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

                                 + + + +

                                 Friday,
                            10 September 1999

                      The workshop took place in the Plaza 

          Ballroom, DoubleTree Hotel, 1750 Rockville Pike, 

          Rockville, MD, 20852, at 8:00 a.m., Regina Rabinovich,

          M.D. and Martin Myers, M.D., Session Chairs, 

          presiding.

                                                                          2

          PRESENT:

          Regina Rabinovich, M.D.       Session Chair
          Martin Myers, M.D.            Session Chair
          David Onions, Ph.D.           Panel Chair
          John Coffin, Ph.D.            Panel Chair
          Philip Minor, Ph.D.           Speaker
          James Robertson, Ph.D.        Speaker
          Joerg Schuepbach, M.D.        Speaker

          Jens Mayer, Ph.D.             Speaker
          Thomas Broker, Ph.D.          Speaker
          Neil Cashman, M.D.            Speaker
          John Sedivy, Ph.D.            Speaker
          Frits Fallaux, Ph.D.          Speaker
          Michael Fried, Ph.D.          Panelist
          Stephen Hughes, Ph.D.         Panelist

          Johannes Loewer, M.D.         Panelist

          Also Present:

          Gary Nabel

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                                I N D E X

          SPEAKER                      PAGE

          Philip Minor, Ph.D.            4

          James Robertson, Ph.D.        32

          Jorg Schuepbach, M.D.         51

          Jens Mayer, Ph.D.             66

          Thomas Broker, Ph.D.          78

          Neil Cashman, M.D.           102

          Panel Discussion             129
                Dr. Onions, Chair

          John Sedivy, Ph.D.           176

          Frits Fallaux, Ph.D.         199

          Panel Discussion             212
                Dr. Coffin, Chair

                                                                          4

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

     2                                               8:04 A.M.

     3                CHAIRPERSON RABINOVICH:  Good morning.  I

     4    would like to welcome you back to Session 6,

     5    Adventitious Viral Agents in Cell Substrates, and

     6    congratulate all those that were here until 10:00 last

     7    night for the latest part of the show, including Mr.

     8    Harris, who put in a grueling 18-hour day in

     9    yesterday, our visual aides person.

    10                I would like to introduce the first

    11    speaker, Dr. Phil Minor, from the National Institute

    12    of Biological Standards and Control, who will be

    13    giving us an introduction to adventitious agent

    14    issues, both reviewing the past and current experience

    15    with adventitious agent contamination of biologicals

    16    in vaccines.

    17                DR. MINOR:  Thanks.  Thanks very much. 

    18    Can I have the slide on, please, or do I just press it

    19    here?

    20                What I am going to do is to review firstly

    21    all of biologicals, if you like, from an adventitious

    22    agent point of view.  So it won't just be vaccines. 

    23    In particular, I will be talking about the range of

    24    source materials that people have used in preparing

    25    biologicals.  There will be a clear message that comes

                                                                          5

     1    out of that, which is that the more you use well-

     2    characterized cells, the better.

     3                I will also be talking about the SV40

     4    story in some detail, which has been gone through a

     5    number of times, but I will be going through it from

     6    a particularly regulatory point of view because again,

     7    there is a message there which says that if you get it

     8    wrong, you will still be working on it 40 years later.

     9                Finally, I will get onto the continuous

    10    cell line bit right to the very end.

    11                So there are a variety of source materials

    12    that you can use if you are preparing biologicals. 

    13    They are sort of listed here, if you like.  There are

    14    biological materials which are made from whole

    15    animals.  That would include things like blood and

    16    blood products.  I will describe that in a moment.  So

    17    you can just go to a whole animal and take something

    18    out and make your biological from that.

    19                You can use your whole animal as a

    20    substrate for growth.  I will discuss that in the

    21    context of things like influenza vaccines and the

    22    like.

    23                You can grow material on primary cells. 

    24    This was the main starting point for things like polio

    25    vaccines in the early days, where the SV40 issue

                                                                          6

     1    arose.  Finally, you can grow materials on well-

     2    characterized cell preparation.

     3                The further down the list you go on this

     4    thing, probably the happier you are from the

     5    adventitious agent point of view.

     6                This shows some examples of contaminants

     7    which have arisen when whole animals have been used as

     8    source materials or the origin of the source material. 

     9    Most of these will be human rather than anything else,

    10    but really an awful lot of the serious adventitious

    11    agent problems that have arisen have arisen because of

    12    materials sourced from whole animals or using pooled

    13    preparation.

    14                The first one on this list here is CJD,

    15    Creutzfeldt Jakob Disease, which was transmitted by

    16    growth hormone.  The growth hormone was produced from

    17    human cadaveric material.  A very unpleasant disease. 

    18    It's almost impossible to detect the agent other than

    19    by standing back and waiting for the incubation period

    20    to go.

    21                In France, there are still a large number

    22    of cases coming through as a result of this.  It may

    23    well be that around 10 percent are recipients of

    24    human-derived growth hormone, will actually wind up

    25    going down with CJD in France.

                                                                          7

     1                Dura mater is another one.  That should be

     2    one T, not two Ts.  Again, this has been shown to

     3    transmit really quite readily when pooled materials

     4    are actually used.  Almost impossible to detect. 

     5    Clear, very, very serious kind of consequence of it.

     6                Scrapie was first shown to be a

     7    transmissible agent by the use of a TBE vaccine, which

     8    was grown in the brains of sheep.  TBE being tick-

     9    borne encephalitis, which then transmitted scrapie to

    10    a large number of the sheep that were actually

    11    inoculated with it.  So again, this is a whole animal

    12    source material, if you like, that had quite serious

    13    consequences, especially if you were a sheep.

    14                Over the last 15 or 20 years or so, one of

    15    the best examples of serious or disease-causing

    16    transmissions of infectious agents has been through

    17    human blood and blood-derived materials, clotting

    18    factors in particular.  In all of these things, the

    19    entire alphabet soup of hepatitis viruses has been

    20    transmitted by blood product.

    21                In the early days at least, some of these

    22    were really regarded as really a hazard, if you like,

    23    of being a hemophiliac.  So, for example, hepatitis C,

    24    in the days when there was a non-A, non-B hepatitis,

    25    it was really regarded as an inevitable consequence of

                                                                          8

     1    using factor 8 to treat hemophilia.  I am not sure

     2    that that is an acceptable way of actually doing

     3    things any more.  I am sure that hemophiliacs would

     4    agree with that.  B-19, paravirus B-19 is still

     5    transmitted by clotting factors.

     6                Finally, this one down at the bottom here

     7    is a classic example of a transmission by a vaccine,

     8    if you like, where hepatitis B was transmitted by

     9    yellow fever vaccine back in the 1940s.  The hepatitis

    10    B actually came from the stabilizers of the albumin

    11    that was actually put in there to keep it stable.

    12                There is a story that Fred McCallum, who

    13    is head of the Public Health Service in the United

    14    Kingdom tells to the effect that he basically won the

    15    war because he prevented Winston Churchill having a

    16    yellow fever vaccine when he was going off to talk

    17    with Stalin around 1944.

    18                So most of the serious consequences really

    19    come from whole animal source materials, if you like. 

    20    You can use whole animals as substrates.  I'm using

    21    the term "whole animals" in a fairly broad sense. 

    22    Eggs in the definition of the Animal Regulated Use Act

    23    in the United Kingdom count as an animal because they

    24    are embryonated.

    25                For many years, rabies vaccines were

                                                                          9

     1    produced in mouse brain or sheep brain.  They have

     2    quite serious consequences, but not necessarily

     3    associated with adventitial agents.  You can get

     4    encephalitis as a result of immune responses to the

     5    non-invasic protein.

     6                The Japanese  encephalitis vaccine, which

     7    is used for travelers in the United Kingdom, is still

     8    made in mouse brain.  So it's not an unusual source of

     9    material, if you like.  Smallpox for a long time was

    10    made on the scarified flanks of calves.  Like I said,

    11    isn't any more.  However, while these things seem

    12    really quite primitive, in terms of how you make

    13    vaccines nowadays, you still have a number of vaccines

    14    that are made in eggs.  Yellow fever is the classic

    15    example, and influenza.

    16                Yellow fever is not required to be grown

    17    in avian leukosis-free eggs.  The reason for that is

    18    that there are a number of sites at which it was

    19    manufactured throughout the world, where yellow fever

    20    is a really very serious problem, such as Nigeria, for

    21    example, South America, whatever, where SPF eggs,

    22    avian leukosis-free eggs even, were really not freely

    23    available.  So yellow fever can in principle at least

    24    be made in avian leukosis containing eggs, and in fact

    25    is.  I think there's no evidence that this has an

                                                                         10

     1    adverse consequence.  But on the other hand, you

     2    wouldn't necessarily want to have a virus in there

     3    that you didn't know about.

     4                Influenza is an actuated vaccine.  Again,

     5    it's not made on SPF eggs, that is, specified

     6    pathogen-free eggs.  They are avian leukosis free, but

     7    they are not free of all the other variety of

     8    pathogens that you would choose to screen for measles

     9    vaccine production system, for example.

    10                So even today then you have to bear in

    11    mind that a large amount of vaccine that's made is

    12    made on really quite crude materials, from an

    13    adventitious agent point of view.  It's not a trivial

    14    usage.  In fact, when you go through and consider what

    15    vaccines are actually made on these days, they are

    16    quite primitive, if you like, in some respects.

    17                Primary cultures as been described

    18    previously around here, are really cultures that are

    19    made directly from the animal.  So they are not one

    20    pass.  They are directly from the animal, if you like. 

    21    Here are a number of examples where agents are

    22    actually being found or at least located in these kind

    23    of cultures.

    24                SV40 is one that I'm going to talk about

    25    in some detail in a minute.  This was in polio

                                                                         11

     1    vaccines in the 1950s and very early 1960s, probably,

     2    a source from rhesus monkey kidney.  Polio vaccines

     3    are still made on monkey kidney, though they are not

     4    usually on rhesus monkey kidney.  It would be

     5    cynomologous or something like that, for reasons which

     6    I'll describe in a moment.

     7                Nonetheless, a great deal of vaccine is

     8    still made in primary monkey kidney cells.  There are

     9    reasons for that.  There's a deep conservatism I think

    10    about changing the vaccine production process if you

    11    have a vaccine that works, largely because you are

    12    dealing with a prophylactic material rather than a

    13    therapeutic material.  So you don't want to mess about

    14    with anything if it's reasonably safe and effective.

    15                I'll mention very briefly the defective

    16    retrovirus story in chick embryos.  I think Jim

    17    Robertson will probably mention this in more detail,

    18    but I will mention that just as I go by.  Finally

    19    recently, the FDA released a talk paper on a

    20    preparation of urokinase, which is used in treating

    21    the heart.  This material was grown from primary

    22    cultures made from aborted fetuses.  I think it was

    23    aborted fetuses or miscarriages, or whatever.  There

    24    were quite a variety of infectious agents were

    25    actually found in this.  I believe this one has now

                                                                         12

     1    been suspended.

     2                The point is that there are still a large

     3    number of materials which are made on really quite

     4    basic culture systems, if you like, where adventitious

     5    agents are a serious consideration, if you like.  So

     6    it's not all continuous cell lines versus the rest. 

     7    I mean there are -- most of the vaccines that are made

     8    in the world probably come from other primary cultures

     9    or eggs or things of that nature.

    10                I will now talk about SV40.  I'm sure in

    11    this audience there are people who know far more about

    12    SV40 than I do.  But nonetheless, I'll talk about this

    13    from what you might call the regulatory adventitious

    14    agent point of view, if you like.

    15                So it's a very common polyoma virus of old

    16    world monkeys, and particularly rhesus macaques.  The

    17    difficulty with this was that when the rhesus macaque

    18    monkeys are sacrificed and a primary monkey kidney

    19    culture is made from him or her, as the case may be,

    20    a silent infection is set up.  So there is on evidence

    21    of infection just by looking at the cultures.  In

    22    fact, these cultures can throw out as much SV40 as

    23    they do polio, when you start infecting it with polio. 

    24    So you wind up with a culture that's just stiff with 

    25    adventitious agent which you really don't want.

                                                                         13

     1                It's able to transform non simian cells in

     2    vitro, and it can be tumorigenic if you have the right

     3    kind of animal that you put it into.  Between 55 and

     4    62, probably at least a third of all the vaccines that

     5    were made on these kind of cultures, because they were

     6    pooled and the like, were almost certainly

     7    contaminated with SV40.  It wasn't a trivial

     8    contamination.  It was really quite a serious

     9    contamination.

    10                Because it was mainly an activated polio

    11    vaccine, there wouldn't have been that much live SV40

    12    in it perhaps.  But SV40 is more resistant to formalin

    13    than polio is.  So almost everybody who received the

    14    shot of inactivated polio in the 1950s, which would

    15    include me, would have received live SV40 in some form

    16    or another.

    17                So the concern is really summarized here,

    18    which is basically that everybody, I mean this is my

    19    own take on it, that everybody -- I mean you can argue

    20    that it might not have been sort of everybody, but I

    21    think it probably was.  But almost everybody who

    22    received the full course of polio vaccine between 1955

    23    and 1965, also got live SV40 stuck into them.  That's

    24    millions of people basically.

    25                There were epidemiological studies that

                                                                         14

     1    were done at the time which really didn't cause much

     2    concern, but they can all be criticized.  Some of the

     3    studies were really quite short-term, about two or

     4    three years or so, looking to see if there were cancer

     5    effects basically, as a result of SV40.  It may be

     6    that two or three years is not enough to actually find

     7    such an effect, if it actually exists.

     8                The longest which was assumed was over a

     9    period of about 19 years.  Most of the individuals

    10    involved in that study would have been oral polio

    11    vaccine recipients rather than inactivated polio

    12    vaccine recipients.  So they have had it by mouth

    13    rather than by injection.  Again, you could argue that

    14    that might not be the right cohort to actually be

    15    looking at.

    16                So while the studies were reassuring, the

    17    most reassuring thing was that there was no sudden

    18    surge of cancers that you can actually trace back to

    19    polio vaccine usage in the United States or in Europe

    20    where these things were used in a big way.  So it

    21    really did seem that in the long term, over about 19

    22    to 20 years, there was no real cause for alarm.

    23                However, in 1992, Michaili Carboni and

    24    colleagues and others, a number of others, including

    25    Janet Butelle down in Texas and the like, identified

                                                                         15

     1    SV40 sequences which were present in a variety of

     2    relatively rare tumors.  So mysathelia, which is the

     3    asbestos tumor, osteosarcomas, pendymonas, actually

     4    the young chorioid plexus tumors of children, these

     5    sequences do appear to be genuine SV40 sequences.

     6                Where they come from is really not quite

     7    clear.  Part of the argument was that you could get

     8    similar types of tumors in experimental animals, like

     9    hamsters.  I think that is probably the only example

    10    where a hamster is cited as a good model for a human

    11    being perhaps.  But who knows?  In fact, this might

    12    actually be an argument that this has got nothing to

    13    do with it.

    14                So the question then arises as to where

    15    did the SV40 sequences come from.  Of course the

    16    classic response really would have been it must have

    17    come from the polio vaccine because why not?

    18                Now SV40 was discovered around 1961 or

    19    1962 or thereabouts, 1960 perhaps.  Directly it was

    20    discouraged.  There were precautions put in place to

    21    exclude it from polio vaccines, because it was known

    22    to be a tumor kind of virus, if you like.  These were

    23    the kind of things that were put in place.  They are

    24    listed in WHO requirements from about 1962 onwards. 

    25    They reached their final fully flowered form, if you

                                                                         16

     1    like, by about 1965.  A number of countries certainly

     2    had put this in place before that.

     3                The first thing you can do is to use

     4    seronegative animals as the source of cells.  So you

     5    can use animals that have no evidence of SV40

     6    infection as your source.  That really is something

     7    which is now very firmly in place, which manufacturers

     8    now do.

     9                The second thing is, you remember I said

    10    that it was the rhesus macaques with the problems. 

    11    The problem was that the cell cultures didn't show any

    12    sign of having defect, when they were actually

    13    infected with SV40.  What you can do then is you can

    14    use species, such as cynomolgus or pattus monkeys,

    15    where the primary monkey kidney culture cell, when

    16    infected with SV40, will actually wrinkle up and die

    17    on you.  So at least you know you have got something

    18    nasty and you can throw it away.

    19                Finally, you can test your control of

    20    production cultures for SV40 by the same kind of

    21    procedure.  That is why using sesetral cells to see if

    22    anything comes through.

    23                Around the period that this was taking

    24    place, wild caught monkeys were being used extensively

    25    in vaccine production.  Up to a half of the cultures

                                                                         17

     1    would have been thrown away because of adventitious

     2    agent contamination, mainly foamy virus, but certainly

     3    other things as well.  I think that just illustrates

     4    the kind of lack of control, if you like, over the

     5    source materials that was going on, and the extent to

     6    which adventitious agents are really a serious problem

     7    in finding monkey kidney cultures or primary cultures

     8    in general.

     9                An alternative way of doing this is to

    10    actually use a validated cell bank.  Certainly many

    11    manufacturers use MRC5, and Mary of course used vira

    12    cells, as we heard last night.

    13                Nonetheless, a significant, if indeed not

    14    a large proportion of the world's supply of polio

    15    vaccine is still made on primary monkey kidney cells,

    16    which should really fit this kind of criteria for

    17    excluding SV40.

    18                One of the questions that then arises is

    19    were these precautions good enough?  What we did at

    20    NIBSC, because we happened to have about 150-odd

    21    batches of vaccine archived from the years, was to go

    22    back and look at them by PCR.  PCR of course is the

    23    cat's pajamas.  It's really the best technique that

    24    anybody ever invented in terms of sensitivity.  It's

    25    probably about as good as infectivity, at least in our

                                                                         18

     1    hands anyway.

     2                But nonetheless, we went back and we

     3    looked by PCR at 133 preparations of polio vaccine

     4    which had been used in the United Kingdom between 1966

     5    and about 1997.  What we had done was looked at all

     6    batches of vaccine which had been used since 1980, and

     7    all of those were free of SV40 sequences.  So that

     8    gives you some reassurance that these precautions were

     9    actually appropriate.

    10                In fact, the only preparation which had

    11    any SV40 sequence in it at all was a seed virus which

    12    was used by a manufacturer for making vaccine from. 

    13    The amount that was in there was around two logs worth

    14    of genome as opposed to seven logs of genomes in a

    15    really full-fledged infected preparation.  So there

    16    wasn't that much in there.  The manufacturer had also

    17    treated this stuff with toluidine blue, which is

    18    supposed to kill of SV40.  This was done on the advice

    19    of Albert Sabin back in 1960-something or other.

    20                But nonetheless, it does seem to me that

    21    it's rather a foolish thing to have a seed that's got

    22    SV40 sequences in it at all.  I think the WHO

    23    requirements have now been changed so the seed has to

    24    be checked to see if it does have SV40 sequences in 

    25    it or not.

                                                                         19

     1                This particular seed was not infectious

     2    SV40.  We did some quite serious studies on it, like

     3    transvecting the DNA into cells to see if it would

     4    work, infecting monkeys with it to see if we could

     5    actually get seroconversion.  There was no

     6    seroconversion.  So there was no infectious virus

     7    there that we could actually detect.  But nonetheless,

     8    the seed did have material in it.

     9                If on the other hand you look at materials

    10    from around the 1960s or from other parts of the world

    11    a little bit later than that, you can pick up SV40

    12    sequences quite easily.  So the method would have

    13    picked it up had it been there.

    14                So our conclusion from that was then that

    15    really as soon as these kind of precautions were put

    16    in place, no SV40 would have been present in all polio

    17    vaccines used, at least in the United Kingdom and I

    18    would guess in the United States as well, because it's

    19    after the same kind of precautions were put in place. 

    20    So the precautions were adequate.  Which means that

    21    SV40 exposure of the population through polio vaccines

    22    would have stopped around 1962.

    23                So what you then have is the problem of

    24    the chorioid plexus and appendinoma tumors, which

    25    occur in children who are around two years of age or

                                                                         20

     1    maybe less.  You have to say well how did they get a

     2    hold of the SV40 sequences?  One possibility, which is

     3    mooted with some enthusiasm is that maybe you are

     4    getting passage of SV40 from parents who did receive

     5    the SV40 contaminated polio vaccine to their children. 

     6    So how this stuff gets around is quite important.

     7                One of the things that we have been

     8    involved in is doing serological surveys of

     9    populations to see who has got SV40 antibodies and who

    10    hasn't.  It is about a five percent seropositivity by

    11    the assay that we're using at least.  It seems to peak

    12    at around age 10 or thereabouts, and doesn't arise

    13    after that.

    14                So what you could argue then is that you

    15    are seeing vertical transmission from parents down to

    16    their children.  What you could also argue is that you

    17    are not picking up SV40 specific antibodies at all,

    18    and they could be other human polyomas like the BK or

    19    the JC, and it's cross-reacting antibodies that we're

    20    picking up.  I think that is still a thing that needs

    21    to be resolved.  This is how we were trying to resolve

    22    it.

    23                We have access to a number of sinomorgous

    24    breeding colonies.  One of them at least is absolutely

    25    riddled with SV40.  It's chronically infected.  They

                                                                         21

     1    are all infected basically.

     2                So this is just four examples of this

     3    particular colony.  There's about another 50 or so. 

     4    This happens all the time.  The mothers here are

     5    highly sera positive to SV40, all of them.  What

     6    happens is that the mother and the baby stay together

     7    for about six months until the baby is weaned.  Then

     8    the babies are taken off, no longer being babies of

     9    course.  They are all banged up together in one

    10    gigantic sort of teenage squabbling colony.

    11                At the time of weaning, the babies are

    12    uniformly negative.  So despite the fact they have

    13    been on the mother for six months, they have not sera

    14    converted to SV40.  Almost immediately you bang them

    15    up together, or at least within about a month or so,

    16    they sera convert.  So we actually have a sera

    17    conversion panel here, if you like, with about 50 or

    18    100 or so sera, where the babies actually were

    19    seronegative and then become seropositive.

    20                My view on this is probably that the

    21    babies don't get infected until you bang them up

    22    together.  But it may be that they are infected, but

    23    they are just not seropositive.  So what we have to do

    24    here is to fish out the virus from these animals here,

    25    and see if it looks like the mother's virus or if it

                                                                         22

     1    looks like the other babies' virus.

     2                The point about this long story which I

     3    have just been telling you about SV40 is that SV40 was

     4    a problem between 1955 and 1962, and it's now 1999,

     5    and we still don't really know what was going on.  So

     6    if you actually make a mistake, it's really quite

     7    serious.  It may keep you occupied for the rest of

     8    your working life.

     9                One last quick thing or two last slides

    10    here.  One is about reverse transcriptase of vaccines. 

    11    Dr. Schuepbach will be talking later and Jim Robertson

    12    will be talking in a moment about detection of reverse

    13    transcriptase in chicken cell grown vaccines, such as

    14    flu or yellow fever or measles, mumps, rubella.

    15                This appears to be due to the presence of

    16    defective non-infectious particles.  There are

    17    sequences from EAV and ALV both in these things, a

    18    ratio of about nine to one as I understand it.  It

    19    does seem to me that you are not really quite sure

    20    what the AV sequence is in there and what ALV sequence

    21    is in there.  It's probably going to vary from chicken

    22    to chicken in so far as these chickens have not been

    23    bred.  In other words, every egg is a new experiment. 

    24    You are really not quite sure what you are dealing

    25    with in that.  I think that is quite an unfortunate

                                                                         23

     1    position to be in.  I'm not sure how you control it.

     2                Finally, this is my last slide, and this

     3    has to do with characterized cells.  The issues that

     4    I have been dealing with really have been to do with

     5    primary cells and primary cell problems where the

     6    virus comes in direct from the animal origin.  I think

     7    there is no doubt in my mind that that's the main

     8    source of concern in terms of human health.

     9                Nonetheless, there are clearly problems

    10    which also arise with characterized cells and the

    11    continuous cell lines, in particular.  We have some

    12    down here.

    13                Now the regulatory authorities in the room

    14    will be well aware of a large number of other examples

    15    of this type which don't actually get published.  I

    16    think that's not so good.  I think this stuff really

    17    should be out there in the public literature.  But

    18    nonetheless, these are the ones which are well known,

    19    I think.

    20                CHO viruses, CHO cells have defective

    21    retroviruses.  Manufacturers take a great deal of care

    22    to actually get rid of them in the final product.  So

    23    they are endogenous.

    24                There are examples of things like BVDV

    25    contaminating cells which are growing in culture, and

                                                                         24

     1    also other bovine viruses contaminating cells in

     2    culture, particularly when they are grown on a very

     3    large scale.  Whether or not that poses a hazard is

     4    another matter, but clearly there must be methods in

     5    place to actually detect them.

     6                The classic example here was the minute

     7    virus in mice, where the tpa had been grown in CHO

     8    cells on a 10,000 litre stove essentially, and then

     9    tiled up for an effect with minute virus of mice.  Now

    10    this was on the order of eight logs, as I understand

    11    it, of virus per mil, and yet a 10,000 litre fermenter

    12    culture.  This is probably more minute virus of mice

    13    in one place on the planet than has ever been the case

    14    before.  You might want to think how you actually get

    15    rid of it actually.

    16                This is a question of actually getting the

    17    cells infected while they are actually burning in

    18    culture.  So while family cells are clearly a major

    19    problem, and while whole animal sources, if you like,

    20    are probably the biggest hazard which is likely to be

    21    raised in terms of human health, biological aspects to

    22    do with well characterized cell banks, where viruses

    23    may be introduced from biological materials or they

    24    may be introduced by mice walking across the top of

    25    the fermenter or whatever, are nonetheless a

                                                                         25

     1    significant matter.  It really is not totally clear

     2    whether these things have an implication for human

     3    health.  But I think you would be wise to make sure

     4    that they are not actually present.

     5                That's where I stop.  Thank you.

     6                (Applause.)

     7                CHAIRPERSON RABINOVICH:  Please identify

     8    yourself.

     9                DR. COFFIN:  John Coffin of Tufts.  That

    10    was a really nice summary actually, Phil.  But some

    11    caution might be called for in translating the results

    12    of vertical transmission experiments from monkeys to

    13    humans.

    14                As far as we know, simian immuno

    15    deficiency virus in monkey populations are not

    16    transmitted vertically.  Yet HIV-1 is transmitted with

    17    reasonable efficiency vertically in human populations. 

