. 

     2    So that gave us a fraction of mutations that are

     3    insertions of 28 over 16,650 or about one in 600.  So

     4    most of the mutations in humans are point mutations,

     5    but occasionally you get an insertion, one in 600

     6    insertions.

     7                So one can then go back and see, well,

     8    what's the frequency of insertions in the average

     9    genome, and one can do that by a couple of

    10    calculations.

    11                The estimated frequency of spontaneous

    12    mutations in man is about ten to the minus nine per

    13    nucleotide per year.  So we can go from there.  There

    14    are three times ten to the ninth nucleotides per

    15    genome, and I used here a generation time of 30 years

    16    for another calculation.  I said it was 25, but let's

    17    say it's 30.  It's a little long, but 30 years, and

    18    times ten to the minus nine per nucleotide per year. 

    19    That's this number.

    20                And that equals about 90 mutations per

    21    haploid genome per generation, or let's go to that and

    22    say it's 90 mutations per sperm producing an

    23    individual upon fertilization.  That's total

    24    mutations, not insertions, total mutations.  Okay? 

    25    Ninety per sperm.

                                                                        228

     1                So the frequency of retrotransposon

     2    insertions per sperm would be one in 600.  Remember I

     3    said one in 600 is the frequency of insertions per all

     4    of the mutations.  One in 600 times that 90 or about

     5    one in six.  So about one insertion per six sperm.

     6                If you use the 25 number for the

     7    generation time, you get one in eight sperm.  Okay. 

     8    So that's about one insertion, new insertion, in every

     9    six haploid sperm.

    10                Since these insertions are random, less

    11    than five percent of the insertions should be

    12    deleterious because exons comprise one percent of the

    13    genome.  Other sequences important for gene expression

    14    likely account for maybe another one or two percent of

    15    the genome, but it should be that most insertions

    16    shouldn't cause a problem.

    17                Okay.  So how about somatic insertions and

    18    what do we know about endogenous somatic insertions? 

    19    And the answer is not much.

    20                Okay.  So I know of one L1 insertion

    21    that's likely to have been involved in the etiology of

    22    cancer.  There was one L1 insertion into an exon of

    23    the APC gene, adenomenous polyposis coli gene reported

    24    by Nakamura and his group about five years ago, and

    25    that particular insertion was only seen in the tumor,

                                                                        229

     1    not seen in the constitutional tissue of the normal

     2    colon, so the colon cancer tumor.

     3                There was one alu insertion intoan MLVI-1

     4    or 2 gene which was associated with leukemia.  It was

     5    in a cell line.  As I recall, the patient was not

     6    available for study.  So it's not clear whether that

     7    was a somatic event or a germ line event.

     8                Okay.  There have been a few germ line

     9    events in tumors of alu insertions, but they're not

    10    somatic events.

    11                Okay.  We do know that L1 transposes at

    12    high frequency in Hela cells.  I showed you that.  All

    13    right.  So that's something.

    14                And we do know from the work of Tom

    15    Fanning that L1 is expressed in ovarian, testicular,

    16    and breast cancers.  Okay.  So it's expressed in

    17    cancers, and not all, but in some cancers.

    18                So that about ends what I know. 

    19    Retrotransposon insertions occur in tumors with some

    20    frequency, although their observation has been pretty

    21    rare, and clearly retrotransposon insertions occur in

    22    cell lines.  We've seen that in the laboratory.

    23                The incidence of endogenous somatic

    24    retrotransposon events is unknown as far as I'm

    25    concerned at this point.

                                                                        230

     1                That's all I have to say.  Thank you.

     2                (Applause.)

     3                DR. ZOON:  Thank you.

     4                Go ahead, Phil.

     5                DR. KRAUSE:  Phil Krause, FDA.

     6                That was a very nice talk.  One mechanism

     7    by which people would like to consider testing lines

     8    for the presence -- cell lines for the presence of

     9    adventitious agents is obviously highly sensitive

    10    reverse transcriptase assays.  I guess two questions

    11    which are related.

    12                One of them is if you were to test a

    13    cancer cell line which you say might be more likely to

    14    contain L1 elements, would that cause it to be

    15    positive in a highly sensitive RT assay?

    16                And I guess the next question is:  is that

    17    a good thing or a bad thing?  On the one hand, does

    18    that tell you that the cell line is more dangerous? 

    19    On the other hand, does it prevent you from detecting

    20    or does it then make the assay less valuable because

    21    that's a potential alternative explanation for a

    22    positive RT result?

    23                DR. KAZAZIAN:  Okay.  First of all, you

    24    should do the assay in such a way that you might

    25    detect an L1 reverse transcriptase so that it's going

                                                                        231

     1    to be different from a -- I mean you could do a

     2    specific assay because the reverse transcriptase with

     3    L1 is going to have different properties than a

     4    retroviral reverse transcriptase.  Okay?  So you

     5    should try to set it up as a relatively specific assay

     6    for the L1 reverse transcriptase.

     7                Secondly, you're going to have a hard time

     8    finding it because people have had a very difficult

     9    time finding endogenous L1 reverse transcriptase. 

    10    Okay?

    11                So it's very likely that if you find it,

    12    it's probably, you know, with your assay, without

    13    looking for a specific L1 reverse transcriptase, that

    14    it's going to be a retroviral reverse transcriptase.

    15                DR. ZOON:  Johannes.

    16                DR. LOEWER:  Johannes Loewer, Germany.

    17                Perhaps I missed the point.  Is the

    18    transposition activity of the construct you have

    19    described restricted to human cells or does it occur

    20    in every cell?

    21                DR. KAZAZIAN:  Okay.  I didn't mention

    22    that, but we have shown that it is functional in mouse

    23    LTK minus cells, even though it's a human element. 

    24    You can put the human element into mouse cells.  You

    25    can also put the mouse element into human cells and in

                                                                        232

     1    mouse cells.  It's functional in LTK minus cells. 

     2    It's functional in hepatocarcinoma cells.

     3                I heard from Maxine Linial's lab that it

     4    was functional in quail cells?  Okay.  So, yes, it's

     5    functional in quail cells.  It's functional in Chinese

     6    hamster ovary cells from another lab that has it.

     7                However, we have not been able to get it

     8    functional in differentiated fibroblast type cells. 

     9    Okay?  That's what I mentioned this morning.

    10                Yes.

    11                DR. ONIONS:  David Onions.

    12                I really enjoyed the talk.  I've got a

    13    quick comment and then a question.

    14                If I remember correctly, I think Don Cohen

    15    showed that there were line elements in a tumor called

    16    transmissible venereal tumor, which is a canine tumor,

    17    and I think he's got a line item in the upstream of

    18    myc, which is in the --

    19                DR. KAZAZIAN:  Yes, that's correct.  Okay.

    20                DR. ONION:  But just another example.

    21                DR. KAZAZIAN:  Right.

    22                DR. ONIONS:  But the question was that the

    23    remarkable transposition frequency that you're seeing

    24    in Hela cells.  Did you follow the fate of any of

    25    these clones that you had?  And ask the question:  was

                                                                        233

     1    there retransposition?  And if so, what was --

     2                DR. KAZAZIAN:  Okay.  I'll tell you what

     3    we have done though.  We haven't done that because all

     4    of the insertions that we saw were highly truncated

     5    insertions.

     6                DR. ONIONS:  Okay.

     7                DR. KAZAZIAN:  So they were missing then

     8    their five prime end, and so they would lack promoter

     9    activity.  Okay?

    10                DR. ONIONS:  Okay.

    11                DR. KAZAZIAN:  But what we have done, an

    12    experiment where we've looked to see if there's,

    13    quotes, heterogeneity of the Hela cells.  So that we

    14    have taken cells that have had a retrotransposition

    15    event and compared those and looked at the frequency

    16    of retrotransposition in those cells and in another

    17    group of Hela cells that have not had a

    18    retrotransposition event to see if the frequencies

    19    were different, and we did not see the heterogeneity. 

    20    We did not see a different frequency.

    21                So the one group of cells was not any

    22    better for retrotransposition than the other.

    23                DR. ZOON:  Thank you, Haig.

    24                We're going to take a 15 minute break.  So

    25    we're going to reconvene shortly after 3:45.

                                                                        234

     1                Thank you.

     2                (Whereupon, the foregoing matter went off

     3                the record at 3:34 p.m. and went back on

     4                the record at 3:54 p.m.)

     5                DR. LOEWER:  Good afternoon.  I think we

     6    should continue with the program, the panel-audience

     7    discussion.

     8                First what I want to do is to ask the

     9    speaker to come to the podium.   

    10                So I think we are now complete.

    11                I would like to welcome you again and

    12    shortly to introduce myself.  My name is Johannes

    13    Loewer.  I'm Deputy Director of the Paul-Ehrlich

    14    Institute in Frankfurt, Germany. 

    15                Paul-Ehrlich Institute is an institution

    16    which has more or less the same duties as the Center

    17    for Biologics Evaluation and Research.  I'm,

    18    therefore, also a regulator, and I will lead this

    19    discussion from the point of view of a regulator, not

    20    of a scientist, which is not much like exclusive, I

    21    guess.

    22                The organizers of this meeting have

    23    prepared quite a number of questions which I have

    24    posed to the chairs of the different panels, and

    25    because the issue we have to deal with is really very

                                                                        235

     1    complex and all of the discussion that we have had so

     2    far show that there are many different facets which

     3    influence each other.

     4                So to be as constructive as possible, I

     5    thought it is maybe helpful to follow the questions

     6    which have been posed directly to me, and copies of

     7    this question have been distributed here.

     8                And I would also like to try to simplify

     9    the questions in order to be able maybe to come to

    10    clearer answers wherever possible.

    11                So I have made a number of slides from

    12    these questions, and these slides should guide us

    13    through the discussion.  I would like to introduce the

    14    question shortly, and then we could open the

    15    discussion, which should not exclude if there are

    16    other urgent questions and contributions.

    17                So the first question posed to the panel

    18    here is, again, of course, the risk of residual DNA,

    19    residual DNA which comes from neoplastic cells which

    20    would be used in viral vaccines, and there are a

    21    number of questions.

    22                Is there a special, I would say,

    23    infectious risk event, a special neoplastic risk

    24    event, and is there a risk for epigenetic changes?

    25                So we should keep these questions apart,

                                                                        236

     1    I guess, and should maybe focus on the question of

     2    infectious risk event.  As we have discussed during

     3    the last two and a half day, one and a half day or two

     4    days, there are different types of infectious events

     5    maybe.  

     6                There may be extraneous agent, and I would

     7    say there is not a different risk between neoplastic

     8    cells and diploid cells for extraneous agent, but we

     9    should ask the question:  does transformation increase

    10    the risk for infectivity for maybe a broader range of

    11    viruses or is this risk for extraneous agents really

    12    similar for diploid cells or not, or does it really

    13    depend on the virus?

    14                That's one question we should discuss.

    15                The second one is there may be, of course,

    16    endogenous virus or endogenous element as we have

    17    heard, and is there an increased risk for the activity

    18    in neoplastic cells compared to diploid cells, and

    19    perhaps we could discuss this question first.

    20                First I would like to invite members of

    21    the panel to give their comments to the questions I

    22    have just posed.

    23                DR. KAZAZIAN:  As far as retrotransposons

    24    go, there's no infectious risk.  They don't go from

    25    cell to cell.  They're stuck in the cell in which

                                                                        237

     1    they're transcribed.

     2                Neoplastic risk, hard to know.  I would

     3    guess it would be low, but I don't think we have

     4    enough data on that as yet.  We'll probably have more

     5    data in the next few years.

     6                And the third, again --

     7                DR. LOEWER:  Let's come to the later

     8    question.

     9                DR. KAZAZIAN:  All right.  Go ahead.

    10                DR. MAJOR:  I'm sitting next.  So I have

    11    to answer this, too, I guess.

    12                I think, you know, as I tried to point

    13    out, of course, if you have an integrated viral DNA,

    14    particularly in a cell line, it can be excised.  You

    15    need to have a functional original of DNA replication,

    16    and in many cases perhaps adventitious viruses don't

    17    have the complete genome present there.  So whether

    18    they're there or not, I think you have to ask the

    19    question whether that DNA integrated is functional to

    20    potentially look at an infectious risk.

    21                I think also something we probably should

    22    be mindful of, too, even though we're looking at these

    23    kinds of risk factors here, is that, for example, in

    24    the viruses I talked about, the human ones, BK and JC,

    25    they're very widely prevalent in the population

                                                                        238

     1    worldwide.  All of us in this room have come in

     2    contact with these agents.  We have antibodies to

     3    them, and the vast majority have these type of agents

     4    already latently infected and potentially, as far as

     5    we know, integrated in some fashion or other in

     6    certain types of organs, as well.

     7                But it seems like my impression is the

     8    consensus seems to be that cell lines or neoplastic

     9    cells do not represent that much of a risk here.

    10                DR. RUPRECHT:  I would concur with that

    11    last statement.  The data I presented were generated

    12    with exogenous primate lentiviruses for which probes

    13    do exist, and neoplastic human cell lines could be

    14    screened to rule out exogenous lentiviral infection.

    15                One issue though that maybe should be

    16    reiterated, we presently do not know what the minimal

    17    infectious doses for lentiviral DNA is in the animal

    18    models.  So that would be something that's relatively

    19    straightforward and information that maybe should be

    20    obtained.

    21                We also do not know whether or not

    22    infections can be induced with chromosomal DNA taken

    23    from infected animals.  I did mention that most of the

    24    profile sequences that accumulate during the course of

    25    a lentiviral infection in the case of this SIV is

                                                                        239

     1    actually defective.  So the chance of getting

     2    replication competent virus out through contamination

     3    seems to me relatively remote, but we should get this

     4    baseline information.

     5                DR. BLAIR:  I think as far as the

     6    infectious agents, I think I would agree that although

     7    you don't know the minimal dose, it would appear that

     8    the efficiency uptake is probably fairly low so that

     9    if you reduce the level to the kinds of residual

    10    levels that people talk about in viral vaccines and

    11    things, that this would not pose a risk.

    12                I think in terms of the neoplastic risk,

    13    I think clearly evidence is that to induce a

    14    neoplastic event in a normal cell of any lineage takes

    15    multiple events, some positive, some negative, and the

    16    attempts to do this even under optimal conditions

    17    requires a fair amount of effort and fairly large

    18    amounts of material.

    19                So, again, the risk would seem to be

    20    minimal with the kinds of residual DNA that you find

    21    in vaccines.

    22                DR. LOEWER:  John.

    23                DR. PETRICCIANI:  Yeah.  There's very

    24    little that I can add to what people have already

    25    said.  Just let me remind you the paper that was

                                                                        240

     1    recently published by Krause and Lewis, taking a worst

     2    case analysis for an infectious event, if you

     3    normalize that down to 100 picagrams of DNA, it falls

     4    right back into the same order of magnitude of risk as

     5    the oncogenic events and  is really no different from

     6    that.

     7                The other thing that Ruth mentioned a

     8    moment ago and I mentioned in my talk I think really

     9    is important.  If as a result of this conference

    10    people feel that yet more information is needed, and

    11    that's debatable, but if more information is needed,

    12    I agree with Ruth 100 percent that one of the most

    13    important elements is some dose response data because

    14    we're operating in Never-Never Land without that, and

    15    whether it's in the model that she was talking about

    16    or if it's in the 3T3 assay or whatever, I think

    17    that's a basic, fundamental piece of data that's been

    18    absent for decades.

    19                And if we're going to pursue this as an

    20    intellectual discussion, we really should have that

    21    background data.

    22                DR. LOEWER:  I have a specific question to

    23    Dr. Ruprecht.  

    24                You mentioned it's not known with

    25    chromosomal DNA which have a complete retrovirus is

                                                                        241

     1    really infectious or not.  Would you accept such a

     2    cell line for production of vaccines?

     3                (Laughter.)

     4                DR. LOEWER:  Even if the possibility is

     5    rather remote?

     6                DR. RUPRECHT:  Well, I would probably try

     7    to get the non-provirus containing equivalent of the

     8    cell line.

     9                DR. LOEWER:  I think this should be clear. 

    10    It's obvious, in my opinion, at least, cell lines

    11    which have a complete virus, genomes are not good

    12    candidates for production of any biologicals.  I think

    13    I could mention a degree on this.

    14                The question is whether these cell lines

    15    contain part of whatever this, for example, oncogenes

    16    or other parts, but this still would exclude -- sorry?

    17                PARTICIPANT:  (Inaudible.)

    18                DR. LOEWER:  Right.

    19                DR. RUPRECHT:  Exogenous virus.

    20                DR. LOEWER:  Yeah, I guess they have only

    21    sometimes part of DNA viruses integrated, not these

    22    retroviruses maybe.

    23                DR. KRAUSE:  Dr. Loewer, how would you go

    24    about proving that a cell line does not contain a

    25    single copy of an infectious genome?

                                                                        242

     1                (Laughter.)

     2                DR. LOEWER:  I think, of course, you can

     3    test directly for certain virus you know.  I think

     4    every cell line should be well characterized in the

     5    cell bank, and especially the use of cell lines over

     6    the years, I think, proves to some extent that they

     7    contain or do not contain any extraneous viruses.

     8                So I think one can be rather sure about

     9    well characterized cell lines, that they do not

    10    contain complete viruses.

    11                DR. KAZAZIAN:  Phil, I gather that if you

    12    PCR'ed it up and found that it was negative, you still

    13    couldn't be sure.  Is that what you're getting at?

    14                DR. KRAUSE:  Well, what primer would you

    15    sue?

    16                DR. KAZAZIAN:  Yeah, right.

    17                DR. KRAUSE:  Right.  So my answer to Haig

    18    is what primers would you use.

    19                DR. LOEWER:  Okay.  I think this question

    20    is not really new and maybe not specific for

    21    neoplastic cells and all people dealing with

    22    regulation of biologicals deal 50 years with this

    23    problem.

    24                DR. KRAUSE:  I agree with you, of course,

    25    and again, for the transcriber, I'm Phil Krause of the

                                                                        243

     1    FDA.

     2                But I guess the question comes up if

     3    there's a cell line that's neoplastic or that's

     4    tumorigenic and we don't know why it's neoplastic or

     5    tumorigenic because of an incomplete history or maybe

     6    it came from a person or something like that, the

     7    question is then:  does that make us think that it's

     8    more likely to contain some of these latent viral

     9    sequences or does it make us a little bit more

    10    suspicious, that being one of the mechanisms by which

    11    a cell like that might have become neoplastic?

    12                DR. LOEWER:  Of course, this is an always

    13    open question.  To test for the unknown is always

    14    rather difficult.

    15                DR. RUPRECHT:  Maybe if I could add one

    16    thing here.  If we knew from a known animal experiment

    17    what the minimal dose is of chromosomal DNA, say, from

    18    an SIV infected animal, and if it turned out that it

    19    is ten micrograms intramuscularly, you know that you

    20    never have ten micrograms in a vaccine dose.

    21                If it turns out that 100 picagrams of

    22    chromosomal DNA will never, you know, even if you test

    23    it on 200 animals, never lead to systemic infection,

    24    even if the DNA were derived from a known viremic

    25    animal, then at least you would have some sort of

                                                                        244

     1    frame of reference.

     2                DR. KRAUSE:  I agree with you.  That makes

     3    a lot of sense.  One think that makes me pause a

     4    little bit is the slide that you showed and that I

     5    showed a couple of nights ago that appear to

     6    demonstrate that polyoma virus DNA, for instance, is

     7    quite a bit more infectious than retrovirus DNA.

     8                And you know, the list of viruses whose

     9    DNA has been tested for infectivity, and in fact, the

    10    study that Andy Lewis and I published was based on

    11    retrovirus infectivity, DNA infectivity, when we

    12    submitted that, we actually weren't aware of the

    13    polyoma virus data.

    14                But so the question is:  are there other

    15    kinds of DNA from other families of virus which

    16    haven't even been looked at which are quite a bit more

    17    infectious?

    18                And so how does one really determine what

    19    that baseline is?

    20                DR. RUPRECHT:  The answer to that is yes. 

    21    I showed a slide that summarized a number of animal

    22    experiments with other agents, including Hepatitis B

    23    in chimpanzee, HTLV-1 in rabbits, FELV, murine

    24    leukemia virus, just to name a couple of the others. 

    25    Those response curves though are not always available.

                                                                        245

     1                DR. BLAIR:  It seemed to me though that

     2    the polyoma data was in the system where the polyoma

     3    replicated, and in a sense, that's biasing.  I mean,

     4    if the virus can replicate in the tissue, in the thing

     5    you expose it to, it's going to -- you're going to

     6    much more easily pick it up than if it's something

     7    that has to go in, you know, and multiply infect many

     8    cells just directly as DNA.

     9                DR. KRAUSE:  I guess, except you're not

    10    putting the virus in to start with.  I'm not sure how

    11    that's different from the SIV mac situation where

    12    you're also putting the DNA, which also then

    13    presumably once you get a virus out of that also

    14    replicates in that animal.

    15                PARTICIPANT:  The yield of the polymer is

    16    about a million particles per cell.  So I think it's

    17    a lot more than the retrovirus.

    18                DR. MAJOR:  As long as we've talking about

    19    polyoma, I'd like to bring it back to the human again. 

    20    JC DNA is not infectious in animals, in the kind of

    21    situations that it causes tumors in non-human

    22    primates.

    23                BK, on the other, is.  The BK DNA is

    24    infectious, and animals will not only make virions,

    25    but they'll make antibody to the BK DNA and that

                                                                        246

     1    antibody will also be specific to chromosomal DNA as

     2    well.  We don't know that in humans, but certainly in

     3    animal situations it is.

     4                DR. LOEWER:  Yes.

     5                DR. HEINEINE:  Walid Heneine, CDC.

     6                No one has mentioned how much DNA we now

     7    have in the licensed vaccines.  I mean, how much are

     8    we being exposed to?  Do we have any idea in the viral

     9    vaccines, like yellow fever, measles, mumps, how many? 

    10    Do the regulators have an idea from the manufacturers,

    11    how much DNA there is?

    12                DR. LOEWER:  I have probably no idea. 

    13    Nobody that I know has mentioned it.

    14                Paul?

    15                DR. KRAUSE:  I promise to sit down for a

    16    while after this.  This actually came up when we were

    17    licensing the chicken pox vaccine, an it's part of the

    18    safety data that was submitted for that vaccine.  The

    19    manufacturer, Merck, did a number of studies

    20    quantifying the amount of DNA in different vaccines

    21    and submitted those to us.  I noticed that there are

    22    people from Merck here if someone from that company

    23    would like to describe those results, which are in the

    24    public domain already.

    25                (Laughter.)

                                                                        247

     1                DR. KRAUSE:  But the answer to the

     2    question is that -- 

     3                DR. SHEETS:  I do have an answer.  This is

     4    Becky Sheets from CBER.

     5                I think that the vast majority of licensed

     6    vaccines, U.S. licensed vaccines, have not been tested

     7    for residual DNA.  Most of them are manufactured in

     8    primary cells and diploid cells and nobody worried

     9    about it.

    10                The few that have been tested are the ones

    11    that have been licensed in the last few years,

    12    including varicella and Hepatitis A.

    13                DR. KRAUSE:  The one that has the most

    14    that I'm aware of is varicella, in fact, which has a

    15    little over a microgram per dose of MRC5 cell DNA, and

    16    in that context several other vaccines besides the

    17    ones that Becky mentioned were also tested and were

    18    lower than that, but that gives you a range of what

    19    one potentially could be dealing with if one is

    20    talking about a live attenuated viral vaccine that

    21    doesn't grow particularly well in tissue culture, for

    22    which you simply can't do anything to get rid of the

    23    DNA without risk to the virus that you're attempting

    24    to deliver.

    25                DR. HENEINE:  So, I mean, the whole thing

                                                                        248

     1    could be an issue and may not be a big issue for some

     2    vaccines, and it could be an issue for some other

     3    vaccines where we know we have some DNA.

     4                We have some premier data from our lab. 

     5    We've looked at the MMR vaccines, and we amplified the

     6    chicken endogenous retroviral sequences basically in

     7    an attempt to type these loci in these vaccines, and

     8    what we found out in the cef produced vaccines,

     9    there's very little genomic gain anyway.  We are able

    10    to amplify some sequences from them, but there's not

    11    very strong signals.

    12                And other vaccines like yellow fever, for

    13    example, there's we found much higher amounts of

    14    genomic DNA and much higher amounts of viral proteins,

    15    as well, which you can immunoprecipitate very easily.