    18    So there may be some underlying biological difference

    19    that perhaps a very subtle one, that promotes this

    20    kind of transmission in people, where you wouldn't see

    21    it necessarily in monkey models.

    22                DR. MINOR:  Yes.  I take you point.  We

    23    are doing the studies for two reasons.  Firstly, to

    24    look at natural transmission to monkey on the grounds

    25    that it might be a model, although I take your point

                                                                         26

     1    entirely.  But also to supply serum conversion panels

     2    so that we can try and sort out specificity of

     3    immunological reactions as well.  I take your point

     4    entirely.

     5                DR. ONIONS:  David Onions, Glasgow.

     6                Phil, when people switched to cynomologous

     7    monkeys, and I can see the reason because you can pick

     8    up SV40.  That's very clear.  But how do you know that

     9    at the same time, you have not invented a new problem,

    10    that you have got another polyoma virus in that

    11    species that you are not detecting.  I mean has anyone

    12    done redundant PCR to look?

    13                DR. MINOR:  I think I would choose to look

    14    at a polyoma man to answer that question.  Anybody?

    15                DR. MAJOR:  Gene Major, Bethesda.  In the

    16    monkeys that we have used for the human polyoma virus

    17    studies, we have screened all those animals for the

    18    conventional SV40, but not have addressed the question

    19    of whether or not there's other additional polyoma

    20    type viruses that are present there.

    21                Clearly by this time, PCR technology has

    22    advanced to the point that perhaps if something were

    23    there whose sequences were somewhat similar to the

    24    ones that are currently expressed in these animals, we

    25    may have picked it up, but we certainly haven't found

                                                                         27

     1    anything yet.

     2                DR. LEWIS:  Phil, I take it that

     3    seroconversion is by neutralizing antibody?

     4                DR. MINOR:  Sorry?  Say that again.

     5                DR. LEWIS:  I take that seroconversion is

     6    by neutralization?

     7                DR. MINOR:  That's right.  Yes.

     8                DR. LEWIS:  Have you had any chance to

     9    look at monkey breast milk to see how long they may be

    10    treating antibodian, so that the newborns can be

    11    passive immune?

    12                DR. MINOR:  Right.  The answer is no.  We

    13    have discussed, for example, getting the urine out of

    14    these monkeys after they are banged up together.  I am

    15    told that actually chasing them around the floor is

    16    insuperable.  I'm not sure you can actually pry a baby

    17    monkey from her mother long enough to actually milk

    18    her.  It's a worthwhile question.  I think we'll have

    19    another go and see if we can do something about it. 

    20    There may be some resistance, however.

    21                DR. BROKER:  Tom Broker, UAB.  I wanted to

    22    follow up on that exact question.  We are facing the

    23    same problem with potential vertical transmission of

    24    human papilloma viruses.  I'll mention it later in my

    25    own presentation, but briefly, it does appear there is

                                                                         28

     1    some protective immunity during nursing.  On the other

     2    hand, removal of an infant, say through adoption to

     3    another family, is the highest risk factor for a child

     4    acquiring laryngeal papillomatosis later in life.  So

     5    a two to three year delay.

     6                DR. MINOR:  So is the assumption then that

     7    the infant is infected, but it's not infected properly

     8    then?

     9                DR. BROKER:  It's infected vertically,

    10    presumably perinatally, perhaps just before or during

    11    delivery, but receive sufficient passive immunity by

    12    nursing that gives it life long protection.

    13                So what I would propose as a potential

    14    experiment is to literally take the, if possible, take

    15    the baby monkey immediately away from the mother, and

    16    don't allow it to nurse, and then just have different

    17    lengths of time of nursing to see if this onset of

    18    seroconversion is affected by a timing mechanism. 

    19    Alternatively, don't ever let that baby monkey be

    20    housed with other baby monkeys in the daycare center,

    21    and keep it with the mother even if it's not nursing,

    22    and see if it fails to seroconvert.

    23                So the question is, is it getting infected

    24    from its playmates.

    25                DR. MINOR:  That's right.

                                                                         29

     1                DR. BROKER:  Or is it receiving a period

     2    of important passive protection from the mom.

     3                DR. MINOR:  I figured we could do that by

     4    looking at the actual strain of viruses the monkeys

     5    get infected with.  We have a number of different gang

     6    rooms, if you like.  If you get a different strain in

     7    each gang room, but it's the same strain within a gang

     8    room, then I think that will answer the same question. 

     9    You can also go back to the mother and see what kind

    10    of strain she's got too.  But it's a valid point.

    11                AUDIENCE MEMBER:  I would like to

    12    reemphasize one of the important points that you made. 

    13    I know you didn't have time to expand on it, but I

    14    think it is extremely important.  That is the need for

    15    those organizations who discover a new virus or some

    16    contaminant, cell population used for vaccine

    17    production or in a production run, to make that

    18    publicly known.

    19                I think that the declaration by Genentech,

    20    who has published this information under their name,

    21    that an NBM contamination occurred in a 10,000

    22    fermenter is an act of great courage.  I think that

    23    that kind of courage, this declaration by other

    24    companies in this field, is very necessary for the

    25    health of this industry.

                                                                         30

     1                I understand from some of the remarks that

     2    have been made that there are others that are known to

     3    a small coterie of people here that have not been

     4    publicly declared.  I urge all of you to think about

     5    this seriously because it can and will have a great

     6    impact on this industry.  Thank you.

     7                DR. MINOR:  I agree totally with that.  It

     8    does seem to me that sooner or later the information

     9    will leak out.  I think the industry looks very bad.

    10                DR. VAN DER EB:  Van der Eb, Leiden.  Did

    11    I understand it correctly that ferrisfaruses were

    12    found in human embryo material that was used for

    13    urokinase production?

    14                DR. MINOR:  I think the FDA can answer

    15    this one better than me, yes.  But I mean that was my

    16    understanding of it.  It's out on the net.

    17                DR. VAN DER EB:  But where does it come

    18    from?

    19                AUDIENCE MEMBER:  I think it's a rea

    20    virus.

    21                DR. MINOR:  It's various rea viruses, plus

    22    others.

    23                DR. VAN DER EB:  I see.  Okay.

    24                DR. FRIED:  Mike Fried.  Was any of the

    25    old vaccines from the 1960s that were contaminated,

                                                                         31

     1    were they PCR'd up to show that the virus was the same

     2    as being found today?  Because it's also possible that

     3    we all have a latent SV40 type virus which likes to

     4    grow in tumor cells, and that's why you find it.  It's

     5    a passenger.  But I mean since there's polymorphisms

     6    in the sequence, if you can go back to the 1960s and

     7    then find out if it's the same thing that we find

     8    today, it would be helpful.

     9                DR. MINOR:  We looked at, when you say the

    10    1960s, I have to emphasize this is very early 60s. 

    11    Certainly the things that we have got which came out

    12    positive weren't used in the UK, or they might have

    13    been used somewhere else.

    14                We had a Russian SV40 and we had an

    15    American SV40, and we had an SV40 of unknown origin

    16    all from the 1960s, and they were all different

    17    basically.  They were different from the 7-7-7, you

    18    know, the cos kind of sequence as well.  So they were

    19    all unique basically, in terms of the region we were

    20    looking at, which was C terminus of t antigen.

    21                CHAIRPERSON RABINOVICH:  Last question

    22    please.

    23                MS. MARCUS:  Carole Marcus Sequora from

    24    Bassey Consulting.

    25                I just wanted to clarify that urokinase is

                                                                         32

     1    produced from cells.  It's not aborted fetuses.  It's

     2    newborns who did not survive.  Just for the record.

     3                DR. MINOR:  Thank you.

     4                MS. MARCUS:  It was rea virus.

     5                DR. MINOR:  I'm sorry about that.

     6                CHAIRPERSON RABINOVICH:  Thank you.  Our

     7    next speaker is Dr. Jim Robertson, speaking on

     8    experiences with retroviruses in avian and mammalian

     9    cell substrates.

    10                DR. ROBERTSON:  Good morning.  For those

    11    of you who don't know, NIBSC is CBER's cousin from

    12    across the pond in the U.K.  What I am going to do is

    13    pick up where Phil left off and concentrate on the

    14    retrovirus aspect of viral contamination.  Initially

    15    I will look at say biologicals in general, but

    16    ultimately focusing down on the vaccine issues.

    17                So I will begin with some direct

    18    information regarding retrovirus situations with

    19    biologicals.  I will go onto look at how some of the

    20    regulatory guidelines deal with the issue of

    21    retroviruses.  I will go into look at RTase testing,

    22    which is a reasonably current them just now, and

    23    finish up looking at the recent situation of the

    24    finding of retroviral-like particles in avian cells.

    25                So to begin with, here is a short list of

                                                                         33

     1    the incidence of retrovirus contamination found in

     2    biologicals in general, not just vaccines.  I have

     3    sub-divided these into two groups here.  You see this

     4    upper half here, this is where we have in the past had

     5    overt adventitious contamination by a retrovirus of a

     6    biological.  For instance, being mentioned earlier,

     7    ALV, that causes virus in yellow fever vaccine by

     8    virtue of producing the vaccine in embryonated eggs

     9    infected with the virus.  The other one that was

    10    mentioned earlier by Phil, HIV and blood products.

    11                The bottom half here is a quite, somewhat

    12    separate type of contamination.  In fact, you might

    13    find it equaler to call it contamination or not. 

    14    Certainly these are not adventitious situations. 

    15    These are situations in which an endogenous

    16    retroviral-like particle is present in the

    17    manufacturing process.

    18                In the first instance here, it's

    19    established that murine hybridomas used in the

    20    manufacture of monoclonal antibodies produced,

    21    secretes C type particles.  These have been tested in

    22    a variety of other mammalian, including human cell

    23    lines, and generally are not infectious.

    24                The titre can be very high for these types

    25    of particles.  You can get 10 to the sixth particles

                                                                         34

     1    per mil.  I've even seen 11 particles per mil in one

     2    instance.  So you can have a very high burden of

     3    direct viral particles.

     4                It is also well established that CHO

     5    cells, which are used for producing

     6    biopharmaceuticals, secretes C type particles.  You

     7    also get intertestinal type particles from these

     8    cells.  These are probably much more characterized, a

     9    bit more work has gone into describing the particles

    10    from CHO cells, sequence information from the

    11    endogenous elements within the CHO genome, which is

    12    producing these particles, give some ideas as to why

    13    they are defective.  The reading frames are

    14    incomplete.  There are stop signals.  So you don't get

    15    a proper infectious virus from these endogenous

    16    elements.

    17                The latter type is the only type here

    18    that's dealing with vaccines, even dry vaccines

    19    produce either an ovo or cef cells.  I'll come back to

    20    that in a few minutes.

    21                From a regulation point of view, how do we

    22    deal with virus contamination and retrovirus

    23    contamination?  There are a couple of guidelines I

    24    would like to bring to your attention.  The first one

    25    here is an ICH guideline, which looks at viral safety

                                                                         35

     1    evaluation.  Admittedly it is only for biotech

     2    products.  The scope of the guideline does comment

     3    that this is not, this guideline is not applicable to

     4    vaccines.  But I think it is worth looking at what it

     5    says about virus contamination.

     6                Within the document, it describes five

     7    different cases of potential contamination, starting

     8    from the most desirable case, where you don't have a

     9    virus present in the process in any way, down to the

    10    worst scenario where you know you've got a virus, but

    11    you haven't a clue what it is.

    12                The guideline goes on to state what is

    13    acceptable and what is not acceptable in the

    14    manufacturing process.  The only two cases which are

    15    generally acceptable of the first two cases, Case A,

    16    where you have got no virus, and Case B, where you

    17    have got a non-pathogenic retrovirus.  The other cases

    18    are only exceptional.  Generally you don't want one at

    19    all.  The manufacturing is not allowed when you have

    20    got a virus contamination.

    21                So for Case B, really what you have here

    22    is a murine retrovirus is probably the only

    23    contaminant acceptable in the bulk harvest.  If you

    24    remember this guideline is applicable only to

    25    recombinant products and not to vaccines in general,

                                                                         36

     1    and these recombinant products are highly purified.

     2                The other guideline is the WHO

     3    requirements which came out recently for use of animal

     4    cells as in vitro substrates.  That does include

     5    vaccine production.

     6                When it comes to testing for retroviruses,

     7    this guideline has several -- many other guidelines in

     8    the past have indicated, the types of assays being

     9    used for retroviruses, specific infectivity assays,

    10    electron microscopy and transcriptase assays, are the

    11    three general approaches for checking for retrovirus

    12    contamination.

    13                There may be the use of specific antigen

    14    detection as is in some particular cases, but these

    15    are the generally recognized methods of going about

    16    picking up retros.

    17                If I can concentrate now on the RTase

    18    assays.  The traditional type of assay involves

    19    incorporating a nucleotide precursor, a labeled

    20    precursor of some kind into an assay using a rather

    21    synthetic type of template.  Then more recently of

    22    course we have the PERTs, PB RT, AMP RT type of

    23    assays, which includes a PCR amplification step, with

    24    the result that these type of assays are incredibly

    25    more sensitive than the more, as I can call it,

                                                                         37

     1    traditional type of assay, and what is often quoted as

     2    up to a million fold times more sensitive by virtue of

     3    incorporating a polymerase chain reaction.

     4                Now using this type of assay, the cat was

     5    set among the pigeons.  When this paper came out, I

     6    might even say that the fox was set amongst the

     7    chicken coop.  Detection of reverse transcripted

     8    activity in live attenuated virus vaccines.  This

     9    quite naturally caused a bit of concern as to what was

    10    going on here.  The vaccines indicated, the one common

    11    feature was that ovine produced in eggs of some kind,

    12    measles vaccine out of CEF primed cultures, similarly

    13    mumps.  Yellow fever and influenza in ovo.  But not

    14    measles vaccine out of human diploid cells or rubella

    15    vaccine out of human diploid cells.  So the common

    16    link here seemed to be the CEF, the chicken source

    17    used in the production of the vaccine.

    18                We joined in the boat here and started

    19    looking at this issue.  Every type of hen fluid that

    20    we have looked at, CF fluids or an type fluid from a

    21    variety of different strains of hen have all been

    22    positive in the assay for reverse transcriptase. 

    23    Summarily, quail, jungle fowl, and pheasants are

    24    positive.

    25                The types of sources of fluids which have

                                                                         38

     1    been negative for reverse transcriptase are listed

     2    here.  Some species are not positive, turkey and duck

     3    cultures, quite a range of human cell lines.  Simian

     4    rabbits have been tested and found to be negative.  So

     5    the clear source of this RTase that was being picked

     6    up in the vaccines is quite clearly of chicken avian

     7    origin.

     8                We would want to look at -- I should add

     9    that this RTase was known at the time to be particles

    10    associated and appears in the supernate of the cells. 

    11    We are going to look at this particle to see if it

    12    would pick up any infectivity.  In all, we looked at

    13    10 different cell lines, mainly human, but including

    14    rabbits and turkey.  Over 21 tests and 116 passages.

    15                In each case, in every test and at every

    16    passage level, the cultures were negative for reverse

    17    transcriptase activity.  There's absolutely no

    18    indication that this particle is infectious.  Since

    19    then, CBER and CDC have also come up with similar

    20    data, including use of PBMCs.  No infectivity

    21    associated with these RTase containing particles.

    22                Where might these be coming from? 

    23    Presumably they are derived from endogenous

    24    retroviral-like elements in the chicken.  The

    25    information to date regarding such elements in the

                                                                         39

     1    chicken genome are quite well characterized EV loci,

     2    which are related to the avian chosis virus family,

     3    and more recently discovered about 10 years ago, EAV-0

     4    family, which is an older element than EV, and then

     5    older still, ART-CH and CH-1 elements.

     6                The information at the time and pretty

     7    much where it still exists is that we knew that

     8    there's a line of chickens which was negative for EV. 

     9    It had been eliminated from the genome.  This line of

    10    chickens, the culture fluids were positive for RTase. 

    11    So we knew that it had to be at least one of these

    12    elements producing RT activity.  At the same time, you

    13    couldn't eliminate the fact that EV might also be

    14    producing RT activity.  The best bet was EAV-0, given

    15    the sequence information that was present at the time.

    16                More recently, in the last year or two,

    17    Joerg Schuepbach's laboratory has produced a good

    18    evidence for the presence of EAV-0 derived RNA

    19    associated with the RT particles secreted from CEF

    20    cells, and then this year, Walid Heneine, CDC, also

    21    produced the presence of EAV and ALV RNA.  When I say

    22    ALV, I mean derived from the EV loci and not exogenous

    23    ALV contaminating RNA.

    24                So what can happen here retrovirus-like

    25    particles, defective particles being produced from

                                                                         40

     1    endogenous elements both from EV and EAV-0 family of

     2    endogenous elements.  The presence of the RNA and

     3    relsconstrictase in a particulate fraction leads one

     4    to come to the conclusion that we have retroviral like

     5    particles in the CF fluids of the chick cells, which

     6    is present in the vaccines measles and mumps.

     7                The absence of infectivity in the current

     8    genetic information, sequence information that we have

     9    on EV loci and the EAV-0 family of endogenous elements

    10    strongly indicates that these particles are defective

    11    viral particles. The only question mark that remains

    12    from the regulation point of view, but also scientific

    13    point of view, the possibility of pseudotype formation

    14    during vaccine manufacture.  The current evidence

    15    suggests the particles that are defective in the

    16    envelope-like protein and so there's a particulate of

    17    pseudotype formation with the glycoprotein of vaccine

    18    viruses being grown in the CF cells.

    19                So to summarize a couple of these issues

    20    then, from the practical point of view, testing for

    21    reverse transcriptase as an indicator of retroviral

    22    contamination, these assays are evolving, changing all

    23    the time.  One has to take into consideration the

    24    strength of the assay and the validity of the assay. 

    25    There may be different requirements within an assay

                                                                         41

     1    for different sources of RT.  It may be necessary to

     2    use some other sort of method to asses the

     3    significance of any RT detected because we know that

     4    RT activity can derive from other enzymes.  Telomerase

     5    is or DNA polymerase, cellular DNA polymerase is. 

     6    These features are not specific to the more recent

     7    sensitive type of assays involving PCR, the parents,

     8    and the PBRT.  These features were also factors that

     9    had to be considered in the more traditional types of

    10    assays.

    11                It is often quoted that the RT levels in

    12    chick cells is very low, given that it was detected by

    13    a very, very sensitive assay, and has not been

    14    detected by the more traditional type of assays. 

    15    Certainly some preliminary data that I have got

    16    suggests that it is not quite as low as we first

    17    thought.  Really this RT activity in chick cells and

    18    ultimately in vaccines is only just below the level of

    19    detection of the more traditional type of assay.  In

    20    fact, this was a relatively novel phenomenon

    21    discovered just a few years ago.  It was in fact first

    22    reported 20 years ago in the late 1970s by Berne and

    23    Hofschneider at the Max Planck Institute in Munich. 

    24    They reported the presence of a novel type of RT

    25    enzyme in chick embryos and in chick cells.  That was

                                                                         42

     1    in the days before PCR.

     2                So the level -- I certainly believe the

     3    level of RT and the level of these particles is

     4    actually quite high in chick cell fluid.  Ultimately

     5    I think what we have to do is look into the need for

     6    standards, standard materials in some way to assess on

     7    a quantitative basis the level of RT activity in chick

     8    cells, in measles vaccine, in mumps vaccine, in order

     9    to come up with some meaningful conclusions regarding

    10    it.

    11                So to look at the RT issues from a

    12    regulatory point of view, a couple of comments I would

    13    make.  First, that these are state of the art

    14    technologies.  When these highly sensitive assays

    15    first came about, it posed very useful from a research

    16    point of view to what use are they in a routine

    17    manufacturing validated type of assay.

    18                I think the time has come where yes, you

    19    would say that these are state-of-the-art techniques

    20    and can be and should be used for detecting the

    21    presence of RT in your manufacturing process. However,

    22    when it comes to, for example, chick cells, and until

    23    we have a greater understanding of what the levels

    24    might mean, and until standards are available, there

    25    is really -- it is difficult to justify any

                                                                         43

     1    requirements to perform RT or PBRT assays on systems,

     2    and basically here I am talking about chicks, which

     3    inevitably will be positive.  We know they are going

     4    to be positive, that there's no great need to actually

     5    require any manufacturer to do these assays.  But

     6    certainly there is a still a strong requirement to

     7    provide evidence for freedom from retrovirus

     8    contamination.  This will have to derive from other

     9    data.  Thank you.

    10                (Applause.)

    11                CHAIRPERSON RABINOVICH:  Thank you, Dr.

    12    Robertson.

    13                Any questions?

    14                AUDIENCE MEMBER:  Just a comment.  For

    15    known endogenous avian retroviruses or exogenous avian

    16    retroviruses, of the cell lines, of the cells that you

    17    tested for infectivity, only the turkey cells would

    18    have given a positive result.  I would urge for avian-

    19    derived -- urge the use of those cells, and a PERT

    20    assay is a sensitive readout, for detection of perhaps

    21    unknown agents in these vaccines, end products, as

    22    being the most sensitive, at least for avian-derived.

    23                DR. ROBERTSON:  Yes, yes.  The turkey

    24    cells are sensitive for rav, because it's virus, but

    25    apparently not for the RTase.  I have no idea, duck

                                                                         44

     1    cells are also negative, but I have no idea if duck

     2    cells are susceptible to the --

     3                AUDIENCE MEMBER:  They are not as good as

     4    turkeys.  Turkeys themselves actually are not

     5    sensitive to all exogenous ALVs, but to most they are,

     6    and all endogenous ones.

     7                DR. ONIONS:  David Onions.  I really

     8    enjoyed that, Jim.  I just want to make a comment on

     9    your comment about standards.  I think as we heard

    10    from Keith last night and what we're doing, and I'm

    11    sure George is doing too, using the tac man technology

    12    where you can actually quantitate the PCR product. 

    13    Then if you actually do EN counts of virus particles,

    14    dilute these out, you can actually quantitate your

    15    assay system and actually determine the number of

    16    particles you can detect.

    17                Now it seems to me that that is a useful

    18    kind of standardization, and that you can then relate

    19    that to if you like, a consistency of your starting

    20    material, in this case the egg.

    21                So I think in that case, applying those

    22    techniques does have value, because it gives you a

    23    kind of lock-to-lock consistency of your materials. 

    24    So that if something goes out of spec, then perhaps

    25    something odd is going on in those materials.

                                                                         45

     1                DR. ROBERTSON:  Whatever we want to

     2    approach this standardization, one would have to do it

     3    on a quantitative basis.  I was sensitive earlier to

     4    quote any actual figures, but to quote you some

     5    figures that I have got so far, in one chick cell

     6    preparation, the culture fluid, there was the

     7    equivalent of the order, and this the first of

     8    investigation, 10 to the 4 focus forming units of rav

     9    in uninfected chick cell fluid.

    10                A large current high level is when your

    11    typical infection goes up to 10 to the 6th, I believe,

    12    focus forming units.  You are only talking about 104

    13    drop, lower value.  So you are not far away.  It is

    14    going to be difficult.  If you have got an overt

    15    contamination going up to 10 to the 6th, I think that

    16    will be quite clear on a quantitative basis.  But once

    17    you drop down a bit, it is difficult to say whether

    18    you have got an infection or whether it's just

    19    background level of endogenous RT-derived activity

    20    that you are picking up.

    21                DR. MYERS:  Martin Myers from National

    22    Vaccine Program.  As I sit and count the number of

    23    immunizations that various populations receive with

    24    these particles in it, repeated immunizations with it,

    25    I wonder if there is any data on sero responsiveness

                                                                         46

     1    in longitudinal --

     2                DR. ROBERTSON:  Well the reason I am

     3    looking over your shoulder, we have Walid Heneine from

     4    CDC.  I'm not sure if you are going to say something

     5    along those lines, but serologically, there is no real

     6    evidence for reaction to ALVs.  Epidemiologically,

     7    when it first came out, we also, not ourselves but

     8    epidemiological colleagues, to provide information. 

     9    There is no evidence again, for any increase in the

    10    incidence of childhood cancers since the onset of

    11    measles, mumps vaccination.

    12                Walid, were you going to say something?

    13                DR. HENEINE:  Yes, I have.  Regarding

    14    transmission risks, so far the data we have where we

    15    have looked at the presence of antibodies to avian

    16    leukosis virus by western lot, I'd say we developed as

    17    well as presence of ALV sequences and EAV sequences in

    18    the peripheral blood lymphocyte from vaccinated kids

    19    as well as in plasma.  So far, the results have been

    20    all negative for both viruses.

    21                I have just one comment regarding

    22    referring to these viruses in general as defective. 

    23    My comment is that the evidence we have so far on

    24    those we have studied in a couple of vaccines between

    25    in Europe and in the U.S. suggests that those, for

                                                                         47

     1    example, ALVs we're dealing with could be defective

     2    because they come from loci that have deletions. 

     3    However, this may not be true for all the contaminants

     4    from it we might find in other vaccines, because these

     5    contaminants reflect the particles expressed from

     6    these loci in the different cell substrates that Phil

     7    mentioned in his talk, that the nature of these

     8    particles and their phenotypes would vary depending on

     9    the presence of the particular loci in these

    10    substrates.

    11                So just a comment, not to generalize that

    12    we always should expect to have defective particles. 

    13    We might or we might not in certain cases.

    14                DR. ROBERTSON:  Yes.

    15                DR. SCHUEPBACH:  Joerg Schuepbach from

    16    Zurich.  I would also like to make a comment regarding

    17    seroconversion.  We have done two vaccination studies,

    18    one with yellow fever vaccines, where about 120

    19    individuals were tested actually for reactive

    20    antibodies against HIV.  The reason for this was that

    21    in 1991, there have been reports that false positive

    22    HIV reactions were found in people that have been

    23    vaccinated against influenza.  Influenza vaccine is

    24    one of the vaccines which contains the EAV.

    25                So we found that statistically highly

                                                                         48

     1    significant the vaccinees which have received the

     2    vaccine which have the higher content, about 80 times

     3    higher than the other vaccine which we used to use

     4    too.  That these patients actually had highly

     5    significantly elevated antibodies to HIV 1 and 2,

     6    although none of these actually became sero positive.

     7    The serious bonds was highest in those individuals

     8    that also had a history of measles vaccine.

     9                In the second study, this was an influenza

    10    vaccine where we tested two different brands, a split

    11    vaccine and a rather crude vaccine.  We also had a

    12    response to HIV bond two in the third generation tests

    13    in the vaccine which contained more in the crude

    14    vaccine, which contained more of the EAV protein, and

    15    again those individuals who had a history of yellow

    16    fever vaccination had the highest type, the highest

    17    increases.

    18                We also tested yellow fever vaccinees by

    19    PCR, RNA PCR and DNA PCR for EAV-0 sequences.  We

    20    found one out of 180 individuals in which both these

    21    tests were positive as plasma on PBNC.  At the moment,

    22    we cannot exclude that this was the result of a

    23    contamination, but we are working on that.  So I think

    24    that the matter is actually not as clear as has been

    25    presented by others.

                                                                         49

     1                MS. SHEETS:  Hello. I'm Becky Sheets from

     2    FDA.  How would you recommend that avian-derived

     3    products be tested for retroviruses?  EM is not very

     4    sensitive.  The conventional test is often inhibited

     5    by the allantoic fluid, and therefore, is not

     6    necessarily a valid test.  How would you recommend, if

     7    you don't use a PCR-based RT des?

     8                DR. ROBERTSON:  At the end of the day, it

     9    would have to be an infectivity assay.  There are also

    10    some antigen, ELISAs for the viral antigen.  I don't

    11    have experience with those.  I don't know the

    12    sensitivity of them.  Ultimately you are looking at an

    13    infectivity assay which can be performed even on chick

    14    cells, which are positive for RTase.  One could assess

    15    for after several passages on chick cells, looking at

    16    an increase in RT activity or increase in antigen.

    17                MS. SHEETS:  When you said infectivity

    18    test, were you talking about those specific for ALV or

    19    were you talking about general tests to detect any

    20    kind of retrovirus?

    21                DR. ROBERTSON:  Well, it would have to be

    22    an avian retrovirus if one is performing the assay on

    23    chick cells.  Propagating the material, the test

    24    material in chick cells but using either RTase in

    25    general or an ELISA specific for ALV test for

                                                                         50

     1    increased presence of either RT or for the presence of

     2    ALV antigen.

     3                CHAIRPERSON RABINOVICH:  Dr. Coffin, the

     4    last question?

     5                DR. COFFIN:  Yes.  I'd like to actually

     6    address Dr. Schuepbach's comment.  Did I understand

     7    that you were basing your sero assays on the rationale

     8    that there might be cross reactivity between ALV and

     9    retro viruses in HIV?  There is no rational basis for

    10    that.  There's virtually no amino acid the same

    11    between those two viruses, except for some extremely

    12    highly -- you know, three or four in pol and some

    13    other places.  Did you actually assay directly for

    14    seroreactivity against ALV?  It would have been a much

    15    more straight-forward experiment.

    16                DR. SCHUEPBACH:  We agree that there is no

    17    sequence homology on the nucleic acid and on the

    18    protein label, but these are the results which we

    19    found.  We have to find an explanation for them.  We

    20    don't have at the moment.

    21                CHAIRPERSON RABINOVICH:  Thank you, Dr.

    22    Robertson.

    23                Our next speaker, Dr. Jorg Schuepbach,

    24    from the Swiss National Center for Retrovirology. 

    25    Induction/activation and detection of occult viral

                                                                         51

     1    agents that are present in mammalian tissues.

     2                DR. SCHUEPBACH:  May I have the first

     3    slide, please?  Okay, from previous remarks, I heard

     4    that I was expected to talk about these avian

     5    retroviruses as well, but actually I was asked to talk

     6    about the induction activation of occult viral agents. 

     7    So I will just have a few remarks on this other stuff.

     8                So occult viral agents are agents you

     9    don't detect or at least do not easily detect.  They

    10    may include two groups: a group that includes known

    11    agents which are present at two low concentrations for

    12    easy detection.  The reason for these may be latency; 

    13    The other group consists of unknown agents.  Since we

    14    do not have good detection methods for these, they may

    15    be present at low or also at higher concentration.

    16                Viruses known for their latency or various

    17    types of the herpes virus, true, they are latent in

    18    various types of non-permissive cells such as neurons,

    19    B cells, monocytes, PBLs, and others.  They are

    20    activated from these latent stages by various kinds of

    21    stimulation of their host cells by differentiation,

    22    agents by the differentiation of precursor cells, to

    23    more mature cells.  Again, by other activating agents.

    24                Other viruses could be considered in

    25    addition to the herpes viruses include the adeno

                                                                         52

     1    viruses, the adeno-associated virus and the pathyloma

     2    and polyoma viruses of which we heard yesterday, and

     3    will hear more in a subsequent talk.

     4                Regarding the RNA viruses, I might discuss

     5    the measle viruses and of course the retroviruses.

     6                When we look at the mechanisms by which we

     7    can activate these various viruses, it is mostly by

     8    activation of their host cells, by cell stimulation,

     9    by induction of cell differentiation of these cells,

    10    and then by co-cultivation with cells which are

    11    permissive for replication.

    12                Now since we have different viruses and

    13    host cells systems, these methods vary greatly among

    14    the different viruses.  If you have unknown viruses,

    15    you really don't know what to do.

    16                So the effect of such activation would be

    17    that from a lonely latently infected cell, by

    18    inducement of replication, a virus would spread

    19    throughout the culture, resulting in virus gene

    20    amplification in production of viral proteins.  So

    21    this would make of course the detection easy.  You

    22    might also have some pathogenicity which is easy to

    23    detect.

    24                However, our goal is actually not

    25    detecting any possible virus that might be present. 

                                                                         53

     1    The principal goal is to provide a virus production

     2    system which is free of such agents.  It is suggested

     3    here the easiest way of achieving this is actually

     4    cellular cloning.  Because if you have an agent that

     5    is present in only a minority of the cells, the

     6    chances that you derive a clone that is free of these

     7    agents is very high.

     8                If by chance you hit an infected cell, the

     9    descendants of that cell will all carry along the

    10    virus and of course then we come into a situation

    11    which makes detection of unknown viruses and also

    12    known viruses much easier because either all of the

    13    cells will be infected or none at all.

    14                So cellular cloning, if we hit an infected

    15    cell, has actually a viral gene amplification effect

    16    which is comparable to virus induction activation if

    17    it's successful.  Most importantly, it is a procedure

    18    that works for all the latent viruses except

    19    endogenous retroviruses, but these are present anyway

    20    in all of the cells.

    21                So going on to the detection methods for

    22    these agents, let's first talk about known viruses. 

    23    Since all the cells will be infected, we actually do

    24    not need the most sensitive procedures.  We do not

    25    need procedures that detect the single viral copy. 

                                                                         54

     1    What we need is broadly reactive methods which go

     2    detect all the different members of a certain virus

     3    group.

     4                So I think techniques, old-fashioned

     5    techniques like hybridization techniques on the low

     6    stringency or if we want to use PCR or nucleic acid

     7    based methods, we should take care that we take a lot

     8    of different probes, use data generated primus,

     9    multiplex PCR and so on.

    10                Of course in addition, we should also do

    11    the classical methods, doing cell activation and co-

    12    cultivation as permissive cells, the routine detection

    13    methods of broadly reactive antibodies which detect

    14    all the different members.

    15                For those who think that what I have told

    16    so far is rubbish, and that we actually do need very

    17    sensitive methods, I offer the mega PCR, which has

    18    also been named catcher PCR by others.  The purpose of

    19    this method is to take very rare sequences among a

    20    very high background of DNA or RNA.  So here we

    21    convert the samples of up to 500, maybe even 1

    22    milligram of DNA or respectively RNA.

    23                The principle is very simple.  We use

    24    biotinylated capture probes which bind to these

    25    sequences inquest.  We isolate these complexes on

                                                                         55

     1    coded beads, wash the rest of the DNA away, and them

     2    amplify these by PCR with primus which are located

     3    outside of this capture probe.

     4                The advantage of this is that we

     5    absolutely do have no carry over because the amplicons

     6    are selected against when we do the capturing.  It is

     7    this type of test which I would actually like to have

     8    been seen when testing in the question of

     9    xenotransplantation where the PERV sequences can be

    10    found in humans which have received pork material.  I

    11    think this will be the test, to test these questions.

    12                Now using this method, it's actually very

    13    sensitive.  You can detect a single copy here of HIV

    14    DNA.  We still have double positive signal, is about

    15    one copy.  This serial dilution was done in the proper

    16    range here.  The fact that in these two, three last

    17    dilutions only one of the two duplicates was positive

    18    clearly demonstrates that we are in a Poisson

    19    distribution.  So we can detect the single copy with

    20    this method in 100 microgram.

    21                DNA, we have actually demonstrated that

    22    there's 95 percent probability we can detect three

    23    double standard HIV copies in 100 micrograms of DNA.

    24                So now going on to the exclusion of

    25    unknown viruses, and I will talk about retroviruses

                                                                         56

     1    later, we can actually use the same procedures as I

     2    have already described previously.  We just have to

     3    take care that we really have broadly reactive

     4    methods.  This is true for molecular based tests as

     5    well as for the more classical procedures.

     6                Now coming to retrovirus detection, of

     7    course also of cell cloning, here we have two

     8    situations, the exogenous retrovirus may not be

     9    present in none of the cells or in all of the cells. 

    10    The endogenous retroviruses were always present in all

    11    of the cells.  The known exogenous retroviruses are

    12    detectible by tests for conserved sequences.  Of

    13    course you might also use universal pool primers for

    14    unknown retroviruses -- because of the endogenous

    15    retroviruses.  Not all of which, or very few of which

    16    are actually harmful.

    17                So I think it is better at this time to

    18    switch from the analysis of cells to the analysis of

    19    particles.  This is best done by the PERT assay which

    20    has been mentioned before by several speakers.

    21                Now when we devised this test in 1992, we

    22    devised it as an anti-family of related tests which

    23    would have in common that reversed inscriptase present

    24    in a sample would be used to create from a template

    25    primer combination and nucleic acid that is to be

                                                                         57

     1    unamplified.

     2                Now in most instances, this will simply be

     3    the cDNA.  There are other possibilities as well.  You

     4    can take any nucleic amplification procedure, not just

     5    PCR.  You may also use ligase chain reaction or NASPA

     6    or you can make use of auto replicated DNAs or RNAs in

     7    order to generate amplification product, which can

     8    then be assayed by different methods.

     9                So since we have provided for all these

    10    different methods already in 1992, we do not think

    11    that it is necessary to invent new names for these

    12    current assays.

    13                Now this test is actually very sensitive. 

    14    This experiment in comparison to classical RT assay. 

    15    It occurred as six to seven orders of magnitude more

    16    sensitive, and in a direct comparison with -- in the

    17    case of HIV, where we compared the method with RT PCR,

    18    detecting one copy of cDNA, we had the same dilution

    19    endpoints for two different samples.

    20                Actually as others, we can detect only a

    21    few particles in the case of HIV.  We believe that in

    22    some cases we can detect even less than one particle.

    23                Now this is one of the theories taken from

    24    the Joerg Koenig paper in 1996, where we demonstrated

    25    that the measles vaccines, the mumps vaccines, the

                                                                         58

     1    yellow fever vaccines, and the MMR vaccines all

     2    contain activity which is about three orders of

     3    magnitude higher than the background here on other

     4    vaccines, and were negative.

     5                Now in order to identify the viruses

     6    behind these activities, we along with the PERT assay,

     7    developed the method for the identification of unknown

     8    retroviruses.  It is based on three properties of old

     9    retroviruses, namely, that they all are

    10    polyadenylated, that R sequences are repeated at both

    11    ends, and that cDNA synthesis has started here at the

    12    primer, binding site, and that for primers, tRNAase

    13    are used and the use of such tRNAase is actually very

    14    much restricted among the various retroviruses.  For

    15    example, is just four PRNA primer equivalence.  You

    16    can start cDNA synthesis for all exogenous

    17    retroviruses known today.

    18                So what we do is that we bind the

    19    retroviral RNA to poly t coated beads.  Then we start

    20    here, the synthesis of the cDNA with one of the

    21    various t RNA primers, synthesizing the strongest of

    22    DNA.  Then adding a tail here, and then with anchored

    23    TCR, we can amplify this sequence and submit the

    24    sequencing directly.

    25                Actually this method has also been used by

                                                                         59

     1    the group of Dr. Loewer at the Paul Ehrlich Institute,

     2    and even published before us.  But we have somehow

     3    optimized this procedure, so in general we need less

     4    than one-thousand RNA sequences, sometimes as few as

     5    20 or 40, 50, in order to generate this sequence here. 

     6    As soon as you have it, you actually know whether you

     7    are dealing with a retrovirus or not.

     8                When you deal with a retrovirus, you have

     9    to R sequence and then you can check with the other

    10    anchored PCR.  Where there is R here, it's repeated at

    11    the three prime end.  If it is, you can then amplify

    12    the entire genome with a little bit of luck by long

    13    PCR.

    14                So this is what we use to identify this

    15    EIV-O sequence.  We have also done some other work. 

    16    For example, we investigated the NIH 323 cell line. 

    17    This was negative by convention RT tests, but positive

    18    by PERT assay.  We had a nice band in sucrose, and

    19    then radiant.  Using this procedure which we call

    20    parar, we identified 23 different products, 15 of

    21    these were actually retroviral sequences from four

    22    different groups.  Three of them were unknown

    23    sequences, at least at that time.  So far we have not

    24    further characterized these sequences, but this is

    25    still awaiting.

                                                                         60

     1                Now staying with retroviruses, as Dr.

     2    Coffin pointed out yesterday, sometimes if you have a

     3    cell line here, you are dealing with melanoma cell

     4    lines which were found to be highly percipated by PERT

     5    assay.  We analyzed what was in there.  It turns out

     6    to be endogenous murine leukemia virus, and later we

     7    were told that these cell lines have actually been

     8    passaged in mice.

     9                If you have low titres of activity, then

    10    that becomes a little bit more complicated.  This is

    11    the analysis of primary samples from a patient with MC

    12    cor cultures.  No actually not cor cultures, just cor

    13    cultures which were found lowly positive in the PERT

    14    assay with activity in the order of two, maybe three

    15    times above background.

    16                Here the patterns is a little bit more

    17    complicated.  You have here a small peak that might

    18    correspond in density to ritualized particles.  This

    19    one might correspond to cor particles.  You have

    20    another identified -- unidentified peak here.  It will

    21    certainly be a challenge to find out what this stuff

    22    is.

    23                Next, please.  This is another example of

    24    a primary culture where we have a very short peak at

    25    the higher density.  This might be for particles,

                                                                         61

     1    could be a different retrovirus, a different virus, or

     2    just a subcellular particles containing some cell or

     3    enzymes.

     4                Now you will say that this test of course

     5    detects only retroviruses that are released.  We are

     6    also worried about retroviruses that are inside the

     7    cells, so stimulation may be necessary.  Actually I

     8    think one important question is or one possibility is

     9    that actually the vaccine virus we would like to

    10    produce in such a cell might activate latent

    11    proviruses.  So I think it is important that we

    12    actually do not just test the virus production systems

    13    while uninfected, but also when this seed virus has

    14    been added, and then we harvest the virus.

    15                Now in some cases, as in the measles virus

    16    or so, this has proven very easy.  We had quite a good

    17    specificity.  But in other cases, it might be more

    18    difficult as indicated in this example, where we

    19    tested a vaccine, experimental vaccinia, recombinant

    20    vaccinia virus vaccine against melanoma.  This was

    21    found highly positive by PERT.  It had actually been

    22    produced by just the lysing, the infected cells by

    23    ultrasonication.

    24                What we now find is here in black, is the

    25    vaccinia virus DNA two peaks.  We have here a major

                                                                         62

     1    peak of RT activity which does not coincide with the

     2    vaccinia virus peaks, and also is not characteristic

     3    of retroviruses.  So I think in this case, we can rule

     4    out the presence of a retrovirus.

     5                Now it may also be interesting to find out

     6    whether upon induction, viruses might come out.  So

     7    this would add an increase of safety to the vaccine. 

     8    As retroviruses are regulated, you have the promoter

     9    in anti sequences in the upstream LTR in the U3

    10    region.  Depending on the cell type, activation state

    11    of the cell and the differentiation, you have various

    12    sets of transcription favors interacting with this

    13    enhancer regions.

    14                In addition to this balance of positive

    15    and negative transcription factors, you may have

    16    positional effects as the chromatin structure or the

    17    DNA methylation.  You may now try to influence this

    18    balance by tipping it by either inducing mitosis cell

    19    differentiation by substances that lock inhibitors or

    20    by alleviating the negative positional effects, again

    21    by inducing mitosis or by inducing DNA de methylation.

    22                The number of induces have been described

    23    in the past.  The most important ones are listed here

    24    at the top, allogenated pyrimidins, the azacytidine,

    25    which only both of them working only in infected

                                                                         63

     1    cells.  I will not mention the others because of the

     2    lack of time.

     3                Now it depends a lot on the virus whether

     4    azacytidine or the deoxy pyrimidine is preferable. 

     5    For example, in experiment in cell line where two

     6    types of different retroviruses are produced, several

     7    type A particles here.  The azacytidine is certainly

     8    better.  But in C-type particles, these cells produced

     9    IdUdr.  Yes, the IdUdr is better.  So you might have

    10    to use a combination of these two drugs.

    11                So in conclusion, I think induction

    12    activation certainly serves to amplify latent viruses

    13    for which improved detection.  I think it is more

    14    important that we early in the process of selecting

    15    virus production systems be cloned B cells, and sub-

    16    cloned, because this will amplify, because this really

    17    facilitates detection very much.

    18                In consequence of this, we do not -- I

    19    think this is very important.  We do not need the most

    20    sensitive procedures.  What we need is broadly

    21    reactive procedures which will detect all the

    22    different agents.

    23                I also think that at the end, the only

    24    important thing actually when dealing with

    25    adventitious agents, not just with DNA, which might be

                                                                         64

     1    infectious, is that the vaccine is free of these

     2    contaminant viruses and for retroviruses I believe

     3    that this can be verified by the PERV assay.  Thank

     4    you.

     5                (Applause.)

     6                CHAIRPERSON RABINOVICH:  We'll take just

     7    a couple of questions because I would like to leave

     8    the rest for the panel discussion.

     9                DR. COFFIN:  John Coffin.  I would agree

    10    that if you get preparations of vaccines that are

    11    negative by all these assays, you can have a pretty

    12    good level of confidence that they are not

    13    contaminated with retro viruses.  The problem is, if

    14    you do these enough, it may well be that no vaccine

    15    will pass these tests.

    16                What I think is very important to add to

    17    this would be one more level to your last slide.  That

    18    is an infectivity step.  As in the example we saw

    19    before when one perhaps collects a panel of cells or

    20    cell lines which are pert negative, and there seems to

    21    be reasonable numbers of those, and then test the

    22    vaccine, the induced stuff and everything else by

    23    infectivity and induction of pert activity on those

    24    cells.

    25                I think that would be a much more useful

                                                                         65

     1    and reliable test for the presence of viruses that

     2    might be problematic than simply looking at the pert

     3    activity in preparations with cell soups.

     4                DR. SCHUEPBACH:  Yes, I agree with you. 

     5    I actually thought that was included in those

     6    conventional methods which I have listed for the known

     7    viruses.  Of course you should also do some studies

     8    for retro viruses.

     9                AUDIENCE MEMBER:  You mentioned results

    10    associated with particles from supernatants of primary

    11    human materials.  Did you try to find retro virus-like

    12    sequences in these particles for para assay?

    13                DR. SCHUEPBACH:  Yes.  These are very

    14    recent results.  We are in the process of doing that.

    15                DR. KRAUSE:  Phil Krause, FDA.  One of the

    16    issues in testing vaccine products is obviously what

    17    tests are available and have been validated and that

    18    we understand the sensitivity of.  So I guess in the

    19    context of thinking of highly conserved sequences to

    20    which we might develop primers that could detect a

    21    broad array of viruses, including some unknown related

    22    viruses, what can you say about the current state of

    23    the art?  How good is that?  How well has that been

    24    validated?  Is that something which if we decided

    25    tomorrow we wanted to apply that to new vaccines

                                                                         66

     1    producing neoplastic cells, we could simply say "let's

     2    do it" or is more work required?

     3                DR. SCHUEPBACH:  I'm actually not very

     4    familiar with other viruses than retro viruses.  But

     5    I think these things, however they exist, should

     6    clearly be developed.

     7                CHAIRPERSON RABINOVICH:  Thank you.  We

     8    will go onto our next speaker, Dr. Jens Mayer, from

     9    the University of Pennsylvania.  The status of HERV in

    10    human cells.

    11                DR. MAYER:  Okay.  My talk will deal with

    12    -- can I have the first slide, please?  Okay.  My talk

    13    will tell you something, I hope, about the status of

    14    these human endogenous retrovirus regarding the coding

    15    capacity and the expressions.  Just again, it was

    16    mentioned before already what is actually an

    17    endogenous retro virus.  HERV is created by the germs

    18    of infection of an exogenous retro virus.  This leads

    19    to radical inheritance of this newly created virus

    20    following generations.  In the course of the

    21    evolution, it will be also inherited to newly arising

    22    species.

    23                The human genome, like all mammal genomes,

    24    and also some invertebrates, invertebrate genomes

    25    where it has been shown, contains several families of

                                                                         67

     1    elements and so on.  It has been estimated that about

     2    one percent of the human genome of such retro origin. 

     3    These elements antiquated already several million

     4    years ago through the genomes of human predecessor

     5    species.  Some present for at least 30 million years. 

     6    Some have been shown to be present for at least 40

     7    million years.  We have several indications of

     8    different various families.  So they were independent

     9    of several exogenous retro viruses.  Some of these

    10    elements that are now present in the human genome

    11    existed.  Single copy, and some have copies, copy

    12    numbers up to 1,000, per haploid genome.

    13                But as I said, most of these sequences

    14    were already present for a long time.  Therefore were

    15    targets for mutations.  Most of these families then

    16    became coding deficient or they do no longer encode

    17    for retro R proteins.  However, even if they are

    18    coding deficient, many of these families are still

    19    transcribed in several human tissues.  Some have been

    20    discolored just by virtue of their expression.

    21                It also seems that the expression of these

    22    sequences is regulated in certain tissues and tumors,

    23    so we heard that there might be an deregulation of

    24    families.  It seems possible that that deregulation

    25    mechanism is not present in certain tumor tissues.

                                                                         68

     1                Just a word regarding the nomenclature of

     2    these sequences.  The tRNA that was originally used in

     3    the priming of the transcription process, the life

     4    cycle of the exogenous vaporized, and according to the

     5    amino acid and tRNA codes for, and this single code

     6    for the amino acid stands dependent.  This is just one

     7    possible nomenclature of perts.  It's still very

     8    confusing.

     9                I said that most retro viruses are coding,

    10    HERVs are coding deficient.  However, there are some

    11    good described examples, especially some new examples

    12    of coding in tact HERV sequences.  At least there are

    13    some in tact genes.  We have already known for a long

    14    time the so-called ERV-3 sequence that belongs to the

    15    R-family.  This, we agreed, pro-virus, or pro-virus

    16    sequence encodes, and 1.9 KBN open reading frame. 

    17    That open reading frame is highly regulated to the

    18    transformation of trophoplasts into sensitio-

    19    trophoplasts in the placenta.  So we have here clearly

    20    an up-regulation during a developmental stage.

    21                We have for instance, you have H-family

    22    and we have about 1,000 copies of that H-family. 

    23    Among them are 100 copies that are still in tact

    24    regarding the pro-virus structures.  They have an LTR

    25    gag pol env, LTR structure remaining 900 lack N gene. 

                                                                         69

     1    There has also been reported that this HERV-H families

     2    are expressed in various cell lines.  We see the

     3    highest expression for these elements has been

     4    reported in cell lines that are derived from germ cell

     5    tumors, and germ cell tumors I guess you will hear

     6    some more about germ cell tumors later on.

     7                Just this year, Lindeskog, Mark Lindeskog

     8    reported the isolation of an intact HERV-H env gene. 

     9    So it is now clear that there is within the human

    10    genome one intact HERV-H evn gene.  It's not know so

    11    far whether there are any among these many sequences,

    12    whether there are any intact gag of pol sequences.

    13                I would like to mention the new discovered

    14    HERV-W family that has originally been reported, has

    15    been isolated from retro virus by particles from

    16    multiple sclerosis patients.  It has also been

    17    reported that these HERV-W sequences are up-regulated

    18    in the placenta.  Joni Blanc also reported this year

    19    the isolation of an intact HERV-W in the genes.  It is

    20    also not known whether there are intact gag pol genes.

    21                I would like to in the second part of my

    22    talk, report about results for our family of clearly

    23    outlines from our other HERV families in the coding

    24    capacity.  This is the so-called HERV-K HML-2 family. 

    25    This is quite complicated.

                                                                         70

     1                The human genome contains several families

     2    that use lycine primer binding site or TRNA for primer

     3    binding.  They were named human MMTV-like sequences,

     4    one through six.  The family that we are talking about

     5    is reported in more detail by Ono and co-workers and

     6    the original sequence was the so-called HERV-K 10

     7    sequence, which is by the new nomenclature is the

     8    HERV-K HML-2 sequence.

     9                We have reached about 25 to 50 copies of

    10    that HERV family is present in old world monkeys, but

    11    not in new world monkeys.  One concludes that family

    12    is present for at least 30 million years in the

    13    genomes.

    14                In the past, there have been reports of

    15    isolation of the isolations of intact HERV HML-2

    16    sequences.  So there were reports about intact gag

    17    sequence and intact protease sequence has been

    18    reported, that is able to process that HERV-K gag

    19    protein, intact pol sequences with RT activity, with

    20    endonuclease activity, and have been reported and also

    21    intact MRNA has been reported from the group from

    22    Johannes Loewer.  And also what we heard yesterday

    23    evening, there is also an additional splicing product

    24    from the N gene, the so-called C-ORF that still has a

    25    rav-like function.

                                                                         71

     1                What is known already for a longer time is

     2    that these particles or the cell lines are derived

     3    from germs of tumors or typically testicular tumors of

     4    the young man.  These cell lines do produce with rav

     5    particles.  Boller and coworkers could show that these

     6    particles are encoded by the HML-2 gag protein,

     7    labeled antibodies, and recognized that gag protein.