    16                So it looks like there's a wide spectrum

    17    of residual DNA and proteins that may be present in

    18    these vaccines.

    19                DR. LOEWER:  I think this is now known for

    20    a couple of years that Dr. Schuepbach, also in the

    21    audience and I guess he will address this question

    22    tomorrow during his talk, and your findings are, I

    23    guess, repeated by a number of laboratories which also

    24    find AMB and EAB sequences as well as DNA in such

    25    vaccines.

                                                                        249

     1                But to my knowledge, at least in our

     2    laboratory we have not quantified the amount of cell-

     3    wide DNA in these vaccines.

     4                DR. HENEINE:  Yes.  The relevance in those

     5    vaccines that have large amounts of DNA is that we

     6    have full length proviruses that are intact that would

     7    be present there, and that could be taken up by the

     8    cells like the mechanism we've been talking about

     9    these two days.  The dose would be enough to initiate

    10    infections.

    11                DR. LOEWER:  Yes, but the point mentioned

    12    earlier is that these DNAs derived from normal cells,

    13    therefore, didn't create any concern so far, and at

    14    least to my knowledge there are no adverse reactions

    15    in the millions of vaccinated people which could be

    16    attributed to the presence of this DNA.

    17                That's the point.  There's a question

    18    here, indeed.  Do we expect the same with neoplastic

    19    or with DNA derived from neoplastic cells or not? 

    20    That is the question we cannot really answer so far.

    21                DR. MAJOR:  Yeah, let me ask something,

    22    maybe a naive question here.  I'm not sure I

    23    understand exactly what the nature of the state of the

    24    DNA that is the contaminant in these vaccines.  Is

    25    this cellular DNA which is encapsulated into virion

                                                                        250

     1    structural particles so that there would be a

     2    difference between enveloped --

     3                DR. LOEWER:  No.

     4                DR. MAJOR: -- versus -- or is this DNA

     5    which is just carried along with the purification

     6    process?  Is that what we're talking about?

     7                DR. HENEINE:  Yes.  I mean, if you take

     8    the vaccine, you resuspend it, and you extract it, and

     9    you can amplify endogenous retroviral sequences from

    10    it very easily, and so it must have been cellular,

    11    genomic DNA that got purified with these particles.

    12                Now, also you can find particle associated

    13    RNA.  That's something else, but in addition to that

    14    you also find genomic DNA.

    15                DR. LOEWER:  But one has to realize that

    16    there is not much purification for live viruses.

    17                DR. HENEINE:  And that's not true, like I

    18    mentioned, for all viruses.  Maybe the yellow fever

    19    vaccine, the way it is produced explains why you have

    20    higher levels of genomic DNA in it.

    21                DR. ONIONS:  Johannes, I wonder if I could

    22    just ask for a clarification because rather as Ruth

    23    was talking about the titration of DNA proviruses to

    24    get a take, to get an infection, we can push FALV down

    25    to around about ten micrograms.  You don't get 100

                                                                        251

     1    percent take at that level, but you do get takes.

     2                And you just said -- and could you clarify

     3    this? -- you just said that you got full length

     4    proviruses; is that correct?  You've actually

     5    amplified the whole --

     6                DR. HENEINE:  No, no, we have not tried

     7    that.  The experiments were originally designed to

     8    type the endogenous ALV loci in the vaccine.  In other

     9    words, if the locus -- are we dealing with defective

    10    loci or full length infectious loci?

    11                So we've used the vaccine itself as a

    12    source of DNA which could be a good representative of

    13    the cell substrate in which it was grown to amplify

    14    and type by PCR based methods the different ALV loci.

    15                DR. ONIONS:  Okay.  I'm sorry.

    16                DR. HENEINE:  But while doing those

    17    experiments, we found out that some vaccines have very

    18    little DNA, but others have much higher levels of DNA,

    19    and the concern is whether those that have high levels

    20    of DNA, whether there is DNA with full length loci

    21    that are infectious, that are known to be infectious

    22    loci like EV2, EV18, EV19, very known, and ALV that

    23    can be taken up by cells and initiate infection.

    24                DR. ONIONS:  I mean that seems to be a

    25    central point, is the state of sharing of the DNA in

                                                                        252

     1    all of these examples, and again, not just making an

     2    average estimate, but actually looking at the

     3    distribution of size I think is something that would

     4    be relevant.

     5                DR. HENEINE:  I guess the other experiment

     6    to do is to see if we can have any evidence of full

     7    length providers that are associated with the

     8    vaccines.

     9                DR. HUGHES:  If we're talking ALV related

    10    avian viruses, rav 0 and its cogenants, they are not

    11    infectious for any mammalian cell on two criteria: 

    12    the envelope, subgroup E does not recognize an

    13    appropriate target on mammalian cell, and the

    14    machinery of the virus even if you transfect in the

    15    intact provirus into a mammalian cell does not produce

    16    an infectious virus.

    17                So while I'm not recommending having those

    18    kinds of full length proviruses present necessarily,

    19    I don't think they prose any particular threat to

    20    human.

    21                DR. LOEWER:  Any further question or

    22    comment?

    23                DR. HAYFLICK:  Hayflick, UC-SF.

    24                I think it's important to keep in mind

    25    that it's not only the cell substrate that might

                                                                        253

     1    contribute DNA and RNA, but in those instances where

     2    serum and trypsin are used, there is a logical

     3    expectation that it could come from one or both of

     4    those sources despite the fact that the final product

     5    might be diluted heavily or no serum might be used in

     6    the final product, but be present in the former

     7    population doublings that led up to the production.

     8                I don't think that this has been

     9    addressed, and I'm wondering whether anyone has, in

    10    fact, determined the residual DNA content or RNA

    11    content in serum.

    12                DR. LOEWER:  Not to my knowledge at least.

    13                DR. SHEETS:  I think there is an answer to

    14    that, but I'm not the one that knows it unfortunately.

    15                This is Becky Sheets, FDA.

    16                I wanted to respond to an earlier question

    17    regarding how purified are live viral vaccines.  Most

    18    -- I'll just generalize -- many, maybe most live viral

    19    vaccines are made by inoculating virus into tissue

    20    culture or into the allantoic fluid of chicken eggs,

    21    and once infectious virus is produced either by cell

    22    lysis or budding, then the harvest is made of the

    23    supernatant or the allantoic fluid, and oftentimes

    24    this harvest is filtered, and that's your viral

    25    vaccine.

                                                                        254

     1                So that we're all on the same page, that's

     2    a live viral vaccine.  It's not impure or unpurified,

     3    as has been said, but it is minimally purified, often

     4    simply by filtration to get rid of cell debris and

     5    bacteria and fungi that might be present, but it is

     6    not purified to the extent that a single protein in a

     7    therapeutic biologic is purified.

     8                DR. LOEWER:  Thank you for this

     9    clarification.

    10                Now I would like to ask a regulatory

    11    question to Dr. Kazazian.  As he mentioned, the

    12    activity of retrotransposons is highest in some type

    13    of tumors.  For example, you mentioned testicular

    14    tumors and, I guess, ovary tumors.  The same is true

    15    for other endogenous retrovirus, and I show a Northern

    16    Blot from our laboratory which shows a number of

    17    different tumor cell lines and diploid cell line. 

    18    This is a terata carcinoma cell line, amnio chorion

    19    carcinoma.  I guess this is a raptomycin sarcoma cell

    20    line.  There's a vero cell line, and one endogenous

    21    human retrovirus, HERV-K, for example.  It is

    22    expressed only in the testicular tumors, and we know

    23    that other endogenous retrovirus, for example, HERV-H,

    24    is also very well expressed in testicular tumors.

    25                Would it be your opinion that this would

                                                                        255

     1    exclude such cell lines for the production of

     2    vaccines?

     3                DR. KAZAZIAN:  Well, first of all, I think

     4    that there are testicular, ovarian tumors of similar

     5    type that don't express retroposons, at least, and

     6    what I guess I'm saying is not all tumors of the same

     7    type will express.  I don't know whether that's the

     8    case with HERV-K or not, and I think that certainly

     9    there are those that don't have the expressions

    10    excluded.

    11                That's a tough question.  That's a tough

    12    question because I don't know that expression is

    13    related to retrotransposition.  Clearly we don't know

    14    that with HERV-K.  We don't know that HERV-K is

    15    retrotransposable at this point, and I don't know that

    16    there is a correlation between expression and

    17    retroposition for sure.

    18                DR. LOEWER:  Now that you mention it,

    19    expression is a prerequisite for transposition.

    20                DR. KAZAZIAN:  Yes.

    21                DR. LOEWER:  Yeah.  John.

    22                DR. KAZAZIAN:  It is requisite, but I

    23    don't know that it completes it.

    24                PARTICIPANT:  Regarding endogenous viruses

    25    like this one and the ALVs, I don't think it's been

                                                                        256

     1    discussed much so far that these viruses as virtually

     2    all of them that's found in the genome are very

     3    extensively methylated and expressed at very low

     4    levels.  The endogenous provirus that gives rise to

     5    the infectious rav 0, in the form in which it's

     6    inherited seems to be expressed at a level of maybe a

     7    tenth of a copy of RNA per cell; if you then allow

     8    that to be replicated, it goes up by a factor of 1,000

     9    or more over that.

    10                So that actually provides -- if you're

    11    worried about these things, that actually provides a

    12    margin of safety because that's the form in which

    13    these kinds of things at least will be passed in

    14    recipient cells if they contaminated the vaccine

    15    products.

    16                DR. KAZAZIAN:  And presumably that's also

    17    what's holding down retrotransposition of line

    18    elements of methylation.

    19                PARTICIPANT:  Presumably.  It's been

    20    argued that this may be a mechanism to protect the

    21    genome against these.

    22                DR. KAZAZIAN:  Right.

    23                PARTICIPANT:  But if so, it's doing a

    24    pretty lousy job.

    25                (Laughter.)

                                                                        257

     1                PARTICIPANT:  In fact, it probably allows

     2    their increase because it reduces the counter-

     3    selective effects.

     4                PARTICIPANT:  As a bit of a follow-up to

     5    an issue I raised this morning about how you culture

     6    the cells may affect genetic events within the cell,

     7    have you ever compare the activation of lines, for

     8    example, in stationary cells versus rapidly cycling

     9    cells?

    10                A lot of, for instance, prophage will

    11    activate as E. coli approach the stationary, and there

    12    may be very, very big differences between the

    13    activation of events in cells depending on how they're

    14    being handled at that time.

    15                DR. KAZAZIAN:  Yes.  We have not, but Karl

    16    Schmid has looked at both alus, principally looking at

    17    alu transcription.  This is only transcription.  It's

    18    not retrotransposition.  So looking at alu

    19    transcription, and he's looked at line transcription,

    20    and it goes up, as I recall now, in serum starvation,

    21    goes up in heat shock, goes up in adenovirus

    22    infection.  That's what I can tell you.

    23                PARTICIPANT:  Yeah.  That's sort of what

    24    I kind of expected.  

    25                DR. KAZAZIAN:  So it's way up.

                                                                        258

     1                PARTICIPANT:  A stress like environment

     2    where somebody wants to bail out of the genome, that's

     3    the perfect trigger for these things to start thinking

     4    about moving.

     5                DR. KAZAZIAN:  Thank you.

     6                DR. LOEWER:  So let's go on in discussion

     7    to come to the neoplastic risk event, to leave the

     8    infectious risk event, and we had quite a number of

     9    talks and discussions already on this issue, and I do

    10    not know whether somebody on the panel would like to

    11    comment again.

    12                We know from the 3T3 assays that there is

    13    a neoplastic risk rendered at least in tissue culture. 

    14    I had the impression from the talk today that NIH3T3

    15    test is mainly a test for activated ras and to a

    16    lesser degree for other oncogenes.

    17                But I think what is intriguing is that is

    18    difficult or impossible to find a neoplastic risk by

    19    injecting animals with neoplastic DNA.

    20                So what about epigenetic and genetic

    21    changes, since this is an issue, I guess, mainly to

    22    the people working on methylation?  Can the

    23    methylation pattern -- I think that's the background

    24    of this question -- can the methylation pattern maybe

    25    change by transfection or by transfection of

                                                                        259

     1    neoplastic DNA?

     2                And is this a risk for the recipient cell

     3    with respect to development of a tumor?  Are there

     4    data available in this respect?

     5                DR. DOERFLER:  Walter Doerfler from

     6    Institute of Genetics in Cologne.

     7                Why you probably approach this question

     8    partly to me at least, it's difficult to answer.  Of

     9    course, we have pursued this possibility very

    10    systematically in some of the transfected cell lines

    11    that we have produced and see changes in methylation

    12    at quite distinct places.  Whether this is a general

    13    phenomenon, of course, we haven't amassed enough data.

    14                On the other hand, I feel one could

    15    probably predict for the foreseeable future that a lot

    16    of people will study this or similar issues with the

    17    increasing availability of DNA chip (phonetic)

    18    technology because DNA methylation is one thing, but

    19    changes in expression patterns is not so difficult to

    20    do.

    21                Say I make a lambda transgenic cell line

    22    and a non-transgenic cell line or take a lambda DNA

    23    transgenic mouse and non-transgenic mouse, same organ,

    24    same mouse strain, and I compare cDNA hybridizing back

    25    to a huge DNA panel, say, having 20, 30, 40,000 DNA

                                                                        260

     1    dots on it, which technically is not impossible to do. 

     2    It's actually quite simple to do.  It's probably

     3    expensive right now.

     4                So I would expect a lot of work in this

     5    direction will be done within the next few years. 

     6    Certainly we plan to do a lot of that sort of work. 

     7    Whether this will answer the question is another

     8    problem, of course, but we will get at least an

     9    overview, and we perhaps will be able to predict in a

    10    more defined and a more scientific way what's going on

    11    in these cells.

    12                And I suppose this will also help evaluate

    13    some of the other because if you found quite distinct

    14    differences, well, maybe your suspicion would be

    15    stronger that also oncogenic events might ensue.

    16                I understand, although I've only heard it

    17    in talks and never seen the data myself, that there

    18    are a number of labs now who have looked in transgenic

    19    in knockout mice, yeah, I think in knockout animals,

    20    and they do find changes in genes other than the one

    21    knocked out.

    22                I cannot swear that this is true because

    23    I haven't seen the data, but I have heard from, as one

    24    says, reliable sources that this is so.  Perhaps other

    25    people have information on that.  It would be

                                                                        261

     1    interesting.

     2                DR. KAZAZIAN:  I would just say that I'm

     3    sure that you all know that with expression analyses,

     4    that when you do these kinds of things, I've heard --

     5    again, I haven't done them myself -- but you get

     6    changes in hundreds of cDNAs both in the up direction

     7    and in the down direction, and it's a very complex

     8    analysis.

     9                PARTICIPANT:  Or a viral infection.

    10                DR. KAZAZIAN:  Or in viral, right.

    11                PARTICIPANT:  I liked your approach of

    12    asking the questions about what kind of cell line

    13    could one rule in or out, and I wasn't sure that there

    14    was a complete answer to this question of specifically

    15    if there were an intact viral genome of some nature

    16    that could be defined by any of you, would that rule

    17    out the use of that cell.

    18                And likewise you asked Dr. Kazazian

    19    whether he thought that the expression of HERV-K would

    20    rule out the use of a cell, but I wasn't sure that

    21    there was a clear consensus on that.

    22                DR. KAZAZIAN:  Well, I said I didn't think

    23    so because I don't know of any HERV-Ks that are

    24    active.  Okay?

    25                PARTICIPANT:  Okay.  So maybe there is a

                                                                        262

     1    consensus on that.

     2                DR. LOEWER:  But the same is true, for

     3    example, for IAPs when, for instance, atag (phonetic)

     4    particles and cell lines producing IAPs are accepted

     5    for the production of biologicals, but not for

     6    vaccines insofar as CHL cell (phonetic), for example.

     7                Yeah.

     8                PARTICIPANT:  We've been thinking about

     9    producing live attenuated virus vaccines in vero cells

    10    for use by the mucosal route.  Give it a couple of

    11    drops in the nose, and we were wondering and I'm

    12    wondering whether you feel that it is safe.

    13                These live virus vaccines will have

    14    cellular DNA in it, vero cell DNA.  I think, Dr.

    15    Blair, you had mentioned that vero cell DNA was given

    16    in your assay and was not found to be tumorigenic in

    17    the 3T3 cells, and I think as far as some of the

    18    examples Phil has been using, if you take polyoma

    19    virus DNA and give it parenterally, it is not only

    20    infectious, but it is tumorigenic.  That same DNA when

    21    given mucosally has no biological activity, you know,

    22    when you give it the maximum amount that you can grow

    23    up and actually give to a mouse.

    24                So would it be reasonable to conclude,

    25    based on these kinds of data that do exist, that

                                                                        263

     1    certain continuous cell lines, such as VEROs, which

     2    are poorly tumorigenic would be perfectly safe as far

     3    as the DNA issue in terms of transferring infectivity

     4    or neoplastic risk, would be perfectly safe to use via

     5    a mucosal route?

     6                I personally believe that it absolutely

     7    would be, and I believe that there are other

     8    neoplastic cell lines that could also be considered

     9    for this route.

    10                What is the feeling of the group in this

    11    first cell line such as this?

    12                This is a very important point because

    13    there are vaccines for very important viruses can be

    14    made in this cell line and almost only in this cell

    15    line.

    16                DR. LOEWER:  So as a member of a

    17    regulatory authority, it wouldn't be wise for me to

    18    give you a clear-cut answer.

    19                (Laughter.)

    20                PARTICIPANT:  So you're being asked as

    21    advisors to the regulatory --

    22                DR. LOEWER:  The members of the panel, of

    23    course, are free to answer what they believe is

    24    correct.

    25                (Laughter.)

                                                                        264

     1                PARTICIPANT:  Maybe in support of what you

     2    said about it's difficult to give a clear-cut answer,

     3    I think we have the practice.  We make oral polio

     4    vaccine, as I will tell later, on vero cells, and it's

     5    purified, and the amount per dose is probably in the

     6    range of one to ten picagrams.

     7                Now, having said that, it doesn't say that

     8    for another virus systems one could reach such figures

     9    because the virus system, the processing, the

    10    downstream processing will probably greatly affect,

    11    and the cost that you want to pay for that downstream

    12    processing, for the losses that were incurred during

    13    the purification process will probably decide how much

    14    residue of DNA will be there.

    15                Besides, that should be put in parallel

    16    place in parallel to the basic decisions of whether we

    17    could or we could not be happy with, say, the presence

    18    of cDNA.  It's not clear-cut.  It's not black and

    19    white.  It's really a quantitative issue.

    20                PARTICIPANT:  (Inaudible.)

    21                PARTICIPANT:  Can't hear.

    22                PARTICIPANT:  Thank you.

    23                I want going to question the assumption

    24    that live viral vaccines necessarily have to be impure

    25    because modern methods of production should blend the

                                                                        265

     1    possibility to provide even conventional whole

     2    particle or live vaccines in a purified form, and

     3    we've hear the answer to that, the vero cells.

     4                Now, over 20 years ago I produce

     5    experimental cold adapted in flame devirus (phonetic)

     6    vaccines in chick eggs, and these, in fact, were

     7    purified products.  It was then purified.

     8                The reason we were only purifying them at

     9    that time wasn't safety.  It was to enable -- we had

    10    more control over the product.  I could adjust the

    11    titre, clinical trials, but there's also a practical

    12    reason.  It enabled me to have product which had

    13    tropic sucrose in it, which improved the infectivity,

    14    but it also improve the palatability for the

    15    volunteers, and I very soon found out that the

    16    volunteers did not like having allantoic fluid put

    17    down their noses, and a purified product was much more

    18    acceptable.

    19                So I just risk the question, the

    20    assumption that the live virus vaccines need to be

    21    pure.  They couldn't be purified.

    22                DR. LOEWER:  So personally I am not

    23    absolutely sure even with new technologies it would be

    24    possible to purify live vaccines or live virus much

    25    better because this may be true, for example, for

                                                                        266

     1    naked viruses, polio or so, but with enveloped

     2    viruses, maybe herpes viruses or even rabies, it may

     3    be more difficult, and they may even incorporate

     4    similar DNA or nucleic acids which cannot be purified

     5    away.

     6                It's at least a possibility which has to

     7    be checked.

     8                So CBER, of course, quite a number of

     9    questions this group, and this is the next question

    10    they asked.  Consider factors that might contribute to

    11    increased safety concerns associated with residual

    12    cell substrate DNA, and one of the question is -- this

    13    was posed several times during this meeting and

    14    wasn't, I guess, really answered -- was aggressiveness

    15    or tumorigenicity of tumor cells is a factor which

    16    increases safety concern.

    17                That means the more aggressive, the more

    18    risky or isn't it true?

    19                So my personal view to say it, having

    20    learned that the NIH3T3 test really recognizes only a

    21    certain spectrum of oncogenes, the question would be

    22    whether these tests in animals also select only for

    23    certain aspects, for certain oncogenes.  The question

    24    is how well it models the situation in human.

    25                And, therefore, personally I would think

                                                                        267

     1    it's difficult to extrapolate from the data in animals

     2    to the possible risk in humans.  So that would be my

     3    argumentation, but I would like to hear other

     4    opinions.

     5                So if there are no opinions, I guess --

     6                (Laughter.)

     7                DR. PETRICCIANI:  Johannes.

     8                DR. LOEWER:  Yeah.

     9                DR. PETRICCIANI:  I think with regard to

    10    the aggressiveness or the degree to which a cell is

    11    tumorigenic in one or another animal system, as I

    12    mentioned during my presentation, I don't see that as

    13    an important criteria with regard to whether or not a

    14    cell should or should be not more or less acceptable.

    15                I think the real issue is as we've said

    16    many times.  The issue is what cell contaminants are

    17    in the final product.  Whether they came from a very

    18    aggressive cell or a less aggressive cell probably

    19    doesn't make any difference.

    20                The example that I gave was you could have

    21    one cell that's very aggressive with a few activated

    22    oncogenes.  You could have one that's much less

    23    aggressive in those systems for whatever reason that

    24    has 100 activated oncogenes, and I don't know that you

    25    can say one is more or less better than the other as

                                                                        268

     1    far as the cell substrate, just as one example.

     2                I think using that as a criteria is

     3    fraught with problems.

     4                DR. LOEWER:  Dr. Hayflick.

     5                DR. HAYFLICK:  Hayflick, UC-SF.

     6                I'd like to return to the point I made

     7    earlier relevant to the use of serum and trypsin in

     8    many, but certainly not all of these vaccines, and

     9    that is the likelihood that there can be carried over

    10    into cell culture sources of DNA and RNA, for example,

    11    from live virus vaccines that are given to most cattle

    12    these days.

    13                There are a number of vaccines that are

    14    used worldwide, certainly in this country, to prevent

    15    a number of virus diseases of cattle.  Those virions

    16    are carried over in the serum that is then used for

    17    the manufacture of vaccines.

    18                In addition to that, most cattle in this

    19    country and in other countries are given anti-

    20    helminths and other chemicals that are known to have

    21    breakdown products identical to known carcinogens that

    22    are also carried over in the serum to a source of

    23    product development or manufacture.

    24                And I think very little attention has been

    25    given to this.  As long as we're talking about

                                                                        269

     1    aggressiveness and potential tumorigenicity, I think

     2    that this matter should be seriously considered.

     3                Thank you.

     4                DR. LOEWER:  Thank you.

     5                I think we have already discussed the next

     6    two questions here, the presence of proviral DNA, the

     7    presence of latent/occult viral genomes, and there is

     8    a question whether there are other factors which could

     9    increase the concern.

    10                I have here a list which was put together

    11    already 15 years ago or so.  One is amplification for

    12    oncogenes, which John already mentioned.  Then there

    13    is maybe the risk for enrichment during purification,

    14    especially when the sequences are incorporated  into

    15    the viruses themselves.

    16                And there may be a certain risk from

    17    substances which could enhance the uptake.  For

    18    example, so far as I know nobody has tested the effect

    19    of adjuvants on the uptake of DNA itself, but of

    20    course, one has to realize that live viral vaccines do

    21    not contain adjuvants.  Only inactivated viruses, and

    22    this inactivation of viruses alters the DNA, may be

    23    inactivated.

    24                So these are other factors which have been

    25    considered at the beginning of the '80s at least.

                                                                        270

     1                I don't know whether somebody wants to add

     2    some other factor or some other possible risk.  So we

     3    will shortly continue.

     4                This was also discussed so far that the

     5    amount of residual DNA, if it it's as low as possible,

     6    which may contribute to a lesser concern.