     8                If we look at patients suffering from germ

     9    cell tumors, we also have some surprising results

    10    regarding that HML-2 sequences.  Namely, if we look at

    11    the antibody status of these patients compared to

    12    controls or other non-germ cell tumor types, we see

    13    that mixed germ cell tumors and here especially,

    14    seminomas, these patients have very high antibodies

    15    directed against HERV-K gag and HERV-K N proteins. 

    16    These tumors or these antibody titres are already very

    17    high if the tumor is clinically detected.  From other

    18    results, we also know that the precursors of these

    19    tumors, the so-called carcinoma in situ, also

    20    expresses already on the RNA level these HERV-K HML-2

    21    sequences.

    22                We were interested to see or to find out

    23    where in the genome are these impact genes located

    24    that are responsible or that cause finally the

    25    production of these gag and env antibodies.  As I

                                                                         72

     1    said, it has previously already been reported that

     2    they are intact genes, but it was not possible because

     3    of the high copy number of these sequences to isolate

     4    or to at least chromosomally assign these intact

     5    genes.  We, therefore, tried to chromosomally assign

     6    these intact sequences using a combination of the so-

     7    called protein truncation test and using a

     8    monochromosomal hybrid panels, or panel of human

     9    rodent fusions, fusion cells.

    10                We were able to show that there are at

    11    least, still at least eight intact gag genes within

    12    the human genome, and at least three intact env genes. 

    13    We did not publish that.  There are also several

    14    intact pol genes within the human genome.

    15                I just want to show you how we got these

    16    numbers.  This is the protein truncation test that has

    17    been described by Roest and coworkers in 1993.  So it

    18    was originally developed for the detection of APC gene

    19    carriers that carry it, the APC gene.  So the APC

    20    lesion is characterized by trends or not completely

    21    translated APC proteins.  It is almost like the 3

    22    prime terminus.

    23                So we have three possibilities.  One is

    24    that both are intact, both genes are intact.  The

    25    carrier will carry one defective APC gene.  The

                                                                         73

     1    defective person would carry the two defective genes. 

     2    The principle of the test is that the coding sequence

     3    is PCI amplified, where the protochomo contains the T-

     4    7 promoter and the translation initiation sequence. 

     5    So if this PCI product is then in vitro transcribed

     6    and translated and impressed and radiolabeled amino

     7    acid, electrophoresed, and then auto radiographed, you

     8    will see according to the status of these donors that

     9    you will have only full-length proteins, the carryover

    10    also show an additional shortened protein and

    11    defective people will only produce defective proteins.

    12                We in principle used the same test because

    13    we in principle have the same situation.  We have some

    14    defective gag genes within the genome.  There must be

    15    at least one gag or env gene because we have the

    16    antibodies.  So we put -- in principle used the same

    17    test.

    18                What we did was we are looking for the

    19    presence of full-length gag genes or env genes on the

    20    human chromosomes and then tested the PCI product we

    21    got from the chromosomes for their coding capacity. 

    22    This is the result for the gag coding capacity.  So

    23    gag protein would result in a protein of about 73

    24    kilodaltons.  So these are controls that give the

    25    respective proteins.

                                                                         74

     1                You see that there are several human

     2    chromosomes that contain or produce a full-length

     3    protein.  There are eight human chromosomes that

     4    contain at least one gag gene that contains four full-

     5    length proteins.

     6                I also would like to mention that we are

     7    also able to demonstrate the defective gag genes if we

     8    see here, these proteins that are just smaller than

     9    expected.  These are very likely the gag genes that

    10    are defective.  Stop codons within the coding

    11    sequence.

    12                We did the same for the HERV-K env genes. 

    13    We see here that three chromosomes produce a protein

    14    of about 76 kilodaltons.  These are the chromosomes 7,

    15    19 in here on the chromosome.

    16                What we also see in the gag experiments is

    17    that there are additional env genes that are only on

    18    the almost intact.  We have here a protein that is

    19    about four kilodaltons more.  So this actually could

    20    also be considered as an intact reading frame.

    21                So we have several human chromosomes that

    22    still contain gag and env genes.  We have three

    23    chromosomes that contain both intact gag and env

    24    genes, the chromosome 7, 19, and the Y chromosome.  We

    25    were interested whether these chromosomes or the

                                                                         75

     1    intact genes on these chromosomes are derived or

     2    located within one provirus or within several or

     3    different positions within the particular chromosome.

     4                I would like to report or tell you

     5    something about what we found out for the chromosome

     6    7.  We were using for addressing that question, we

     7    were using a chromosome-specific, chromosome-7

     8    specific cosmid library.  We were screening for clones

     9    that contained both gag and env sequences.

    10                What we finally found out, that we

    11    isolated the so far least defective human endonuclease

    12    on chromosome 7.  We were able to characterize the

    13    proviral sequence within one cosmid clone that still

    14    has intact LTRs.  So they regulate to the elements. 

    15    They are able to transcribe, as you will see.  We have

    16    an intact gag gene.  We have an intact protease gene

    17    that protease is able to cut itself from a gag

    18    protease, polymer precursor protein, and is

    19    furthermore able to process encoded gag proteins.  So

    20    it's typical retro-ized protease.

    21                We know just from sequence comparison, one

    22    can deduce that the endonuclease within the polymer

    23    genes also acted just by sequence comparison, no

    24    significant changes compared to recently described

    25    active K in the nuclease.  We have an intact env gene. 

                                                                         76

     1    This intact env gene sequence has already been

     2    described by Johannes Loewer's group as an MRA, which

     3    also shows that this sequence is actively transcribed.

     4                So this is actually an expressed provirus. 

     5    We have spliced on the inceptors sides the

     6    corresponding position that would allow to splice an

     7    M on A, and what we heard yesterday also, to splice an

     8    additional soft M RNA.

     9                What we see is that this proviral sequence

    10    is only defective in the RT domain.  It has a single-

    11    based permutation within the YXDT motif.  So very

    12    likely, this highly important catalytic motif is -- so

    13    only in reverse transcription function this probably

    14    missing from that proviral.

    15                Okay.  We have here almost intact proviral

    16    sequence.  But now regarding infectivity, we had that

    17    already several times I guess before.  We have HERV-

    18    encoded retro of particles, several cell lines, even

    19    in tissues, the placenta tissue for instance.  We find

    20    HERV-RNA in these particles.

    21                We have no infectivity so far shown for

    22    any of these HERV sequences.  We do not really know

    23    why.  There are several reasons that can be mentioned

    24    for the HML-2 family.  It has to be reported that the

    25    env protein cannot be cleaved into the auto membrane

                                                                         77

     1    transmembrane domains.  It is conceivable that they

     2    are defective genomes that are packaged into these

     3    particles, so only if they would be able to get a new

     4    cell, they would only deliver defective genomes.

     5                It is also not clear whether the

     6    receptors, that they were once used by that, retro

     7    families are still present and would still be used.

     8                So what you should take home I guess is

     9    that human endogenous are expressed in several tissues

    10    tumor types that are highly up-regulated in certain

    11    tumor types.  Several HERV families are still able to

    12    encode proteins, and among them, the HML-2 family that

    13    still encodes all essential proteins.  We have almost

    14    intact HML-2 provirus within the human genome.  Thank

    15    you.

    16                (Applause.)

    17                CHAIRPERSON RABINOVICH:  I think we will

    18    hold questions at this point.  We are going to take a

    19    10-minute break now.  We are going to come back and

    20    finish with the last two speakers.

    21                I need to figure out how to catch up time,

    22    and yet leave the time for the panel discussions.  I

    23    ask you to do two things.  Check-out time from the

    24    hotel is 12:00.  You should know that.  They have

    25    already called in a bunch of the taxis so that if you

                                                                         78

     1    need taxi arrangement, please let them know so they

     2    can do that for you.  Ten minutes we will start again.

     3                (Whereupon, the foregoing matter went off

     4                the record at 9:55 a.m. and went back on

     5                the record at 10:10 a.m.)

     6                CHAIRPERSON RABINOVICH:  Is Dr. Broker

     7    here?  Great.

     8                If you could take a seat please.  The next

     9    speaker is Dr. Thomas Broker from the University of

    10    Alabama at Birmingham speaking on viral latency-

    11    papilloma virus model.

    12                DR. BROKER:  Thank you very much.  I would

    13    like to deal with two subjects under this topic.  The

    14    first is a study of the prevalence of HPV in the

    15    general population, and then following on Dr. Mayer's

    16    pattern that you just heard, a study of some

    17    endogenous sequences in papilloma virus transformed

    18    cell lines with some surprising results.

    19                We have done some inside 2 hybridization

    20    studies of the expression of human papilloma viruses

    21    in biopsies from women with HIV/AIDS who were

    22    moderately immuno deficient.  This is one example, but

    23    fairly typical.

    24                What you are seeing is a full thickness of

    25    across the cervix.  The various probes that we used

                                                                         79

     1    reveal the expression of one of the major early

     2    transcripts of papilloma virus, the E4, E5.  You are

     3    seeing it here in bright field illumination and dark

     4    field, matched pairs, basal layers right there.

     5                As I indicated yesterday evening,

     6    papilloma transcription is differentiation dependent

     7    and occurs typically in the upper half of the skin. 

     8    E6, E7 messages, the delayed early oncogenes are hard

     9    to see in bright field, but fairly easy to see in dark

    10    field.  You can see they follow a comparable

    11    distribution.

    12                The capsid component, L1 or L2, again, is

    13    right at the very top of the last live layers of the 

    14    epithelium.  Also to the point, the vegetative

    15    amplification of viral DNA is in the upper half of the 

    16    epithelium.

    17                Papilloma infections of the genital tract

    18    in fact have been designated an official AIDS-defining

    19    illness in the syndrome because of the significant

    20    upregulation of HPV gene expression in women who have

    21    AIDS or other immuno deficiencies.  With that

    22    knowledge in hand, and pictures like this, we

    23    undertook the following study.

    24                We decided to investigate the prevalence

    25    of HPB in the population by focusing on immuno-

                                                                         80

     1    deficient groups.  The three that we have chosen so

     2    far are: women who are in enstay renal failure and in

     3    need of a kidney, and most clearly ill; those then who

     4    get a kidney and are pharmacologically immuno-

     5    suppressed beyond their underlying illness; and those

     6    with AIDS.

     7                The strategy that we're using is an

     8    extension of the techniques Steve Wolinsky and I

     9    developed really 12 or 13 years ago when we first

    10    proposed the use of degenerate primers for looking at

    11    related genomes.  The pair that's most commonly used

    12    in the papilloma field is our original design called

    13    MY911, but Louise Chao and I moved right next door. 

    14    We found that this region is a little too long to use

    15    in form one fixed tissues, and this particular pair

    16    has some wonderful restriction fragment polymorphisms

    17    available that will allow us to do genotyping after

    18    amplification.

    19                So basically we start with the nested PCR

    20    approach, outer primers and inner primers.  The

    21    starting material is cervico vaginal lavage, which

    22    harvests cells from throughout the lower genital tract

    23    of the women.  We amplify and then we put it through

    24    several different assays.  Initially, agarose gel

    25    electrophoresis to look for a 278-base amplimer.

                                                                         81

     1                Secondly, restriction fragment

     2    polymorphisms which usually can tell us which genotype

     3    is present.  But if it's a pattern that we cannot

     4    recognize, we will put it through sequencing.  As you

     5    are going to see, about half of the fragments that we

     6    amplify we need to sequence.

     7                The results of this study, I am going to

     8    summarize.  It's absolutely mind-boggling.  Seventy

     9    four percent of all women in the AIDS cohort have

    10    clearly identifiable HPVs.  We have managed to type

    11    over 85 percent of these so far.  Fifteen percent are

    12    still under investigation.  In more than half the

    13    cases, the individuals yield multiple HPV types.

    14                The study is longitudinal, and has been

    15    going on for three-and-a-half years now.  Many of the

    16    members of the cohort have been sampled two up until

    17    seven different times at six to 12 month intervals. 

    18    So that's our biggest cohort.

    19                These are the renal transplant cohort.  We

    20    have statistically significant numbers.  I would like

    21    to point out that in instage renal failure, but no

    22    pharmacologic suppression, about 59 percent of those

    23    women have detectible HPV.  Again, quite a few, a high

    24    percentage have multiple infections.  This carries

    25    over to that portion of this group who go onto actual

                                                                         82

     1    transplantation.

     2                Some of the remarkable outcomes of the

     3    care with which we undertook the genotyping is the

     4    following.  In the pre-transplant population, the

     5    prevalent types are those that are commonly seen in

     6    the general population as causing disease, namely HPV-

     7    6, 11, and 16.  Those types persist in those women who

     8    were pharmacologically immuno-suppressed.

     9                We see a scattering of other types, but

    10    the common types from prior studies are those that

    11    predominate in the renal transplant cohorts.

    12                In contrast, those women who are in

    13    various stages of immuno-deficiency as a result of

    14    AIDS, do not show the same genotype profiles.  The

    15    only member in common is in fact most common of all

    16    genital HPVs, HPV-6.  What we see instead are niche

    17    homologs of the common types.  For example, HPV-45, as

    18    you are going to see, if a close relative of HPV-18,

    19    which is often cited as a common virus.  But we don't

    20    see that in the AIDS cohort.

    21                HPV-52 is our most common virus.  It is a

    22    close homolog of HPV-16, which we don't see amplifying

    23    in this cohort.  Most notably are the ones that I

    24    indicated by stars, which are a very rare detection

    25    within the general population, but in fact are most

                                                                         83

     1    common viruses in the AIDS cohorts.

     2                In particular, we have identified 13 new

     3    HPV types based on less than 75 percent sequence

     4    homology to each other or to any other known papilloma

     5    virus.  They are all members of what has been

     6    designated group A-3, which appear to be an AIDS-

     7    defining subset of HPVs.

     8                These can be at least considered in the

     9    context of phylogenetic trees based on sequence

    10    alignments in the L1 region.  So, for example, HPV-16,

    11    the main cause of cervical cancer in the world, is

    12    seen in the renal cohort, but a very close relative,

    13    52, is seen in AIDS.

    14                Six and 11, that cause benign genital

    15    warts and laryngeal papillomas are here.  One of the

    16    main groups coming up in AIDS is this group of cousins

    17    of these guys.

    18                HPV-70 is one of our most common types, as

    19    well as 45.  They are in the HPV-18 family, but

    20    represent new members of this niche.  The group I just

    21    mentioned, A-3, that is so commonly seen in AIDS,

    22    include our members jyn 2, 3, 4, all the way up to 13,

    23    MM8 and 61, 72, and 83.  That cluster seems to be an

    24    AIDS defining group.  The other ones that we have seen

    25    abundantly are 51 and 53 in this arm.

                                                                         84

     1                Overall, in the Birmingham and generally

     2    Alabama population, every virus types seen with the

     3    star we have found one up to 23 times, indicating that

     4    we have universal presence and also detectibility of

     5    all of the known viruses within our immediate

     6    population.

     7                While this is up here, I also wanted to

     8    point out the very large huge group of epidermal

     9    dysplasia formus viruses that other labs have studied. 

    10    Again, it is a very rare group of illnesses, in fact,

    11    only defined a few hundred times in all of medical

    12    history in terms of individual patients.  However,

    13    there is this huge ramification of somewhat related,

    14    but clearly distinct genotypes that comprise the

    15    family or subgroup of viruses responsible for EV.

    16                It is known that these patients all have

    17    particular cell-mediated immune deficiencies.  Again,

    18    suggesting that particular arms of the immune system

    19    are responsible for either containing or failing to

    20    contain different subgroups of the papilloma viruses.

    21                As we look at these women over a period of

    22    time through these six month or so samples, what we

    23    also find, and other labs have exactly the same

    24    results, is every time we sample, you may or may not

    25    see the type you saw before.  It may switch.  For

                                                                         85

     1    instance, we have this patient who had 6 plus 16, and

     2    then 11 plus one that was minor and we couldn't tell,

     3    then jyn 2, and then type 40, and then we had a type

     4    53, but the others disappeared.

     5                Everyone's experience in the field has

     6    been that the viruses rise above a detectibility

     7    threshold, stay there for a while, days or weeks or

     8    months, and then fall below detectibility, only to be

     9    replaced by a different HPV type.  These are not new

    10    infections.  They are basically cryptic or latent

    11    persistent infections that fluctuate in their levels

    12    of replication and detectibility.  Pretty much anybody

    13    is showing that flexibility.

    14                What I want to state at this moment before

    15    showing the correlation with disease may sound

    16    controversial, but I will stick by it.  We have found

    17    a brand new HPV type for every 10 people that we have

    18    looked at.  Philodelius and Ethel Michelle Diveres and

    19    zur Hausen and Shamen in European study of tutanius

    20    papilloma viruses have found a new papilloma virus for

    21    just about every other person they have looked at when

    22    they use the combination of nested PCR and DNA

    23    sequencing.

    24                Robbie Burke's group, Jill Polefski's

    25    group, have very comparable experiences looking at

                                                                         86

     1    anal papillomas or female genital tract.

     2                It is my contention right now that instead

     3    of 80 HPV genotypes or 150 that have been officially

     4    named, that there probably are millions of variants,

     5    virtually a continuum.  We feel that basically

     6    everybody has their own personal micro flora, that

     7    these are passively acquired or vertically acquired,

     8    not necessarily sexually, but certainly possibly

     9    sexually, and that they simply are part of the human

    10    condition as are microflora, just as we have

    11    microflora composed of bacteria and many other

    12    viruses, and that they basically are utterly

    13    ubiquitous.  I will come back to that point in a

    14    moment.

    15                We did try to correlate the various other

    16    medical parameters in these cohorts, especially the

    17    AIDS cohort, with CD4 count, HIV virus load, other

    18    infecting known STDs like herpes, chlamydia,

    19    trichomonas, so forth.  The one correlate that held up

    20    and not surprisingly at all, was that the degree of

    21    pap smear abnormality from normal, abnormal cells of

    22    unknown significance, low grade dysplagias or high

    23    grade dysplagias, is with CD4 count.

    24                The medians, these are all the people who

    25    had multiple infections, a high risk virus type, a low

                                                                         87

     1    risk, no virus at all, and had either normal or these

     2    various abnormal pap smears.  These bars here are the

     3    median CD4 count in each of these groups.

     4                The one place where we saw active disease,

     5    low and high grade dysplagia, these by median, is when

     6    people fell below the CD4 count of 200 cells per cubic

     7    millimeter.

     8                In summary of that data, we found that

     9    it's very very possible to have negative pap smears,

    10    but definitely have HPV infections.  We feel these are

    11    people who have not yet reactivated long enough to

    12    have resulted in cytologic change as a result of

    13    infection.  We have on the other hand, the people with

    14    overt disease by biopsy or by cytology, and the higher

    15    the grade lesion, the more likely it is to see either

    16    single infection or especially multiple virus types

    17    present within that patient at that time.

    18                So the more that we can detect the virus,

    19    that is, the more it has replicated or amplified

    20    throughout the population, the more cells that are

    21    shedding the virus in effect, the more likely we see

    22    disease.

    23                So to summarize this part of the talk, I

    24    feel that they are virtually ubiquitous.  they are

    25    typically sub-clinical, persist in or latent

                                                                         88

     1    infections.  There are staggeringly large number of

     2    genotypes if we take the care to look.  I might say

     3    that the reason these are typically not found is that

     4    people use generic cross-hybridizing probes or have

     5    cut off their probe sets.  If you're not probing for

     6    something, you are not going to see it.

     7                Most of the viruses in this number 60, 70,

     8    80 and above, are not even present within the

     9    commercial probe sets.  So if you aren't probing, you

    10    are not going to see them, and you are going to get

    11    lower numbers.

    12                They can be found throughout the genital

    13    tract in 60 to 75 percent of the people that we have

    14    looked at who are admittedly good yielders, because

    15    they are immuno-compromised, but I think this simply

    16    represents the general infection in the population.

    17                They can be found in oral and esophageal

    18    mucosa.  Utaneious types persist in hair follicles. 

    19    There's a wonderful study from Amsterdam by Tershaget

    20    and Ingebor Boxman.  She plucked hair follicles, both

    21    eyebrow hairs and pubic hair, and 60 to 70 percent of

    22    all people harbored EV viruses or other rare virus

    23    types in their hair follicles.  No disease, it's just

    24    part of the human condition.

    25                I believe they are vertically transmitted

                                                                         89

     1    perinatally, mother to baby.  Some of them are clearly

     2    pre-natal infections.  As we know, there's long-term

     3    maintenance that requires viral replication in concert

     4    with host replication in the cell cycle.

     5                So what I would like to do now is tell you

     6    a little bit about a very unexpected observation we

     7    made in Hela cells.  This goes back to last night's

     8    talk regarding the structure of the replication

     9    complex of HPVs.

    10                As you know, cyclin E is one of the key

    11    checkpoints or entries into S phase.  Ectopic

    12    expression of cyclin E can speed up entry into S

    13    phase, and it can even bypass the need for some of the

    14    RB phosphorylation by cyclin D.  It's simply one of

    15    the key steps that needs to follow the induction of

    16    the DNA replication enzymes.

    17                HPV E7, the viral oncogene that in fact

    18    binds RB and can help bypass that step, among the E2F

    19    enhenca protein regulated genes is cyclin E itself. 

    20    In other words, HPV infection upregulates cyclin E.

    21                So we asked whether the induction of

    22    cyclin E is essential for the reactivation of

    23    unscheduled cellular DNA synthesis in the upper

    24    stratum of squamous cepathelium that differentiated

    25    caratinocyde.  I'll just summarize that data.

                                                                         90

     1                I got you to the point last night where

     2    the E1 diheximer, the double helicase held together by

     3    the HSP-70 cochaperone protein, is there.

     4                The next thing that loads in the study we

     5    did with Theresa Wong at Stanford, is the recruitment

     6    of the cellular DNA polymerase, and showed direct

     7    interactions between the helicase and the catalytic

     8    sub-unit of pol alpha, P-180, as well as its P-70 sub-

     9    unit.  This was the first indication of what P-70 does

    10    in the four sub-unit complex of pol alpha, which

    11    includes two primary sub-units.  The answer is, it

    12    brings the polymerase to the ora itself.

    13                The next thing that comes in is cyclin E,

    14    CDK-2 complex, that critical S phase entry point.  AS

    15    a result of that, what happens is upon cyclin E

    16    finding an appropriately assembled pre-initiation

    17    complex, five target proteins are phosphorylated. 

    18    They include: the E2 protein, which appears to be

    19    displaced by that event; in addition, P-70 helps

    20    displace E2.  So the loading of this and the

    21    phosphorylation kicks this guy out.

    22                Secondly, E1 is phosphorylated.  These two

    23    subunits of preliminary salpha that bind directly to

    24    E1 are phosphorylated.  When all four of those have

    25    been successfully modified, the kinase phosphorylates

                                                                         91

     1    cyclin E itself, which is displaced and degraded by

     2    ubiquitination.  That enables the pre-initiation

     3    complex to convert to the elongation complex.

     4                In studies with Wade Harper and Jien-Ling

     5    Ma at Baylor, two things were done.  The first is

     6    together we found that there's a cyclin binding motif

     7    that the amino terminal have at the E1 protein, which

     8    in fact is shared with a number of other things that

     9    bind the cyclin E.  That motif involves an RXL.  That

    10    is, an arginine something leucine motif right there.

    11                In addition, their candidate

    12    phosphorylation cites, the series of serine,  serine,

    13    serine, and threonine, mutation of any of these, the

    14    motif or any of the target phosphorylation cites,

    15    diminishes the capacity of cyclin E to convert the

    16    pre-initiation complex to an initiation complex.  So

    17    the functional requirement for phosphorylation has

    18    been verified.  But keep in mind this location.  We'll

    19    come back to it in a second.

    20                So we assumed that the consequence of

    21    upregulation of cyclin E by E7 gene expression would

    22    identify those cells that are capable of supporting

    23    papilloma replication.  To our amazement, we found the

    24    opposition.  This is our epithelial raft model.  We

    25    have done the same in natural papilloma lesions.  Here

                                                                         92

     1    we monitored cyclin E expression, over expression in

     2    the tissue.  Here's bromo deoxy uridine incorporation

     3    or PCNA upregulation.  These are the match.

     4                What we found is the cells that had high

     5    cyclin E could not replicate.  In fact, they are

     6    mutually exclusive with those capable of supporting

     7    DNA synthesis.  Conversely, PCNA, which is upregulated

     8    by papilloma 7 and cyclin E do co-localize.  But we

     9    see a number of cells where PCNA is present and there

    10    is no cyclin E.  So we have a reciprocal pattern to

    11    what we expected.

    12                I am just going to very briefly tell you

    13    that P-21 cip, one of the inhibitors of cyclin D and

    14    cyclin E, is also upregulated by E-7 expression in

    15    natural condylomas or in our E-7 expression raft

    16    cultures.  You can see those signals in the upper

    17    strata again.  So we have P-21 upregulation, again, in

    18    a subset of cells.

    19                When we look in rafts or in natural

    20    papillomas, we see that those cells that have high P-

    21    21 are mutually exclusive from those capable of

    22    supporting either viral or cellular DNA synthesis. 

    23    When we did the third pairwise combination and looked

    24    at cyclin E and P-21, we found perfect colocalization

    25    of those two.

                                                                         93

     1                So ironically, the cells that have high

     2    cyclin E also have high P-21 and do not support

     3    replication.  This was really perplexing, except we

     4    did know this inhibited that.  But we assumed cyclin

     5    E was in the licensing factor for engaging in

     6    replication.

     7                So what we came to feel is the following

     8    model: that in the course of unscheduled DNA synthesis

     9    reactivation, if cyclin E appeared in the appropriate

    10    timing or sequence or amount, once a pre-initiation

    11    complex formed, you would successfully phosphorylate

    12    the target proteins, polymorases and E-1 and E-2

    13    proteins, and successfully engage in elongation

    14    replication.

    15                Conversely, if too much cyclin E appeared

    16    and it appeared in an untimely fashion, its inhibitor,

    17    P-21, would recognize misassembled complex.  They

    18    would cross stabilize.  They would both pile up to

    19    high levels, and those would be defective in engaging

    20    in elongation.

    21                Now we put this all together by asking how

    22    does this play into the establishment of immortalized

    23    and transformed cells and cancers.  What Wade Harper

    24    had found is that when he did pull-down assays with

    25    cyclin E to ask in hela cells what binds to cyclin E,

                                                                         94

     1    almost all the things that came down in the assay was

     2    that E-1 protein from the resident HPB-18 in the cell

     3    lines.  Up until that point, people had thought the E-

     4    1 gene was deleted from hela.  In fact, it's present. 