     7                Then there is mentioned the possibility of

     8    the physical state of residual DNA, whether

     9    incorporated in viruses or not, but it's of high

    10    molecular weight or it's cut in pieces, and personally

    11    I'm not sure what the influence of the physical state

    12    really is.

    13                The larger the DNA molecule, the higher

    14    the probability that a complete genus is coded for. 

    15    It contains a complete gene, but maybe less

    16    effectiveness of incorporation, and also some less

    17    possibility of integration.

    18                And the shorter the pieces, and we have

    19    heard yesterday that even very, very small pieces can

    20    integrate and cause tumors; that means the smaller the

    21    DNA, the more single DNA molecules, the higher the

    22    risk for indication maybe.  So there may be two sides

    23    of the coin.

    24                And the question of DNA clearance removal

    25    was also addressed yesterday, I guess, and we have

                                                                        271

     1    discussed it shortly here.

     2                Is there any further comment from the

     3    panel or from the audience to these questions?

     4                So it's not the case.  So this was a

     5    question, I guess, which was not addressed in our

     6    session or the session as the members or the  speakers

     7    have presented their data, namely, the usefulness of

     8    the model system, and so I will switch this, and we'll

     9    come to maybe as a last point of this discussion to

    10    the quantitative aspects.

    11                And you may have read the paper by Krause

    12    and Lewis with some considerations with respect to the

    13    quantitative issues, and the first question here is: 

    14    would it be acceptable that in a frequency of one

    15    pair, one million doses, if it is an acceptable level

    16    of risk?  In other words, would it be acceptable that

    17    one of one million vaccines developed a tumor because

    18    of the tumor DNA, which may be --

    19                DR. KAZAZIAN:  By the way, how are you

    20    going to prove that?  How are you going to prove that,

    21    you know, one in a million is due to the tumor -- I

    22    mean due to the vaccine as opposed to a natural cause?

    23                DR. LOEWER:  You are completely right.  I

    24    will not defend this.  I just mention what he has

    25    written.

                                                                        272

     1                (Laughter.)

     2                DR. LOEWER:  And indeed --

     3                DR. KAZAZIAN:  So who wrote that anyway?

     4                (Laughter.)

     5                DR. LOEWER:  Indeed, that is the major

     6    question.  This would never be detected in the

     7    background of tumors.  That's absolutely clear, and it

     8    may be only a theoretical value, which could be a goal

     9    to reach.  It will never be detected in --

    10                DR. KAZAZIAN:  The problem is that the

    11    jury might pay the suer for that one million even

    12    though we don't know.  It's part of the background.

    13                DR. LOEWER:  So, indeed, I think this

    14    highlights the problematics or the problems with all

    15    of these theoretical calculations.  It's questionable

    16    whether it's worse to do so.

    17                Phil, you make a comment?

    18                DR. MINOR:  Well, I'm not sure you

    19    wouldn't pick up one in a million per vaccine dose. 

    20    When you think how many doses children get and you're

    21    talking about in my country it's like half a million

    22    children a year get this stuff, and you can pick up

    23    adverse reactions at one in 100,000, and vaccines get

    24    withdrawn for that kind of reason.

    25                DR. LOEWER:  But it depends.

                                                                        273

     1                DR. MINOR:  But, yeah, there are things

     2    like meningitis is huge, as well, and you can pick

     3    that up, and you can withdraw vaccines on the basis of

     4    that.

     5                DR. LOEWER:  It may depend, I think, on

     6    the patient period.  If it takes 40 years to develop

     7    the tumor you will never detect it, but if it takes

     8    only two years, it may be possible.

     9                DR. MINOR:  Well, I think alternatively

    10    you might get a very nasty surprise in 40 years.

    11                DR. LOEWER:  Yeah.

    12                PARTICIPANT:  Yes.

    13                (Laughter.)

    14                DR. LOEWER:  Okay.

    15                PARTICIPANT:  I think the idea is to try

    16    to think about these issues conservatively based on

    17    the idea that if one is going to start manufacturing

    18    vaccines in new cell substrates, you would like to be

    19    able to assure the public that you've considered the

    20    risks down to a very low level, and so even if it

    21    occurred at this level we would not be able to detect

    22    it against the background, that doesn't mean that we

    23    as a regulatory agency don't have an obligation to the

    24    general public to be able to tell them --

    25                PARTICIPANT:  At least tell them that.

                                                                        274

     1                PARTICIPANT:  Well, we'd like to know

     2    that, too, that the risks are at negligible levels.

     3                DR. LOEWER:  So usually the figure one to

     4    one million is more or less accepted figure in

     5    biologics.  For example, the risk for transmission of

     6    HIV by blood donations is also in this order, and it's

     7    more or less accepted or the risk of polio OPV

     8    associated, poliomyelitis is also a disorder.

     9                PARTICIPANT:  On the other hand, just to

    10    follow up, I'm not sure that right now the public is

    11    willing to accept one in a million risk of vaccine

    12    associated paralytic polio, and I'm certain that the

    13    public is not willing to accept a one in a million

    14    risk of HIV infection among healthy children who are

    15    just coming in to get their routine shots for school.

    16                DR. LOEWER:  Okay.

    17                DR. LEWIS:  Yeah, I think that those

    18    figures are based on what we could possibly generate

    19    from an experimental or a validated test system, not

    20    what we're going to expect in the population at large. 

    21    In other words, the issue is how to come to grips with

    22    estimating the risk or the perceived risk, not

    23    necessarily the risk, because at this point in time

    24    I'm not sure we have any risks here, but these are

    25    perceived risks, and if we're going to go out and

                                                                        275

     1    convince people to give these products to their

     2    children and to their patients, then we have to have

     3    some level of information upon which to make a

     4    decision.

     5                At least from my own personal perspective

     6    to say "I think" or "my opinion is" in a situation

     7    like this is not going to be good enough.  We have to

     8    have data.

     9                Now, the question is:  how do we generate

    10    that data and what do we use that data to do?

    11                And I think an estimation like this is

    12    based on a simple attempt to come to grips with how to

    13    estimate this level of risk so that when we try to

    14    convince ourselves as a regulatory body that it is

    15    safe and we want to put the stamp of FDA approval on

    16    this or, to quote myself the other night, the FDA says

    17    this is okay, we need to have a concrete set of

    18    information upon which to make that decision. 

    19    Otherwise it becomes a simple exercise or article of

    20    faith.

    21                And at this point in time I don't think

    22    I'm willing to sign my name to something that's an

    23    article of faith.  We need some data, and the question

    24    is:  how do we generate that data?

    25                And what we're, you know, hoping to get

                                                                        276

     1    from discussions like this is how to proceed to get

     2    that information.

     3                DR. LOEWER:  Dr. Hayflick.

     4                DR. HAYFLICK:  Hayflick, UC-SF.

     5                We already have a baseline for the

     6    exception of what I read up there to be infectious

     7    risk events in respect to the fact that we are living

     8    today with an attenuated polio virus vaccine that, if

     9    my memory serves me well, has resulted in one

    10    acceptable case of paralytic polio per three million

    11    doses or something in that range.

    12                And, in fact, there are more cases of

    13    paralytic polio occurring in this country per year

    14    from the vaccine than from the wild type.  We already

    15    have such a baseline.

    16                DR. LOEWER:  Thank you.

    17                I would like to come to a final point

    18    here, and this will address as the problems with all

    19    of these calculations, and what I've shown here is the

    20    calculation for the activation of, quote, oncogenes by

    21    intracellular foreign DNA, and this calculation was

    22    published by Reinhardt Kurth after some discussions in

    23    our institute with Phil Minor sitting over there and

    24    starting to laugh.

    25                It demonstrates, I guess, when you run

                                                                        277

     1    into problems by all these calculations, and I would

     2    like to go through very quickly to the difficult

     3    aspects here.

     4                The calculation starts with a quantitative

     5    aspects of the confection of the zarc gene (phonetic)

     6    in the wing of chickens, which are already mentioned

     7    here and the probability to create a tumor with

     8    respect to the per molecule of DNA or three times ten

     9    to the minus 12.

    10                And the efficiency of the cellular uptake

    11    was taken from some studies to be ten to the minus

    12    nine.  That means that the probability that an

    13    intracellular DNA may integrate in the chromosome at

    14    three times ten to the minus three, but anybody can

    15    easily recognize that there's many assumptions, and

    16    I'm not sure how representative these data really are,

    17    especially the uptake of injected DNA in the cell as

    18    more attachment.

    19                The probability that if an integration

    20    occurs it's integrated into or close to a portal

    21    oncogenous, easier to calculate from the size of the

    22    genome and the size of the portal oncogene, and this

    23    is in the range of ten to the minus five.

    24                The probability that this integration

    25    leads to an activation of the gene is taken here as

                                                                        278

     1    ten to the minus two, and this, I guess, is  pure

     2    judgment.  It's not based really on data, but now you

     3    can multiply all of these probabilities, and you will

     4    end up with the probability of a single intracellular

     5    DNA molecule to activate a single portal oncogene in

     6    a size range of three times ten to the minus ten.  

     7                It's pretty low, but we all know that the

     8    way -- that is a multi-step way to tumor information,

     9    and this calculation it was assumed that two

    10    independent activation events are required, and this

    11    leads to a number of ten to minus 19.

    12                And if you'll look in the recent

    13    publication of Nature, we have seen the picture, their

    14    claims that at least four different steps would be

    15    necessary, and then we apply this figure to this

    16    calculation, and we end up with ten to the minus 38

    17    here in this case.

    18                And then one starts to wonder why tumors

    19    at all occur if this is such a rare event, and this

    20    again shows that all of these calculations are very

    21    difficult, and one has to take great care to take

    22    these figures and to draw conclusions from this.

    23                And please also, my problem for you if you

    24    want to find figures to convince the public on the

    25    risk, it will be always difficult to have figures and

                                                                        279

     1    to rely on these figures.  They may lead to a false

     2    sense of security or knowledge.

     3                So I would always be very care of these

     4    type of calculations.

     5                Comments?

     6                DR. BROKER:  Tom Broker, UAB.

     7                I want to kind of approach these kinds of

     8    numbers in a very different way, and that is from the

     9    perspective of human papilloma virus infections that

    10    lead to cervical or penile carcinomas.

    11                One of the more remarkable facts of this

    12    family of diseases is that since 1980 more people have

    13    died of HPV disease than have died of AIDS.  I mean

    14    this is a disease that takes out three-quarters of a

    15    million people per year.

    16                Now, the kinds of calculations, they just

    17    don't seem to square with what we know is the reality

    18    of that disease process.  For example, vast numbers of

    19    people, hundreds of millions, harbor HPVs, and the

    20    process through which neoplasia occurs is, first, the

    21    establishment of the viral genome in the infected

    22    tissue, followed some time later in a small

    23    percentage, usually one percent of the patients, where

    24    an integration event takes place, and that's where I

    25    think we're interfacing with this kind of table.

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     1                The integration, it turns out, is somewhat

     2    selected.  It's going into an actively transcribed

     3    region of the genome.  There's further selective

     4    events for the active expression loci, which then

     5    leads to an up regulation of genes that foster

     6    somewhat more rapid cell cycling.

     7                In the presence of mutagenic agents, and

     8    over a period of time, probably ten years or more,

     9    these genes get more and more activated.  There's a

    10    little bit of increase in P53, of E6 taking out P53,

    11    and so forth, and it's a progressive neoplastic

    12    process.

    13                And so I think you can't have

    14    instantaneous numbers, but the bottom line is three-

    15    quarters of a million people die every year of this

    16    family of diseases.

    17                DR. LOEWER:  I personally believe that

    18    such type of calculations cannot be applied for virus,

    19    tumor viruses.  In the case of HPV, of course, virus

    20    produce actively oncogenes, E6 and E7, and this is not

    21    taken into account in all of these calculations. 

    22    These were just calculated, passive activation of an

    23    oncogene.

    24                PARTICIPANT:  Johannes, I was just going

    25    to re-echo that last point.  I mean the assumption

                                                                        281

     1    here is that they are independent events, and I don't

     2    think that is the case necessarily in transformation

     3    events.

     4                For instance, if you introduce nat and myc

     5    (phonetic) into a cell, the probability that that cell

     6    population then goes on to cumulate the other hits is

     7    heightened because we're into a replicated advantage.

     8                So I think that final assumption is just

     9    not correct.  Once you've initiated a process, you're

    10    on a cascade of other events are required, but they're

    11    not necessarily independent anymore.

    12                DR. LOEWER:  That's a chronic observation,

    13    yeah.

    14                PARTICIPANT:  Maybe I can ask you to

    15    clarify.  Are you rejecting the use of a quantitative

    16    approach?  Because as Andy just came up here and

    17    pointed out, the alternative to at least attempting

    18    some kind of a quantitative approach is to simply go

    19    based on your gut feeling, and our question is:  is

    20    that good enough?

    21                So while maybe these numbers give us too

    22    much reassurance, which I think is what you're

    23    implying, the question is if these numbers came out

    24    differently and this number were ten to the minus five

    25    or something like that, wouldn't that mean we need to

                                                                        282

     1    go back and work a little harder?

     2                DR. LOEWER:  So that's really my personal

     3    opinion.  I would be very careful with numbers in this

     4    respect because we are so many ambiguities, and I

     5    personally prefer more qualitative approach to this

     6    question, but this, of course, can be discussed.

     7                John.

     8                DR. PETRICCIANI:  I think the issue of

     9    trying to quantitate to get a more firm fix on a given

    10    situation is attractive and it's seductive.  The

    11    problem in applying a quantitative approach in this

    12    area to me lies in the uncertainties of all the things

    13    that go into that final number so that when you come

    14    out with a number, it's mushy because you have so much

    15    uncertainty in virtually every variable that you put

    16    in it.

    17                It doesn't mean that it's of no value, but

    18    I think at least in my mind what you'd come out with

    19    is a qualitative impression of whether you're on one

    20    end of the spectrum or in the middle or at the end.

    21                But I agree with you, Johannes, that in

    22    this arena I'd be pretty reluctant to use a number

    23    because it doesn't have enough meaning to it.

    24                PARTICIPANT:  That may very well be right,

    25    but then the implication of that is that if you can't

                                                                        283

     1    say that you think these risks are very low, less than

     2    one in a million or something like that, but you all

     3    say, "Well, these risks exist.  You know, we see these

     4    things happening in tissue culture," there's a chance

     5    that this stuff can happen.  What you're really saying

     6    then is if we don't have a way of getting a handle on

     7    what these risks are, that really if the best we can

     8    do is qualitative, then can we take these risks?

     9                DR. PETRICCIANI:  Well, let me go back to

    10    two points that I made in my talk.  Number one is that

    11    in this analysis and all of the other ones, they are

    12    absolute worst case.  They're not best case by any

    13    stretch of the imagination, and if you used what I

    14    think is perhaps a more relevant piece of data, and

    15    that is human tumor DNA as opposed to viral oncogene

    16    DNA, you'd have zero up on the top because there isn't

    17    a shred of evidence that I know of that human tumor

    18    DNA causes tumors in any animal system.

    19                The other point is that it would be

    20    helpful -- you're getting tired of me saying this --

    21    but it would be helpful to have some basic generic

    22    pieces of information like DNA dose response to help

    23    actually get a better handle on those quantitative

    24    questions.

    25                PARTICIPANT:  Yeah, I was going to save

                                                                        284

     1    this for tomorrow, but I'm getting more and more

     2    irritated in a sense over actually the last ten years

     3    when I last attended a meeting to discuss these issues

     4    at which this kind of calculation was done by both me

     5    and Howard Clemens' calculation.  The same thing was

     6    cited  using Hsing-Jien Kung's data, and here we are

     7    ten years still using Hsing-Jien's classical data when

     8    for more than ten years the basic experiments to get

     9    a handle on some of these numbers have been obvious

    10    and doable.

    11                They're not rocket science.  It is really

    12    very simple, basic stuff of taking this and that DNA

    13    and treating it this and that way and injecting it in

    14    this and that animal.  Any one of us in this room

    15    could design a suitable set of experiments, but nobody

    16    has done it for ten years.

    17                Why not?  Why not?  It could probably be

    18    done on less than 1,000 mice and probably for less

    19    than a million dollars spread out over three or four

    20    years.

    21                That may be a lot of money to the FDA, but

    22    to the NCI this is what you ought to be supporting,

    23    this kind of experiment.  This is a very small amount

    24    of money.

    25                (Applause.)

                                                                        285

     1                DR. ONIONS:  So again really echoing --

     2    David Onions -- echoing what John has just said, I

     3    mean I do think those systems do exist, and I made a

     4    case yesterday for a variety of transgenic models, and

     5    I think those do offer the prospect of giving you the

     6    kind of sensitivity that you need and to give you the

     7    kind of dose response curves that John has referred

     8    to.  I think that is important.

     9                I mean, you can force a system rather like

    10    the V-SARC experiments and take the worst case, but

    11    then you can go and do the experiment that I think

    12    John has wisely said.  That is, you take tumor DNA

    13    from a tumor cell line or cell lines where you

    14    actually use the substrates, inject those in, and

    15    you're using systems that are highly primed to develop

    16    neoplasia, and because of the time scale and because

    17    of the sensitivity, you don't have to go into very

    18    high numbers either.

    19                So I mean, I think those systems exist. 

    20    You might have to use multiple models, but I think

    21    they exist, and I think the reason they have not been

    22    done is actually because the academics like me who

    23    develop these systems do it for other reasons.  We're

    24    looking for new oncogenes.  So that kind of experiment

    25    just gets in the way.

                                                                        286

     1                But, I mean, if somebody now said that's

     2    a useful experiment to do, then we should do it.

     3                PARTICIPANT:  I'd just like to say one

     4    more thing about the numbers and the models.  I'm a

     5    very poor person to be talking about mathematics

     6    because I can't add two and two, but I think from a

     7    simply perception point of view, as an experimentalist

     8    and now as a regulator, to get at the issues that Dr.

     9    Petricciani was mentioning, we have to start

    10    somewhere.  We have to think about how to approach

    11    these things from a quantitative perspective because

    12    that's where the future is.

    13                If we continue to do this on an ad hoc,

    14    believe type basis, we're not going to make any

    15    progress, and 15 years from now we're going to be

    16    right back here addressing the same questions again. 

    17    We have to start somewhere.

    18                As crude as the numbers may be, if we

    19    begin to make the attempt, we can find out what we

    20    know.  We can find out what we don't know.  We can

    21    pose the questions to ask to improve the numbers as we

    22    go along.

    23                If we don't make that attempt, we're not

    24    really making progress, and I think that has been the

    25    sort of thing that has been driving us for the past

                                                                        287

     1    five or six months, is we've tried to struggle with

     2    how to come to grips with thinking about these issues

     3    and putting together some sort of approach that made

     4    sense.

     5                This is simply a beginning, and we're

     6    going to make mistakes, but the thing that is, we have

     7    to be very careful about the kind of mistakes we make. 

     8    We can make mistakes in calculations, but if we make

     9    mistakes in delivering products that are unsafe, the

    10    consequences of that are going to be incalculable,

    11    both for the regulatory process and for the public

    12    health and for the companies that are unfortunate

    13    enough to be associated with that.

    14                So I would, again, I would make the plea

    15    that our job is to try to figure out the best models

    16    we can at the time, realizing that they're not

    17    perfect.  They probably will never be perfect, but the

    18    job is to start somewhere and attempt over time to

    19    improve them as information becomes available.

    20                DR. LOEWER:  I didn't want to say that

    21    quantitative considerations wouldn't be helpful.  I

    22    think they are definitely helpful to find out and to

    23    evaluate the different factors which may influence the

    24    whole process, but I have to admit the same ideas like

    25    John came into my mind.

                                                                        288

     1                These issues have been discussed in the

     2    early '80s, and in the meantime the number of

     3    experiments which help to clarify these issues is very

     4    limited, and maybe one of the outcomes of the

     5    quantitative approach may be to design adequate

     6    experiments to perform and to find solutions to the

     7    questions we have posed.

     8                I think we are already half an hour late,

     9    and I think I shall conclude this afternoon's panel-

    10    audience discussion.  

    11                I would like to thank the speakers again

    12    and also all the members of the audience who have

    13    contributed.

    14                Thank you very much.

    15                (Applause.)

    16                (Whereupon, at 5:10 p.m., the afternoon

    17    session was concluded, to reconvene at 7:00 p.m., the

    18    same day.)

                                                                        289

     1                       EVENING SESSION

     2                                            (7:08 p.m.)

     3                DR. PEDEN:  Good evening.  I think we'd

     4    better get started since we have to be out of here by

     5    ten o'clock.

     6                This is a miscellaneous session, and the

     7    topics are diverse, and at the end of the formal

     8    presentations of the program, there are going to be

     9    some shorter presentations, but so we'll see how we

    10    go, how many we get through.  We have to be out of

    11    here by ten.

    12                And I'm going to ask the speakers this

    13    time to abide by the lights that now are going to

    14    appear on the podium.  So I've brought it down to 15

    15    minutes.  So perhaps we can get everything done by 15

    16    minutes.

    17                So I'm going to use the Chairman's

    18    prerogative, although I have one other thing to say. 

    19    This is a very special day today.  It only occurs once

    20    every decade, right, and the question, I suppose, is

    21    where were you when it last occurred.  So you can be

    22    thinking about that.

    23                What I want to do first, if I may have the

    24    first slide, I'm going to take the Chairman's

    25    prerogative and -- is someone over there?  Could I

                                                                        290

     1    have the first slide?  All right, and the lights down

     2    a bit.

     3                Because part of the thing we have to deal

     4    with is quantitation of things, what I want to do

     5    first is just give a couple of slides briefly on

     6    methods that we've been developing at the FDA on two

     7    topics.

     8                The first is the sensitive reverse

     9    transcriptase assay called PERT that Joerg Schuepbach

    10    coined as the product enhanced reverse transcriptase

    11    assay, and we've adapted that to the real time

    12    quantitative PCR machine, the taq man machine, and

    13    just go on and give another example of an assay we're

    14    developing to look at primate polyoma viruses as an

    15    example of just what sort of quantitative assays we

    16    can do.

    17                Since it hasn't been covered yet, this is

    18    the quantitative real time PCR assay, and it's a very

    19    convenient assay, and what it depends on is a reporter

    20    and a quencher, and when these are on the same probe,

    21    the reporter does not fluoresce.

    22                But then after PCR, with the taq DNA

    23    polymerase, it gets excised because of the five prime

    24    and three prime nucleus activity.  It releases the

    25    quencher, releases the fluorochrome, and the machine

                                                                        291

     1    registers that.  So it's a real time PCR, and the

     2    assay detects the cycle at which the product is

     3    detected.

     4                So in the first example we use the PERT

     5    assay, and since, again, no one has presented this, I

     6    just run it through briefly.  It's the standard RT

     7    assay where the reverse transcriptase either comes

     8    from a retrovirus or purified RT, and the presence of

     9    a known RNA sequence, for example, MS2 RNA and a

    10    primer, you get a cDNA synthesized, and then the taq

    11    polymerase by PCR, you can amplify this sequence since

    12    it's known, and you get double strand cDNA products,

    13    and then you can assay it by various methods.

    14                And what we're talking about now is the

    15    taq man PERT assay, the TM PERT assay, and I just want

    16    to show you what this assay can do.

    17                When we look at three different purified

    18    reverse transcriptases from AMV, Maloney murine

    19    leukemia virus RT and HIV RT, this assay -- and make

    20    dilutions -- this assay, as you can see, is linear

    21    over about six orders of magnitude, and when you use

    22    equal amounts of the RT according to the manufacturer,

    23    these two enzymes fall on the line, and the HIV, in

    24    fact, is off the line, indicating that the assay, in

    25    fact, if the units are correct as determined by the

                                                                        292

     1    manufacturer, is probably more sensitive on the MS2

     2    RNA than it is on the synthetic templates.

     3                But in any case, the assay is highly

     4    sensitive, and it's linear over, let's say, about six

     5    or seven logs, and we can detect, going down here to

     6    make the calculation, we can detect the equivalent of

     7    RT of a single virion, not infectious virion, just a

     8    single virion.

     9                So the sort of things we want to do or use

    10    this assay for us you can assess the retrovirus

    11    contamination in cell substrates, biologicals, and

    12    vaccines.  You can use this assay, and we're testing

    13    to see how efficient this is, to monitor retroviral

    14    clearances from such products as monoclonal

    15    antibodies, and also detect and identify unknown

    16    retroviruses.

    17                So it's a very useful assay.  The problem

    18    with this assay, as everybody who's worked with it

    19    knows, is it's so sensitive and it detects DNA

    20    polymerase's activity.  So you're never sure that the

    21    assay you're detecting is, in fact, pure retrovirus,

    22    a true retrovirus or DNA polymerase.

    23                And we have not been able yet using this

    24    assay to discriminate between those two.  We're still

    25    working on that.

                                                                        293

     1                In the second example using the taq man,

     2    we've designed primers and probes for the primate

     3    polyoma viruses for 40 BK and JC-V, in this case in

     4    the late region of the genome, and then when we show

     5    an example of this, we're using dilutions of SV 40 DNA

     6    down to about in this case you can register ten

     7    molecules of SV 40 DNA.  This is a cloned SV 40 DNA. 

     8    Viral DNA is the same.

     9                And in these blue dots are dilutions of

    10    cos cell DNA which carries a single copy of SV 40

    11    genome, and this should be one copy, two copies, ten

    12    copies, and 100 copies, and as you can see, it falls

    13    on that line.  So from this we can conclude that, in

    14    fact, cos cells do have a single copy of SV 40 DNA.

    15                So what we've done so far is test the

    16    sensitivity and specificity of each of these primer

    17    sets.  With SV 40 we can detect about one to ten

    18    molecules reproducibly.  With the BK-V primers we're

    19    only at the moment at about ten to 100 copies per

    20    assay, and in the JC-V it's about one to ten.  No the

    21    JC-V primers pick up also BK-Vs.  So now we have to

    22    both increase the sensitivity of this assay and try to

    23    improve the specificity of this assay.

    24                So just in summary, we can say that the

    25    developed primers for the three primate polyoma

                                                                        294

     1    viruses, it can detect reproducibly between ten and

     2    100 copies and sometimes down to one.  The reaction is

     3    linear over at least seven to eight logs, and primers

     4    so far specific for SV 40 and BK-V, and JC-V primers

     5    cross-react with BK-V.

     6                And using a series of SV 40 transformed

     7    mouse and hamster lines, we've shown that, in fact,

     8    the assay can detect different quantities of the SV 40

     9    genome per cell.

    10                So this is just an example of sort of

    11    quantitative assays that are being developed and are

    12    going to be very useful in determining the level of

    13    quantitation -- the level of contamination of various

    14    products in cell substrates and vaccines.  So that's

    15    what I'm going to say, and other people will cover the

    16    assay a bit later on.

    17                I think we should move on to the first

    18    talk, and that's by Bernard Meignier from Pasteur

    19    Merieux, according to the program, and he's going to

    20    talk about industrial experience with live polio

    21    vaccine prepared in the VERO cells.

    22                And, again, I would try to ask you to keep

    23    to the 15 minutes.

    24                MR. MEIGNIER:  Can I have the first slide,

    25    please?

                                                                        295

     1                I would like to thank the organizers for

     2    their invitation to share with you this example of the

     3    use of -- no, that's not the one.  I'm Bernard

     4    Meignier -- so to share with you that example of the

     5    use of continuous cell line applied to the

     6    manufacturing of vaccines.

     7                When we have the slide, I wish also to

     8    acknowledge the colleagues of mine that helped me

     9    assembling that presentation, and I would also like to

    10    say as an introduction that I was not personally part

    11    of the development of the VERO cell system, nor of the

    12    manufacturing of those vaccines.

    13                However, I will take the liberty of using

    14    the "we," "we do this," "we don't do this," "we did

    15    this," or "we did that" --

    16                (Laughter.)

    17                MR. MEIGNIER:  -- just to express how

    18    collective an activity could be.  It's just the

    19    liberty of language, nothing else.

    20                Forward.  It looks like this time ring. 

    21    Good.

    22                So the VERO cell story starts in 1962. 

    23    The two Japanese scientists there on March 27th, one

    24    to establish a system, a cell system where they could

    25    grow SV 40 virus in a lytic system to work more easy.

                                                                        296

     1                They could establish one line.  From that

     2    line there was a derivation of various other sub-lines

     3    in Japan, and then two years later Simizu brought one

     4    of those branches to NIH, and again, in the States

     5    there were various lines that were spread among some

     6    laboratories.

     7                One of those branches, LT3 and VDOT

     8    (phonetic) ATCC was deposited by Drs. Hann and Riem at

     9    pass 113, as you can see here.  ATCC, characterized at

    10    cells scale, the top (phonetic), and two, 121, and

    11    from there can supply cells -- you have the code

    12    number there -- supply cells at the level of per such

    13    one in 24 (phonetic), and that's where in 1979

    14    Institut Merieux, which then became Pasteur Merieux

    15    Connaught for various mergers, that's where we

    16    started; they started at the time.

    17                And from that passage 124 derived as shown

    18    here, primary cell bank, and then from there working

    19    cell bank, and established a scheme of production,

    20    production being done at the passage one and 42.

    21                In parallel to that, one bank was

    22    established at the level of the working cell branch

    23    that was made available to WHO.  That bank is

    24    deposited, in part, in ATCC, in part at ICAC, and is

    25    available or could be supplied upon request through

                                                                        297

     1    WHO.

     2                Now, these cells, just a few words on the

     3    technology.  These cells are grown on microcarrier

     4    cells.  Microcarrier is a small particle on which the

     5    cells are grown.

     6                There were cells encourage dependent. 

     7    They would not grow unless you got them -- especially

     8    they could not grow in suspension.  So in our case,

     9    the microcarrier is made of DEAE dextron with an

    10    optimized charge.  The brown (phonetic) name is called

    11    cytodex, and it's made by Pharmacia.

    12                And what you're seeing here is one of

    13    those small particle sizes, about, say, 100 microns or

    14    slightly more, and actually you don't see the

    15    microcarrier.  What you do see are the cells.  This

    16    was taken from a culture, and you do see the cells

    17    that are entirely covering the beads.

    18                The prime scale at which we are operating

    19    is 2,000 liters, and a full fermenter contains when

    20    it's ready for being effective, contains only half a

    21    dozen trillion cells, which is a fairly large amount.

    22                Now, those cells, to maintain the safety

    23    subjected to quality control and all the activity of

    24    development of the banks and all of the QC that goes

    25    with them and all the characterization that was done

                                                                        298

     1    was established in relationship -- I should say in

     2    collaboration -- with authority BWHO that has the

     3    asset of regulations and guidelines with national

     4    authorities, including FDA in this country.

     5                The sets of QC that you do see here, it's

     6    a busy slide, but just to say that some of QC are just

     7    done once in a while.  We are still working with the

     8    same primary cell bank from the beginning.  We make

     9    seven working cell banks, while some other controls

    10    are done routinely on each and every batch of

    11    production.

    12                And as you can see from the list -- I

    13    won't read it -- but most of those assays which are

    14    done both on the cell themselves and on -- I'm sorry

    15    -- on the supernatants; most of those cells really

    16    have to do with looking for adventitious agents, be

    17    they bacteria, be they fungi, and mostly be they

    18    viruses.

    19                The basis is hem (phonetic) absorption

    20    inoculation to animals and inoculation to cells.  ADC

    21    means human diploid cells.  The PMKC are primary

    22    monkey cells, and NPCC is cell, a continuous line from

    23    monkey kidney.

    24                Attention is paid also to assays to look

    25    for tumorigenicity as shown here and here, and that's

                                                                        299

     1    done only theoretically when we make a working cell

     2    bank.  The cells are assayed, of course, on the cell

     3    bank itself and are also assayed on a special pool of

     4    cells that is made ten passage doubling after the

     5    production level.  That is to optimize the chance or

     6    the probability that we have to pick up

     7    tumorigenicity.

     8                And, by the way of tumorigenicity, out of

     9    the number of assays that were done, I selected this

    10    table because it fits with things that were said and

    11    discussed on several occasions during this meeting.

    12                The test here consists of injecting

    13    newborn rats with various amounts of cells, one of ten

    14    million for rats, and keeping the rats

    15    immunosuppressed by injection of antithymocytic

    16    antibodies.

    17                And they are then monitored for the

    18    development of nodules at the site of injections or

    19    metastases at the draining lymph node or metastases in

    20    the lung.

    21                And what you can see, there are two

    22    messages basically.  One is that -- it's not

    23    unexpected, by the way -- but one is that the amount

    24    of cells that are injected do actually influence the

    25    results.  This is really typical here of the sort of

                                                                        300

     1    limit of positivity of metastases, this one here, and

     2    that always causes the question for the sort of

     3    arbitrary design that you can take for a test.  That

     4    was said before.

     5                And the other message is that clearly

     6    something happens.  The cells change with the level of

     7    passages, and something happened between that level of

     8    pass -- the passage that was tested and that level of

     9    passage in the sense that before the subcu. nodules

    10    were -- there were no metastases and histological --

    11    it's not historical.  It's also historical, but it was

    12    histological.  The pathologist could see here

    13    regressing signs on the lesions, on the nodules wide

    14    beyond that passage or from that passage on, they do

    15    see progressive nodules, and you do see metastasis.

    16                That's not really original.  It's been

    17    said before, but it's always interesting to see

    18    numbers.  What I showed is really the set of pre-T

    19    controller assays (phonetic) that are mandated by

    20    regulations or guidelines or points to consider. 

    21    There are specifications attached to them, and they

    22    have to pass for the banks to be qualified or for the

    23    production to be accepted for release.

    24                Now, in addition to that, a number of

    25    assays were done on the banks at various levels using

                                                                        301

     1    one of several of the techniques that are shown here

     2    to look for viruses or for viral sequences.

     3                And the next slide shows the set of

     4    viruses that were specifically searched.  Most of the

     5    QC assays are rather broad.  You go for the unknown. 

     6    In that case, those studies were looking specifically

     7    for viruses of simian region.  For obviously reasons

     8    we are dealing with simian cells and for some human

     9    viruses that, say, were causes of concern for us and

    10    for health authorities.  All of those searches were

    11    negative.

    12                I did not mention that there is another

    13    set of analyses that goes with your in-process

    14    analyses, like the monitoring, the visual inspection

    15    of the cell 12 passages day after day, et cetera. 

    16    That also brings useful information as far as the

    17    status, the cleanliness, if you wish, of the cultures.

    18                A few years ago, Florian Horaud published

    19    in 1992 his attempts or his -- his attempts to look

    20    for the expression by Northern Blotting of some of the

    21    oncogenes.  He was really trying to -- he wanted or he

    22    wished at the time to try and explain how come that

    23    those cells have, let's say, a normal profile and,

    24    however, have an indefinite life span as far as we

    25    know.  In his analyses, there was no signal that was

                                                                        302

     1    beyond the background.

     2                Now, just a few words on the way we use

     3    those cells for the manufacturing of OPV.  We choose

     4    OPV for tonight because we also make IPV, inoculated

     5    polio and rabies with the VERO cell system, but those

     6    have been described several times before, published. 

     7    So it's a bit more fun with this one.

     8                In principle it's very simple as

     9    manufacturing.  You take the VERO cells, and as was

    10    mentioned from the working cell bank, say, about 200

    11    million cells through five passages.  The cells are

    12    just trypsinized like regular culture from the beads

    13    and then plate seeded, if you wish, on the more beads. 

    14                You go in five passages to the level of

    15    use for the production.  Once the cells, the culture

    16    is established, it is seeded.  It is inoculated with

    17    one or the other of the virus types taking from the

    18    working seed that has been also controlled for being

    19    clean, what it claims, et cetera, and you just keep

    20    the culture for a couple of days until the cells are

    21    lysed and progeny viruses are released, and that

    22    constitutes the harvest.

    23                And there is a set of steps to concentrate

    24    and purify that virus.  That's a code for filtration,

    25    two steps of concentration through pettycon (phonetic)

                                                                        303

     1    and ion exchange per chromatography.

     2                The purpose of that is to concentrate so

     3    that industrially we have manageable volumes of virus

     4    suspension and purify so that we have clean material.

     5                This suspension is tested or titred for

     6    infectious titre, and based on the result of that and

     7    knowing what regulation asks to be put in the

     8    monovalent vaccine or in the trivalent vaccine, then

     9    there is dilution to adjust the potency.  It is

    10    filtered, mixed with other valences and that is the

    11    vaccine.

    12                Again, the viruses or virus suspension are

    13    subjected to a number of assays, and here, again, it's

    14    the same pattern.  There is a set of assays that are

    15    done during the process as in-process control that

    16    contain things that you will not see here because

    17    these are the ones that are asked or requested by

    18    regulation.

    19                For example, in-process control has -- we

    20    monitor the amount of DNA.  It's not requested

    21    officially, but it is monitored as a way to follow the

    22    efficacy of the purification.  We do monitor the

    23    content of protein.  We do monitor the mayput

    24    (phonetic) test, et cetera.

    25                The set of QC assays that were done, I

                                                                        304

     1    will just focus on the prediction level.  We are not

     2    really dealing with the virus itself tonight -- has a

     3    set of analyses to show that we have the right virus,

     4    what we claim we have, in terms of its polio on the

     5    right site.

     6                It is also to determine that we have the

     7    right quantity, and then there is a number of analysis

     8    to show that we have no more than what we claim. 

     9    There is no adventitious agents, and there is the two

    10    analyses, RCT4 and level virulence in monkeys.  The

    11    purpose of those analyses is to show or to be sure

    12    that throughout the manufacturing process the polio

    13    virus, which is a live attenuated virus, again, did

    14    not change in the  pattern of attenuation.   

    15                Another important feature in the

    16    development was to show that the virus, the vaccine

    17    that was produced on VERO cells was equivalent to the

    18    vaccine produced in primary monkey kidney cells, and

    19    that, again, was done through a variety of approaches,

    20    the clinical monitoring antibody references and the

    21    pattern of isolations.

    22                Just the last or the one before last, this

    23    slide is just to show the power of that technology

    24    which enables us now to produce 440 -- more than 400

    25    million monovalent doses per year.  That's the number

                                                                        305

     1    for '98.  The number for '99 will be in the same

     2    range.

     3                More important than that, at the scale

     4    that we operate, 2,000 liter scale, we have a capacity

     5    for about ten times more.  That will be impossible to

     6    run with monkey primary cells.

     7                And the last thing I would like to add is

     8    that beyond suppressing monkeys for the production,

     9    using that scale, using the scale that permeates the

    10    VERO systems reduces also the use of monkeys for QC

    11    because the neurovirulence test can be applied to much

    12    larger batches.  We need to use less monkeys on the,

    13    say, dose or median dose basis.

    14                And I think I will stop here.  Thank you

    15    for your attention.

    16                (Applause.)

    17                DR. PEDEN:  Thank you.

    18                Does anybody have a question they wish? 

    19    David?

    20                DR. CASHMAN:  Neil Cashman from Toronto.

    21                A trivial question.  Is the entire batch

    22    of vaccine per year -- does that come out of the 2,000

    23    liter production hit?

    24                MR. MEIGNIER:  One fermenter makes one

    25    batch.  That's the practical and the regulatory rule.

                                                                        306

     1                DR. CASHMAN:  How many fermentations do

     2    you do a year?

     3                MR. MEIGNIER:  In principle one a week. 

     4    One week provided you have the right set of fermenters

     5    to feed the final fermenter.

     6                DR. ONIONS:  David Onions.

     7                Bernard, incredibly impressive performance

     8    in producing those number of doses.

     9                MR. MEIGNIER:  I tell them.

    10                (Laughter.)

    11                DR. ONIONS:  But as this meeting is about,

    12    you know, concerns of perhaps new adventitious agents,

    13    could I just ask?  We now know that lots of serum

    14    batches have contained fetal calf serum batches and

    15    calf serum batches contain bovine polyoma virus, and

    16    we know that VERO cells are permissive, and there's

    17    also good serological evidence that that virus is

    18    potentially zoonotic.

    19                Do you do any testing for that virus now

    20    or what was the position?

    21                MR. MEIGNIER:  Yes.  (a)  It's the sort of

    22    multi-level process.  (a)  The calf serum is obtained

    23    from donor calves that are monitored for their health

    24    and that fermenting (phonetic), as we say, hervs or

    25    frons (phonetic) with sanitary status that is

                                                                        307

     1    monitored.

     2                These batches of serum are tested for a

     3    number of agents, BVD.  I'm not sure for you -- BVD

     4    testing.  They have a set of --

     5                DR. ONIONS:  Yeah.

     6                MR. MEIGNIER:  -- bovine viruses that they

     7    look for, and third, the calf serum is irradiated

     8    before being used.

     9                DR. ONIONS:  Okay.  Oh, sure.

    10                MR. MEIGNIER:  We hope that if you go down

    11    that way, it turns out without problem.

    12                PARTICIPANT:  Could I ask very quickly

    13    what your seed virus is grown in, and is that in

    14    monkey kidney or is that VERO cells?

    15                MR. MEIGNIER:  That's a tough one.  

    16                PARTICIPANT:  I mean the point is if

    17    you're going -- if you have a VERO cell system, you're

    18    introducing whatever is in your monkey kidney cells if

    19    that's your seed.  On the other hand, if you have a

    20    VERO cell seed, you've undergone a couple of passage

    21    levels from the Sabin strains.  So it's of interest

    22    from two points of view.

    23                MR. MEIGNIER:  I must say I do not

    24    remember.  I believe, but I'm not sure.  If you want

    25    the exact information, I'll be glad to --

                                                                        308

     1                PARTICIPANT:  Okay.

     2                MR. MEIGNIER:   -- convey the question.

     3                I believe it's done on -- it was done --

     4    new burns (phonetic) were prepared on VERO cells, but

     5    my recollection is also that they kept the same level

     6    of passages of the virus because of the concerns for

     7    changes in givens (phonetic).

     8                PARTICIPANT:  Bidion (phonetic) from

     9    Provirus.

    10                Have you tried looking to see how much of

    11    the virus which is produced by the VEROs are active?

    12                MR. MEIGNIER:  Active in terms of?

    13                PARTICIPANT:  In terms of nucleic assay

    14    and also particles.

    15                MR. MEIGNIER:  No, I don't think that was

    16    done.

    17                PARTICIPANT:  It was not, and have you

    18    tried an NCO-free medium for the production?

    19                MR. MEIGNIER:  I missed the question.

    20                PARTICIPANT:  NCO-free medium for the

    21    production?

    22                MR. MEIGNIER:  No.  The cells are grown in

    23    serum, in a medium that contains serum, and serum is

    24    removed for the virus production.

    25                PARTICIPANT:  And comparing the adduced

                                                                        309

     1    matter with like using eggs or other kind of systems

     2    for growing, how would you compared those, in vivo and

     3    in vitro?

     4                MR. MEIGNIER:  I don't think eggs were

     5    ever used for the polio production.

     6                PARTICIPANT:  Or any kind of --

     7                MR. MEIGNIER:  If you compare the sort of

     8    standard that other companies or other institutes

     9    still use are primary monkey kidney cells or human

    10    diploid cells.  Now, the comparison is clearly in the

    11    scale.  In the capacity -- the main feature of the

    12    system is that using the VERO cells of microcarriers,

    13    one can apply the ferment of the technology so that

    14    you can keep the cells because those particles are

    15    small enough; you can keep them stirring culture

    16    medium, and you can scale up relatively easily I would

    17    say, not to say that it's easy, but relatively easy,

    18    while future in retrovirals, for example, is extremely

    19    tedious, and when you want to scale up, you just have

    20    to multiply the number of bottles, the broken

    21    incubators (phonetic), the people to handle them, all

    22    by robots, but still it's tedious, cumbersome.

    23                PARTICIPANT:  So this is showing that all

    24    of the virus which is produced is active more or less?

    25                MR. MEIGNIER:  I really cannot -- I don't

                                                                        310

     1    have data to really answer that one.

     2                PARTICIPANT:  Okay.  Thank you.

     3                PARTICIPANT:  Do you have any evidence

     4    that you have an interseception (phonetic) in  oral

     5    polio vaccine?

     6                MR. MEIGNIER:  I missed the question.

     7                PARTICIPANT:  Interseception rate in

     8    children that received the polio vaccine.

     9                MR. MEIGNIER:  The immunization rate?

    10                PARTICIPANT:  No, interseception is

    11    adverse event in vaccination regard.

    12                MR. MEIGNIER:  I don't think it was looked

    13    specifically at the time.  The only thing I can say is

    14    we have, as mandated by law, there is a special

    15    department that recalls side effects that all may be

    16    related to vaccines, and despite the high number of

    17    doses that are sold and used -- I guess they were used

    18    if they were sold -- no record was made of side

    19    effects associated with those vaccines.

    20                But I don't think they ever -- there was

    21    a specific study to look to address that question.

    22                DR. MYERS:  Martin Myers, the National

    23    Vaccine Program.

    24                Relative to what we're discussing here, do

    25    you have any long-term follow-up on a cohort of

                                                                        311

     1    children specifically looking at malignancy attack

     2    rates?

     3                MR. MEIGNIER:  No, there is no follow-up.

     4                DR. EGAN:  Bill Egan from FDA.

     5                That was also my question about what the

     6    clinical safety profile was that was studied with the

     7    vaccine, sort of in keeping with this current meeting.

     8                MR. MEIGNIER:  Well, at that time the only

     9    follow-up that there is is the regular market -- post

    10    market, if you wish, surveillance and the reports that

    11    doctors may make that they found one or the other

    12    strange association.

    13                And I agree that it does not account for

    14    possible putative evidence that occur not only in

    15    people who received the vaccine ten, 20 -- ten years

    16    ago or more.

    17                DR. EGAN:  Okay.  Thank you.

    18                DR. PEDEN:  Last one.

    19                DR. MERTEN:  Merten from Genethon in

    20    Paris.

    21                When I worked in the institute in Paris,

    22    I developed  different media for different cell lines,

    23    and I want to respond to the question concerning cell

    24    free (phonetic) media.

    25                The virus that grow rather easily in cell

                                                                        312

     1    free media and stay attached as cells, NCO-free media,

     2    and so you can grow them in serum free medium  and

     3    they stay always attached and you can grow them in

     4    microcarriers.

     5                And the titres you will get out with polio

     6    virus, for instance, are more or less the same as when

     7    you use a class production process from -- from area

     8    (phonetic) for instance.  The titres are about, for

     9    the Type 1 virus, about ten to the eight P few

    10    (phonetic) per liters.

    11                DR. RUSSO:  I think we need to discuss the

    12    scale of your growth because in our experience in

    13    Merck -- I'm Carlo Russo from Merck -- the VERO cells

    14    in the serum free medium, large scale, are now growing

    15    very well.

    16                PARTICIPANT:  Okay.  This was a laboratory

    17    scale because in Pasteur Institute and in Paris we had

    18    only laboratory scales, and in the scales of 1.5

    19    liters, using five grams per liter of cytotex 1,

    20    cytotech 1, microcarrier, they attach very well, and

    21    they grew very well.  We had no problems with this.

    22                DR. PEDEN:  All right.  Thanks.

    23                Do you want to respond?

    24                MR. MEIGNIER:  The only comment is we have

    25    tried to, too, to use the medium, and the difficulty

                                                                        313

     1    still, as you know -- I really wasn't aware -- the

     2    difficult is still the passaging of the cells.  

     3                It's true that there was some good growth

     4    in cell free medium, but in the industrial scale, the

     5    kind of handling, the passaging or associating the

     6    cells and passing them still would be difficult to

     7    really master, if you wish.

     8                DR. PEDEN:  Okay.  Thank you, Dr.

     9    Meignier.

    10                So the next talk is by Girish Vyas on the

    11    proteins of replication-incompetent virions for HIV

    12    vaccination.

    13                DR. VYAS:  This is a cartoon of the famous

    14    virus, HIV.  There's a lot more known about it because

    15    our government has been spending almost $1 billion on

    16    everything that we can think of about biology, the

    17    structure of the virus.  

    18                Three things have been learned in the last

    19    ten years.  First is that this grows in unlike

    20    Hepatitis B and Hepatitis C virus -- this virus can be

    21    cultured both in cell lines, as well as in normal,

    22    peripheral blood mononuclear cells, or PBMCs.

    23                In fact, all of the structure of proteins,

    24    particularly the envelope proteins, the gp120 and

    25    gp41, these two proteins are critical in design of

                                                                        314

     1    vaccines, and the first generation of vaccines have

     2    been designed with cloned proteins which have an

     3    inherent problem of antigenic radiation that results

     4    from traditional mutations that occurred in gp120 or

     5    all replicating RN viruses have a propensity to

     6    mutate.  So that's the built in handicap with some of

     7    the recombinant vaccines.