     5    The entire length of the E-1 gene is still present in

     6    hela.  In fact, is expressed.

     7                Now the functional assay that our lab did

     8    was that we found that hela cell extracts could not

     9    support HPB replication in our cell-free system, that

    10    there was a missing factor in hela that the extract

    11    needed.  We could put 293 cell extracts or any other

    12    cell line that we could find, they would easily

    13    complement papilloma replication in vitro.  But

    14    anything from hela cHa caski or any other HPB

    15    transformed cell line could not support it.

    16                The upshot of the whole thing is that

    17    every papilloma transformed cell that we studied

    18    expressed a full length E-1 transcript, but in all

    19    cases, the transcript had an either frame shift or a

    20    stop code on partway through the gene or miss sensed

    21    mutations in this vicinity, so that in tact E-1 could

    22    not be made.  But in all cases, it made the RXL

    23    portion that interacts with cyclin E.

    24                So we added a little cyclin E back to hela

    25    cell extracts and immediately restored full

                                                                         95

     1    complementability to those extracts, establishing that

     2    the missing thing in hela cells was cyclin E.

     3                So our conclusion is the following.  I

     4    think we feel very confident about it.  In the process

     5    of immortalizing cells, either natural cancers or

     6    attempts at making cell lines, substrates in effect,

     7    it's good to have all the upregulation of DNA

     8    polymerase, topasomerases, PCNA and so forth, that

     9    help rapid cell cyclin.  But the one thing that E-7

    10    upregulates you don't want, is cyclin E.

    11                So what these natural experiments did, is

    12    figured out a way to sequester part, but not all, of

    13    the cyclin E by putting in retaining fragments of E-1,

    14    capable of mopping up that one product that's

    15    upregulated that you don't want to have.  That is,

    16    cyclin E.

    17                Brian Van Tine, last night, also indicated

    18    there's evidence of some antisense in other papilloma

    19    lines like cHa caski, which would again, modulate the

    20    amount of E-1 that you could translate from the

    21    messages that are clearly there.

    22                Together we believe that to establish

    23    these cell lines, whether you make them in the lab or

    24    whether nature has made them for you through cancer,

    25    you need to diminish the amount of cyclin E to achieve

                                                                         96

     1    an equilibrium where you have enough cyclin E to

     2    support cellular replication, but not too much so that

     3    it's an unsuccessful high level.  Thank you very much.

     4                (Applause.)

     5                Oh, I did want to acknowledge -- could I

     6    have the slides for one second -- a very, very large

     7    number of collaborators.  I'll leave them up here. 

     8    But we're very grateful for our own students and

     9    collaborators at UAB, Jeff Engler, Doug Seer, Sean Van

    10    Tine, Kim Towns.  At Baylor, Wade Harper's lab, UNC,

    11    Jack Griffith's lab, who did the things with the HSP-

    12    40, and at Stanford, Theresa Wong, and our

    13    collaborators at the Free University, who did a lot of

    14    work on some transcriptional control that we

    15    collaborated on for several years.  Thanks.

    16                CHAIRPERSON RABINOVICH:  Thank you.  Are

    17    there any questions?

    18                DR. FRIED:  How could you be so sure that

    19    with the evolution of the HPVs, are not due to new

    20    infections, but to pre-existing sequences to cover?

    21                DR. BROKER:  I think one is the repetity -

    22    -

    23                DR. FRIED:  Mike Fried.

    24                DR. BROKER:  With which these viruses are

    25    appearing, especially in women who at least declare

                                                                         97

     1    they are not engaged in much, if any, sex.  A number

     2    of the epidemiologic studies have traced either the

     3    frequency of recent sexual activity.  Everyone who has

     4    looked feels its an emergence of pre-existing

     5    subclinical infections.  There is some evidence in new

     6    infection, but an awful lot of it simply appears.

     7                Women in that stage renal failure, for

     8    example, who are 38 years old and married basically,

     9    are not suddenly acquiring new infections.

    10                CHAIRPERSON RABINOVICH:  Back microphone,

    11    please.  Why don't you come up to the front.

    12                DR. RUSSO:  Hi.  Carlo Russo, from Merck. 

    13    Very interesting talk.

    14                I have a couple of questions.  One is, how

    15    can you be sure that what you are sampling by PCR is

    16    really an infective virus, it's not just a transient

    17    presence with a virus due to the fact that you can't

    18    control sexual behavior, and perhaps the woman has

    19    just been exposed to a virus?

    20                DR. BROKER:  Well, I think one way is that

    21    in a very high percentage of the people, there are

    22    clear dysplasia, low and high grade.  All the women

    23    who have any degree of dysplasia are also biopsied,

    24    and the inside 2 hybridization, as you can see, is

    25    showing clear effects in the tissue.

                                                                         98

     1                So I don't think there's surface

     2    adventitious contaminants.  These are within, at least

     3    a fair number, if not all, are within the cells, and

     4    causing various degrees of actual overt illness.

     5                DR. RUSSO:  I may have missed the data. 

     6    Did you show the types that are associated with high

     7    grade lesions, the HPV types?  I didn't see on the

     8    table.

     9                DR. BROKER:  Yes.  Well basically, in this

    10    immense spectrum of what's now 37 different viruses

    11    that we found, those that are most typically

    12    associated with low and high grade dysplasia, the

    13    actual diseases, are the higher risk types.

    14                DR. RUSSO:  So you are not suggesting that

    15    if you want to prevent cervical cancer, we should

    16    focus on different types of the one already

    17    identified?

    18                DR. BROKER:  Well the real problematic

    19    thing for any clinical management, either vaccination

    20    programs or small molecule drugs, is this absolutely

    21    exploding number of virus types.

    22                The one thing that I think is going to --

    23    and I commented a day or two ago that in the U.S.

    24    alone today, there are over 250 to 300,000 people

    25    immuno-suppressed just due to organ transplants,

                                                                         99

     1    steroid use, or bone marrow transplants or AIDS.  So

     2    there is an immense reservoir of particularly high

     3    risk patients.

     4                Nonetheless, most of the diseases are

     5    still being caused by a handful of viruses like 16,

     6    18, 52.  So I think, at least the ones we have to

     7    worry about today, are still manageable in number.

     8                AUDIENCE MEMBER:  I would like to ask a

     9    little bit about cell substrates.  Considering that

    10    hela has multiple HPV integrants, I guess, are any of

    11    those infectious?

    12                DR. BROKER:  No.

    13                AUDIENCE MEMBER:  Can you get them back

    14    and make them infectious?

    15                DR. BROKER:  No.

    16                AUDIENCE MEMBER:  What are they missing?

    17                DR. BROKER:  They are all truncated within

    18    E-2, at best.  Although there are 30 to 50 copies,

    19    depending on the hela sub-1.  All the integrated

    20    copies are truncated.

    21                AUDIENCE MEMBER:  Would that DNA be

    22    transforming, even not infectious?

    23                DR. BROKER:  They do contain the E-6 and

    24    E-7 genes.  Expression of those genes, as shown by

    25    studies primarily in zur Hausen's lab, must be

                                                                        100

     1    maintained or you no longer can cycle hela cells. 

     2    That is antisense to E-6 and E-7 in hela makes them

     3    not cycle any more.  So the driving force of hela is

     4    the overt expression of E-7.

     5                AUDIENCE MEMBER:  Would hela cells qualify

     6    as an example of a cell that should never be used to

     7    make a vaccine?

     8                DR. BROKER:  I don't know that I would go

     9    that far.  It's obviously wonderful as a producer of

    10    all sorts of biomedical products.

    11                DR. MURPHY:  I actually wanted to ask you

    12    the same question, but I would phrase it in a

    13    different way.

    14                (Laughter.)

    15                DR. MURPHY:  This is Murphy from NIAID. 

    16    That is, do you see any reason why, you know, having

    17    an intimate knowledge of hela cells and human

    18    papilloma viruses, that hela cells should not be used

    19    as a substrate for making live attenuated virus

    20    vaccines?

    21                DR. BROKER:  I don't know of any evidence

    22    of these genes being transduced out or in any way

    23    posing a risk.  I was going to save it for the panel,

    24    but it occurred to me last night, I had challenged one

    25    of last night's speakers about the use of psorilins as

                                                                        101

     1    a cross-linking agent.  It gives me great concern that

     2    it is a known carcinogen.  However, Brian and I were

     3    talking.  Brian van Tine and I were talking last

     4    night, and he reminded me that there are biotinylated

     5    psorilins.

     6                So for all the debates regarding how to

     7    remove contaminating DNA, one strategy in principle is

     8    throw in a biotinylated psorilin, cross link it, and

     9    pass the whole thing over avidin magnetic beads or

    10    batch subtraction of the DNA.

    11                So in fact, that strategy may actually

    12    help you deplete adventitious contaminants very, very

    13    readily.  So it is an alternative at least.

    14                AUDIENCE MEMBER:  Can I just ask you about

    15    hela again?  We learned last night that not every HPV,

    16    if there's 30 to 50 copies, are not all active, I mean

    17    in caski only one was active.

    18                DR. BROKER:  Yes.

    19                AUDIENCE MEMBER:  What is the state of

    20    hela?  Are they --

    21                DR. BROKER:  Very, very few are active. 

    22    We and Wade Harper's lab are both sequencing all the

    23    transcripts.  This actually was done by Elizabeth

    24    Schwartz and others in zur Hausen's lab in 1985, and

    25    a variety of groups since then in Japan and elsewhere

                                                                        102

     1    have looked at the expression loci in copies in hela.

     2                There appear to be three or four different

     3    transcripts made from different positional integrants,

     4    but the majority are silent.  A few of them are

     5    active.  But so far, all the ones that are active have

     6    truncated E-1s.  They have the cyclin E binding motif,

     7    but they don't have their normal carboxy terminus.

     8                CHAIRPERSON RABINOVICH:  Thank you very

     9    much.

    10                Let's go on to Dr. Cashman.  Thank you for

    11    being so patient.  Transmissible spongiform

    12    encephalopathies: vaccine issues.

    13                DR. CASHMAN:  It worked.  My friendly A/V

    14    guy explained how to do this.

    15                I am Neil Cashman.  I am predominantly at

    16    the Center for Research and Neuro Degenerative

    17    Diseases at the University of Toronto.  I have a

    18    special interest and a long-term research effort in

    19    the expression and function of the normal cellular

    20    isoform of the prion protein.

    21                I am also obliged to mention that I am the

    22    chief scientific officer one day a week of a little

    23    biotechnology company in Montreal called Caprion.

    24                I want to spend a few minutes talking

    25    about prions and prion disease.  We had a speaker

                                                                        103

     1    yesterday who said "and now for something completely

     2    different."  Well, how does a genomeless infectious

     3    agent grab you?

     4                Creuzfeldt-Jakob disease is the most

     5    common human prion disease that we run into.  I do

     6    want to spend a few minutes talking about this so that

     7    we are all on the same page with regard to public

     8    health risks.  Creuzfeldt-Jakob disease or CJD is

     9    basically a disease you wouldn't want to wish on your

    10    worst enemy.  It is a completely untreatable uniformly

    11    fatal disease resulting in death within six to nine

    12    months of presentation.  Survival over a year is

    13    recorded, but it is not very frequent.

    14                The presentation is usually that of a kind

    15    of Alzheimers-like syndrome, with problems in memory

    16    and intellectual function, but it can also present as

    17    a disorder of gait and balance as well.  Most people

    18    have mild clonus, which is twitching of the muscles,

    19    sufficiently forceful to move a joint.  Other features

    20    of the neuro-degenerative syndrome are reminiscent of

    21    other neuro-degenerative diseases like Lou Gehrig's

    22    disease and Parkinson's disease.  Basically it's like

    23    having every neuro-degenerative disease at once,

    24    telescoped into an unmercifully short period of

    25    decline.

                                                                        104

     1                Fortunately, it is rare.  Sporadic

     2    Creuzfeldt-Jakob disease occurs at about one per

     3    million population per year.  Also, somewhat

     4    fortunately, it's not a disease of children.  The

     5    average incidence of CJD is in the 60s.

     6                There are three recognized forms of CJD. 

     7    The most common being sporadic.  This is a spontaneous

     8    onset of CJD in an individual for which we have no

     9    clue why they have developed it.  There are familial

    10    variants, which seems to be passed as an autosomal

    11    dominant in families.  That constitutes about 15

    12    percent of the cases of human prion disease that

    13    occur.  There are iatrogenic prion diseases, which are

    14    caused essentially by treatments and surgeries, well-

    15    meaning, but nonetheless transmitting the disease.

    16                Of course the transmissible spongiform

    17    encephalopathy that even my kids know is bovine

    18    spongiform encephalopathy, or so-called mad cow

    19    disease.  Since the early 1980s, this disease has

    20    affected about 200,000 cattle in the U.K. and Republic

    21    of Ireland, and a few hundred across continental

    22    Europe.  About 2 million cattle have been killed in an

    23    attempt to stem the epidemic.  This culling, as well

    24    as change in policies, such as feeding ruminant to

    25    ruminant -- we turned cattle into neo-cannibals -- is

                                                                        105

     1    resulting in a rapid decline of new cases, predictions

     2    being that the epidemic in cattle may be essentially

     3    stamped out in the early part of the new millennium. 

     4    I won't even say the new century.  I'll say the new

     5    millennium.

     6                Unfortunately, this disease is unique,

     7    unlike every other known naturally occurring prion

     8    disease.  It doesn't seem to obey species barriers, or

     9    at least obeys them to a much lesser degree.  There is

    10    an outbreak of feline spongiform encephalopathy in

    11    house cats.  There is spongiform encephalopathy in zoo

    12    animals, including primates.  The primates that we are

    13    most concerned about are also vulnerable to this

    14    disease.

    15                To date, 44 people have developed a new

    16    variant of Creuzfeldt-Jakob disease, which is

    17    clinically and pathologically distinct from classical

    18    CJD.  The statisticians predict there will be

    19    somewhere between a few hundred and maybe 80,000

    20    cases.  This does not include the chicken little

    21    predictions of the extent of the epidemic.

    22                The disease unfortunately seems to strike

    23    the young.  There have been teenagers involved.  It is

    24    a relatively slower progression than classical CJD. 

    25    There are clinical features that are distinctive, but

                                                                        106

     1    I won't bore you with them this talk.  The pathology

     2    is also absolutely distinct, including a preter

     3    natural accumulation of PrP Sc, which is this abnormal

     4    amyloid protein that's been linked to infectivity. 

     5    This occurs both in the brain and in peripheral

     6    lymphoid tissues.

     7                Well, before we leave the clinical stuff

     8    about CJD and prion disease, I want to kind of set the

     9    stage with a sobering statistic, which is there is

    10    iatrogenic transmission of this group of diseases. 

    11    Considering the penetrants and the young age of

    12    vaccinees, this is a scary possibility.  This would

    13    dwarf every other iatrogenic transmission known to

    14    date.

    15                In humans, basically a few hundred cases

    16    have been attributed to iatrogenic transmission, from

    17    hormones extracted from cadaver pituitaries, from dura

    18    mater transplantation, which is the tough lining of

    19    the brain.  But incredibly, the largest iatrogenic

    20    transmission known to date, also the first documented,

    21    was that in passage with a vaccine, which was a

    22    vaccine for looping ill of sheep.  Formal and

    23    inactivated brain preparations passed sheep scrapie to

    24    about 1,000 sheep.  So hopefully this will not be a

    25    pattern with human vaccines.

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     1                Well here is the prion hypothesis.  This

     2    has gone from being an object of ridicule to the

     3    middle of the road interpretation of prion

     4    infectivity.  It has been sanctioned by the Nobel

     5    Prize committee, garnering the prize for Stanley

     6    Prussiner, the investigator whose ferocious work with

     7    this group of disorders and with this agent has given

     8    him, in my opinion, a well-deserved Nobel Prize.

     9                The basic tenants of the prion hypothesis

    10    are that there's a normal cellular protein, which is

    11    called PrPC, which has been cloned and recognized. 

    12    It's expressed by just about every organism down to

    13    drosophila.  It is a very old gene.  It's incredibly

    14    well conserved in evolution.  It is predominantly

    15    alpha helical in secondary structure.

    16                Now this normal cellular protein can adopt

    17    an alternate confirmation, which is rich in beta sheet

    18    structure.  When this protein is in this alternate

    19    confirmation, it acquires many unique physical

    20    chemical properties.  It becomes partially protease

    21    resistant.  It tends to aggregate.  It's very poorly

    22    soluble.  Plus, it then seems to act as a catalyst for

    23    recruiting more confirmational copies of itself.

    24                Now whether this occurs by a kind of

    25    enzymatic confirmase activity or whether this is kind

                                                                        108

     1    of a biological crystallization phenomenon is being

     2    actively investigated.  But it is clear that this

     3    abnormal confirmation isoform of the protein, called

     4    PrP Sc for scrapie, is capable of recruiting more

     5    confirmational copies of itself from the normal

     6    cellular isoform.

     7                So onto vaccines.  There are some concerns

     8    about vaccines.  I will mention three areas that need

     9    to be considered.  I will dwell most of the time on

    10    cell substrates, which is nice of me considering this

    11    is a cell substrate meeting.  I will also talk briefly

    12    about potential prion infectivity coming over in media

    13    supplements for those cells, and in excipients, which

    14    are compounds used to stabilize vaccines in their

    15    final formulation.

    16                In this case, luck seems to be at least

    17    partially on our side, because it's not easy to infect

    18    cells in vitro.  It is possible to infect primary

    19    neuro cultures.  It is even possible to infect

    20    neuronal cell lines neural blastoma.  But there is not

    21    much infectivity, and basically each successful

    22    infection of a cell line is worth a publication or 10.

    23                This may be due to the fact that cell

    24    lines have very little PrPC, which is the precursor

    25    for PrP Sc.  The conversion of the protein from PrPC

                                                                        109

     1    to PrP Sc forms occurs at the surface or a post-

     2    surface compartment.  So in general, cell surface

     3    abundance of the protein correlates with infectivity. 

     4    Most cell lines in my own laboratory, including hela,

     5    express no more than one-tenth of the amount of cell

     6    surface PrPC that a primary neuron does.

     7                It has also been thought that the very act

     8    of cell division itself can kind of sterilize a

     9    culture because cell division can out pace the

    10    relatively slow conversion and processing of PrP Sc. 

    11    So if you have a couple units of infectivity, they get

    12    progressively diluted by having huge numbers of cells

    13    that bear no infectivity.  Finally, those cells may

    14    die, the infected cells.

    15                There is also a poorly quanitifiable role

    16    for cell biology, which I put in quotation marks here. 

    17    Things that we really cannot quantify at this point,

    18    like proper trafficking, post-translational

    19    modifications of PrPC that are important in

    20    conversion, and even sub-cellular distribution.  The

    21    protein seems to accumulate in this glycosal

    22    phosphatindinol rafs at the cell surface.  Some cell

    23    lines don't seem to support these sort of rafs.

    24                Now to make some kind of estimate about

    25    the spontaneous development of prion infectivity in a

                                                                        110

     1    cell culture, especially a vaccine cell culture that

     2    may have hundreds of trillions of cells, I am going to

     3    back up and try to explore some assumptions about the

     4    spontaneous development of prion disease in humans,

     5    which is the species for which we have the best

     6    numbers.

     7                According to the prion hypothesis, an

     8    occasional accidental mis-folding of PrPC to PrP Sc is

     9    what triggers the recruitment process which proceeds

    10    on an exponential basis.  Each molecule that's

    11    converted converts to more, da, da, da, da, da, da,

    12    which happens on a post-translational level.  No

    13    genome involved.  "Look, ma, no genome."

    14                But sporadic disease in humans is

    15    incredibly rare, one per million people per year. 

    16    Humans have something on the order of 100 billion

    17    neurons.  So one can make the kind of interesting

    18    calculation that a productive infection arising from

    19    a single neuron, you need about 10 to the 17th

    20    neurons, 100 million neurons across a million people

    21    in order to develop spontaneous scrapie.

    22                But of course that's not the only way one

    23    could potentially develop spontaneous CJD.  Discovered

    24    by familial CJD and familial prion diseases, there are

    25    mutations within the open reading frame of the prion

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     1    protein that apparently predispose to this accidental

     2    misfolding, such that somebody with a prion protein

     3    mutation that actually results in an amino acid

     4    substitution is basically guaranteed of developing the

     5    disease over the course of a lifetime if he or she

     6    lives long enough.

     7                So could this occur in vitro?  Could

     8    certain cells in vitro acquire a somatic mutation

     9    which is then propagated to infect an entire culture,

    10    again, on a post-translational level?  Well, let's run

    11    some numbers on this one.

    12                The mutation rate in man is about one per

    13    billion basepairs per year.  I thank Dr. Kazazian for

    14    yesterday for pointing me to this reference.  Thank

    15    you very much.

    16                The prion protein open reading frame is

    17    really less than 1,000 basepairs.  It's a relatively

    18    small protein that's all contained in one exon.  This

    19    gives rise to a kind of pseudo calculation that a cell

    20    can develop a mutant prion protein gene, a cell in

    21    vivo, and a human can develop a mutant prion protein

    22    gene in about one out of a million cells per year, if

    23    you take one out of a billion and multiply it by

    24    1,000, that's one out of a million.

    25                This, just as an aside, this gives a rise

                                                                        112

     1    to a kind of startling calculation that all of us in

     2    the audience are generating about 100,000 prion

     3    mutants in our brain per year.

     4                However, there must be a safety factor

     5    here because the rate of prion disease arising from

     6    somatic mutation cannot exceed the calculation of

     7    prion disease arising from individual neurons that we

     8    just went through in the last slide, which is 10 to

     9    the 17th neurons per year.

    10                So incredibly, somatic mutation is a

    11    seriously flawed non-efficient process for producing

    12    prion disease.  Something on the order of one out of

    13    100 billion mutations are productive of infection. 

    14    This may give us some comfort when we turn to the in

    15    vitro scenario.

    16                So let's talk about cell substrates.  Is

    17    it possible that spontaneous prion infectivity could

    18    arise de novo in a culture?  I told you that substrate

    19    cells have usually less than one-tenth PrPC than

    20    neurons.  So if we run these calculations, by

    21    misfolding one substrate cell per 10 to the 18th

    22    years, and I think that is a quadrillion or something

    23    like that, it's certainly comfortably larger than the

    24    projected age of the universe.

    25                Substrate cells, however, are less

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     1    genomically stable than primary neurons.  If one says

     2    that there's say 1,000-fold greater rate instead of

     3    one out of a billion basepairs, one out of a million

     4    basepairs can be mutated per year in a substrate cell,

     5    this gives rise to a calculation suggesting that you

     6    need 10 to the 14th substrate cells per year in order

     7    to have one productive, i.e. spontaneous infectivity

     8    arising in a culture.

     9                Now this number looks incredibly large,

    10    but when we think about the numbers we heard last

    11    night about the production of polio virus from vero

    12    cells, according to my calculations, 300 million cells

    13    are used per year to generate all those vaccine lots. 

    14    Ten to the 14th is only 100 -- sorry, 300 trillion,

    15    and 10 to the 14th is only 100 trillion.  Did I get

    16    that right?  Please forgive me and correct me if I

    17    didn't get it right.

    18                So perhaps it is possible, considering the

    19    enormous scale of substrate cell culture, that prion

    20    infectivity could arise through somatic mutation in a

    21    substrate cell, and could contaminate a vaccine

    22    destined for human beings.

    23                Well, there are some things to talk about

    24    with this model.  If this is so, how come we haven't

    25    seen any vaccine transmissions yet?  One of the

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     1    factors is that very few human cell line vaccines have

     2    gone into humans, certainly not on a scale of vero

     3    cells or primary cells that are used for culture.

     4                Another unsettling thing is that if indeed

     5    there is somatic mutation in a culture of human

     6    substrate cells, would we ever detect it?  We are

     7    talking about something that would occur in one out of

     8    a million cells or even one out of a thousand cells,

     9    would ever be able to find by PCR or SSEP or anything

    10    you could think of, a mutation at this level.

    11                So aside from substrate cells, I did want

    12    to touch upon a few potential sources of infectivity. 

    13    The media coming in contact with substrate cells are

    14    potentially carrying prion infectivity.  Bovine serum,

    15    fetal calf serum, and newborn calf serum is used as a

    16    supplement for proteins and growth factors and

    17    hormones for most cell lines.  Some cell lines are

    18    also supported by human serum albumin.  I am not aware

    19    of a vaccine cell line that's supported in this

    20    manner, but many recombinant proteins are supported

    21    with human serum albumin.

    22                There's also potential prion infectivity

    23    in excipients, this last compound that's added to the

    24    preparation to keep it stable before use.  Many

    25    childhood vaccines are stabilized with pig skin

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     1    gelatin.  Pigs don't seem to be a species which

     2    spontaneously develop prion disease or a species that

     3    can catch prion disease via the oral route, although

     4    deliberate intracranial injection of BSE infectivity

     5    can produce a prion disorder.

     6                Human serum albumin is also an excipient

     7    in measles, mumps, rubella, and rabies vaccines.  I

     8    would like to spend just a few seconds talking about

     9    human plasma proteins before I close, and give my

    10    final advice, such as it is.

    11                Human serum albumin of course comes from

    12    humans.  Of course it's a plasma fractionation

    13    product.  There has been a great deal of work trying

    14    to identify potential risks of transmission of CJD

    15    from human to human through blood or blood products. 

    16    Suffice it to say that population studies, case

    17    control studies, and cohort studies have proven

    18    universally negative.  There is no documented

    19    incidence of human CJD, classical CJD being passed

    20    through blood or blood products.

    21                There are of course case reports of people

    22    getting a transfusion and developing CJD, but one

    23    should not expect that transfusion or administration

    24    of a blood product is protective against CJD.  The

    25    incidence of CJD in the transfused or treated

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     1    population is the same as in the non-transfused or

     2    treated population.

     3                However, we're in more difficult territory

     4    with variant CJD.  This is, I told you, an unusual

     5    agent.  It seems to cross species lines with impunity. 

     6    There are other features that are quite scary with

     7    regard to human blood and human plasma products,

     8    including albumin that might be used as an excipient.

     9                The agent starts of course in the

    10    periphery through oral exposure, suggesting a

    11    prionemia.  There is a huge accumulation of PrP Sc,

    12    our only surrogate for infectivity, our only

    13    biochemical surrogate for infectivity, in not only

    14    brain, but in lymphoid tissue.