     8                The background is that limited progress in

     9    the development of a safe and effective AIDS vaccine

    10    has been accomplished.  The structure of HIV-1

    11    envelope and some antibody neutralizing epitopes has

    12    been defined.  A successful AIDS vaccine should in

    13    this, both CTL, cytotoxic T responses and neutralizing

    14    antibody responses against primary virus isolates and

    15    not laboratory virus isolates, and finally, expanded

    16    studies of the vaccine's alarming progress in non-

    17    human primates and in Phase 1 and Phase 2 clinical

    18    trials.  These include vaccination with DNA and

    19    attenuated pox viruses which induce virus specific CTL

    20    in non-human primates, and in combination with the

    21    booster immunization with recombinant subunit vaccines

    22    which induce neutralizing antibodies against primary

    23    isolates.

    24                Again, this background some naive guy like

    25    us from blood banking field though that perhaps it may

                                                                        315

     1    be a good idea to have a perfectly human blood based

     2    vaccine as an alternative, and let me just

     3    recapitulate what are the ideal requirements for an

     4    AIDS vaccine.

     5                First is the efficacy in preventing

     6    transmission by mucosal and parenteral routes.

     7                Secondly, safety in profile with minimum

     8    risk of adverse reactions, even unscreened, real world

     9    populations.

    10                Third is long-lived protective effect of

    11    many years after successful immunization.  This is a

    12    wish list.

    13                Low cost, allowing widespread vaccination

    14    in developing countries.

    15                Stability and ease of administration,

    16    facilitating mass immunization campaigns in developing

    17    countries with minimal infrastructure.

    18                And finally, protective immunity against

    19    diverse virus isolates, preventing need for many virus

    20    isolate specific vaccines.

    21                Now, we thought it's rather simple to take

    22    advantage of the biological property of replication in

    23    competent viruses or what John Holland used to call

    24    defective, interfering particles, or DI particles. 

    25    Any RN virus each time it replicates makes 100,000

                                                                        316

     1    times perhaps more noninfectious viruses than a

     2    replication competent virion, and this replication

     3    incompetent virion or RIV is an acronym that we have

     4    given predominantly all of the replicating virions in

     5    all viral isolates.

     6                So prepared, whole virus vaccine is our

     7    approach, and triple inactivation of this primary

     8    isolates of prevalent HIV strains, pooled and expanded

     9    in vitro by full culture with peripheral blood

    10    mononuclear cells as the cell substrate, and I

    11    emphasize the word PBMC as a cell substrate.

    12                As you know, each time you get a unit of

    13    transfusion, four million American get 14 million

    14    units of blood and blood products, and each time you

    15    get a transfusion, you get basically 400 micrograms

    16    equivalent of human DNA because these cells

    17    disintegrate.  They are perfectly useless and,

    18    therefore, some time in near future blood bankers are

    19    going to be actually excluding the peripheral blood

    20    mononuclear cells from blood for transfusion.

    21                So these cells as a substrate becomes

    22    cheap, available, widely donated, and blood bankers

    23    can uniquely do this.  My own background is actually

    24    in blood banking.

    25                Secondly, instead of fetal calf serum in

                                                                        317

     1    culturing the virus, we have shown that human AV serum

     2    works equally well and, in fact, probably slightly

     3    better than fetal calf serum in HIV cultures.  So it's

     4    human blood based vaccine.

     5                The prestored leukodepletion (phonetic)

     6    makes sense because immunological and virological

     7    hazards of transfusions are minimized, and quality

     8    control systems are now in place in blood centers in

     9    the country.  Ten percent of blood donated in this

    10    country is leukodepleted.  I understand that in

    11    England, they are going to start routine depletion of

    12    leukocytes from blood for transfusion.

    13                So this is a ready resource that's

    14    available for using culturing virus.

    15                Let me just focus that, please.

    16                Okay.  Well, I want you to focus your

    17    attention on the fact that any virus isolate is

    18    composed of the replication incompetent virions or in

    19    vitro virions and the natural virions of the

    20    replication incompetent virions, and for the purpose

    21    of the vaccine, the most important things are gp120 on

    22    the surface and gp41, the trans-brain antigen.

    23                Both antigens are present in the

    24    replication incompetent virions as well as in

    25    replication competent virions.  The built in advantage

                                                                        318

     1    of making a blood based vaccine is that in a given

     2    unit of plasma, for every virus that is replication

     3    competent, there are 100,000 virions of this type that

     4    are replication incompetent, and by in vitro expansion

     5    of this virus each time that we do the expansion, we

     6    are creating more of this replication incompetent

     7    virions in vitro, and that's really the principle that

     8    we thought would be an advantage in making a blood

     9    based human vaccine or a blood based HIV vaccine.

    10                And the genetic analysis can be done with

    11    prototype relevant strains by interlooplets (phonetic)

    12    analysis, and this would be done at Uttering

    13    (phonetic) Memorial Blood Bank in San Francisco.

    14                The principles are that plasmas is

    15    procured as the primary source of HIV, negative for

    16    HCV and HBV, and the PBMCs -- I'm sorry.  I'll come to

    17    that -- plasma pooled and concentrated for viral

    18    expansion in peripheral blood mononuclear cells

    19    prepared from CMV negative blood donors and AB serum

    20    is used as a supplemental medium.

    21                So this is basically the principal

    22    components of the culture system.  The HIV is

    23    cultured, is concentrated from primary virus

    24    inactivation using a compound called Sino-wiring N

    25    (phonetic).

                                                                        319

     1                Now, blue-green algae is a bacterium which

     2    has an 11,000 Dalton protein that it makes, and this

     3    has been cloned and sequenced and large quantities are

     4    being produced by National Cancer Institute.  Its

     5    unique property is that it interacts with gp120 almost

     6    irreversibly, but it only selectively inactivates the

     7    replication incompetent -- I mean, sorry --

     8    replication competent virions.

     9                So after the expansion is achieved, the

    10    Sino virion is bound to magnetic particles used

    11    Steptoff (phonetic) in biotin.  That is, this is

    12    biotylated.  It's bound to Steptoff and encoded, and

    13    it removes the infectious virions so that the leftover

    14    virions, which are lot of viral protein, is actually

    15    culture negative in vitro.

    16                This work has been presented.  Some of you

    17    who may have been at the meeting that we organized in

    18    San Francisco in March on blood safety know this data

    19    that were presented there.

    20                The secondary inactivation is specific for

    21    inactivating residual RNA that is still present in

    22    some of the replication incompetent virions, and this

    23    can be achieved by nucleophilic means or with dyes,

    24    such as dimethyl methylene blue or psorlens.  Psorlens

    25    is now in advanced area clinical files, and perhaps a

                                                                        320

     1    preferred compound that does not bind to any proteins,

     2    but specifically binds to the RNA.

     3                And then finally, the purification of the

     4    inactivated virus from all the components in the

     5    culture system, as well as the inactivating chemicals

     6    is achieved by molecular C ring (phonetic).

     7                After this is put into final containers

     8    for terminal viral inactivation, the pressure cycling

     9    technology, this is basically a hydrostatic pressure

    10    that is repeatedly applied to the virions in the final

    11    container that disrupts the virions and disintegrates

    12    into nonprevalent bond proteins.

    13                So basically that's where the word or the

    14    title comes, that HIV proteins are produced through

    15    this pressure cycling technology.

    16                After that we put the adjuvant and the

    17    immune response tested in mice shows that such

    18    antigens are quite immunogenic.

    19                Safety and efficacy testing is going to be

    20    done in chimpanzees, and finally this is what I say we

    21    are going to be doing.  CGMP facilities for

    22    manufacture of the RIV vaccine, as we call it, in an

    23    FDA approved facility, and finally Phase 1 and Phase

    24    2 clinical trials in humans are planned.

    25                And finally, I want to show the

                                                                        321

     1    investigators at UC-SF are myself; Cliff Lawless,

     2    Chief of Immunology; Manish Gandhi is a pathologist

     3    and transfusion medicine specialist at our place. 

     4                Mike Boyd at NCI is the one who cloned and

     5    provides the Sino virion N.

     6                Mark Manak at the BBA Biotech is the one

     7    who has the pressure cycling technology for

     8    inactivation of the final product.

     9                Mike Bush and Eric Diewart were both

    10    responsible for purifying for genetic analysis of the

    11    pool against standard strains of HIV.

    12                And finally, the safety testing in

    13    chimpanzees will be done in Southwest Foundation by

    14    Chris Mouffy (phonetic).

    15                And finally, I want you to make any

    16    thoughtful suggestions you can make so that this

    17    project has the maximum benefit of peer input.

    18                Thank you.

    19                (Applause.)

    20                DR. PEDEN:  Thank you for keeping on time.

    21                Are there any questions?

    22                PARTICIPANT:  Can you say something about

    23    where you HIV is actually going to come from Girish? 

    24    I mean, it seems to me that the blood banking people

    25    have spent the last 15 to 20 years getting rid of

                                                                        322

     1    intravenous drug abusers and HIV infected gays, and

     2    you're going to be out there hunting for them again;

     3    is that right?

     4                (Laughter.)

     5                PARTICIPANT:  I mean, are they saying

     6    thank you to you for this?

     7                DR. VYAS:  Well, actually we have more

     8    than adequate resource of plasma that is HIV positive.

     9                Yes?

    10                DR. BROKER:  In the protocol, you had

    11    mentioned possibly putting psorlens into the

    12    preparation near the end; is that correct?

    13                DR. VYAS:  That's correct.

    14                DR. BROKER:  That's a potent mutagen.  It

    15    seems like you're kind of going backwards on this in

    16    terms of safety.

    17                DR. VYAS:  Well, there are three

    18    alternatives, and any one of the three, which is RNS

    19    specific inactivation otherwise.  That is, there is

    20    RNA there.  Nobody would simply accept final virion as

    21    a primary inactivating agent as adequate.  Certainly

    22    my colleagues at FDA would not approve of it.

    23                So we wanted to be sure that we include

    24    one step which is specific for nucleic acid and does

    25    not in any chemical way alter the proteins, and either

                                                                        323

     1    it can be a means that has been used for foot-mouth

     2    disease, virus, for many, many years or it could be

     3    psorlens.

     4                Now, we have not completely made a

     5    decision as to which of the three alternatives we are

     6    going to go, but putting inactivation of HIV has been

     7    successfully done with psorlens, and then in the final

     8    stage, they are able to remove the free chemicals to

     9    molecular series.

    10                DR. BROKER:  Yeah, I just would point out

    11    that there have been some adverse effects in using

    12    psorlens in dermatology for psoriasis.  It tends to be

    13    used only in the extreme.  It's an interpolating dye,

    14    and if it for any reason passes through to the

    15    patient, I'd be quite concerned.

    16                DR. VYAS:  Your point is well taken.  In

    17    fact, the amount of psorlens that's found to the

    18    residual nucleic acid is so exquisitely small that it

    19    has no adverse effect so far, in fact, much less than

    20    a unit of blood.

    21                The clinical trials for virally

    22    inactivated blood is in Phase 2 and Phase 3 clinical

    23    trials, and those trials have not had any adverse

    24    reactions as far as I know.  So I'm not discouraged by

    25    all the points that you made.  We are aware of it, and

                                                                        324

     1    I think that since we are removing the chemical

     2    inactivating agents, we probably are safe.

     3                And in the amount that is there in the

     4    residual inactivation, it's probably trivial.

     5                DR. BROKER:  Okay.  Thank you.

     6                DR. VYAS:  Sure.

     7                DR. PEDEN:  That question was by Tom

     8    Broker.

     9                Please give your name when you ask the

    10    question.  In the back.

    11                DR. RUSSO:  Carlo Russo from Merck.

    12                How stable is your inactivation?  How

    13    stable do you think it is going to be?

    14                And you know, in the context of this

    15    meeting where there is lots of concern in the way we

    16    validate the product that we're going to use for a

    17    vaccine, how are you going to validate your release

    18    assay for your preparation?

    19                DR. VYAS:  I'll come to Merck for doing

    20    that work.

    21                (Laughter.)

    22                DR. RUSSO:  That's not a bad idea.  We

    23    have a terrific validation procedures, but I would

    24    like to have an announcement from you.

    25                DR. VYAS:  I just want to mention that the

                                                                        325

     1    primary work of this nature has been done in my lab. 

     2    The major NIH support for this study is impending, and

     3    if that gets approved, then the R&D work will be done

     4    at the University of California and BBA Biotech.

     5                The validation issues and the manufacture

     6    issues are tertiary, and we have not yet come to grips

     7    with those issues yet.

     8                DR. LEWIS:  Yeah, Lewis, FDA.

     9                Have you given any possible thought to the

    10    use of human stem cells as a source of substrate?  It

    11    might be less of a problem.  You might be able to

    12    generate them in large volumes and from a very limited

    13    source that would be a more amenable testing than

    14    would be pooled human peripheral blood monocells --

    15    mononuclear cells.

    16                DR. VYAS:  Our major consideration was

    17    actually having the Third World countries' ability to

    18    make this vaccine.  I think the stem cell biology and

    19    propagation in vitro is still not available in the

    20    majority of Third World countries.

    21                So leukofiltration is one way of getting

    22    rid of leukocytes, and we have actually been able to

    23    get leukocytes out of the filters and been able to

    24    culture them and grow HIV into that.  So that's a

    25    first step.

                                                                        326

     1                The point out stem cells is a very good

     2    point, and I think ultimately that would be one of the

     3    ways that we want to go about it.

     4                DR. JOHN LEWIS:  Lewis from Merck.

     5                I'm curious to know if you know what the

     6    physical product of your high pressure dissociation is

     7    and whether you have quality control assays that would

     8    assure the consistent manufacture of that.

     9                DR. VYAS:  Unfortunately you were not in

    10    San Francisco in March when this work was presented by

    11    the BBI people, and they have the protect technology

    12    and the quality control in place for pressure cycling

    13    technology and validation that introduces six logs of

    14    HIV infectivity.

    15                As such, after the primary inactivation,

    16    in vitro we cannot demonstrate any infectivity in the

    17    vaccine.  The second inactivation with psorlens or MEs

    18    is an added, and I call it tripolene (phonetic)

    19    activated with special cycling technology.

    20                But the immunogenicity is the main

    21    criterion we are using in the animal model, and as

    22    long as it is immunogenic and it produces neutralizing

    23    antibodies, those are our standard criteria for useful

    24    product.

    25                DR. PEDEN:  Okay.  Thanks.  We have to

                                                                        327

     1    move on.

     2                The next talk is by Alex van der Eb,

     3    unusual response to apoptin of diploid fibroblasts

     4    from cancer prone syndromes.

     5                DR. VAN DER EB:  I would like to talk

     6    about apoptosis producing protein that we call

     7    apoptin, and which some of you may have heard, and the

     8    unusual effects of apoptin on diploid cells of certain

     9    cancer prone individuals.

    10                However, before I do that, I have to

    11    summarize some of the properties of apoptin, and if I

    12    can have the first slide, please, apoptin  is a

    13    protein of the chicken virus, chicken anemia virus. 

    14    The chicken anemia virus is a small avian pathogen

    15    that causes a lot of economic damage in the poultry

    16    industry.  It causes clinical signs in young chickens,

    17    although older chickens are immune for the virus.

    18                Anemia, it causes anemia, and by

    19    destruction of the erythroblastoid cells,

    20    immunodeficiency by depletion of the thymocytes.

    21                And in the early stages of the work, which

    22    was actually intended to try and make a vaccine

    23    against the virus, we found that CAV causes these

    24    disease symptoms by inducing apoptosis.

    25                In order to clone the virus in the final

                                                                        328

     1    DNA was a circular, single stranded genome which

     2    encodes a single MRNA, and on this MRNA there are

     3    three open reading frames, coding for what we call

     4    VP1, VP2, and VP3.

     5                So we asked the question:  which of these

     6    three open reading frames causes the apoptosis?  And

     7    we transfected cells with each of the open reading

     8    frames and found that it was VP3.  So the smallest of

     9    the three proteins causes apoptosis.

    10                So how does VP3 causae apoptosis?  There

    11    are many pathways of apoptosis, and the next slide

    12    shows you the major pathway that is mediated through

    13    P53, when P53 is properly activated will cause a

    14    signal that causes activation of the CAV spaces.  IS

    15    is one of the CAV spaces, and that will then

    16    eventually lead to apoptosis.

    17                There are several proteins that regulate

    18    this process, like VCL2 and BAG1, which are negative

    19    regulators.

    20                So we asked the question which does

    21    apoptin, as we call this protein, that is encoded by

    22    VP3 -- does that need VP3?  Is it inhibited by VCL2? 

    23    Does it need half spaces for apoptosis induction?

    24                And the answer was initially rather

    25    surprising.  P53 is not needed.  Also cells without

                                                                        329

     1    P53 go in apoptosis, VCL2 and BAG 1, which are strong

     2    anti-apoptotic proteins, do not inhibit apoptin

     3    induced apoptosis, and a number of CAV space

     4    inhibitors, including CrmA, did not prevent apoptosis

     5    induction by apoptin.

     6                So that was surprising and indicated that

     7    apoptin had an apoptosis pathway of its own.  However,

     8    recently we found that the P35 gene, an anti-apoptotic

     9    gene or maculovirus and which is an inhibitor of CAV

    10    spaces, of the downstream CAV space also inhibit

    11    apoptin, indicating that apoptin also activates the

    12    CAV spaces.

    13                When we did these studies, we realize that

    14    what we ha tested so far, what we had tested in

    15    initial studies was a number of cell lines that all

    16    went in apoptosis, and all of these cell lines were

    17    actually neoplastic cells.

    18                And so what about the non-neoplastic,

    19    normal, diploid cells?  And then came a surprise.  It

    20    turned out that the normal cells, diploid, fibroblast

    21    endothelial cells, T cells, smooth muscle cells, et

    22    cetera do not go in apoptosis.  They are completely

    23    resistant.

    24                That was a surprise, and why would apoptin

    25    not cause apoptosis?  Well, it turns out to be the

                                                                        330

     1    localization probably of the protein.

     2                The next slide shows you a tumor cell, an

     3    osteosarcoma cell which is transfected two days ago

     4    with apoptin, and you can see here the protein, the

     5    fluorescence of the protein in the nucleus, and it is

     6    just present in the nucleus throughout the nucleus,

     7    and the DNA staining of the same cell shows that

     8    everything is still normal.

     9                However, the days after this second day,

    10    an increasing number of cells appeared that have a

    11    condensed and fragmented fluorescence as you can see

    12    here, and if you look at the DNA staining, you can

    13    also see that it is abnormal, and this cell is an

    14    apoptotic cell.

    15                So this happens in cancer cells.  So what

    16    about the normal diploid cells?  Well, it turned out

    17    that the apoptin is not present in the nucleus, but

    18    stays in the cytoplasm, and even if you looked many

    19    days after transfection, it still stays in the

    20    cytoplasm.

    21                They even have transgenic mice now that

    22    express apoptin in a number of tissues and everything

    23    seems normal.  So it is not toxic for normal cells.

    24                Now, what would happen if you transformed

    25    these cells with SV 40, for example?  We have the same

                                                                        331

     1    cells formed by SV 40, and then we are completely

     2    sensitive to apoptin.  So what would happen if you co-

     3    transfect normal diploid cells now with SV 40, the

     4    antigen and transforming gene that completely

     5    transforms the cell?

     6                And then it turns out that these cells

     7    become sensitive.  So here is the diploid VH10

     8    fibroblasts that are co-transfected with apoptin and

     9    the SV 40 P antigen, and here you see that the cells

    10    that were transfected with apoptin 3T3 alone and

    11    neofactor did not go in apoptosis, whereas cells that

    12    were transfected with VP3 and actually 4T, large T,

    13    here went in apoptosis and will eventually reach the

    14    100 percent if you wait long enough.

    15                So this means that also a transient

    16    expression of an oncogene can cause sensitization of

    17    normal diploid cells to apoptin.

    18                The next question that we asked was:  what

    19    would happen if you treat normal diploid cells with a

    20    carcinogenic agent?  And since we have an irradiation

    21    program in the lab, we irradiated with ultraviolet

    22    light or with actually ionizing radiation, and we

    23    found that if you do that, the diploid cells still

    24    remain completely insensitive to apoptin, and this is

    25    actually what was expected because a single radiation

                                                                        332

     1    exposure will not transform the cells, of course, in

     2    a single step, and maybe you induce a few mutations

     3    and no more than that.

     4                So up until recently we have found a

     5    category of diploid cells from cancer prone patients

     6    that do react with apoptin, and before I go into that

     7    part, I have to briefly introduce something about

     8    irradiation or radiation program or radiation

     9    research.

    10                Oh, incidentally, this is what happens if

    11    you transfect with T antigen a normal cell, T antigen,

    12    and apoptin, and this is two days after transfection. 

    13    In the normal cells after one day the apoptin is still

    14    present in the cytoplasm, but after two days -- and

    15    this is after two days -- there is a kind of a

    16    transfection.  You see that it moves into the nucleus. 

    17    So it is actually the movement of the protein, the

    18    transport of protein to the nucleus that may be

    19    essential, and one day later, everything is in the

    20    nucleus.

    21                And now radiation.  It is, as you probably

    22    know, that radiation of cells, particularly UV

    23    irradiation, causes a large number of responses, most

    24    of which are transient, such as activation of plasma

    25    membrane, activation of genes that are normally

                                                                        333

     1    induced by growth factors, secretion of growth factor,

     2    stabilization of P53, and so on and so on.

     3                Also, ultraviolet light induces so-called

     4    SOS-like phenomena, such as a reactivation, an aus

     5    (phonetic) mutagenesis, ER and EM.  They are called

     6    like that because these phenomena they resemble, the

     7    SOS responses that were first described in E. coli in

     8    response to irradiation.

     9                Now, we asked the question, and this is

    10    several years ago:  do these, in particular, EM

    11    somehow contribute to carcinogenesis?  And we decided

    12    to try and study that.

    13                First I would like to show you what ER and

    14    EM are.  ER, an ounce (phonetic) reactivation, and EM

    15    are both measured with viruses.  They are either

    16    measured with DNA viruses, like HSV, or actually for

    17    virus and ER, an ounce (phonetic) of reactivation, is

    18    the phenomenon that your re-irradiated virus survives

    19    better in your re-irradiated cells than in

    20    unirradiated cells.  In fact, it survives about two

    21    times better.  And that means that ER by definition is

    22    two.  

    23                EM also makes use of the virus, but in

    24    this case the virus is not irradiated, and if you

    25    allow it to replicate this unirradiated virus and you

                                                                        334

     1    re-irradiate it or unirradiated cells, it turns out

     2    that the mutations that are accumulated in the virus

     3    are about two to three times more higher in your re-

     4    irradiated cells, indicating that your re-irradiated

     5    cells have a kind of mutator activity, and that is

     6    about two or a little more than two.

     7                The effect is transient, and here you see

     8    ER, an ounce reactivation, in a cell.  It returns

     9    after a few days going back to one, and EM also

    10    returns to one, and they have a maximum expression of

    11    one day after irradiation.

    12                So do any of these phenomena play a role

    13    in carcinogenesis?  So we decided to measure ER and EM

    14    in cells and diploid cells from cancer prone

    15    individuals, and we have measured two different cancer

    16    prone individuals, and in one of which we saw

    17    abnormalities, and not with EM, as we expected, but it

    18    was with ER.

    19                And ER turned out to be extremely high in

    20    diploid fibroblasts from individuals who are cancer

    21    prone as a result of a germ line mutation in a tumor

    22    suppressor gene, and here you see, for example,

    23    Aniridia cells from an Aniridia patient, just normal

    24    diploid cells who have a mutation in one of the

    25    Willems tumor genes, and as you can see here, the ER

                                                                        335

     1    is extremely high.

     2                Now, this is an only single point, but you

     3    can believe me that this is reproducible, and the

     4    correct slide with more points in the curve I couldn't

     5    find, and it's always disappeared in the lab.

     6                So surprisingly this was ER and not EM

     7    that was correlating with cancer prone.  So we then --

     8    this is a list of the syndromes from which the diploid

     9    fibroblast show the extremely high ER phenomenon, ER

    10    super plus, as we call it.  Retinal blastoma, Li

    11    Fraumeni, Euro Frondromitosis (phonetic), Willems

    12    tumor, et cetera, et cetera, et cetera, and we have

    13    measured now more, and all of them show this

    14    abnormally high ER phenomenon.

    15                And then we decided to test these cells

    16    also for their sensitivity to apoptin without and with

    17    radiation, and we have -- and this is quite recent --

    18    we have three of these cell strains in culture:  Li

    19    Fraumeni cells that have one inactive LEU (phonetic)

    20    and one wild type LEU, and they are ER super plus.

    21                A Lynch Type 2 syndrome familiar in breast

    22    and ovarian cancer, ER super plus.  The gene is in

    23    this case, as far as I know, still unknown.

    24                And there was a dysplastic Nevus syndrome,

    25    a familiar melanoma, which is also ER super plus and

                                                                        336

     1    which has mutated P16.

     2                Each of these cells, these two cell lines,

     3    we have two different individuals from the same

     4    family, and for the Li Fraumeni syndrome, we had one

     5    patient who was affected and one individual from the

     6    family who had two wild type alleles.

     7                The first experiment already showed that

     8    the normal irradiated fibroblasts are completely

     9    resistant to apoptin.  This was actually not

    10    unexpected, of course, but after irradiation with

    11    ultraviolet light, we found quite a surprise, and that

    12    was that these cells become completely sensitized to

    13    apoptin just as tumor cells.

    14                Here you see the reaction of the PH10

    15    normal diploid cells, which remained resistant to

    16    apoptin, and they remain so much longer, as long as

    17    you can keep the cells alive.

    18                However, this is a Li Fraumeni cell, and

    19    that is the Lynch Type 2 syndrome, ovarian/breast

    20    cancer syndrome.  They become completely sensitive

    21    within four to five days.

    22                And the next slide shows the more complete

    23    study of all the cells together, and in the left panel

    24    you see the cancer prone syndromes here, fibroblasts

    25    from the cancer prone syndromes, and here first the

                                                                        337

     1    cells, the normal cells, which have nothing special or

     2    the Li Fraumeni patient, the Li Fraumeni individual

     3    who had two wild type, family member who had two wild

     4    type P53 molecules, genes.

     5                You can see that these remain completely

     6    resistant to apoptin after re-irradiation, whereas the

     7    other ones go completely in apoptosis as if they are

     8    cancer cells.

     9                In contrast, if you transfect and you re-

    10    irradiate the cells with a desmid control gene instead

    11    of apoptin, then all of the cells remain completely

    12    resistant and do not go into apoptosis, indicating

    13    that the effect is specific for the 3T3 or apoptin

    14    gene.

    15                The next slide shows just a dose response

    16    and where you can see that at ten Joules you already

    17    get the maximum reaction of the Li Fraumeni cells in

    18    this case, whereas at five Joules there is still

    19    nothing.

    20                If you transfect with desmid, again,

    21    nothing happens, and if PH10 normal cells, even at 25

    22    Joules, show no reaction whatsoever compared to

    23    transfection with desmid.

    24                And then the last slide shows you here

    25    that the effect on these cells from cancer prone

                                                                        338

     1    individuals is transient, like many of the other

     2    injury induced phenomena.  It completely disappears

     3    again after four to five days and returns to zero, to

     4    the same level as the  PH10 control cells.

     5                So in conclusion, the diploid cells of

     6    these cancer prone syndromes that have one allele of

     7    a tumor suppressor gene have two abnormal properties

     8    and have lost one allele tumor suppressor gene, have

     9    two abnormal properties.

    10                First, they show very high ER response,

    11    and, second, they express what are also found in

    12    cancer cells and which render them susceptible to

    13    apoptin, which is a cancer specific protein.

    14                And essentially it means that apoptin is

    15    allowed to be transported to the nucleus, and both

    16    properties, irrespective of the tumor suppressor gene

    17    because it's found in many different tumor suppressor

    18    genes now also irrespective of the type of

    19    irradiation.

    20                We are trying now to identify what is

    21    induced by radiation in these cells and that is

    22    different from what is caused by irradiation in normal

    23    cells.

    24                (Applause.)

    25                DR. PEDEN:  Thank you.  

                                                                        339

     1                And we have time for a couple of

     2    questions.

     3                DR. FRIED:  Did you say you had a

     4    transgenic with --

     5                DR. VAN DER EB:  Yes.

     6                DR. FRIED:  Does it get cancer?

     7                DR. VAN DER EB:  This is something that we

     8    still have to study.  The transgenic mice exist, and

     9    it is expressed particularly in the lymph nodes and

    10    other lymph organs, and what we have to do is see what

    11    happens if you, for example, try to induce lymphoma by

    12    leukemia viruses.  They should be resistant.

    13                PARTICIPANT:  That was Mike Fried.

    14                Could you give your name, please?

    15                DR. CASHMAN:  Neil Cashman.

    16                You've adroitly avoided any mention of the

    17    sequence of this gene.

    18                DR. VAN DER EB:  Right.

    19                DR. CASHMAN:  Can you comment at all?

    20                DR. VAN DER EB:  Yes.

    21                DR. CASHMAN:  Are you free to comment?

    22                DR. VAN DER EB:  I have the slide with me,

    23    but of the protein sequence of the gene.  It's the

    24    small protein, 120 amino acids.  It does not resemble

    25    any other known protein in the cell bank.  It has two

                                                                        340

     1    nuclear localization signals, in the right-hand C

     2    terminal part, and it has a nuclear export signal in

     3    the internal part, and that's about it.  There's

     4    nothing else, no.

     5                I have it.  It's published also.

     6                DR. COFFIN:  John Coffin.

     7                Ordinarily one thinks of induction of

     8    apoptosis as an antiviral mechanism.

     9                DR. VAN DER EB:  Yeah.

    10                DR. COFFIN:  Can you speculate on why it

    11    might be of value to the virus to do such a thing?

    12                DR. VAN DER EB:  I have no idea.  This is

    13    a good question.

    14                One possibility is that this virus is a

    15    very simple virus.  It has only three genes, and it

    16    has no other means to get out of the cell, and then

    17    the best way to get out of the cell or to infect other

    18    cells is to induce apoptosis.  Then the cell fragments

    19    and is taken up by other cells, and maybe that's the

    20    way it's expressed.  I don't know.  No, I don't know.

    21                DR. PEDEN:  Thank you, Alex.

    22                The next speaker is Tom Broker from UAB,

    23    and his title is "Human Papillomaviruses:  a Window

    24    into Eucaryotic Cellular DNA Replication Mechanisms

    25    and Regulation."

                                                                        341

     1                DR. BROKER:  Okay.  Thank you very much.

     2                I'd like to share with you several things

     3    regarding  papilloma virus DNA replication, and what

     4    I'd like to do is start by very briefly  reviewing two

     5    aspects of HPV biology.

     6                The first is that the virus infects a

     7    squamous epithelium through a wound allowing direct

     8    access of mature virus particles from the environment

     9    to the germinal layers of the skin, typically the

    10    basal cells or the parabasal cells.

    11                The virus would be harbored in these cells

    12    and maintained more or less indefinitely by

    13    replicating in synchrony with the normal cell cycle,

    14    and it would be latent in that capacity in most cases.

    15                Occasionally induction of viral early, as

    16    well as late gene expression can commence at about the

    17    third layer up where it becomes so the so-called

    18    spinus epithelium with the delayed early, late

    19    transcription, viral DNA replication occurring here,

    20    and assembly into mature virus particles, and then

    21    naturally a release through desquamation of the

    22    cornified envelopes.

    23                Now, periodically the genes that normally

    24    affect the onset of reentry into S phase can

    25    inadvertently become expressed in cells that have not

                                                                        342

     1    yet withdrawn from the cell cycle, and that can lead

     2    to dysplasias, and through an accumulation of cellular

     3    mutations and probably some viral mutations, you can

     4    progress over a period of years toward higher grade

     5    dysplasias and carcinoma in situ.

     6                I want to emphasize that there are several

     7    modes of replication of HPVs.  They're in the wound

     8    healing phase when there is literally a stripped off

     9    epithelium.  There is a re-epithelialization across

    10    the wound bed that originally allowed access in the

    11    virus to these layers, and at that time there's a

    12    transient up regulation of viral oncogenes E6 and E7.

    13                However, once the full thickness skin has

    14    been reestablished, E6 and E7 transcription stops at

    15    this level, and I'll show you that, and the E6 and E7

    16    oncogenes only are transcribed from this layer up in

    17    a full thickness epithelium.

    18                So we have establishment replication

    19    during the first phases of wound entry; secondly,

    20    maintenance replication here; and third, vegetative

    21    replication there higher up.

    22                This is the HPV18 genome.  It's pretty

    23    typical of most viruses.  The proteins that I'll be

    24    discussing today are the origin binding protein E1 and

    25    E2.  What you'll also see is E2 plays a role in

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     1    transcriptional regulation at the promoter right

     2    there, and that the E1 is the only enzyme encoded by

     3    this virus and is the preferred target, therefore, for

     4    drug discovery.  It is a helicase with some remarkable

     5    properties.

     6                It together as a multimer binds to the E6

     7    promoter region, which is synonymous with the

     8    replication origin right here.  E7 protein, which I'll

     9    talk about a bit today and a bit tomorrow, interacts

    10    with the retinoblastoma protein and triggers S phase

    11    entry, and as many of you know, the E6 protein

    12    interacts with P53 and blunts some of the repair

    13    responses and also can blunt movement toward

    14    apoptosis, allowing the virus time to replicate.

    15                Now, in a normal, full thickness, squamous

    16    epithelium, in this case cervix, without any viral

    17    infection, I want to point out that the only DNA

    18    replication that the normal cells are engaged in are

    19    the parabasal or transid amplifying cells, as marked

    20    by PCNA stain.

    21                All cells above the parabasal layer do not

    22    have the oxyribonucleocide triphosphates, DNA

    23    polymerase, topoisomerases, PCNA, or any of the other

    24    replicative enzymes.  Yet the virus replicates up

    25    here, and that is the dilemma the viruses have to

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     1    face.  How do they reactivate S phase entry and the

     2    reinduction of all the replication machinery to

     3    support their own replication?

     4                This is one piece of evidence from a

     5    natural laryngeal papilloma virus from a child in dark

     6    field illumination, and the probe we used was 40-67

     7    transcripts, and as you can see, they're basically

     8    excluded from the basal cells.  This is a dermal read,

     9    and the signals are in the spinus layer of the

    10    epithelium for E6 and E7 once the wound healing has

    11    been complete.

    12                Okay.  Now, I very briefly want to remind

    13    you of the normal cell cycle and that it is controlled

    14    by the retinoblastoma protein being bound to the E2F

    15    enhancer protein, which itself is in complex with a

    16    cofactor called the DP1 or DP family of E2F cofactors.

    17                That would be the normal state in the G1

    18    phase or G0.  However, upon mitogenic signals, cyclin

    19    dependent kinases, primarily cyclin D and DCK4, can

    20    phosphorylate RB, causing it to be released from E2F,

    21    and then E2F DP1 complexes can up regulate the

    22    transcription of the replication enzymes and certain

    23    cell cycle progression proteins like cyclin E.

    24                Conversely, the papilloma viral E7 protein

    25    can disrupt this association in the absence of the

                                                                        345

     1    kinase activity, and therefore, the virus can trigger

     2    the up regulation of the necessary enzymes for its own

     3    synthesis.

     4                And this is shown here in a couple of

     5    examples.  This is a genital condyloma, and I want you

     6    to see in this cross-section of the papilloma that,

     7    again, the basal cells do not have PCNA.  The

     8    parabasal cells do.  Then the lower spinus cells do

     9    not have PCNA, and the upper spinus cells, once again,

    10    do have PCNA.

    11                So you have this natural cell cyclin here,

    12    and the viral induced S phase reentry toward the upper

    13    layers of the epithelium.

    14                This phenomenon is also seen with other

    15    important enzymes, in this case DNA polymerase alpha

    16    in the natural laryngeal papilloma; again the tritium

    17    or S35 silver grains are predominant in the upper

    18    layers of the skin, and I want to particularly

    19    emphasize that there's a massive over expression of

    20    the polymerase, as there is with PCNA, in the upper

    21    layers.  That will become particularly important. 

    22    It's something I'll have to say probably in tomorrow

    23    morning's talk.

    24                Now, the second thing Andy asked me to

    25    comment on is some of the model systems that we have

                                                                        346

     1    for investigating in an experimental way.  This is the

     2    epithelial raft culture, and very briefly, one makes

     3    a collagen matrix, imbeds dermal fibroblast in this,

     4    and then you can take either dispersed keratinocytes,

     5    primary human keratinocytes or established

     6    immortalized keratinocytes, and spread them on the

     7    surface, and at that point you can raise the entire

     8    thing up into the air.

     9                Think about your own skin.  You have an

    10    air surface, and you have a blood surface, and when

    11    you do this, the exposure to air sets up a capillary

    12    action, calcium gradients and other gradients of

    13    probably mytogens, and so forth, and you quickly

    14    develop a stratified and differentiated squamous

    15    epithelium.

    16                To show that this actually can work not

    17    only with disbursed cells, but with punch biopsies,

    18    we've been able to take skin from any anatomical site

    19    in the body, place it on the raft, which is right

    20    there.  This is a punch biopsy from a excess cervical

    21    epithelium, and these cells will grow out across the

    22    collagen surface and very rapidly stratify and

    23    differentiate appropriate to the tissue of origin.

    24                You can do the same thing with papilloma

    25    itself, and you always recapitulate the type of skin

                                                                        347

     1    you started with.  In this case, the natural laryngeal

     2    papilloma will grown out and become an in vitro

     3    papilloma.

     4                The third thing we've been able to do is

     5    introduce papilloma virus genes or reporter genes into

     6    the keratinocytes through retrovirus mediated gene

     7    transfer process.  Denise Galloway and Jim McDougall

     8    did this in which they placed the papilloma virus

     9    E6/E7 oncogenes directly under the control of the

    10    upstream LTR.

    11                With Louise Chan in my laboratory, we

    12    chose to place the reporter gene or the E6 or E7 genes

    13    under the control of their own promoter at a rather

    14    downstream location, in which case we were able to

    15    very exactly recapitulate the differentiation

    16    dependent express of the E6 promoter.

    17                Parenthetically I would note because it

    18    reflects a change in thinking, people had always felt

    19    that the loss of an E2 gene upon HPV integration would

    20    invariably lead to the up regulation of the E6

    21    promoter, which it can repress in basal cells.  This

    22    construct has no E2 gene in it, and indeed, the E6

    23    promoter driving LAX-E (phonetic) in this case is only

    24    in the upper layers where we expected it to be.

    25                So E2 has absolutely nothing to do with

                                                                        348

     1    the repression of the E6 promoter in these types of

     2    cells, unless other cellular changes have occurred,

     3    which in this case they're PHKs, and they haven't

     4    engaged in those mutations yet.

     5                To show that this system works and that we

     6    can do genetic dissection of HPV, we have an empty

     7    vector here.  We have an E7 expressing vector here,

     8    and as you saw in the natural papillomas, polymerase

     9    alpha is up regulated; PCNA is up regulated; and

    10    bromodeoxyurasyl incorporation, all occur in the upper

    11    compartment of the full thickness skin, which

    12    developed in just one week after the viral infection

    13    and lifting of the rafted air surface.

    14                And notice again the basal cells tend to

    15    have low grade expression of these induced genes. 

    16    They're highly up regulated in the differentiated

    17    compartment.

    18                What we noticed upon doing a variety of

    19    analyses is that while many of the cells do have up

    20    regulated PCNA, only a subset of them are capable of

    21    incorporating bromodeoxyurasyl in response to these up

    22    regulated genes.  This will become important for

    23    tomorrow's talk, but notice that we always compare a

    24    natural papilloma with an E7 raft culture, and only a

    25    subset of the superficial cells become competent for

                                                                        349

     1    DNA replication.  In other words, all warts, as well

     2    as rafts, are a mosaic of cells that are capable of

     3    engaging in DNA synthesis and S phase entry, and

     4    others which appear to be inhibited for reasons I'll

     5    discuss tomorrow.

     6                Now, I want to give the bottom line of

     7    what we now know about the assembly of the replication

     8    complex so you know where I'm going with several of

     9    the data slides.

    10                Very briefly, the viral E2 protein is the

    11    main ORI binding protein, and as I'll show you, it

    12    associates with the nuclear matrix.  It then recruits

    13    the viral E1 protein, the helicase, and one of the

    14    most remarkable observations that we've made recently

    15    in collaboration with Doug Seer and Jack Griffith is

    16    that the assembly of this complex depends on heat

    17    shock protein 70 and heat shock protein 40, and in

    18    fact, heat shock protein 40, the co-chaperon for

    19    HSP70, in fact, remains a permanent component of the

    20    active replication complex.  We presume it's true also

    21    in general eucaryotic replication, but it certainly is

    22    with the virus.

    23                The E1 protein, in turn, can recruit DNA

    24    polymerase alpha, and when that assembly has occurred,

    25    cyclin E, CDK2 moves in, and as you'll see

                                                                        350

     1    phosphorylates this complex.

     2                I would like to show you some of the data. 

     3    Very briefly, the E1 protein has a series of domains. 

     4    At this end is an ATPase, which is essential to the

     5    helicase action.  There's a DNA binding motif, and as

     6    you'll see, very, very critical, a cyclin E binding

     7    motif right here which has great bearing on our

     8    understanding of Hela cells.

     9                The E2 protein has three basic domains,

    10    the DNA biding domain, a hinge, and a transacting

    11    domain.  As I'll show you in a moment, the hinge

    12    anchors this entire complex to the nuclear matrix.

    13                What we did is put green fluorescent

    14    protein tags on each of the domains of the E2 protein,

    15    full length protein and the various combinations of

    16    one or two portions of the E2 protein.  We were able

    17    to show function was retained despite the GFP tag, and

    18    bottom line is the hinge unexpectedly was the thing

    19    that targeted the E2 protein to the nucleus.

    20                If you didn't have the hinge domain, as in

    21    this column, the proteins remain cytoplasmic.  If we

    22    did have the hinge, they got nuclear.  If we had the

    23    N terminal domain and the hinge together, we began to

    24    see nuclear foci.  If we had the whole protein, the E2

    25    protein went to the pods or nuclear domain 10s of this

                                                                        351

     1    protein.

     2                And we were able to identify the anchor to

     3    the nuclear matrix as a basic motif in the center of

     4    the hinge that's highly conserved among all papilloma

     5    viruses.  If we mutated that domain, we got a

     6    cytoplasmic distribution.  If it was wild type, it

     7    went to the nuclear foci.

     8                And this is the model then.  The hinge

     9    anchors the E2 protein to the matrix.  The C terminal

    10    domain binds to the viral DNA.  The N terminal domain

    11    associates with the matrix and also helps recruit the

    12    E1 protein into the complex, as you can see here.

    13                E1 protein and the bromodyoxyurasyl

    14    replication foci are coincident.

    15                Now, I want to show you one really

    16    remarkable thing about the nuclear domain, about the

    17    assembly, yeah, yeah, and that is that the DNA J

    18    protein of E. coli and K protein are the things that

    19    help the lambda ONP proteins assemble at the lambda

    20    ORI.  DNA J and DNA K also help the assembly of the E.

    21    coli replication components, and they're highly

    22    conserved all the way up to eucaryotic and human

    23    cells.

    24                This domain right there helps the helicase

    25    assembly.  This was done with Jack Griffith's lab at

                                                                        352

     1    UNC.  In the absence of the J protein, the E1 protein

     2    by itself can form a hexamer ring around the DNA,

     3    either a super coil or an open circle.  You'd throw

     4    the HSP40 in, and you get a dihexamer, which we

     5    believe is a bidirectional helicase.

     6                Secondly, the E2 protein helps recruit the

     7    RPA, the single stranded binding protein, and we feel

     8    this assembly of E2, E1, the J domain right here

     9    creates the preinitiation bubble at which you end up

    10    with four replication strands, two leading strands,

    11    two lagging strands, and the complex bound with RPA.

    12                I think I'll stop at this point, and I'll

    13    be able to show you the role of cyclin E tomorrow.

    14                Thank you.

    15                (Applause.)

    16                DR. PEDEN:  Thank you, Tom.

    17                We'll have maybe one question.

    18                Well, I guess we have to move on.  So the

    19    next talk is by Paul Sandstrom, "Facilitated Detection

    20    of Adventitious Agents Using Genetically Engineered

    21    Cell Lines."

    22                DR. SANDSTROM:  What I'm going to present

    23    tonight is somewhat of a revisiting of some previously

    24    published data in an attempt to try to develop it in

    25    the direction of the theme of this meeting, and that's

                                                                        353

     1    the detection of adventitious agents which may be

     2    present in cell substrates that's used for vaccine

     3    production.

     4                Largely speaking, it's a proof of concept

     5    model which demonstrates approximately a practical

     6    application of a cellular phenomenon that's received

     7    considerable attention over the last ten years, that

     8    being program cell death.

     9                I'm not sure what I'm pointing at here.

    10                Okay.  This project was done in the

    11    laboratory of and under the direction of Dr. Tom Folks

    12    from the HIV and AIDS and retrovirology branch of CDC,

    13    and I myself have the, I guess, notoriety of being, I

    14    think, probably the only Canadian citizen who's

    15    employed by the American Public Health Service and who

    16    is working for the Canadian equivalent of the Public

    17    Health Service and stationed in Canada with all of the

    18    rights and privileges that come with working for the

    19    two largest bureaucracies in the Western Hemisphere.

    20                (Laughter.)

    21                DR. SANDSTROM:  Okay.  It's readily

    22    obvious, I think, to probably everybody here that when

    23    you go about trying to  isolate a potential

    24    adventitious agent, that the failure to isolate

    25    doesn't necessarily mean the agent isn't there.  It

                                                                        354

     1    just means that possibly the culture system wasn't

     2    sufficient adapted to allow for the agent to be

     3    isolated.

     4                So what's needed for isolation is, number

     5    one, it would be good if you had an adventitious

     6    agent, or at least good depending on which side of the

     7    fence you're on, that the virus has the ability to

     8    bind, fuse, or otherwise gain entry into the cell;

     9    that the wiring and forming of the cell are such that

    10    it can support a complete cycle of virus replication;

    11    and of particular importance or I should say of equal

    12    importance, I guess, is that the cell is a good host,

    13    and it stays alive long enough for the virus to

    14    complete its replication cycle.

    15                Apoptosis or program cell death is

    16    involved, in part, not exclusively, but in part as a

    17    first line host defense against pathogenic viruses,

    18    and efficient and rapid apoptosis followed by viral --

    19    following viral infection possibly acts to restrict

    20    virus replication and, if unchecked, will ultimately

    21    result in an aborted viral infection, and this is sort

    22    of the ultimate goal that the cell had in mind when

    23    developing the programs.

    24                So as a result of this intense restrictive

    25    pressure which apoptosis places on virus replication,

                                                                        355

     1    many viruses have evolved to develop their own

     2    repertoire of borrowed proteins, which act to block

     3    the cellular programs.

     4                It's this that I think we're interested in

     5    here, that when trying to isolate an adventitious

     6    agent, along with all the other elements that I

     7    identified earlier, you're always going to be running

     8    up against the possibility that the cells that you're

     9    trying to isolate it in are attempting to undergo

    10    apoptosis and, therefore, limiting your ability to

    11    isolate the virus.

    12                So to get around and to take a look at

    13    this, what we did is to use the, I guess, historical

    14    adventitious agent of HIV, coming from the HIV and

    15    Retrovirology Branch, and we took a cell line which

    16    was exquisitely sensitive to support HIV replication. 

    17    This is a sub T1 cell line.

    18                One of the reasons we chose sub T1 is that

    19    it is, or at least the clones that we were using

    20    lacked expression of the very strong anti-apoptosis

    21    gene, VCL2, and so what we did, quite simply, was to

    22    take VCL2, place behind a constitutive promoter, and

    23    to transfect it into the cells, and then go on to

    24    select clones.

    25                So what we have here is our positive

                                                                        356

     1    control.  This is a Western Blot, positive control,

     2    and two control clones which lack VCL2 expression,

     3    which are paired with two clones which express

     4    significant amounts of VCL2.

     5                And we went on to analyze these and

     6    characterize these in terms of the ability of VCL2 to

     7    block apoptosis in sort of the nonviral, but

     8    traditional, apoptosis models, and we were able to

     9    identify that the expression of VCL2 in this case was

    10    associated with enhanced resistance to program cell

    11    death.

    12                Now, intuitively it would appear as if you

    13    over express a cell, a protein which would block

    14    apoptosis, that you would, in essence, increase the

    15    viability of those cells.  If anything you would

    16    increase the viability of those cells upon infection.

    17                What we found, in fact, was the opposite,

    18    and this graph illustrates viability which was

    19    characterized by using a probe for mitochondrial

    20    function.  So it's not actually measuring cell lysis

    21    in this case.