    15                The agent itself has odd properties.  It

    16    is stable across species.  It may in fact be

    17    specialized or selected.  I realize that these terms

    18    are not often applied to a protein.  I'm borrowing

    19    terms from agents that contain a genome.  It suggests

    20    that this agent may be more virulent, especially with

    21    regard to peripheral exposure.  In other words, one

    22    unit of classical CJD infectivity will not cause

    23    disease when injected intramuscularly.  One unit of

    24    variant CJD infectivity may very well.  There is no

    25    data.

                                                                        117

     1                Another thing that has been noted is the

     2    young age of onset of variant CJD.  This has been

     3    attributed to kids eating hamburgers and all kinds of

     4    weird meat products.  But it could also be attributed

     5    to host factors which would promote infectivity in the

     6    young.  Since vaccinees are usually young, we have to

     7    take this in mind.

     8                But our greatest risk factor here is that

     9    this is a new disease and we have no data.  So how do

    10    we minimize the risks from vaccine transmission? 

    11    Basically there's three ways that I can think of.  I'm

    12    sure that other people can think of more.  It is

    13    important to add prion validation to the list of

    14    agents and microbes which are tested for in vaccine

    15    lots.

    16                This could be done two ways.  The

    17    biochemical marker of infectivity is PrP Sc.  This

    18    technology is in evolution.  It appears that capillary

    19    electrophoresis, some types of optimized immuno

    20    blotting, and even ELISAs are reaching the point where

    21    one unit of infectivity will be detectible.

    22                Another important way of assay for

    23    infectivity is called the bio assay in the field, in

    24    which selected samples are injected into a species

    25    which is capable of supporting that infectivity.  That

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     1    would be non-human primates, and again, a technology

     2    in evolution, transgenics engineered to express human

     3    and perhaps bovine PrP.

     4                There is also the possibility of trying to

     5    prophylax cultures, substrate cultures with chemical

     6    agents.  This is also in evolution, but the classic

     7    molecule in this regard is congo red, which not only

     8    seems to bind to PrP Sc, but seems to dissolve

     9    infectivity in vitro.  New discoveries out of Byron

    10    Kohe's lab that tetrapyrrole, including porphyrins and

    11    phthalocyanines, can block infectivity.  Perhaps some

    12    of these compounds can be utilized at appropriate

    13    concentrations to use as a kind of antibiotic for

    14    substrate cultures.

    15                Finally, the slam dunk in this area would

    16    be to develop a cell line that lacks a prion protein

    17    gene.  The prion infectivity, whatever the hell it is,

    18    seems to be absolutely dependent on the presence and

    19    expression of PrPC.  So if one were able to ablate the

    20    prion gene out of a substrate cell, that didn't come

    21    with 300 other bad pathogens, this may be a strategy

    22    of obviating any prion infectivity in vaccines.

    23                So I would like to summarize by saying

    24    that it is possible, although not favorable, for

    25    substrate cells to be infected with prions.  It is

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     1    possible, considering the huge bulk of cells that are

     2    cultured, 300 million a year for one vaccine, that

     3    prion infectivity could potentially emerge by

     4    misfolding and/or somatic mutation in vitro.

     5                I will note that prion components and

     6    excipients may contain prion infectivity.  Although

     7    this is an old story with regard to classical CJD, we

     8    don't have the information for the BSC variant CJD

     9    agent.  We should worry, at least for the time being.

    10                The remedies for this are selective

    11    sourcing, avoid animals and people that could

    12    potentially be brewing prion infection, biological

    13    manipulation in vitro, including anti-prion agents,

    14    and maybe ablating the prion gene, and then validate,

    15    validate, validate.  Prion infectivity should be added

    16    to the list of infectivities that are excluded in

    17    vaccine lots.

    18                I thank you for your attention, and I

    19    would be glad to answer any questions.

    20                (Applause.)

    21                DR. KRAUSE:  Phil Krause, FDA.  In keeping

    22    with the idea that one presumably wants to find cell

    23    substrates which carry the least risk, I guess if one

    24    presumes that tumor cells have a greater risk of

    25    genomic instability than non-tumor cells, are you then

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     1    implying that there's a greater sort of spontaneous

     2    mutation than prion risk from tumor cells than for

     3    instance primary or diploid cells?

     4                DR. CASHMAN:  That's a good question.  I

     5    guess this is basically not quantifiable.  If one

     6    takes a rock solid cell that enjoys all kinds of DNA

     7    repair mechanisms then yes, that is less likely to

     8    give rise to the mutation in the prion protein gene.

     9                One area which should be investigated, I'm

    10    realizing from this meeting, is to take some cell

    11    lines and look at 1,000 clones a piece and see if any

    12    of the prion copies have acquired mutations.  So this

    13    would be a piece of data that we could use to actually

    14    discuss this issue.  Right now, I don't have any.

    15                AUDIENCE MEMBER:  Along the same lines, I

    16    guess the implication would be that if somebody wanted

    17    to produce a vaccine in cells that are derived from

    18    neurons, given the fact that they are making a lot

    19    more of this, you have the potential for greater RNA

    20    polymerase mutation rates, and perhaps also greater

    21    risks?

    22                DR. CASHMAN:  Well, neurons are the best

    23    factoring for making infectivity, both in vivo and in

    24    vitro.  Part of that is due to high levels prion

    25    protein.  Part of it is due to factors that haven't

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     1    been identified as of yet, prion receptor, trafficking

     2    of prion protein.  So I guess one would want to stay

     3    away from neurons unless they came from an animal or

     4    human that had the prion gene locked out or not

     5    transcribed or translated.

     6                CHAIRPERSON RABINOVICH:  Dr. Egan?

     7                DR. EGAN:  As Dr. Minor mentioned before,

     8    Japanese encephalitis virus is made in mouse brains. 

     9    Can the PrP SC of a mouse catalyze the conversion of

    10    human PrPC to PrP Sc?

    11                DR. CASHMAN:  That is an extremely good

    12    question.  I neglected to mention in my talk, my

    13    oversight, that there is a very prominent species

    14    barrier between most prion agents, something like one

    15    to a thousand, one to a million, even higher for

    16    generating infectivity, especially between widely

    17    differing species.

    18                Now as we move to human cell substrates,

    19    we will no longer be protected by the species barrier. 

    20    Even non-human primates have a sequence similarity to

    21    convert human PrP and vice versa.

    22                So yes, I think that answers it.  The

    23    exception to that is of course this new variant agent 

    24    which we're all frightened about because it doesn't

    25    seem to obey species barrier.

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     1                DR. ONIONS:  David Onions.  Could I just

     2    ask the converse of the question that you posed for

     3    vaccine substrates, which is I think one that has been

     4    discussed.  That is, the idea of knocking out the PrP

     5    gene.  We know that PrP mice are viable, so it looks

     6    like perhaps an interesting way to go.

     7                But can you also engineer cells over

     8    expressing normal PrP and use those as substrates for

     9    infectivity?  You mentioned that one of the problems

    10    was the low level of PrPC in most of the cell lines

    11    you have used.  Can you not over-engineer cells so

    12    that they become susceptible?

    13                DR. CASHMAN:  The only -- that's never

    14    been done.  The only data that really pertains is

    15    transgenic mice.  In fact, if you have knocked out the

    16    prion gene, you are absolutely resistant.  If you have

    17    one normal copy, you have disease with a longer

    18    incubation and a shorter rate of -- or a longer rate

    19    of progression.  If you have two normal copies, you

    20    have disease at the normal time.  If you have

    21    transgenic 10, 20, 100 copies, then the disease

    22    presents at an earlier age and is more rapidly

    23    progressive.

    24                As far as I know, nobody has moved that

    25    observation to an in vitro paradigm.

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     1                DR. LOEWER:  Johannes Loewer, Frankfurt. 

     2    I would like to challenge, to some extent, your

     3    calculation on the risk of mutations, because they do

     4    not take into account the similar biologies of prion

     5    protein that are secreted from the cell, as they can

     6    spread to other cells.  That there's really a short

     7    time induction of new PrP Sc.

     8                For example, at least to my knowledge, if

     9    you infect so to say cell cultures who use prion via

    10    PrP, you get another multiplication of infectivity. 

    11    It stays more or less.  If it disappears, it may be

    12    more or less there's no real spread.

    13                The question I have is, you are asking for

    14    validation studies.  What would be the material you

    15    would recommend to spike?  Should we spike with

    16    scrapie brain material?  Is this relevant for

    17    purification in these cases?  What would be the ideal

    18    spike?

    19                DR. CASHMAN:  I would very much appreciate

    20    some challenge from my numbers.  They are new numbers,

    21    so don't hold back.

    22                The question of spiking, there have been

    23    experiments performed at least with a purification of

    24    albumin and other plasma proteins.  Paul Brown and

    25    Robert Roewer published an article last fall in

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     1    Transfusion detailing the amount of infectivity if you

     2    start from a high amount in whole blood, what you end

     3    up with in albumin.  In fact, there's at least a 10 to

     4    the 6th loss of prion infectivity if one follows the

     5    normal protocols for purification of albumin.

     6                The question is whether a single

     7    infectious particle is there.  The other question is

     8    what happens if this single infectious particle is

     9    variant CJD.  If the single particle is classical CJD,

    10    nothing would happen.  The disease would not take

    11    hold.  We don't have enough information about the

    12    virulence of variant CJD to be able to answer that

    13    question with any confidence.

    14                Did that answer your question?  I have a

    15    feeling it didn't.

    16                DR. FRIED:  Mike Fried.  I think your

    17    numbers also don't take into account modifier genes,

    18    which could be just random mutation.

    19                For instance, I understand that all the

    20    people with the new variant CJD have a polymorphism of

    21    one type in the PrP, the protein.  Is that not true?

    22                DR. CASHMAN:  In fact, that is partially

    23    right.  There is a normal polymorphism in the prion

    24    protein open reading frame at codon 117.  One can

    25    either have a valine or a methionine.  To date,

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     1    everyone -- well, there's a nice distribution in the

     2    normal population, about 50 percent are heterozygotes

     3    and about 25 percent are met met, and 25 percent are

     4    val val.

     5                In a new variant CJD, everyone who has

     6    contracted the disease to date has been met met.  But

     7    that in fact may be due to the fact that the met mets

     8    are more susceptible to the agent.  It may not be due

     9    to the fact that val vals or heterozygotes are

    10    resistant.

    11                DR. FRIED:  Sure.  I am just saying that

    12    that goes into your numbers, whether there's

    13    modifications.

    14                DR. CASHMAN:  The point of modifier genes

    15    is a very important point.  I tried to -- 129, I'm

    16    sorry.  That's David Asher who was keeping me honest

    17    in more ways than one.  It's the polymorphism is at

    18    codon 129.

    19                Now what was I going to say?  I was going

    20    to say something.

    21                CHAIRPERSON RABINOVICH:  Modifier genes.

    22                DR. CASHMAN:  Modifier genes, yes. 

    23    Modifier genes have of course been proposed from

    24    animal studies of infectibility and experimental

    25    scrapie.  Dr. Prussiner has hypothesized a protein X,

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     1    which may be a receptor or may be a chaperon that

     2    somehow modifies susceptibility of an animal to prion

     3    diseases.  There is also a protein Y that Dr.

     4    Prussiner has hypothesized.

     5                I agree with you from the bottom of my

     6    heart that there will be modifier genes affecting

     7    susceptibility to prion diseases and the propagation

     8    of prion infectivity in vitro.  But we don't know what

     9    they are yet.  At the crude operational level of being

    10    able to infect cells, yes.  We can infect cells in

    11    vitro.  So at least some of those modifiers have to be

    12    there.  Did that make sense?

    13                CHAIRPERSON RABINOVICH:  Dr. Hayflick,

    14    final question.

    15                DR. HAYFLICK:  Hayflick, UCSF.  I was

    16    intrigued by your observation that the species barrier

    17    for prion transmission is less, is reduced between

    18    non-human primates and humans, which would raise some

    19    additional concern about the use of primary tissue,

    20    and particular, primary monkey kidney tissue for the

    21    production of human virus vaccines, because contrary

    22    to popular belief, that tissue and any primary tissue

    23    does not contain -- and I'll use primary monkey kidney

    24    as an example, only cells that are derived from a

    25    particular part of the kidney.

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     1                A primary monkey kidney culture consists

     2    of an enormous variety of differentiated cell types

     3    that compose the vascular system and neurons.  So that

     4    monkey neurons do play a part in the production of

     5    polio virus, for example, derived from monkey kidney.

     6    So that I think that it's important to mention that

     7    neurons are not only a part of brain tissue in

     8    considering various cell substrates.

     9                Also I was wondering whether there's some

    10    reason why you omitted the mention of trypsin as

    11    potential source in your discussion of substrates or

    12    media supplements for prion transmission.  I didn't

    13    see trypsin as a component.  Was there some reason for

    14    that omission?

    15                DR. CASHMAN:  Just blanking out.  So thank

    16    you for adding that to the list.

    17                DR. HAYFLICK:  The final question I have

    18    is I may have misunderstood your slide in which you

    19    were addressing the question of utilization of human

    20    cell line, that a human cell line had not been used

    21    for the production of enormous numbers of doses of

    22    vaccine, for example, in order to support one of your

    23    contentions.

    24                Did you mean it in that respect, a human

    25    cell line that is defined to be immortal and

                                                                        128

     1    transformed?  Or did you mean any human cell

     2    population?

     3                DR. CASHMAN:  Perhaps you could educate

     4    me.  Has any human immortal or neoplastic cell line

     5    been used to develop large penetrants, large

     6    population vaccines?

     7                DR. HAYFLICK:  No.  Not as you have

     8    defined that cell population.  I was interested

     9    particularly in normal human cell populations, which

    10    have been used for the production of up to a billion,

    11    with a B, doses of virus vaccine.  But you are not

    12    including in that?

    13                DR. CASHMAN:  Say it again.  I'm sorry.

    14                DR. HAYFLICK:  Normal human cell

    15    populations have been used for the production of about

    16    three-quarters of a billion doses of human virus

    17    vaccine world wide, but these are not continuously

    18    propagable abnormal heteroploid cell populations. 

    19    These are normal finite lifetime cell populations.

    20                DR. CASHMAN:  So these are cell strains?

    21                DR. HAYFLICK:  Yes, as I defined them.  I

    22    realize there's a problem in understanding these

    23    terms.

    24                DR. CASHMAN:  Which vaccines?

    25                DR. HAYFLICK:  Virtually all pediatric

                                                                        129

     1    vaccines, polio, rubella, mumps, measles, rabies,

     2    adenovirus, some rhinovirus vaccines, are all produced

     3    on a semi-continuous human diploid cell strain like

     4    WI38 or MRC5.

     5                DR. CASHMAN:  Thank you.

     6                CHAIRPERSON RABINOVICH:  I think you

     7    better clarify.

     8                DR. LEWIS:  Yes.  To my knowledge, there

     9    are really no nerve cells in the kidney.  The nerve

    10    cells lie on the spinal cord and porosises go down

    11    there.  I don't believe there are any nerve cells in

    12    the kidney.  Even if they are, once the nerve cell is

    13    differentiated, they basically do not grow on tissue

    14    culture.  I think that needs to be corrected.

    15                CHAIRPERSON RABINOVICH:  Okay.  I would

    16    like to move rapidly to bring the panel members up to

    17    the podium, and to invite Dr. Onions to come over and

    18    run things.

    19                DR. ONIONS:  I notice that we now have

    20    actually 45 seconds for discussion according to the 

    21    program.

    22                (Laughter.)

    23                Brilliant as this panel are, I don't think

    24    they could do that.  So could I have some guidance on

    25    when you would like to close this panel session? 

                                                                        130

     1    Perhaps somebody could give me some guidance.

     2                CHAIRPERSON RABINOVICH:  Forty five

     3    minutes.

     4                DR. ONIONS:  Forty five minutes.  Thank

     5    you very much.

     6                Okay.  We were charged in this panel to

     7    answer a number of questions.  I'll come to those and

     8    try and go through and cover the areas that the panel

     9    will discuss.  I would be very grateful for as much

    10    participation from the audience as possible.

    11                I thought it was just useful to pick up

    12    two strands that I think came out of some of the

    13    comments yesterday.  One I think that's important to

    14    make, and that is that vaccine production is a very

    15    pragmatic process, and that once there have been lots

    16    of theoretical objections to particular cell

    17    substrates, particularly autogenic cell substrates,

    18    there are very practical reasons for the use of cell

    19    substrates that might be immortalized or neoplastic

    20    from a new generation of vaccines.  I don't think we

    21    should lose sight of that.  There are very practical

    22    reasons in scale up and use that I think we should

    23    bear in mind to produce therapeutic vaccines.

    24                The second concern was that came out, and

    25    perhaps didn't get enough airing as it should have

                                                                        131

     1    done, and that is there clearly is a trend in society

     2    at the moment about concern in safety of vaccines. 

     3    That perhaps therefore focuses particularly on the

     4    item we are going to discuss today, which is the

     5    possibility of adventitious agents.

     6                The question that the FDA asked us to

     7    evaluate, or at least one of the first questions, and

     8    they would like the panel to take a look at is do

     9    neoplastic cells represent the greater equivalent or

    10    lesser risk for the presence of adventitious agents

    11    than primary cells, diploid cells, or non chunogenic

    12    continuous cell lines.

    13                I am not sure, given my previous comments,

    14    whether I think necessarily that you can answer that

    15    in a simple sense, but does anyone in the panel want

    16    to sort of pick that one up first of all?

    17                What I could perhaps do is to prompt

    18    people, is perhaps to put up those which is just my

    19    suggestion, of some of the factors that might

    20    influence the risk of adventitious agent testing in a

    21    variety of cell substrates.

    22                DR. MINOR:  I am personally convinced that

    23    they are going to be better than primary cultures.  I

    24    am not sure whether they will be necessarily better

    25    than human diploid cells or anything else if we even

                                                                        132

     1    get a decent banking system going.  It seems to me

     2    that when you get to that kind of stage, it's the

     3    concern about how you find what's in there rather than

     4    anything else.  I think the actual extent to which you

     5    can characterize them is clearly to me, it's very

     6    similar.

     7                DR. ONIONS:  That would be a generally

     8    universal statement that primary cells are likely to

     9    be more difficult to characterize and therefore, if

    10    you can use a cell line, that is probably the way to

    11    go.

    12                But I think it's also worth making a

    13    countervailing point that there are still vaccine

    14    strains that are very successfully produced in primary

    15    cells.  There are others that have been passed in

    16    primary cells and therefore change them into a cell

    17    substrate, the genetic stability of them.  So there

    18    are nevertheless countervailing arguments.

    19                I think the statement is correct.  That

    20    is, adventitious agent testing is clearly going to be

    21    more in the region --

    22                DR. ROBERTSON:  Another point which one

    23    could consider.  Where neoplastic cells might be

    24    considered more susceptible than primary cell

    25    cultures, in that the primary cells are derived

                                                                        133

     1    specifically for vaccine production.  Whereas the

     2    neoplastic cells have probably been kicking around at

     3    least one, if not several laboratories, before being

     4    put into use as manufacturing of vaccines.

     5                Because of that, they may well have picked

     6    up something that you don't want to be there.  Nothing

     7    to do with the cell type or the origin of the cell. 

     8    So a virus of some different species all together

     9    which you really have got to check for.

    10                So if you are actually setting up a cell

    11    bank of a neoplastic cell, you shouldn't just be

    12    considering species of origin of that particular cell,

    13    whether it's porcine, human, murine whatever.  You

    14    have really got to consider any virus under the sun. 

    15    We know there have been instances of this happening.

    16                This morning there was the comment about

    17    I think it was a human endogenous retrovirus which was

    18    actually of murine origin, been picked up from passage

    19    somewhere.

    20                DR. ONIONS:  There is a good example in a

    21    commercial product.  That of course was the Glaxo

    22    Wellcome's novalma cell line which was used to produce

    23    interferon, which in fact contained SNRV, and probably

    24    picked up SNRV in George Kahn's lab at some point

    25    during its history, I think was the general consensus. 

                                                                        134

     1    But clearly that was unknown and the whole system was

     2    used in the production for several years before it was

     3    realized that perhaps this was contaminated by an

     4    adventitious agent that you just would not expect in

     5    this cell line.  So I think that is a very good point.

     6                There are issues about tumor cells.  One

     7    of the things that occurs to me is that -- actually it

     8    does concern me quite a lot about adventitious agent

     9    testing.  That is that it is rather traditional in

    10    character still.  It is not very directed in terms of

    11    its specificity in looking for certain viruses.  That

    12    is changing I think, but I think until recently that

    13    has been the case.

    14                For instance, we have known that in

    15    certain tumor cell lines, that viruses that we have

    16    only recognized in the last decade are certainly

    17    found.  For instance, well HHV-6 isn't a transforming

    18    virus, but there are cell lines that carry HHV-6 that

    19    have been used in the lab for many years.  The same is

    20    true for HHV-8, which is a transforming virus.

    21                So there is a concern that we may have

    22    cell substrates that are contaminated by other tumor

    23    viruses.

    24                Tom, would you like to pick up?

    25                DR. BROKER:  I think we have actually a

                                                                        135

     1    wonderful opportunity for a so-called natural

     2    experiment.  That is solid organ transplantation.  It

     3    turns out, as we all know now, virtually all kinds of

     4    organs, not only the corneal we have just heard about,

     5    but kidney, liver, pancreas, part of the intestine,

     6    heart, lung, so forth, have all been transplanted.  I

     7    think the opportunity is that the recipient is

     8    invariably immuno-suppressed until the transplant

     9    takes, and then they are slowly weaned off the drug

    10    like cyclosporin.

    11                Yet on other occasions, the transplant

    12    fails for one or another reason.  One could go back

    13    into failed transplants to look for the reactivation

    14    of agents that came from all these different tumor or

    15    tissue types I mean.

    16                One example I could cite that we recently

    17    encountered in the course of our kidney transplant

    18    study is a pair of kidneys that went in from a five-

    19    year-old boy to a 19-year old female.  Within a few

    20    days, the kidneys had completely become destroyed,

    21    necrotic.

    22                It turned out -- they suspected CMV

    23    infection, but it turned out to be adenovirus.  The

    24    presumption, and I'm being completely hypothetical, is

    25    the five-year-old boy who had died in a bicycle

                                                                        136

     1    accident, the donor, probably was in the age bracket

     2    where adeno was just a natural infection in his

     3    airway, and that these cells say from his tonsils or

     4    adenoids, which were in the midst of processing the

     5    adeno, became circulating, were in the kidneys, and

     6    the recipient female then acquired adeno-infected

     7    kidneys, and upon transplant to her, the virus

     8    reactivated and just wiped out the tissues.  I might

     9    also say the different individual who received the

    10    boy's liver also lost the liver.

    11                So presumably these were entering through

    12    B cells that were in any of these remote organs. 

    13    Nonetheless, the basic opportunity to look at organ

    14    recipients I think is the experiment to ask how much

    15    infectious agent is being transferred.

    16                DR. RUSSO:  Carlo Russo from Merck.  I

    17    think as you indicated, these patients are profoundly

    18    immune-suppressed.  Therefore, is going to be very

    19    difficult to assess where this agent came from.  In

    20    your case, it's very well possible that the woman was

    21    exposed to adeno virus.  Since she was immuno-

    22    suppressed, that's the reason why she got the

    23    infection.

    24                DR. BROKER:  Well, in that case, it's why

    25    I did also point out that the liver who went into a

                                                                        137

     1    completely different boy also wiped out.  But I agree

     2    with you.  One doesn't know whether it's endogenous,

     3    but it does give some indication of infectious agents

     4    in these organs.

     5                DR. ONIONS:  Could I just actually take a

     6    backup actually?  I was about to go back to the

     7    primary cell issue again.  Phil gave I thought a

     8    wonderful presentation.  I hadn't heard parts of this

     9    before.  It actually started to worry me a little bit

    10    actually.

    11                To what extent do you need now to control

    12    the kinds of colonies of these particular primary

    13    colonies?  I'm not sure, I mean I don't know what kind

    14    of testing goes on in these colonies for a range of

    15    adventitious agents.

    16                Can you maybe just comment on that?  I

    17    mean are we dealing with inverted SPF animals?

    18                DR. MINOR:  Well with respect to the

    19    primate, you will certainly not.  But they are

    20    increasingly heavily monitored.  It depends very much

    21    on the manufacturer and how much monitoring they do.

    22                One manufacturer, for example, has only

    23    recently, well in the last four or five years I guess,

    24    started using colonies of monkeys that were monitored

    25    for foamy virus.  The result of that is being

                                                                        138

     1    revolutionary in terms of the number of cultures that

     2    you get surviving to production.

     3                You would have thought you might have

     4    started this a bit earlier perhaps.  But you couldn't

     5    call them SPF, but they are increasingly closely

     6    monitored I think.  Certainly some manufacturers have

     7    them more closely monitored than others.

     8                But one of the difficulties with the whole

     9    of adventitious agent business of course is you only

    10    really find what you are looking for.  That's an

    11    ongoing problem.

    12                Things like chickens are a different

    13    matter.  I mean I think this would establish what you

    14    need to do to make an SPF chicken colony.  But

    15    primates are much more tricky.

    16                DR. ONIONS:  There are other cell

    17    substrates out there that are used, cells like primary

    18    hamster kidney cells in JV vaccines and various other

    19    things.  So there are I think other vaccines out there

    20    that are going to come to attention because they use

    21    primary cells.  I think we perhaps ought to start

    22    thinking of the kinds of procedures that are needed,

    23    like closed colonies and embryo derivation of these

    24    animals in some cases.

    25                Could I move to perhaps the third element. 

                                                                        139

     1    That is, we have heard a lot about retrovirus. 

     2    Retroviruses always come back to focus when we deal

     3    with cell substrates.  What is the panel and the

     4    group's feeling here in general about the concerns of

     5    using either immortalized cells or transformed cells,

     6    because frequently those -- well, that's not an

     7    accurate statement.  Activation of transcription of

     8    endogenous genes is more frequent in such cells.  Is

     9    that of concern or not a concern?  Or do we have to go

    10    cell by cell, species by species, to answer that

    11    question?