    22                And what we saw is that if you gave these

    23    -- infected these cultures at the low MOI of

    24    infection, and this is about one in every 10,000

    25    cells, although we were able to go significantly lower

                                                                        357

     1    than this, what we saw is that for about the first

     2    four or five days everything appeared pretty much the

     3    same in the control and the VCL2 cultures.

     4                However, at about five days, there is this

     5    precipitous drop in viability that was observed in

     6    both of the VCL2 forms, whereas the control forms kept

     7    on going out past ten days before they, too, started

     8    to die due to viral mediated cytopathic effects.

     9                And this effect on cell viability was

    10    recapitulated on what we saw with regard to the effect

    11    on viral replication, where both of the VCL2 clones

    12    that we were looking at seemed to support a much more

    13    robust viral kinetics, peaking significantly in

    14    advance of what we saw in the case of the control

    15    clones.

    16                This larger spike in virus here is

    17    probably due to just more cells being present in the

    18    culture at that point.

    19                So it's somewhat counter intuitive until

    20    you sort of sit back and look at what was going on in

    21    the culture, and what we observed was that -- this

    22    doesn't show up all that well -- but these two clones

    23    here represent -- all four of these cultures are

    24    infected with the same low MOI of HIV, and what we saw

    25    was in the two control clones you had a very nice

                                                                        358

     1    confluent lot of single cells, no evidence of viral

     2    mediated cytopathic effects taking place, whereas in

     3    the case of the cells which were over expressing VCL2,

     4    we had these very large syncytia appearing.

     5                Just to show that it wasn't due to a cell

     6    specific effect, we carried out similar experiments on

     7    another cell line, this being the A301 T cell line,

     8    exact paralleling the experiments I just outlines, and

     9    once again saw the exact same effect, that the VCL2

    10    expressing clones showed a significant enhancement and

    11    cytopathic effects going on in the culture.

    12                What was significant though was the nature

    13    of the typopathic effects.  Using a slightly different

    14    model, and this one here was an attempt to get away

    15    from the issue that comes up with viral kinetics that

    16    would happen during a spreading infection, what we did

    17    was we took a cell line which constitutively expressed

    18    HIV.  This is the A301 cell line, which is quite

    19    commonly used in HIV research, and this cell lines

    20    throws off just gobs and gobs of virus and expresses

    21    on its surface viral proteins, and we cultured that

    22    with our sub T clones, and what we found is that all

    23    the clones would roughly make about the same amount --

    24    that in all of these cultures we could identify about

    25    the same amount of syncytia.

                                                                        359

     1                However, in the case of the control cells,

     2    and the black represents viability here; in the case

     3    of the control sub T cells, that most of these

     4    syncytia were dead, and this was done initially by

     5    just trypan blue, whereas in the case of the VCL2

     6    clones, syncytia were maintaining their integrity. 

     7    Their membranes weren't being disrupted.

     8                We also ran a routine for -- the cell line

     9    itself is RT negative.  So what we did is use other

    10    measures for virus presence in the supernatant, and

    11    found that in both of these cultures.  We've seen

    12    roughly equivalent amounts of virus.

    13                Now, if we examined -- this is the tunnel

    14    method for measuring apoptosis, which basically is an

    15    in situ measurement of DNA fragmentation.  What we

    16    found was that red cells here represent cells in which

    17    the DNA hasn't defragmented, whereas this green

    18    appearance is through a number of steps is

    19    representative of cells where the DNA has been

    20    fragmented through apoptosis.

    21                What we found was -- and these are

    22    syncytia here -- in the case of the VCL2 cultures we

    23    have these nice, health looking syncytial masses,

    24    whereas in the case of the sub T clones we've seen

    25    that these cells are undergoing apoptosis.  So this is

                                                                        360

     1    probably more graphically represented.

     2                In the next slide, if we look at actual

     3    EMs, and once again this isn't really showing up all

     4    that well, but this is a syncytia, and if the

     5    reproduction was a bit better, what you'd see is that

     6    the cellular nuclei are complete intact.  They're sort

     7    of wedged together, crushed together inside of the

     8    cell, but each nuclei generally represents what you'd

     9    see in a single cell, whereas in the case of the

    10    control cell, we see this very characteristic

    11    condensation and marginalization of the chromatin,

    12    which is diagnostic of apoptosis, as well as

    13    vacuolization of the  cytoplasm and membrane webbing

    14    going on.

    15                And this below just represents sort of a

    16    more general view of the field, showing that we're not

    17    just looking at one cell that in the case of VCL T

    18    have multiple, health looking syncytia with no

    19    apoptosis, whereas in the case of the control

    20    cultures, most of the syncytia that are present are

    21    undergoing apoptosis.

    22                And this is just to add in that no matter

    23    whether you have VCL2 expression or you don't, if the

    24    syncytia reaches a significantly large size, what

    25    happens is you start to witness necrosis taking place

                                                                        361

     1    with rupturing of the cellular membranes, release of

     2    the cytoplasmic content, swelling of the nuclei.

     3                So are the effects that we're looking at

     4    here the enhancement of HIV replication and

     5    cytoplasmic effects strictly due to syncytia

     6    formation?  Apparently not.

     7                If we take a look at -- in this case here

     8    what we did is we went back to our original sub T

     9    clones, and these are our two controls, which lack

    10    VCL2 expression, and these are our two clones that

    11    have enhanced VCL2 expression and effective MOI of

    12    one, and this essentially means that every cell in the

    13    culture receives an infectious hit, and 48 hours later

    14    took a look at the level of viral replication, and

    15    what we see is in the case of the VCL2 cultures we

    16    have significant -- and this is a Western Blot looking

    17    for the HIV protein and the cell associated HIV

    18    proteins -- that we have significantly higher levels

    19    of HIV protein present in the clones, in the VCL2 over

    20    expressing clones.

    21                So overall the point that I'm trying to

    22    make here is that in the case at least for HIV and

    23    possibly other lentiviruses, we have looked at other

    24    retroviruses and see similar effects; that the

    25    engineering of cell lines which over express in this

                                                                        362

     1    case VCL2, although there is a range of anti-apoptosis

     2    change, both cellular and viral, which probably gives

     3    similar effect and, in fact, we have seen this exact

     4    same phenomena with other cellular genes which are

     5    VCL2 mimetics, such as glutathione peroxidase, as well

     6    as just throwing in chemical mimetics of VCL2, but we

     7    see a similar phenomenon taking place.

     8                So the engineering of cell lines, the

     9    changing of cell culture conditions in a way to block

    10    apoptosis may allow facilitated isolation of

    11    adventitious agents' presence in cellular substrates.

    12                The other point that sort of came out of

    13    this, and I had forgotten about it until I was

    14    actually putting the slides together this afternoon,

    15    was that when it comes to designing a cell substrate

    16    for the purpose of vaccine production and deciding

    17    what components need to go into the recipe, it may be

    18    wise to look at apoptosis and apoptosis inhibitors. 

    19    Just looking at the data set which I presented, the

    20    over expression of VCL2, we see remarkably higher

    21    levels of viral replication, and in addition, we see

    22    significant inhibition of DNA fragmentation, which are

    23    two issues which I think are of interest at least from

    24    this afternoon's talk.

    25                So that's it.  Thanks.

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     1                (Applause.)

     2                DR. PEDEN:  Thank you, Paul.

     3                Any questions?  

     4                PARTICIPANT:  Very interesting data. 

     5    Indeed, it's fascinating.  I have a couple of

     6    questions.

     7                One is when you say that the over

     8    expression of VCL2 render the cells resistant to

     9    apoptosis, what stimulus do you use for apoptosis

    10    besides the virus?

    11                DR. SANDSTROM:  I have to think about

    12    this.  I know that we use serum starvation, and we

    13    used a flavonoid dose called Genestine, which is --

    14    I'm trying to think of the mechanism by which it

    15    produces apoptosis.  I think it has something to do

    16    with mitochondria.

    17                But with these VCL2 clones, we have looked

    18    at a number of other cell lines that people had

    19    engineered, but the effect is just remarkable, these

    20    VCL2 clones, and I think what was most remarkable is

    21    that Genestine would inhibit cellular proliferation in

    22    the control cells.  You'd see the same pattern. 

    23    Cellular proliferation would cease.  Control cells

    24    would die.

    25                The VCL2 expressing cells would just

                                                                        364

     1    continue to live or at least stay intact in culture,

     2    and this would go on for days or longer, weeks with

     3    cells lysing and releasing their components.

     4                PARTICIPANT:  Yeah.  My question was how

     5    generalized is this observation.  I assume those are

     6    T cells, and perhaps using mechanism of apoptosis that

     7    are specific to T cells, such as engagement of CD3 of

     8    the T cell receptor may allow you to dissect whatever. 

     9    This is a generalized phenomenon that will apply at

    10    different cell lineages or it's specific to T cells

    11    and perhaps to HIV virus.  Do you --

    12                DR. SANDSTROM:  Yes, BCL2 isn't specific

    13    for T cells.

    14                PARTICIPANT:  No, no.  I know the BCL2,

    15    but the T cells that you use in HIV are T cells and

    16    the virus that is tropic for T cells.  So I wonder if

    17    you have other information that will allow to

    18    generalize this information to other cell lineage.

    19                DR. SANDSTROM:  No.  The only cell line

    20    that we've personally looked at was T cells.

    21                DR. LEWIS:  Lewis, FDA.  Am I on?

    22                The development of syncytia in cultures

    23    infected with retroviruses, lentiviruses, whatever you

    24    want to call them, is very much reminiscent of the XE

    25    phenomenon that was discovered by Rowe in Wally's

                                                                        365

     1    laboratory back in the late 60s.  Do you have any

     2    evidence that there's any proportion dilution with

     3    these syncytia?

     4                DR. SANDSTROM:  I'm sorry?

     5                DR. LEWIS:  Do the syncytia develop in

     6    response to the dilution of the multiplicity of the

     7    HIV that you put on there?  Have you looked at that?

     8                DR. SANDSTROM:  No, we didn't.  I'm trying

     9    to think if maybe I serendipitously looked at it, but,

    10    no, not specifically.

    11                DR. LEWIS:  Gosh, I mean, the question

    12    goes through my mind of sort of being of the classical

    13    oralgia (phonetic) modes, whether you finally have a

    14    CPE assay for HIV infection.

    15                DR. SANDSTROM:  Oh, I see.  Yeah.  No, I

    16    think the only thing I could say to that is that in

    17    our own experience we would see better syncytia at the

    18    lower multiplicity of infection, but I always think of

    19    it just as a more controlled infection, a more

    20    controlled spreading infection rather than at the high

    21    ones that just went too fast.

    22                Does that answer your question?

    23                DR. LEWIS:  Well, no.  I guess I would

    24    poke at you a little bit and say suppose you went

    25    below the lower multiplicity that you're using because

                                                                        366

     1    if the titre is ten to the seven or ten to the eight

     2    in this assay and you're looking at ten to the four,

     3    then you know, you're up here.

     4                DR. SANDSTROM:  Right.

     5                DR. LEWIS:  You haven't looked at the

     6    range.

     7                DR. SANDSTROM:  Yeah.  One of the problems

     8    is that just because of the culture when you get down

     9    too low, you reach the mathematical endpoint.  There's

    10    no virus for the number of cells that you have there. 

    11    So I think we were able to bring it down to somewhere

    12    in the range of about, so to speak, five viruses in a

    13    culture class, and we would see the effect.

    14                The effect actually is sort of -- the

    15    differences between those clones was enhanced at the

    16    lower multiplicity of infection.

    17                DR. LEWIS:  Well, I guess if it's not, if

    18    the virions are not doing it and it's not proportioned

    19    dilution, then what is it?

    20                DR. SANDSTROM:  Yeah.

    21                DR. PEDEN:  It's certainly proportioned to

    22    the dilution, Andrew.  It's certainly proportional.

    23                Is it pressing?

    24                DR. COOK:  Jim Cook.

    25                So you're putting BCL2 into the cells. 

                                                                        367

     1    The nuclei remain intact.  The cells die early, and

     2    you said your indicator of cell death was a

     3    mitochondrial marker of some sort.  It sounds a lot

     4    like cell necrosis.

     5                Did you look at anything in terms of

     6    mitochondrial function, mitochondrial respiration, and 

     7    BCL2, as you know, is a mitochondrial membrane active

     8    agent.

     9                DR. SANDSTROM:  Right.

    10                DR. COOK:  And the question is whether

    11    somehow or other this is sensitizing the cells to

    12    injury induced necrosis rather than apoptosis, and

    13    that's why you get early cell death.

    14                DR. SANDSTROM:  I don't think that's

    15    what's going on.  I mean, we were using MTS, which is

    16    largely -- I mean, basically that measures

    17    mitochondrial function, and --

    18                DR. COOK:  So if the mitochondria are not

    19    functioning, the nuclei remain intact.  That's almost

    20    the definition of necrotic cell death, right?

    21                DR. SANDSTROM:  Well, I think it's a lot

    22    of things.  I mean, number one, necrosis and apoptosis

    23    are endpoints on a Gray scale.  So there's a certain

    24    amounts of overlap, certain features that they have in

    25    common.

                                                                        368

     1                But largely speaking, when we looked at it

     2    in terms of the lack of chromatin, fragmentation,

     3    marginalization, and the fact that the cellular

     4    membranes stayed intact would to us say that it was

     5    apoptosis that was going on or the blocking of

     6    apoptosis that was taking place.

     7                DR. PEDEN:  All right.  Thank you.

     8                The next and last of the formal program is

     9    by Brian Van Tine.  I hope that's the correct

    10    pronunciation.  "In Situ Transcriptional Analysis of

    11    Integrated Viral DNA."

    12                DR. VAN TINE:  I'd like to introduce

    13    myself.  My name is Brian Van Tine, and I work in the

    14    laboratory of George Shaw in the Center for AIDS

    15    Research at the University of Alabama.

    16                I work on a technique called FICTION by

    17    tyramides, and FICTION stands for fluorescence

    18    immunohistic chemistry  and in situ for the study of

    19    neoplasms, or in our case for the study of RNA DNA in

    20    proteins in a single cell at the same time.

    21                Briefly, what I'd like to go over is just

    22    a method which is I realize basic for most people in

    23    here, but is highly informative.  You pick a target,

    24    whether it's a protein, an RNA or a DNA.  We design

    25    either an antibody or an RNA or DNA probe to it; come

                                                                        369

     1    in with a detection scheme, which leads to an HRP;

     2    come in with a fluoro-4-tyramide and cause a covalent

     3    deposition to the slides that we're interested in, and

     4    why this is important is because you can kill that HRP

     5    and start over.

     6                So if you're interested in studying, say,

     7    your HIV-1 DNA, the RNA it produces, and some of the

     8    proteins it's associated with, you can now do this in

     9    a single cell.

    10                This work was done in collaboration with

    11    NEN because the experts that went to microdetections

    12    were there, and as a result a collaboration was

    13    involved with Phillip Moen and a papilloma project was

    14    developed.

    15                And so in general, this research that I'm

    16    presenting today is on papilloma HV16, which

    17    predominates in cervical cancers.  It's, in general,

    18    widespread and prevalent, and it's a causative agent

    19    in both benign papillomas and condylomas.

    20                It's multiply spliced, and the early

    21    proteins that we'll be concerned with today are E6,

    22    E7, E1, and E2.

    23                What this shows is a general map of HPB16,

    24    but what's important to note is it's only 8 KB, and if

    25    this virus is to integrate not in tandem, but to be

                                                                        370

     1    just a single copy, it is very, very difficult with

     2    any sort of sensitivity to detect a single copy by

     3    normal methods, and that's why this method becomes

     4    important.

     5                I'd like to introduce our cell line

     6    players for today.  The CaSki cell line has

     7    approximately 600 copies of HPV integrated into

     8    various places in a very complex genome.

     9                The SiHa cell line has one to five,

    10    depending on the literature, copies of HPV, both on a

    11    Chromosome 13, and we have designed probes either to

    12    E6, E7, E1, all the way through E1, and we have a

    13    probe just to E2.  We also have full length probe

    14    specific for each cell line.

    15                Can we make it any darker in here?

    16                What we are -- what I'm demonstrating in

    17    the top figure here is the CaSki cell line, and this

    18    is a hybridization for DNA and in tat cells, and as

    19    you can see, there's many, many sites in each and

    20    every cell, but down here in the SiHa cell there are

    21    two cites that grow up on both 13 chromosomes in this

    22    genome.

    23                What we did was to ask the question, you

    24    know:  are all the copies in the SiHa gene on?  And

    25    what you do is you begin with a hybridization for RNA. 

                                                                        371

     1    What you can see, we have two RNA centers.  This is a

     2    G2 doublet indicative of cells in G2.  This is a bit

     3    brighter in intensity and is probably an overlap G2

     4    signal.

     5                We then RNAase the slides, denatured them,

     6    and detected the DNA, and what you end up with is you

     7    can see that both copies of the HPV associated with

     8    SiHa cells are both on.

     9                This is a metaphase analysis of the CaSki

    10    cell line.  It has approximately 78 chromosomes and

    11    approximately 16 integration sites ranging from what

    12    is probably 100 down to, based on HIV size

    13    comparisons, one copy of HPV.

    14                What became interesting is we then

    15    proceeded in the CaSki cell line to ask which of the

    16    DNA integrants were active, and surprisingly it was

    17    one.  Here is the DNA in situ.  This is a single cell. 

    18    It was pre-extracted, fixed, and hybridized for RNA,

    19    then denatured, and the DNA was detected, and we find

    20    that this minor site right here, an emerged picture

    21    down here, is the only site that is transcribed in

    22    this cell line.

    23                We then went on an exploration to see if

    24    we could discover exactly which of the sites was

    25    active, and if it was the same.  So we did this by a

                                                                        372

     1    variety of paints where in a yellowish color on this

     2    slide we have a Si-3-tyramide to HIV and we have a

     3    chromosome paint in this case to nine.

     4                We at the end of the day came down with

     5    three minor sites, one on Chromosome 3, one on

     6    Chromosome 14, and one on a Chromosome 21 and 22.  We

     7    haven't, because of the paints, decided which one it

     8    is.

     9                We then went in, back to an interphased

    10    intact cell, and just to show you that there are many

    11    colors of tyramides.  If you work with NEM, there's

    12    now about eight.

    13                Here we have in blue an integrated DNA. 

    14    Red, we have the RNA, and in green we have Chromosome

    15    13 territories, and we were looking for association,

    16    and we found that there's an association between

    17    Chromosome 14 and the active integrant (phonetic).  We

    18    have run controls on chromosomes known to contain

    19    larger tandem repeats and not see that association,

    20    and on some of the few chromosomes that didn't have an

    21    HPV integration and found that uniformly we find that

    22    only the integrant associated with Chromosome 14 was

    23    active.

    24                So that led us to our next question, which

    25    was:  is there something special about 14 and its

                                                                        373

     1    position in nucleus that would suggest that it be

     2    specifically transcribed?

     3                And so we began by looking at an SC35

     4    splicing complex oligo to RNA, and we didn't find an

     5    association there.  

     6                We looked at a U2SN RNA and did not find

     7    an association, but what you see here in these darker

     8    regions are nucleoli, and consistently we have an

     9    association between this being turned on and the

    10    nucleoli.

    11                We also looked at coiled bodies and found

    12    no association, but we're not quite sure to this point

    13    yet why there's only one copy turned on in the cell

    14    line.

    15                We also found in our process of running

    16    controls that not only could we detect the sense

    17    transcript in CaSki cells, but there's also an anti-

    18    transcript which has been previously reported in the

    19    literature.  Just to show you the sensitivity and the

    20    specificity of this technique, this is the E6/71

    21    riboprobe in the SiHa cells, and we did a riboprobe

    22    here to the E2 protein which is missing in the SiHa

    23    integration to show you just how clear and specific

    24    this technique is.

    25                What becomes important is we have now gone

                                                                        374

     1    to a cervical carcinoma, and this is actually a

     2    formalin fixed tissue section where in red we detected

     3    DNA and in green we detect RNA, and we found that

     4    we're seeing the same effect in vivo where we have

     5    nuclear -- we have DNAs that are both on and off, and

     6    we're starting to dissect exactly if there's an

     7    association between position and being on or off.

     8                So, in general, in SiHa cells, HPV16, DNA

     9    and RNA, they're both on, and they colocalize.

    10                In the CaSki cells, the RNA colocalizes

    11    only with a single integration site, and at the time

    12    the slide was made, we weren't sure exactly what it

    13    was, but we now know it to be 14.

    14                The RNA and the DNA does not associate

    15    with splicing factor domains.  The RNA and the DNA

    16    does not associate with coiled bodies, but they do

    17    tend to localize near nucleoli.

    18                It was initially assumed that all

    19    integrants were transcribed and that all transcription

    20    occurred in the same direction, and the CaSki cell

    21    line shows evidence for transcription from one minor

    22    locus, and we detected the E2, E6, and E7 transcripts,

    23    were detected from both strands -- I believe that

    24    should be E1 -- and we also have anti-transcriptions

    25    seen in vivo.

                                                                        375

     1                And going with the themes that we've

     2    picked up in the meeting, we have suggested some

     3    mechanisms for the HPV silencing, which may either

     4    deal with methylation, the chromosome positioning

     5    effects, or maybe just histos.

     6                And so with that I'll conclude.

     7                (Applause.)

     8                PARTICIPANT:  Can you -- what is the

     9    sensitivity?  The viral promoter may be very strong. 

    10    Can you use this to detect the cellular RNA?

    11                DR. VAN TINE:  Cellular RNA, yeah.  If

    12    it's colocalized in one spot, you can probably, if you

    13    talk to Robert Singer, who does really fancy

    14    deconvolution, you can see individual copies of RNA

    15    and map the direction.

    16                PARTICIPANT:  Is that right?

    17                DR. VAN TINE:  That was reported in

    18    Science about six months ago.

    19                PARTICIPANT:  I was not too clear about

    20    the fixation.  Why do you not see RNA in the

    21    cytoplasm?

    22                DR. VAN TINE:  They're pre-extracted

    23    before they're fixed.  So the only RNA that remains in

    24    the cell is that associated with the DNA as it's being

    25    transcribed.

                                                                        376

     1                DR. FRIED:  Mike Fried.

     2                If you put in five As of cytidine, do the

     3    other sites come on?

     4                DR. VAN TINE:  As I recall they don't.

     5                DR. FRIED:  So you don't see

     6    inspotsylation (phonetic)?

     7                DR. VAN TINE:  Not on the first

     8    experiments that we've done.  They're still in

     9    process.

    10                DR. PEDEN:  Okay.  Thanks very much.

    11                That ends the formal presentation.  Now,

    12    as far as I know, there are a couple of people who

    13    want to present -- what's this?  Just one?  Yes.

    14                So Dr. Arifa Khan will present first, and

    15    I think Johannes Loewer will present after.  Is that

    16    right, Johannes?  Do you still want to present?  It

    17    depends on the stamina of the audience.

    18                DR. KHAN:  Can we have the first slide,

    19    please?

    20                DR. PEDEN:  Press it.

    21                DR. KHAN:  Oh, Dr. Khan.

    22                DR. PEDEN:  It's K-h-a-n.

    23                DR. KHAN:  Although my talk was to make it

    24    very short, but I think it's going to get longer than

    25    I --

                                                                        377

     1                DR. PEDEN:  It's the one with two people

     2    in it, Peden and Khan.

     3                DR. KHAN:  I just wanted to give a brief

     4    presentation of our recent work on protein analysis of

     5    chemically induced k BALB 3T3 cells and to present

     6    this as a potential strategy for the protection of

     7    infectious retroviruses in vaccine cell substrates.

     8                Okay.  The k BALB 3T3 cells are derive

     9    from BALB 3T3 cells.  These are Christian Maloney

    10    sarcoma virus transformed.  BALB 3T3 cells are non-

    11    producers, and the reason that I selected this

    12    particular cell line is that extensive work has been

    13    done on this cell line actually by Stu Aaronson's

    14    group on the NIH campus in the '70s, and the

    15    endogenous retroviruses that can be induced from this

    16    cell line have been well characterized biologically.

    17                I should also mention that in terms of

    18    retrovirus induction, extensive studies have been done

    19    on mouse cells, and most of the studies have been done

    20    by labs on the NIH campus in the early '70s, and in

    21    terms of optimizing conditions for other cells, very

    22    little, as far as I know, has been done, and basically

    23    the conditions that have been optimized and determined

    24    for the mouse cells in most cases are just directly

    25    applied to other cell lines from other species.

                                                                        378

     1                The purpose of initiating this work was to

     2    optimize -- to understand the kinetics of retrovirus

     3    induction in this well characterized cell line, and

     4    then to extrapolate to see whether the results from

     5    this study can be used to investigate other vaccines,

     6    potential vaccine cell substrates from other species.

     7                In terms of the amount of the BALB 3T3

     8    cells, it's known that these cells contain or con

     9    produce retroviral particles of three types.  One is

    10    the IAP, which are the defective Type A particles

    11    which are present in numerous copies in the mouse

    12    genome, and these are defective sequences that are

    13    associated with internal Type A particles.

    14                In addition, there are two proviruses that

    15    can be induced as infectious virus from these cells. 

    16    They have been designated as BALB virus Type 1 -- BALB

    17    Virus 1, which is an entropic, ecotropic Type C virus,

    18    and BALB Virus 2, which is a xenotropic virus based

    19    upon infection on rat kidney cells, which is also a

    20    Type C virus.

    21                So these two viruses, Virus 1 and Virus 2

    22    have previously been shown to be induced from these

    23    cells.

    24                So what we wanted to do was to revisit

    25    these induction studies, applying the highly sensitive

                                                                        379

     1    PERT assays to study the kinetics of retrovirus

     2    induction from the k BALB cells.

     3                There has been extensive work to

     4    investigate a variety of agents in terms of their

     5    ineducability in mouse cells and in some cases even in

     6    avian cells.

     7                Yeah, it's from your book, John.  It's

     8    your table, and you're right here.  Good.  Any

     9    comments, direct it to Dr. Coffin, please.

    10                (Laughter.)

    11                DR. KHAN:  As you can see, there's a

    12    variety of agents that can induce retroviruses,

    13    chemical agents, especially halogenated pyrimidines,

    14    IBU and BRDU, also other mutagens, and a bit of the

    15    protein synthesis, neurological agents, and even

    16    viruses can induce retroviruses.

    17                So the reason I've listed this is just so

    18    that one should be aware that during the production of

    19    a vaccine, you know, there may be a concern that under

    20    certain circumstances the use of certain agents that

    21    may be useful for producing a high vaccine virus titre

    22    may inadvertently also be activating a retrovirus.

    23                And, therefore, it is very important to be

    24    able to detect very early on if the cell substrate has

    25    a potential infectious retrovirus using sensitive

                                                                        380

     1    techniques.

     2                We investigated using IDU since it is a

     3    potent retrovirus inducer, and extensive studies have

     4    been done with this, some of them.

     5                In this study, the k BALB 3T3 cells were

     6    exposed to various concentrate -- to 20 microgram or

     7    30 micrograms of IDU at 24 hours or at 48 hours, and

     8    the control cells -- this is using the taq man PERT

     9    assay that Dr. Peden has described earlier.

    10                The control cells were negative in the

    11    assay. 

    12                As you can see, the 30 micrograms produced

    13    high RT activity early on.  This is day two time

    14    point.  This is day four, and this is day six here. 

    15    So early on the high dose, even the 24 hour post

    16    induction resulted in high RT production, whereas the

    17    lower dose at either 48 hours or even 24 hours, did

    18    produce low levels, but they were not detectable at

    19    the later time points.

    20                On further culturing, we saw another P

    21    come up later on on day 17 with the higher dose and

    22    was not detectable by the PERT assay  with the lower

    23    dose.

    24                I should mention that  these two peaks, an

    25    early peak and a later peak, has previously been

                                                                        381

     1    identified, induced from these cells using co-

     2    cultivation and marker rescue experiments.  So we

     3    confirmed that we could, you know, repeat these

     4    studies, and using the PERT assay.

     5                However, the additional data is that the

     6    higher dose was necessary to be able to pick up the

     7    virus early on.

     8                I should also mention that in the earlier

     9    studies done by Joe Anderson's group they were able to

    10    also detect RT using the traditional assay.  However,

    11    they had to use samples that were 100-fold

    12    concentrated, whereas in our case this represents one

    13    microliter from a T-75 flask.  So this demonstrates

    14    the sensitivity of the assay.

    15                And in this case, the supernatant was

    16    harvested daily up to seven days, and then after every

    17    three days.

    18                To further investigate the particles that

    19    were induced, we also analyzed some of the time points

    20    by EM.  This is the untreated sample.  At day two

    21    there is your typical Type A particle, which is at low

    22    levels in our induced cells.

    23                At 20 micrograms, even at 48 hours at day

    24    five the only particles that were detected by EM were

    25    the A particles.  We did not detect any C particles,

                                                                        382

     1    although we know that C particles are present at this

     2    concentration because we did detect it by the PERT

     3    assay.  The As would not be released into the medium. 

     4    So we would only be detecting exogenous C.  So this is

     5    just the sensitivity of the assay.

     6                With the 30 micrograms at 24 hours at day

     7    two, we saw the As.  In addition, we could see a few

     8    Cs being produced, as you see this one budding out,

     9    but there were very few Cs at 24 hours, whereas it was

    10    increased at 48 hours.

    11                However, I want to mention that the higher

    12    concentration and the long time of treatment, as you

    13    can see, there was cell toxicity.  So, however, to

    14    assess that the RT that we were detecting was due to

    15    retroviral particles and not due to cellular lysis, we

    16    also have tested lipid in the PERT assay, and the RT

    17    levels that were detected at this time point were

    18    threefold above that of cellular background,

    19    concentrated cell lysis.  So this RT -- this doesn't

    20    go back? -- anyway, so the RT that we were picking up

    21    were associated with the Type C particles and not cell

    22    lysis.

    23                Okay.  So in summary, spontaneous release

    24    of virus was not detected in the untreated k BALB 3T3

    25    cells.  However, it has been shown that under, I

                                                                        383

     1    guess, certain culture conditions this can occur. 

     2    However, we were careful not to overgrow the cells or

     3    stress the cells to avoid that.

     4                Induction of endogenous Type C retrovirus

     5    was dose dependent, and we found that evaluation of

     6    several early and later time points was necessary to

     7    completely analyze all of the inducible retroviruses.

     8                And in conclusion, I would just like to

     9    propose this strategy for consideration for detection

    10    of endogenous infectious retroviruses in cell

    11    substrates used for production of biologics.

    12                The first step would be to optimize the

    13    induction conditions, which would be to identify and

    14    use a potent general retrovirus inducer or it may be

    15    more than one, I think, depending on your cell

    16    substrate and what you might expect from it.

    17                To determine the optimum dose and exposure

    18    time, you want to achieve high viral induction with

    19    low cell toxicity.

    20                Then you want to combine this with

    21    sensitive detection of retroviruses from induced

    22    cells.  The PERT assay is very useful in this aspects,

    23    and you would want to test several early and late time

    24    points.

    25                And of course, if you have a positive PERT

                                                                        384

     1    result, it then would be necessary to further identify

     2    the source of the activity by EM or by co-cultivation

     3    to see whether you have an infectious retrovirus.

     4                I would just end with that.

     5                (Applause.)

     6                DR. PEDEN:  Thank you, Arifa.

     7                Any short questions?

     8                Dr. Coffin.

     9                DR. COFFIN:  I would suggest adding to

    10    this some attempt to characterize the genome of the

    11    virus that's induced.  Do you know what you're

    12    actually seeing from the BALB C cells?  I mean, there

    13    are 60 or so proviruses that might give rise to this

    14    activity.

    15                DR. KHAN:  Well, the BALB C cells, I

    16    think, is very well characterized because, you know,

    17    the two proviral genomes that give rise to these

    18    viruses, I think, are known in terms of genetic --

    19                DR. COFFIN:  Those are the infectious

    20    ones.

    21                DR. KHAN:  Yes.

    22                DR. COFFIN:  But the particles that you're

    23    seeing may actually be coming from any of a number of

    24    other ones as well.  It would be of some interest

    25    actually to look at the envelope genes and see what's

                                                                        385

     1    going on.

     2                DR. KHAN:  As you know, we've been both

     3    down this path in terms of, you know -- I think for a

     4    long time -- in terms of trying to characterize all of

     5    the endogenous retroviral sequences, and I think the

     6    majority of them are defective, and the question

     7    actually, a broad question I get with your -- which

     8    you could present is that, you know, in terms of RT

     9    activity or in terms of particles that are produced,

    10    what is the real public health concern, and I think,

    11    you know, would it be a defective particle?  Would

    12    it --

    13                DR. COFFIN:  We don't know that.  I mean

    14    to be fair, we don't know that for most of the

    15    endogenous C type viruses that you can see in mouse. 

    16    We know that you don't see whole virus that

    17    corresponds to most of those, but we don't know that

    18    they're defective --

    19                DR. KHAN:  No, I agree.

    20                DR. COFFIN: -- in any real way.

    21                DR. KHAN:  So in this case, the next step

    22    that we need to do was to do infectivity studies to

    23    see whether those peaks correspond to any infectious

    24    virus, and the reason I did not -- you know, I went

    25    ahead and presented this data is that for this

                                                                        386

     1    particular cell line, these two time points, day three

     2    and day 20, are associated with infectious virus.  The

     3    early time point is with the phenome, BALB Virus 2,

     4    and then they have infectious BALB Virus 1, endotropic

     5    virus.

     6                But in general, for a novel cell

     7    substrate, if you had RT activity, the next would be

     8    to see whether there's infectious virus.

     9                DR. COFFIN:  But that echovirus has to be

    10    the product of recombination or mutation because the

    11    proviruses themselves are defective.

    12                DR. KHAN:  Right.  That's why you see it

    13    over a long period of time.  So I think --

    14                DR. COFFIN:  There must be other -- there

    15    may well be other defective things inducing that might

    16    contribute biologically to the final product.

    17                DR. KHAN:  Right.  You would propose that

    18    once you have RT activity, then you need to follow it

    19    up with infectivity experiments?  Yes?

    20                DR. COFFIN:  Well, I would do some PCR and

    21    see what sort of sequences you get in there.  It's

    22    fairly simple with oligo probes and so on to just the

    23    PCR amplify envelope genes and see what's being made.

    24                DR. KHAN:  Right.  You can do it, and

    25    then, you know, wraps it and stuff, yeah.

                                                                        387

     1                DR. PEDEN:  Okay.  The next speaker is

     2    Damian Purcell.

     3                Is Purcell's carousel in the -- it is. 

     4    Thank you.

     5                DR. PURCELL:  Okay.  This is going to be

     6    fairly fast since half the slides have been presented

     7    and duplicates in some ways a story we're already

     8    heard from Ruth Ruprecht with slide  differencing in

     9    some of the outcomes which I'll show you.

    10                So what we've done here, as Ruth did, we

    11    evaluated the logistical problems of delivering a live

    12    attenuated HIV vaccine strain, and we saw that this

    13    would be difficult with the virus strain in our minds

    14    being at the time a strain of HIV that emerged in

    15    Sidney, Australia, know as the Sidney Blood Bank

    16    Cohort, which is essentially a nef deleted virus,

    17    which for a long time appeared to be attenuated and

    18    not causing disease in patients, but just recently

    19    some of the cohort of that group of patient have begun

    20    to lose immune function.  So it's probably following

    21    the same story as the simian cases that progressed to

    22    full pathology.

    23                Anyway, we saw that the possibility of

    24    having to culture the isolate of this virus strain

    25    that we had in the lab by necessity in PBMC, we

                                                                        388

     1    thought that this would be a problem rather than an

     2    opportunity as other people have found, and the

     3    necessity to produce large lots even if we could find

     4    a human cell line would cause problems in terms of the

     5    approval of the vaccine, a live attenuated vaccine.

     6                So we figured that the best way of dealing

     7    with these issues would be to deliver a truly

     8    attenuated virus, if this was one, as provirus DNA.

     9                So we thought to model this in the SIV

    10    system, and we took the sequence of the Sidney Blood

    11    Bank Cohort patients and identified the minimal

    12    deletion, which is a region in U3, and the patients

    13    had other deletions in and around nef and NU3, but we

    14    chose to take the minimum region of the deletion.  

    15                We made an alignment against the SIV MAC

    16    239, which through this region is not a very strong

    17    alignment, and we made a deletion that sort of

    18    represented roughly the maximum amount of sequence

    19    deleted out of the Sidney Blood Bank viruses, and just

    20    as a comparison against the equivalent deletion, which

    21    I guess we're more familiar with, the second of the

    22    two deletions in SIV made by Ron Desrosiers.  It sort

    23    of accommodates half of that second deletion.

    24                Now, we made two forms of this proviral

    25    strain, one bearing the deletion in just the three

                                                                        389

     1    prime end, which we call SIV SBBC Delta 3.  So the

     2    deletion is 105 base pairs, and after some coercing

     3    with the provirus, we managed to make the double

     4    deleted form with the deletion also in the five prime

     5    LTR with the provirus.

     6                We're unsure when administering these

     7    proviral constructs into the animals whether it would

     8    be necessary to retain the five prime U3 promoter

     9    function, but even with the deletion in the three

    10    prime end, as we've heard in the meeting earlier, RNA

    11    transcription commences at the R region, so effective

    12    excising the first of the U3 region in this singly

    13    deleted form, so that after one round of replication

    14    of the virus, there's a duplication of the deletion,

    15    and all progeny virus then will have the double

    16    deletion after one round of replication.

    17                So our design was to administer plasmid

    18    DNA into eight juvenile macaque Nemestrinas.  We've

    19    already done one titration point.  Compared to Ruth

    20    Ruprecht here we're down.  She used 500 micrograms. 

    21    We used 300 micrograms IM.

    22                We've also taken the gene gun and

    23    administering 15 micrograms with wild type SIV and

    24    with the two different attenuated strains, the Delta

    25    3; so that's with the just three prime deletion in U3. 

                                                                        390

     1    We did an IM 300 microgram injection of this one, and

     2    with the double deleted version, we loaded this up on

     3    the gold beads and administered 15 micrograms of DNA

     4    by gene gun.

     5                So the wild type delivery in each case was

     6    successful.  What we're looking at is plasma RNA. 

     7    Three of the animals -- well, this one actually died

     8    at ten weeks.  This one was euthanized here with very

     9    low CD-4 counts, as you'll see, and the other one also

    10    was euthanized with very low CD-4s.

    11                But what I want to draw your attention to

    12    is with the singly deleted form of the construct that

    13    we administered, one of these two animals or both of

    14    them scored high virus loads, but then by between five

    15    and ten weeks one of these animals, Monkey 16, had a

    16    high level of virus coming back in the plasma.

    17                The time points we have on the double

    18    deleted virus were short.  They haven't had this virus

    19    in the monkeys as long, but it dwindles down very

    20    rapidly.  It's certainly detectable, to begin with.

    21                Just, again, looking at the CD-4 counts in

    22    the wild type monkeys, they lose their CD-4 very

    23    rapidly.  So it's a very efficient way of

    24    administering virus, just as Ruth Ruprecht showed.

    25                And this Monkey 16, as well as retaining

                                                                        391

     1    high viral load, has begun to lose CD-4 cells.

     2                So just following up, the changes to the

     3    sequence in the two monkeys, this is the one that does

     4    not regain high viral load nor lose CD-4 counts.  This

     5    is the kind of changes that we commonly see when you

     6    administer genuine virus with deletion.  You see extra

     7    deletions within the nef to increase fitness, little

     8    duplications of sequence.

     9                But at week five we did find a wholesale

    10    duplication of 63 base pairs from the wild type part

    11    of U3 into this, but it was lost through time, but the

    12    other monkey, Monkey 16, by the third week we found a

    13    complete replacement of the U3 here in a small

    14    fraction of the clones, and as time went by, all of

    15    the clones had reverted to wild type.

    16                So the question is:  how did this event

    17    occur?  It's a different form of reverse to what we

    18    typically see, and we strongly suspect the presence of

    19    the complete U3 within the DNA, the plasmid that was

    20    administered to the animals as a bolus of 300

    21    micrograms.

    22                So just in conclusion, wild type SIV DNA

    23    is certainly pathogenic.  As low as 15 micrograms of

    24    DNA administered by gene gun is also very efficient

    25    even with the attenuated double deleted form of virus.

                                                                        392

     1                Probably the most interesting thing is

     2    that the evidence of the reversion in one of the

     3    animals implicates the DNA as a potential source of

     4    recombination with the virus.

     5                And I'll leave it at that.

     6                (Applause.)

     7                DR. PEDEN:  Thank you, Damian.

     8                Any brief questions, comments?

     9                DR. PATIENCE:  Damian, thanks a lot.

    10                DR. PEDEN:  Clive Patience.

    11                DR. PATIENCE:  Yes.  Sorry.  Clive

    12    Patience, BioTransplant.

    13                I don't know if I saw it through a haze. 

    14    It's getting late for me, but did you see quite a

    15    severe dropoff in the intradermal administration of

    16    you variants after one week in the CD-4 count?

    17                I think it was just in the intradermal if

    18    I --

    19                DR. PURCELL:  Yes.

    20                DR. PATIENCE:  And if so, if I did read it

    21    right, do you have any explanation why the CD-4 count

    22    went down about fourfold, I think it was?

    23                DR. PURCELL:  It was probably the way --

    24    this was not numbered, the CD-4s.  We were measuring

    25    the percent of CD-4s.  So it's a percent of the pre-

                                                                        393

     1    inoculation percentage, if you like.  So rather than

     2    counting total CD-4 numbers, we're looking -- we run

     3    the samples through the fax (phonetic), and we look at

     4    the ratio of CD-4s at the time point that we examine

     5    and compare that back to the pre-inoculation time

     6    point.

     7                So it's really looking at the ratio more

     8    than the actual numbers.

     9                DR. PATIENCE:  Okay.  Do the real numbers

    10    follow the same sort of pattern as well or is it

    11    disturbance of the ratio of your CD-4 --

    12                DR. PURCELL:  At the time we started this,

    13    we didn't have the technology to count the numbers in

    14    the primates, but we'll have to look at that in the

    15    next set of animals, yeah.

    16                DR. PATIENCE:  Thanks.

    17                DR. BERKHOUT:  I guess the --

    18                DR. PEDEN:  Berkhout.

    19                DR. BERKHOUT:  Ben Berkhout, yeah.

    20                The unusual recombination event that you

    21    see may still have occurred through RNA, although, you

    22    know, I guess there are two ways.  One is that perhaps

    23    the transcription is fired from your plasmids starting

    24    at the three prime LTR, and of course, you can also

    25    have transcripts that are started at the five prime

                                                                        394

     1    LTR, but you get read through transcription.

     2                DR. PURCELL:  The possibility that we're

     3    initiating transcription from here and reading through

     4    an RNA transcript to here, which would then be

     5    terminated, polyadenylated without requiring an

     6    action.

     7                DR. BERKHOUT:  But if polyadenylation is

     8    not efficient then --

     9                DR. PURCELL:  True, and the other probably

    10    more likely possibility is a read through the

    11    polyadenylation site here down to this, the second one

    12    that appears, making it, you know, quite a long RNA,

    13    which might be code packaged.

    14                So we haven't completely excluded it.  We

    15    need a second experiment, I think, but certainly the

    16    possibility is there, and I think that the selective

    17    pressure we're picking up, that piece of DNA would be

    18    greater than any other cellular DNA.

    19                DR. PEDEN:  All right.  Thank you, David.

    20                I think Johannes wanted -- do you still

    21    want to speak?

    22                DR. LOEWER:  I would, but I guess --

    23                DR. PEDEN:  Did you turn off the machine?

    24                So if the audience is able to take one

    25    more talk.

                                                                        395

     1                (Pause in proceedings.)

     2                DR. LOEWER:  What I wanted to do is to

     3    present very quickly the most recent experiments we

     4    have performed to understand and moditate (phonetic)

     5    molecular biology of the human endogenous retrovirus

     6    families, K family.  At least to my knowledge, it's

     7    the only retrovirus, human endogenous retrovirus

     8    family with codes for all structural viral proteins.

     9                These proteins are expressed with the zone

    10    (phonetic) virus family, which is able to form wider

    11    particles which you can see here on the micrograph.

    12                This virus is expressed mainly in terata

    13    carcinoma cell lines, as I've shown this afternoon. 

    14    This is a Northern Blot describing as full length and

    15    singly spliced N message in addition to these four

    16    messages.  We see also two.  There is more messages,

    17    which is very unusual for endogenous retrovirus.  This

    18    resembles the more complex retroviruses.

    19                Analysis shows that one of the smaller

    20    length RNAs is doubly spliced, encodes for a small

    21    protein, which is located in this area which was

    22    called because of this localization central open

    23    reading frame.

    24                And this protein resembles to some extent

    25    the left protein from HIV or the next protein from

                                                                        396

     1    HIV-1 in the sense that it has nuclear localization

     2    signal, as well as a domain which resembles the

     3    (unintelligible).

     4                And the question we ask, of course, is

     5    that if this is a functional protein which enhances or

     6    allows the export of full length simply spliced RNAs,

     7    to study this we used a system developed by Barbara

     8    Felmer and Charles Papalakus.  It's a construct which

     9    expresses HIV only if there is a responsive element

    10    like the RRE and if ref or a corresponding protein is

    11    provided in (unintelligible), we have replaced RRE by

    12    different parts of the three prime part of the K

    13    genome  and have also provided the C orf protein in

    14    trans.

    15                The HIV expression was first -- or what I

    16    have to say, we have checked here several pieces, and

    17    what turned out is that there is a responsive element. 

    18    It's a three prime LTR which we called RcRE, analogy

    19    to our XIA from HIV-1 because it has very similar

    20    localization.

    21                And this is shown by more fluorescence

    22    data here, for example, only in the presence of what

    23    we call RcRE HIV that is produced with other parts of

    24    the nonresponding fragments.  You can only see as a C

    25    orf protein localized in the nuclei.  

                                                                        397

     1                This can be quantified by P24 antigen

     2    measurement.  Here is RRE, the HIV system, and if raf

     3    is present at RRE, you get a high expression of P24,

     4    and the same is true if you have the responsive

     5    element for C orf and C orf is present, but raf can

     6    also go up to some extent on RcRE, but C orf cannot

     7    work on RRE and other parts of the genome are not

     8    active.

     9                Next, we looked at the different domains. 

    10    We mutate nuclear localization signal and also mutated

    11    the positive nuclear export signal, and we check the

    12    shortened -- if the C terminal shortened, C orf would

    13    also be active.  In this case, all this construct,

    14    they were used to keep lost protein (phonetic).

    15                And these are the effects of the different

    16    mutations.  This is wild type.  This is C orf

    17    localized in the nuclei, and P24 in the cytoplasm with

    18    mutation of the nuclear localization signal.  The C

    19    orf cannot enter the nucleus.  It's found only in

    20    cytoplasm, and there's a mutation of the nuclear

    21    export signal as you can expect.  The mutant is

    22    localized in the nuclei as well as the glial line

    23    (phonetic), but no P24 is formed.  The same is true

    24    with the one which is a protein which is truncated.

    25                Shortly, the next question of course is

                                                                        398

     1    whether the same export pathway is used for HIV.  HIV

     2    uses the CREM pathway, and this can be blocked by a

     3    type called leptomycin B, and this is also active in

     4    the RcRE orf system, for example, in the presence of

     5    six nanomolar leptomycin, no P24.  In this case it's

     6    the major core protein of rev K itself, is formed, no

     7    N protein in this construct, but C orf is still

     8    produced and present in the nuclei and in the

     9    cytoplasm.

    10                So in summary the endogenous retrovirus

    11    family, we have K codes for protein which is

    12    equivalent to ref and rex.  That means that the

    13    nuclear export pathway was not only detected by

    14    reasoned lentiviruses or reasoned HIV-1 BLV viruses,

    15    but already 30 million years ago when this family most

    16    probably was active.

    17                What also makes this family interesting,

    18    observations by Nicholas over lunch and his group in

    19    Germany, he has indications that this virus may be

    20    orf'ed in oncogenesis and only in situation where C

    21    orf is also present.

    22                So the next study in the end, to find out

    23    whether this look like nuclear export protein may play

    24    a role without the conditions in carcinogenesis.

    25                Thank you.

                                                                        399

     1                (Applause.)

     2                DR. PEDEN:  Thank you, Johannes.

     3                I think because time is very short and we

     4    have to get out of the room I'd like to express my

     5    gratitude to the audience for staying so late, and

     6    especially to the speakers.

     7                So I think we should terminate this

     8    evening and be back tomorrow.

     9                Thank you.

    10                (Whereupon, at 9:55 p.m., the meeting was

    11    adjourned, to reconvene on Friday, September 10,

    12    1999.)

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