    12                John, would you like to make a comment?

    13                AUDIENCE MEMBER:  Well, I think clearly

    14    there are some famous cases of activation of

    15    transcription of endogenous retroviral genes and

    16    genomes and tumor cells.  We heard about germal

    17    tumors.  We have heard about recently in the news

    18    about mammary tumors and probably a variety of others.

    19                It is not entirely clear to me whether

    20    this actually represents activation of transcription

    21    of these cell lines or a fixation of a differentiated

    22    state which itself is what's activating the

    23    transcription.  I am inclined to think probably both

    24    are true in different cases.

    25                One of the things that we have learned, at

                                                                        140

     1    least from human tumor cell lines, is that none of

     2    these things that are activated have ever been shown

     3    to be infectious, despite the fact that as came out in

     4    the earlier talk, the probability for recombination

     5    between a large variety, for example of the HERV-K

     6    sequences, would seem to be rather high.  If these

     7    were other types of retroviruses, such as MLVs where

     8    you do get that endogenous, you do get that kind of

     9    thing.

    10                So there's probably something else that's

    11    protecting the people in the cell against actual

    12    infectivity in this particular case.  We don't yet

    13    know what it is.

    14                DR. ONIONS:  I was just going to bring in,

    15    I was very struck by the HERV-K story.  That is to a

    16    very pragmatic level.  I take the point that it does

    17    seem to me that it doesn't much recombination or much

    18    adjustment to me, at least into a more functional

    19    virus.  I mean should we simply be screening any human

    20    cell substrate for the expression HERV-K?  Would that

    21    be something that would be useful to do?

    22                DR. PALLEY:  That's a problem, in that you

    23    will I guess you will have a hard time finding a human

    24    cell line that would not express any human retrovirus,

    25    so I report that it's the case for HERV-K, the special

                                                                        141

     1    HERV-K family.

     2                DR. ONIONS:  But that particular locus, I

     3    mean that is not expressed, as I understood you, in

     4    all?

     5                DR. PALLEY:  We do not know whether that

     6    particular locus is expressed in some cell lines.  We

     7    know that if this, at least this HERV-K family, is

     8    activated for some reason, that there is very likely

     9    not only one locus activated within the genome, but

    10    that there are several loci that will be activated. 

    11    So it's possible that this locus is also activated,

    12    but we also then have the problem that if that locus

    13    would not be activated, that there's some

    14    transcomplementation.  So one gag, intact gag gene

    15    would complement another intact pol gene and so on. 

    16    So that could be a problem, but we see it then in

    17    several, even in normal peripheral blood lymphocytes,

    18    we have HERV-K expression.  It obviously is not a

    19    problem.

    20                So I wouldn't see so much a problem in

    21    that we have HERV expression if defective sequences

    22    are expressed, and that are not coding the intact.

    23                AUDIENCE MEMBER:  I think we have to focus

    24    on infectivity here, because if we just go looking for

    25    expression of defective stuff, you always find it, and

                                                                        142

     1    we all go home and won't be able to do anything.  It

     2    might not be a bad idea perhaps, but --

     3                (Laughter.)

     4                AUDIENCE MEMBER:  Sounds good to me.  But

     5    just to push this point a little bit further on the

     6    HERV-K, you know, about two-and-a-half years ago,

     7    there was a report in Cell, which is a better journal

     8    than I usually publish in, which claimed that HERV-K

     9    env can act as a super antigen that then stimulates

    10    diabetes mellitus in some people.

    11                DR. ONIONS:  I think that story -- just

    12    before you go on, I could be wrong, but Johannes might

    13    know.  Is that story being modified?  I'm not quite

    14    sure that's -- yes.  It's no longer supported.

    15                AUDIENCE MEMBER:  Okay.  I think it would

    16    be worth explaining how it's been modified.

    17                DR. ONIONS:  I think the retraction is in

    18    fourth hit.  So before you sort of put that out as a

    19    paradigm.

    20                DR. LOEWER:  I think the main point is

    21    that a couple of groups tried to repeat this data, and

    22    they were not able to repeat.  So it seems not to be

    23    specific and even not effects on the T cell lines

    24    could be repeated.  So let us depart from this idea.

    25                DR. ONIONS:  Can I maybe get your opinion? 

                                                                        143

     1    I really would like to get some feeling because we

     2    have heard a lot, and it's scientifically really

     3    interesting by the expression of these human

     4    endogenous retroviruses.  I think John has probably

     5    just summarized it.  It looks like we're saying the

     6    list, this collected group here of retro, are saying

     7    that as far as we are aware, at the moment these are

     8    not of concern and uninfectious, and probably

     9    therefore there is not a great deal of point in

    10    looking for expression of these in cell substrates,

    11    the very pragmatic practical point.

    12                Would that be your opinion too, Johannes?

    13                Johannes is nodding.  That's a "yes," I

    14    assume.

    15                AUDIENCE MEMBER:  From a research

    16    standpoint, it's absolutely worth pursuing to see if

    17    one can find these things eventually.  But in terms of

    18    vaccine issues, I don't see how we could possibly deal

    19    with it now.

    20                DR. SCHUEPBACH:  Yes.  I also would like

    21    to make a comment regarding that super antigen

    22    activity because we are coauthors in that paper.  It

    23    is true that the presence of these sequences, RNA

    24    sequences in the serum we can not repeat, so it's not

    25    specific for IDDM patients.  But to my knowledge, our

                                                                        144

     1    data regarding the super antigen activity and the

     2    stimulation of VP cells has not been disputed by any

     3    other group.  So that is still around.

     4                I think that the real important topic here

     5    is whether these endogenous viruses actually give rise

     6    to infectious particles.  I believe that with the PERT

     7    assay, we actually can contribute very much to this

     8    question.  I think, as I pointed out, of course the

     9    easiest thing is to test the super natants for

    10    reversed inscriptase activity.  But I think with a

    11    little bit of additional work, it should also be

    12    possible to define that profile of RT activity and

    13    cellular, DNA polymerase activities along the

    14    different fractions of the sucrose gradient and tend

    15    to recognize any abnormal pattern that might be

    16    associated with infectious rate of viruses.

    17                AUDIENCE MEMBER:  Let me just go back then

    18    because obviously the question comes up if the public

    19    is asked to accept a vaccine that's made in cells that

    20    express HERV-K.  Even if one part of the story has

    21    been refuted, the question comes up.  You know, are

    22    there potential immunological consequences of the

    23    expression of antigens from these kinds of cells which

    24    are not expressed in human diploid cells?  Even if

    25    this story is wrong, I guess, and it sounds like it

                                                                        145

     1    hasn't all been refuted, the question then comes up of

     2    what level of scientific data in the literature is

     3    necessary to completely refute it?

     4                I can imagine the outcry that could occur

     5    if people believe the story.  If there appears to be

     6    some controversy about what part of this story has

     7    been refuted, then I think one might have a public

     8    confidence problem as well.

     9                DR. HENEINE:  David, I have a comment

    10    which could be redundant, but go ahead and say it. 

    11    While thinking about all these questions from my

    12    simple mind, it looks like if you want to compare cell

    13    lines versus primary or diploid cells, the two

    14    questions that were raised is which ones transmit less

    15    adventitious agents or transmit less neoplasms to

    16    vaccine recipients.

    17                What we have heard so far about the

    18    mechanisms of the neoplasms, many of those are

    19    mediated by viruses or viral-like elements.  So it

    20    looks to me that the majority of the concerns are

    21    rising from the adventitious agent group rather than

    22    from other elements.

    23                So therefore, in trying to make up our

    24    mind, based on the available data, which one is the

    25    more suitable substrate, maybe we can go very simply

                                                                        146

     1    with a checkpoint list on these different cells,

     2    targets, which one we can test for the presence of

     3    these adventitious agents known, unknown, and which

     4    can be better monitored, which can be for practical

     5    reasons of culture as well, and make up our mind,

     6    rather than jumping right and left with different

     7    issues.

     8                If you can say cell lines, primary and

     9    diploid, and then go one by one, all these concerns

    10    that we have been talking about, and say which one is

    11    more suitable for each of these points so that we can

    12    conclude at the end.  I mean it's one suggestion.

    13                DR. SCHUEPBACH:  I would like to come back

    14    to the human endogenous viruses.  Since we all carry

    15    them and have them expressed in one or the other part

    16    of our body, I do not think that this presents a

    17    particular risk.  Independent of whether you have a

    18    super antigen activity or not in some of them, I think

    19    is just the same as receiving blood from any person,

    20    because they also would have these endogenous

    21    retroviruses.

    22                So I think the only thing that matters is

    23    endogenous retroviruses from other species, not from

    24    humans.

    25                DR. LEWIS:  This is a question.  I think

                                                                        147

     1    we are sort of being faced with something of a dilemma

     2    here, because as you very correctly pointed out, the

     3    one way to look for a retro virus, adventitious retro

     4    viruses or endogenous retro virus, whatever, is by the

     5    PERT assay.  Now if we take a situation in which our

     6    HERV-K is expressed and there's RT activity in there

     7    with the PERT assay, from a regulatory perspective,

     8    what do you do about that?

     9                I don't think you can dismiss it.  I don't

    10    think at this point in time that you could dismiss the

    11    use of the PERT assay for looking for adventitious

    12    retroviruses or any type of retrovirus activity in

    13    there.

    14                So the question is going to be, when you

    15    find something, what do you do about it if the assay

    16    is positive?

    17                DR. SCHUEPBACH:  I think once you have

    18    activity, then you have to characterize what it is. 

    19    Depending on whether this is exogenous or endogenous

    20    virus, steps will be taken.  I think identification is

    21    important.

    22                DR. LOEWER:  I think we shouldn't continue

    23    to discuss use of this type of cell lines in absolute

    24    terms, because the other side of the coin of course is

    25    a product which could be made from it.  This is always

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     1    to be judged in conjunction, in my opinion.  For

     2    example, there's the question of endogenous sequences

     3    which may be active, just retro virus or line

     4    elements.  So far as I know, this is not the same in

     5    all cell lines.  It's mainly expressed in tumor cell

     6    lines, and so far as I know, there is so far no need

     7    to use these cell lines for vaccine production.

     8                If somebody believes it's necessary, must

     9    be a very special virus which can only replicate in

    10    these cells, then perhaps we do not have another

    11    choice to use them.

    12                DR. ONIONS:  Could I just go to Arifa? 

    13    She has been waiting very patiently.  Then Steve.

    14                DR. KHAN:  Yes, thank you.  I think it is

    15    important to clarify the word "expression" in terms of

    16    human cell lines and human cells.  I think we all

    17    expect that there will be some RNA expression in the

    18    human cells from endogenous retroviral sequences. 

    19    However, I don't believe that you are going to get

    20    particle production in the majority of the cells under

    21    normal conditions.  So therefore, I think the use of

    22    the PERT assay would be very helpful to evaluate

    23    particle production from human cells, which can then

    24    further be investigated for infectivity, as opposed to

    25    looking at RNA expression, which I think you would

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     1    find at some level in all human cells.

     2                DR. ONIONS:  I would like to endorse that. 

     3    I think really we're making it too complicated.  I

     4    think it's very simple.  You use that kind of assay

     5    system which has a very high sensitivity developed by

     6    Yumascript here in the audience.  Then if you get a

     7    positive, then you go and look and see if there's

     8    something there that's infectious.  It is a hierarchy

     9    of testing strategies, it seems to me.

    10                DR. HUGHES:  I am willing to take an even

    11    stronger line here, which is I think akin to the line

    12    that was espoused by John Coffin.

    13                If your technology is sensitive enough,

    14    all cells from all vertebrates are going to have

    15    endogenous viruses in them, either intact or

    16    defective.  The question really devolves down to not

    17    whether they are there, because they are.  If we use

    18    the most stringent criterion, are they there, you are

    19    not going to be able to do anything.  We'll be

    20    paralyzed in terms of making vaccines.

    21                So the obvious criteria is not whether or

    22    not these agents are there, and in particular, I think

    23    not whether they would make physical particles,

    24    because my strong prejudice is if you look hard

    25    enough, you are almost certain to be able to find it. 

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     1    There are almost certainly ways of looking even more

     2    stringently than we look now.  In very large batches

     3    of material prepared for vaccines, if you look hard

     4    enough, you are going to find something.

     5                The question is whether there is an

     6    infectious agent which represents any kind of

     7    pathologic threat when its present in a vaccine.  We

     8    have, as has been pointed out earlier, given from

     9    chicken cells, some of which were clearly contaminated

    10    with agents which are infectious for the chickens, if

    11    not for humans, that hundreds of millions of doses

    12    have been given, with as far as anyone can tell, no

    13    untoward effects.

    14                DR. ONIONS:  Okay.  I would like to bring

    15    Jim in, because I am not sure that statement is --

    16    well, it could theoretically be true.  Maybe if you

    17    get a more sensitive technique, maybe you are correct.

    18                But there is a very strong distinction if

    19    you look at cell lines like MRC-5 and compare them to

    20    what you see in say AB, you get signals, if you're

    21    looking at MRC-5 you don't.  If you look in tetra

    22    anacells, you get a signal.

    23                DR. HUGHES:  I would argue that is with a

    24    particular assay that has been tuned up to detect RT

    25    in a particular way.  I would be willing to wager that

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     1    if we look hard enough, we could certainly find

     2    evidence of particle production in any of these cells,

     3    simply because they are full of endogenous viruses

     4    that -- I mean the very fact that there's obviously

     5    expression is RNA present.

     6                DR. SCHUEPBACH:  But the important thing

     7    is I think the number of particles.  You have very,

     8    very low number of particles, and you do a test, a

     9    PERT assay from a concentrated material where you

    10    pellet virus, let's say, from one liter, and assay

    11    that and find a very low activity.  You know for

    12    production of the viral vaccine, the cell harvest, the

    13    vaccine harvest will be diluted 50 to 100.  You also

    14    realize that most of the particles even of infectious

    15    retroviruses are non-infectious.  Then you certainly

    16    can come to some calculation which permits you to

    17    establish a level of safety where you have a very high

    18    probability that this vaccine is safe.

    19                DR. HUGHES:  But the safety is not

    20    predicated on whether or not there are physical

    21    particles.  The safety is predicated on whether or not

    22    the particles are infectious.

    23                If the particles are infectious, a very

    24    small number is very important.  If the particles are

    25    not infectious, in particular, if the particles are

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     1    not infectious for humans, the presence of a

     2    relatively large number of particles is probably also

     3    irrelevant.

     4                DR. ONIONS:  My only caution about that is

     5    that you can make assumptions about affectivity that

     6    also are not true.  Since Clive Patience is here --

     7                DR. HUGHES:  I think you have to do the

     8    test.  I don't think you can make assumptions about

     9    it.

    10                DR. ONIONS:  Well, the point I was going

    11    to make actually was that Clive's group and our group

    12    showed that you could actually infect cells with PERV,

    13    yet those experiments have been done 20 years ago, and

    14    been done by very good people, including George Tadai,

    15    and were unable to show infectivity.  It's just the

    16    techniques have changed slightly and we could get

    17    infectivity.

    18                So I think that you are making -- there is

    19    a straight yes or no about infectivity.  That is not

    20    always the case with these retroviruses.

    21                DR. HUGHES:  I am not trying to suggest

    22    that the assays that we have for infectivity are

    23    necessarily always 100 percent accurate.  But what I

    24    want to get away from is the idea that the presence of

    25    a physical particle is somehow a measure of safety or

                                                                        153

     1    lack of safety.

     2                I certainly agree with you that there are 

     3    numerous technical problems in determining

     4    infectivity.  But what I believe we should focus on is

     5    better ways of doing infectivity assays rather than

     6    better ways of doing physical assays.

     7                The physical assays can be very useful if

     8    they are coupled to infectivity.  In fact, I believe

     9    that was a statement that John Coffin made.  What I

    10    would propose is what I think John Coffin proposed,

    11    that we actually use these really wonderful sensitive

    12    techniques, but in the context of measuring whether or

    13    not the viruses are infectious, not whether the

    14    particles are physically present.

    15                DR. SHEETS:  Can I ask a very pragmatic

    16    question of Dr. Hughes?

    17                DR. HUGHES:  Sure.

    18                DR. SHEETS:  I'm Becky Sheets, FDA.  What

    19    I hear you suggesting is that rather than testing for

    20    RT activity by a physical assay, as you called it, a

    21    PERT assay or a conventional RT assay, you think that

    22    it would be preferable to test vaccines for

    23    infectivity assays?

    24                DR. HUGHES:  The ability to transmit that

    25    RT assay to a reasonable recipient cell.  I believe

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     1    this is exactly what John --

     2                DR. SHEETS:  The pragmatic question is

     3    that would you do this testing lot by lot on vaccines? 

     4    For instance, if you were making a vaccine in a

     5    primary cell substrate, for instance an egg, would you

     6    test each lot of vaccine or each batch of vaccine for

     7    infectivity assays?  Then the really pragmatic part of

     8    it is, if you are making a flu vaccine, where the

     9    timing of production, the timing of testing, and the

    10    timing of lot release is very tight, would you

    11    recommend these infectivity assays on lot by lot for

    12    primary source?

    13                DR. ONIONS:  We're running out of time. 

    14    Do you want to answer that?  You have been asked a

    15    question, do you test lot by lot?

    16                DR. HUGHES:  Very simply, if we're talking

    17    avian systems, I think there are reasonable ways of

    18    determining that endogenous avian viruses are not

    19    infectious.  My personal bias, and I mean it no more

    20    broadly than that, is in the case of avian viruses, as

    21    long as you carefully establish that the avian viruses

    22    that are present are not infectious for human, that

    23    that's not necessary.  But that is my prejudice.

    24                DR. SHEETS:  That's fine for SPF

    25    situations.  I am asking this question because this is

                                                                        155

     1    what sponsors ask FDA.  So they want to know do we

     2    need to do this lot by lot, or if a cell bank, you can

     3    do a one-time characterization.  Of a primary system,

     4    you can't do it that way.

     5                DR. HUGHES:  Use SPF chickens and don't

     6    ask.

     7                (Laughter.)

     8                DR. LOEWER:  I would like to make a

     9    comment to Dr. Hughes' comments.  They are very sound

    10    in a scientific meaning, but they face regulatory

    11    problems, the main problem indeed.

    12                Regulatory authorities have to show that

    13    there is no infectivity and the proof of non-activity

    14    is always nearly impossible in a scientific sense. 

    15    You will always find reasons to say you were not able

    16    to find infectivity.  Look at HIV.  If you would use

    17    MRC-5, for example, or a lot of other animal cells,

    18    you would never find infectivity of HIV.  The same is

    19    true for many situations.

    20                So there a fundamental problem is to test

    21    for non-activity or noninfectivity.

    22                DR. ONIONS:  I would like to stop this

    23    because we are running out of time and there are other

    24    issues.

    25                I am going to take the Chairman's

                                                                        156

     1    privilege and just say that I actually think you need

     2    multiple techniques, because I think as Johannes has

     3    just said, if you have complete infectivity, you will

     4    miss things as we would have missed cell lines

     5    producing ver.  I think you really need to have a

     6    combination technique.  So I think it's a belt and

     7    braces situation.  That's a personal view.

     8                What I would like to move onto, is were

     9    asked by the FDA also to consider species of origin. 

    10    I think really you end up in very general statements

    11    here.  You can argue that if you are worried about

    12    adventitious agents, then clearly there are species

    13    barriers to the transmission of some agents.  On the

    14    other hand, other agents do go across species

    15    barriers, sometimes in abortive replication.  They can

    16    be very nasty.  We of course know that herpes B going

    17    across species barriers is actually lethal.  Ad 12 in

    18    hamsters is oncogenic.  Equine herpes veras, which is

    19    an alpha herpes veras is oncogenic in hamsters.  There

    20    are natural examples of cross-species transmission,

    21    the ovine herpes veras 2 is innocuous in sheep, but it

    22    kills cattle.  So there are examples of these

    23    heterolic transmissions being worse than natural

    24    infections.

    25                Is there anything that we can say, the FDA

                                                                        157

     1    have asked us, in a general statement about species of

     2    origin?  My own view is I don't think you can, but

     3    does anyone want to make a statement?

     4                DR. MINOR:  I think sometimes it is better

     5    and sometimes it's worse.

     6                (Laughter.)

     7                DR. ONIONS:  Yes.  That's exactly what I

     8    think.  Thank you, Phil.

     9                I would like to drop the discussion now,

    10    because I think that sums it up.  I'll turn the phrase

    11    back on the edge and say it's a case-by-case, it seems

    12    to me.

    13                I don't want to trespass really on

    14    yesterday's, but I think maybe just to come back to --

    15    we're going to move onto assay systems in a second,

    16    but I think one of the issues we're coming around to

    17    in a second is latent viruses, because those seem to

    18    be the real concern.  It may be worth just remembering

    19    some of the things that were partly discussed over the

    20    last two days.  That is, that the complementation of

    21    defective viruses can occur.  For instance,

    22    adenoarectus can be complemented by HPV in hela cells.

    23                We talked about psuedotype formation both

    24    today and yesterday.  I think just I would like to

    25    make the point about pseudo formation.  We talked a

                                                                        158

     1    lot about retrovirus retrovirus pseudotype formation,

     2    but this can occur across viral species.  For

     3    instance, paramyxovirus is rather badly, but they can,

     4    pseudo type retro viruses.  So you could alter the

     5    host range when endogenous agent is expressed in your

     6    cells.

     7                Of course there are recombinants.  Some of

     8    these recombinants, and we have got representatives

     9    who did the work here, interesting recombinants like

    10    SV-40 adnivos recombinants.

    11                One of my concerns, we'll come on in a

    12    second, I think is like polyoma viruses in cell

    13    substrates and that potential for interaction with

    14    other cells.

    15                So if we can take that as a kind of

    16    background, can we turn to a question of -- this had

    17    come up and was discussed by several candidates,

    18    unknown viruses.  What are the potential candidates

    19    and what kind of systems do we use to try and go

    20    looking for those unknown viruses.

    21                Anyone want to comment on what we should

    22    be doing about novel cell substrates and you have got

    23    a virus there that you don't know anything about. 

    24    What sort of technique should we be applying?

    25                DR. PALLEY:  I would like to make a

                                                                        159

     1    comment from that HERV field.  I think if we talk

     2    about possible recombinations of HERVs with some other

     3    viruses that might be a little difficult, I think it

     4    could be conceivable.

     5                We do not know, however, it's very hard to

     6    predict the outcome of whether it is possible at all. 

     7    We basically have or we might have situations where a

     8    HERV is expressed where another virus, retrovirus is

     9    also present in the same cell.  I think that has to be

    10    discussed or taken into consideration, that there

    11    might be possible recombinations between HERVs and

    12    some retrovirus status put into a particular cell. 

    13    However, I think it's very hard to predict whether

    14    there's any possible recombination and what the

    15    outcome of that recombination might be.

    16                But I think there are examples where retro

    17    viruses indeed recombine with each other and produce

    18    some productive outcome.  But it's very hard to

    19    predict what, in which ways HERV case for instance, or

    20    whatever HERV sequence within human genome could

    21    recombine with something else.

    22                DR. ONIONS:  I was just going to point out

    23    I think one of the things I have been very struck by

    24    by some of the talks here, particularly the

    25    polymerized talks, it does strike me, the comment I

                                                                        160

     1    made to Phil earlier about the use of cynomologous

     2    monkeys and perhaps have these been screened for other

     3    polyoma virus.  That seems to be of concern.  We know

     4    that polyoma viruses are coming in as contaminants in

     5    bovine serum into the primate cells, the bovine

     6    polyoma virus.  They worry me as potential

     7    adventitious agents.  I am just wondering again

     8    whether we should be doing more in terms of redundant

     9    PCR approaches to look for these agents in both

    10    primate and non-primate cell lines.

    11                Any views on that?

    12                DR. ROBERTSON:  This is potentially the

    13    most important door, also the most difficult to deal

    14    with.  If you think back to what Phil was saying in

    15    the first talk this morning, all these instances of

    16    vital contamination, generally they occur with viruses

    17    unknown at the time, viruses expected, the presence of

    18    viruses in vaccines or biological preparations.

    19                Potentially is not something we're talking

    20    about today, it's not an endogenous virus or

    21    recombination between an endogenous virus, but

    22    something unknown that's going to leap up at us out of

    23    the dark.  Of course almost impossible to deal with.

    24                But Joerg was saying this morning about

    25    this is what we should be looking out for, the

                                                                        161

     1    unknown, and if possible using a more broadly reactive

     2    type of assay rather than highly specific type of

     3    assay to look for something.

     4                If we knew to look for something, that's

     5    fine, we can deal with it.  It's what's not there

     6    which causes the problem.

     7                DR. SCHUEPBACH:  May I add something to

     8    this?  I think our chances to detect such unknown

     9    agents are really much better if they are present at

    10    high concentration.  So I don't know whether you

    11    accept that concept of cellular cloning in order to

    12    either get rid of these agents or to have them at the

    13    very high concentrations so that their detection is

    14    actually much easier.  In the meantime, you can try to

    15    activate the host cells by all kinds of different

    16    agents.  You do EM studies, you do serological

    17    studies, use broadly cross-reactive antibodies.  I

    18    mean this is a wide field actually of methods you can

    19    employ.

    20                So I think using such an approach, we

    21    should actually be quite capable of detecting such

    22    agents.

    23                DR. MINOR:  Can I ask for a definition

    24    here?  What do you actually mean by a virus?  I mean

    25    what I would understand by a virus is something that

                                                                        162

     1    actually grows in the cell and increases in numbers. 

     2    If you think back to the very, very early days of

     3    polio vaccines, amongst others, there was an awful lot

     4    of effort put into trying to make sure that you put

     5    your supernatant or whatever, into all sorts of

     6    potential different systems where a virus might grow.

     7                So you put it into different cell

     8    substrates and you look for cytopathic effect, where

     9    you can say maybe you have got a virus that doesn't

    10    cause a cytopathic effect.  You put it into mice, you

    11    put it into eggs, you put it into everything.  So it's

    12    like evidence of actual growth.  You see?

    13                I think there's actually quite a lot of

    14    effort that goes into trying to detect viruses that

    15    you don't know are there, but you suspect might

    16    actually grow in some system that you are going to

    17    check it on.  If they don't grow, I'm not sure certain

    18    that you are worried about them, or even if they are

    19    viruses.  So what do you actually mean by an unknown

    20    virus?

    21                DR. ONIONS:  Well, okay.  Let me give you

    22    an example.  I think one of the areas that is going to

    23    emerge as a concern are going to be circaviruses.  The

    24    reason I say that is that TGV may be a circavirus.  We

    25    now know of others.  Thomas was talking about the kind

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     1    of normal flora of HPVs.  It looks like we all have a

     2    normal flora of these circaviruses.  Certainly when we

     3    started now looking in animals, we find these all over

     4    the place.

     5                So I think they may come up as potential

     6    cell substrate contaminants, but also in our serum, in

     7    our trypsin, and so on.  Of course trypsin is full of

     8    of course circoviruses.

     9                What we know is if you look in a cell

    10    substrate like PK-15, which has used in the pig

    11    vaccine industry, that carries a circovirus genome. 

    12    It looks, it's possible semi-defective, but you can

    13    introduce it as an infectious agent, but most of the

    14    time it isn't inducible as an infectious agent.

    15                So I think I'm not quite sure I take your

    16    distinction.  I think there are latent viruses there

    17    that are reactivatable under certain circumstances and

    18    are a concern.  In fact, in the vetmurines vaccine,

    19    you have to get rid of that virus.

    20                DR. PALLEY:  Yes.  I just had sort of an

    21    alternative.  Virtually all the conversation has

    22    revolved around using higher eukaryotic type cells as

    23    the producer cell source.  There is an alternative

    24    that's being actively pursued in the papilloma vaccine

    25    business, which is to make virus-like particles either

                                                                        164

     1    with a baculo virus expression system in insect cells

     2    or even in E. coli.

     3                Very briefly, what's done is the L-1 and

     4    L-2 capsid proteins of papilloma virus self assemble

     5    no matter where they are over produced.  Four

     6    different groups are pursuing these now.  I am sure

     7    they are engaged in some corporate relations.

     8                But one of the neat strategies that John

     9    Shiller here at NIH and others have done, is to fuse

    10    peptide epitopes or even intact other genes to the L-2

    11    protein at their n-terminus.  It turns out that when

    12    L-2 assembles into the virus-like particle, but brings

    13    this n-terminal protein in with it, that other protein

    14    is on the inside of the virus-like particle.  So it

    15    will ultimately be presented to the recipient of this

    16    virus infection.  It's non-genetic, but it will be

    17    taken up by cells.  So there is in effect no risk of

    18    some bacteria phage infection running rampant in our

    19    bodies.  So it's a complete alternative.

    20                DR. ONIONS:  I think obviously there is a

    21    move to sub-unit vaccines in this form of vaccines. 

    22    But I think it's still going to be a long time before

    23    our traditional eukaryotic cell production is going to

    24    be lost.  But I take your point.  That is a valid

    25    point.

                                                                        165

     1                 Could I just ask perhaps questions about

     2    we had examples indeed from Thomas toady about using

     3    redundant PCR techniques.  It would be feasible to

     4    screen for a number of viruses that are of concern,

     5    like herpes viruses, circoviruses, polyoma viruses,

     6    herpes viruses retro viruses by redundant PCR

     7    techniques.  These are not in FDA terms, validated

     8    techniques.  On the other hand, they are very powerful

     9    techniques.  For instance, one of my colleagues just

    10    developed a herpes virus redundant PCR technique with

    11    112 primer combinations that to date has picked up all

    12    of the herpes viruses that's been challenged with both

    13    human and animal origin.  So you could go looking for

    14    herpes viruses by that kind of technique.

    15                Robin Weiss' group in London picked up a

    16    new human herpes virus, HRV-5 by a redundant PCR

    17    technique.  So do any of you think that these kinds of

    18    technologies should be implied to cell substrates?  If

    19    so, which viruses should we use?  Because it is a lot

    20    of work to do this kind of thing.  So should we be

    21    doing this or is it not necessary?  Can I have

    22    comments?

    23                Jim, you look like you are about to say

    24    something, but not quite sure.

    25                DR. ROBERTSON:  One would not expect to

                                                                        166

     1    see this type of assay risen up in a pharmacopeial

     2    recommendation of any kind.  But certainly they do

     3    have uses at the investigational level, especially

     4    with some incredibly novel cell types.

     5                Everything is kind of going molecular

     6    these days.  All these assays are looking at things

     7    from the very molecular point of view, and picking out

     8    a signal say with your herpes primers, need not

     9    necessarily say that you have got an infectious herpes

    10    virus.

    11                So go back to this argument we have been

    12    having this morning about it's infectivity which

    13    potentially is what we're concerned about, and not

    14    picking up a fragment of a genome.

    15                These are assays, again I mentioned that

    16    Joerg mentioned them this morning, broadly reactive

    17    molecular assays.  Phil rightly pointed out that we

    18    have had in place for eons broadly reactive

    19    infectivity assays using tissue cultures, suckling

    20    mice eggs, looking for signs of infectivity of any

    21    kind.

    22                DR. ONIONS:  But don't you think -- my

    23    feeling about that is, I tend to share, I don't think

    24    one would ever use these kind of assays on a routine

    25    basis.

                                                                        167

     1                DR. ROBERTSON:  No.

     2                DR. ONIONS:  They might be useful in

     3    establishment of a master cell bank or something, you

     4    know, the first one.  But I mean I would criticize, I

     5    don't think that current infective assays do pick up

     6    everything.  I think that's the whole problem.  I

     7    think, for instance, that it would miss -- well,

     8    polyem virus has perhaps been used.

     9                DR. ROBERTSON:  At the end of the day,

    10    that's the weakness of anything, that you will not

    11    pick up something that's not designed to pick up.  You

    12    will potentially miss viruses in an infectivity assay. 

    13    But you also potentially miss viruses with a redundant

    14    PCR.  If you don't pick something up, you can't say

    15    it's there or not there.  It's a bit of a

    16    philosophical argument.

    17                DR. ONIONS:  That's why I'm just saying

    18    shouldn't you have an adjunct to these?  I mean I

    19    think should you not have at least an adjunct in terms

    20    of broadening the kinds of assay systems that you are

    21    using?

    22                DR. ROBERTSON:  Sure.  Oh yes.

    23                DR. ONIONS:  Does it have anything to do

    24    with the -- I mean there are people out there who have

    25    to do this for a living, rather than us who can just

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     1    sit here and pontificate about it.  How does this go

     2    down with the industry?  What does the industry feel?

     3                DR. PALLEY:  Just one point regarding

     4    again HERVs.

     5                DR. ONIONS:  Sorry?

     6                DR. PALLEY:  I mentioned in my talk that

     7    HERV-W family that has been reported for the first

     8    time this year and has been isolated from vitro by

     9    particles from multiple sclerosis patients.  It turned

    10    out that it at least codes for an env gene.  So it's

    11    certainly worth -- the human genome I guess is among

    12    the genomes that regarding endogenous retroviruses is

    13    among the best characterized genomes besides mouse,

    14    for instance.

    15                I think it is certainly worth to continue

    16    and even by such redundant PCR approaches and so on to

    17    further characterize HERV sequences on endogenous

    18    retrovirus sequences, and to see, to give then an

    19    estimate whether there are any additional sequences

    20    that could be harmful.  So far, we did not find any

    21    sequences, but it's certainly worth doing that.

    22                AUDIENCE MEMBER:  I think if you just kind

    23    of look back at the history of biological products,

    24    and maybe even going way back to when hepatitis B was

    25    discovered, and then there became the ability to look

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     1    for hepatitis B in blood and blood products.  There is

     2    always concern about what are you going to find, and

     3    what are the implications, and what are the costs, and

     4    all of those things.

     5                But the bottom line is as technology

     6    evolved, then the discovery of reverse transcriptase,

     7    and when I was still here at the agency, we applied in

     8    a research setting to vaccines and first demonstrated

     9    RT in yellow fever.  You get concerned about again,

    10    what are you going to find when you look at all these

    11    cell substrates, and then with the more enhanced

    12    sensitivities of these systems.  You are always in the

    13    same muddle.  That is, is this appropriate to apply

    14    across the board?  Where should it be properly

    15    applied?  Should it be done more in the initial stages

    16    of characterizing something versus a routine quality

    17    control test?

    18                Those things, you can't sit here in a

    19    meeting or on a panel and give specific answers to

    20    those questions.  I think the bottom line is as

    21    technology evolves, and it's going to continue to

    22    evolve with more sensitivity and specificity

    23    hopefully, it needs to be explored and it will find

    24    its appropriate place in the overall testing, whether

    25    it's in characterization or perhaps in some cases, if

                                                                        170

     1    it's appropriate, on a routine basis.

     2                But I think the general principle of

     3    applying new technology as it begins to be available

     4    to look at these issues, particularly as they relate

     5    to safety and the presence of adventitious agents, is

     6    unassailable.

     7                DR. ONIONS:  I agree.  Can we move on,

     8    because I would like to just cover TSEs just before we

     9    have lunch time.  I'm desperate for some lunch.

    10                If you have got something, sorry to

    11    inhibit you.  If you can be brief.

    12                AUDIENCE MEMBER:  I agree completely with

    13    what John says, but would add that if we are faced

    14    with a decision of whether to approve the use of

    15    different types of cell substrates that are

    16    tumorigenic or derived from tumors from which we don't

    17    know the mechanism of transformation, we are faced

    18    with not only the question of should technology be

    19    applied, but is the technology as it exists today and

    20    can be applied today, good enough to permit us to say

    21    that it's okay to use these cells.  So that is a very

    22    practical question which perhaps could be answered.

    23                DR. ONIONS:  Can we have a view on that? 

    24    That is, given the technology we have today, is it

    25    acceptable to use the kinds of substrates we have been

                                                                        171

     1    talking about, that is tumorigenic or immortalized

     2    cell substrates?  Are we confident that with the

     3    technology we have, that we can use these cell

     4    substrates safely?

     5                DR. BROKER:  I would just basically say I

     6    think so.  I think if we combine PCR with these

     7    microchip or microprobe arrays, DNA chip technologies,

     8    and we have the growing human genome base and the

     9    analogs and a number of other species, I think we have

    10    got the tools at a level of sensitivity far beyond

    11    what would probably would be more than adequate.

    12                DR. ONIONS:  Okay.  I would just like to

    13    finish up, because we heard a really I think important

    14    interesting talk from Neil.  While perhaps the risk of

    15    spongiform encephalopathies in the kind of cell

    16    substrates we are concerned with is probably extremely

    17    remote, the consequences of being wrong about this

    18    issue are potentially devastating.  So it is certainly

    19    worth cautious consideration.

    20                Really I think Neil in his talk, already

    21    summed up these key issues about the possible origins,

    22    are the mutations spontaneous or infection, and the

    23    kinds of cell substrates of concern might be, it seems

    24    to me, are the neuronal cells.  Since it's recently

    25    shown that in the peripheral introduction of TSEs, the

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     1    B-lymphocyte might be important to carriage, then

     2    perhaps lymphoid cells, particularly B cells that are

     3    invariably used, and since we're looking at the

     4    possibility of using HIV and T cells, maybe that

     5    suddenly becomes an issue.  Maybe we should be looking

     6    at lymphoid cells for the potential of there being

     7    spongiform encephalopathies.

     8                Which brings you back to the question that

     9    Neil finished with.  That is, what should we do?  It

    10    did strike me that one of the possibilities was that

    11    there are now very good, very interesting new cell

    12    lines being based on retinal cells, which we heard

    13    from Dr. van der Eb and others, which look very, very

    14    promising for the generation of anti-viral vectors.

    15                But as they have, as I understand it in

    16    theory, I mean just at the simplistic level, should

    17    not one thing be done and just sequence the PrP gene

    18    in that?  The probability of having a key mutation

    19    seems to me extraordinarily remote.  But then it's a

    20    relatively simple thing, cheap thing to do is to go

    21    and sequence the PrP gene.  Is that something that we

    22    should do in that kind of a situation?  Should we also

    23    do that in T lymphocytes?  It's a trivial thing to do?

    24                DR. CASHMAN:  I would say in this case,

    25    you get what you pay for.  It is a trivial experiment,

                                                                        173

     1    but if one out of a million or one out of a thousand

     2    cells could be harboring a mutant prion protein gene,

     3    the technology is a little more dicey.  So yes.

     4                DR. ONIONS:  I was thinking of excluding

     5    the origin of the familial form, which clearly occurs

     6    to some inherited disorders.  I mean I think you are

     7    right.  You can't cope with a somatic mutation.

     8                DR. CASHMAN:  Okay.  You can't cope with

     9    somatic mutation, I agree.  But certainly one can cope

    10    with a mutation that's in every cell, yes.

    11                DR. ONIONS:  Neil touched on validation

    12    technology.  I think that's important.  I think there

    13    are new techniques for doing validation of TSE

    14    removal, but using a disrupted PrP protein.  But I

    15    don't think that's going to be applicable to quite a

    16    lot of the processes that are used to produce vaccines

    17    at the moment.  It is by technology products, but not

    18    to vaccines.

    19                I just wanted to touch on testing because

    20    I know that Neil has an interest in that area.  It

    21    seems to me that we were sort of rather optimistic a

    22    couple of years ago, and indeed, there have been

    23    publications by Bruno Esch and others on specific

    24    antisera of PrP Sc.  But those haven't held up.  They

    25    actually pick up aggregated protein and not, as I

                                                                        174

     1    understand it, strictly PrP Sc.

     2                We can use, and we have been using,

     3    treatment of protease followed by immuno blotting.  It

     4    certainly works, but it isn't that sensitive.  The

     5    problem, it seems to me, is that we really don't have

     6    a specific test that's an in vitro test.  The only

     7    thing that you are left with, at the moment, it may

     8    change, but at the moment is animal inoculation.

     9                Would you like to comment?

    10                DR. CASHMAN:  I think that the bio assay,

    11    with no species barrier, can detect one unit of

    12    infectivity.  It's the most sensitive thing we have to

    13    date.  But in fact, there is new technology.  Mary Jo

    14    Schmirr and her colleagues have developed a very

    15    sensitive capillary electrophoresis technique which

    16    is, if one can believe the papers, including one in

    17    press, is as sensitive as bio assay.  So technology is

    18    evolving, and there may in fact be a specific and

    19    sensitive test for PrP Sc right around the corner.

    20                DR. ONIONS:  Okay.  On that optimistic

    21    note, I am going to wrap the session up.  I think

    22    we're all getting edgy for lunch.  Unless anyone has

    23    got some burning issue that they wish to go into

    24    before we go.

    25                If not, I think the panel members, and

                                                                        175

     1    thank you all for participating.

     2                CHAIRPERSON RABINOVICH:  Thank you, Dr.

     3    Onions.  There is a light repast outside for those

     4    that have been so patient.  I would like to get

     5    everybody back in here in 15 minutes.

     6                For those of you who would like to avoid

     7    the wholesale garage sale that's going to go with your

     8    luggage, for those of you that haven't checked out of

     9    your room, I encourage you to complete that now. 

    10    Thank you.

    11                (Whereupon, the foregoing matter went off

    12                the record at 12:33 p.m. and went back on

    13                the record at 12:53 p.m.)

    14                CHAIRPERSON MYERS:  Back to order.  I

    15    would like to introduce a co-chair and a new person we

    16    are very pleased to have attending the meeting, Dr.

    17    Gary Nabel, who is the new Director of the Vaccine

    18    Research Center at NIH.  So he is going to join me in

    19    chairing this session.  I am probably going to

    20    disappear before the end of the session to make a

    21    plane.

    22                The first night I got here, as some will

    23    recall, I came in a little late.  I ran into a couple

    24    of you in getting a beer because it was after the time

    25    of the close of the meeting.  The discussion ensued as

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     1    to what is a designer cell substrate.  What do we mean

     2    by that?  My first reaction to that of course was it's

     3    anything that I happened to have made.  Clearly at

     4    this point in the meeting, it's not primary cells.

     5                I suppose from a strictly semantic

     6    perspective, it would be a cell substrate created with

     7    specific characteristics.  It could be immortal or

     8    not.  But I think over the last couple of days, at

     9    least my thinking on this and for the purposes of this

    10    discussion, by a designer cell substrate, we mean a

    11    cell substrate of defined origin and with a defined

    12    pedigree.

    13                It is probably immortalized because it is

    14    likely to have been cloned.  It will be validated as

    15    specific pathogen-free and at least specific pathogen

    16    sought and perhaps in certain circumstances, defined

    17    as non-infectious.

    18                For the purposes of the next discussion,

    19    we are really talking about immortalization.  Jim

    20    McDougall, as you know, presented his paper yesterday.

    21                So we'll start this session with the first

    22    paper by Dr. John Sedivy from Brown University, who

    23    will talk about differences in the capacity to

    24    immortalize rodent, primate, and human cells by tissue

    25    culture passage or viral transformation.

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     1                DR. SEDIVY:  Thanks very much for the

     2    invitation.  I am sorry that Jim gave his talk

     3    yesterday because -- well, maybe it will jive all

     4    together.

     5                I was asked to give somewhat of a

     6    historical overview on the issues of replicated

     7    cellular senescence, and obviously the topic of

     8    cellular immortalization.

     9                So from a historical point of view then,

    10    this is the Hayflick phenomenon.  This experiment has

    11    been performed in numerous labs and always with the

    12    same result.  This happens to be an experiment in my

    13    lab.  You will see a number of slides like this from

    14    me today.  What we're plotting here is replicated

    15    lifespan, the doublings of the culture versus days. 

    16    We see a culture growing and then reaching a non-

    17    proliferative plateau.  This is what we define as

    18    senescence.

    19                Really the interesting point here is that

    20    the correlation here of this plateau is with the

    21    number of cell divisions as opposed to chronological

    22    time.  The question that has been plaguing this field

    23    ever since its inception is well, is this really some

    24    type of a terrible artifact.  I don't really want to

    25    get into this discussion.  It really revolves around

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     1    the issue of media and media artifacts, and have these

     2    really been adequately resolved today.  I don't think

     3    they have, especially for some more specialized cell

     4    types.  I think they have been pretty well resolved

     5    for keratinocytes, maybe breast epofelial cells,

     6    fibroblasts, et cetera.

     7                One really has to keep in mind that if one

     8    sees a culture that is slowly declining in its

     9    proliferation, this could simply mean that

    10    increasingly a larger and larger fraction of those

    11    cells are withdrawing from the cell cycle.  This could

    12    be perfectly explained by inadequate culture

    13    conditions, such that eventually on the macroscopic

    14    scale, the culture has ceased proliferating.

    15                There are really three arguments that have

    16    been used historically to justify the claim that

    17    replicated senescence is a biologically interesting

    18    phenomena.  Here we are plotting, again very simply,

    19    the mean-like span of a species versus fibroblasts

    20    replicated life span in tissue culture.  As you can

    21    see, there is a rather striking correlation, such that

    22    animals that don't live for very long don't have cells

    23    that live for very long in tissue culture.

    24                The next phenomenon that one often sees

    25    cited is the age of the donor plotted against -- here

                                                                        179

     1    is the age of the donor, and the remaining life span

     2    of the cells, in this case fibroblasts taken from that

     3    donor.  As you can see, the points are all over the

     4    place.  In fact, more recently, this view has been

     5    challenged by a recent paper in PNAS from Vince

     6    Cristofalos, who actually claims that this correlation

     7    doesn't exist.  But if you read the literature, you

     8    will see this coming up over and over again.

     9                The one fact that seems to remain, at

    10    least to my knowledge, and that is if you look at

    11    these points down here, these are fibroblasts taken

    12    from individuals that suffer from premature aging

    13    syndromes.  These are called progerias.  Typically,

    14    these cells have a very short life span.

    15                So this really is the issue here.  How do

    16    we differentiate between senescence, quiescence, and

    17    differentiation.  I think that for the purpose of

    18    discussion today, this is really not a point of major

    19    interest, but for historical reasons I'll go through

    20    it rather quickly.

    21                Quiescence is defined as a reversible

    22    process.  So what we are talking about here is

    23    essentially a cell cycle phenomenon.  That is, we can

    24    have a culture that is cycling or contains a large

    25    fraction of cycling cells.  Then these cells can

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     1    withdraw into the quiescence state.  Then when they

     2    are induced with the proper growth factors, and here

     3    of course the key phrase is what are the proper growth

     4    factors to elicit this phenomenon.  At any rate, we

     5    are talking about a reversible process.  Whereas

     6    senescence by definition is irreversible.

     7                So then of course the very interesting

     8    next question is how do we differentiate senescence

     9    from terminal differentiation.  I don't really have

    10    answers here because in many cases, this is very

    11    difficult to do in many specialized cell types.  What

    12    one would like to see in general is the absence of

    13    features that are characteristic for terminally

    14    differentiated cells.  But this is not possible in

    15    many cases.

    16                So really this has given the impetus to a

    17    search for molecular events.  So then if we pose the

    18    question are there molecular events that are unique to

    19    senescence versus quiescence versus differentiation,

    20    again, the picture is not very clear cut.  I don't

    21    want you to absorb this whole slide.  Suffice it to

    22    say that this is well, not all, but the major part of

    23    the regulatory circuitry in G-1.  Here you see the D-

    24    type cyclance.  CDK-4 and CDK-6 driving RB

    25    phosphorylation, which in turn drives the second

                                                                        181

     1    phase, which is cyclin E production, activation of

     2    CDK-2.  Of course there are a lot of modifying

     3    proteins here, CDK inhibitors, kineses that activate

     4    the basal CDK kinase, et cetera.

     5                Now this is an area that is receiving a

     6    lot of attention.  The general theme, at least to me,

     7    it seems that there's a high degree of overlap between

     8    mechanisms that regulate quiescence, senescence, and

     9    differentiation.  I don't think this is really

    10    surprising because all these three states are

    11    characterized by the absence of cell cycle

    12    progression.  In most cases, by an arrest in the G-1

    13    or a G-0 state.

    14                The one central theme is that the

    15    regulation of cyclin dependent kinase activity is

    16    necessary to achieve a physiological cell cycle

    17    arrest.  In addition to the cyclins, which are the

    18    positive affecters, there is a number of CDK kinase

    19    inhibitors that have been shown to play a key role.

    20                The two major inhibitory pathways that act

    21    on this basal cell cycle machinery are the RB pathway,

    22    shown here, and also the pathway regulated by the

    23    tumor-suppressor protein P53.  In both of these

    24    pathways, CDK kinase inhibitors have been shown to

    25    play key roles.

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     1                So let me turn to the issue of

     2    immortalization.  We all know that senescence can be

     3    overcome because quite obviously, there are many cell

     4    lines out there that are very immortal.  So in a very

     5    simplistic and general sense, we can think of cell

     6    culture in three broad categories.  We can have

     7    primary cells or cell strains that have a limited life

     8    span and senescence after several passages.  We have

     9    a category of cell lines that are immortal, not

    10    necessarily by the 3T3 protocol, but in general, they

    11    display the characteristics of unlimited lifespan,

    12    non-malignant phenotype, and in most cases by the

    13    ability to become quiescent.

    14                Finally, we have the large group of cell

    15    lines that are derived from either tumors or have been

    16    transformed by one process or another.  These of

    17    course also have an unlimited lifespan, but they have

    18    a malignant phenotype as defined by one or more

    19    criteria.  They also usually cannot become quiescent.

    20                This again is the Hayflick plot.  What I

    21    am showing here is a rodent culture, mouse in this

    22    case, and human.  This little bump on the curve in

    23    fact is senescence for a mouse fibroblast culture.  So

    24    it's been known for a very long time that rodent cells

    25    can overcome senescence spontaneously.

                                                                        183

     1                You can also see the great difference

     2    between the replicated lifespan in vitro of human

     3    cells that go on for a very long time.  If this

     4    experiment here was continued, it would level off and

     5    you would see the typical Hayflick phenomenon.  So the

     6    human plateau up here in fact is corresponding to this

     7    rather short plateau senescence in rodent cells.

     8                So the relatively low frequency of

     9    immortalization -- I should point out that this

    10    doesn't really seem like a low frequency, but on a per

    11    cell basis, it actually is an event that has a

    12    frequency of 10 to the minus 5, to 10 to the minus 6. 

    13    It's just that the X axis is plotted in days here.

    14                The fact that this immortalization can be

    15    stimulated by mutagens has led to the hypothesis that

    16    this in fact is a mutational event in nature.  This is

    17    supported by the existence of several viral genes,

    18    such as, and we have heard about them here, SV40 large

    19    T antigen, polyoma large T antigen, animal virus E1A,

    20    HPV E6 and E7, that can cause immortalization.  In

    21    fact, when these genes are introduced into rodent

    22    cells, they are sufficient to cause immortalization in

    23    a single step.  In other words, if you take a rodent

    24    culture and you put SV40 large T into those cells at

    25    this point, the curve would look like this.  No

                                                                        184

     1    apparent senescence under the right culture

     2    conditions.

     3                So what are these viral oncogenes doing to

     4    promote immortalization?  Without going into a lot of

     5    detail, there is a large body of evidence that now

     6    indicates that these proteins interfere with the

     7    function of the P53 and/or RB growth inhibitory

     8    pathways.  In agreement, there's a lot of data from

     9    knock-out mice now recently that has shown that the

    10    elimination by gene knock-out of a variety of

    11    negatively acting affecters can result in apparent

    12    one-step immortalization, as shown here for example.

    13                To date, embryo fibroblasts from strains

    14    deleted for P53, P16 inc 4A, P19 arf 1 in P21 cip 1

    15    have displayed this apparent immortalization

    16    phenotype.

    17                So what happens in human cells?  Normal

    18    human cells have never been observed to spontaneously

    19    immortalize.  Senescent cultures do not give rise to

    20    sub-populations that resume proliferation as shown

    21    here.  Treatment with mutagens has been shown to

    22    sporadically give rise to immortalized derivatives,

    23    but the frequency of these events is significantly

    24    lower than that in rodent cells.

    25                Let me now talk a little bit about the

                                                                        185

     1    phenomenon of crisis.  So what happens when we put,

     2    for example, SV40 large T or E1A into a human

     3    fibroblast?  What we get instead of immortalization,

     4    is a phase of so-called extended lifespan.  So here we

     5    see a primary cell, the initial proliferative phase. 

     6    This is senescence or the Hayflick limit.  The

     7    introduction of a viral oncogene is going to cause an

     8    extended lifespan for variable duration, typically in

     9    human fibroblasts of 20 to 30 divisions.  Then one

    10    sees a second proliferative decline.  This has been

    11    designated as crisis.

    12                Now this decline at the end of this

    13    extended lifespan which we call crisis, this word is

    14    somewhat ambiguous, because it has also been applied

    15    to rodent cells.  These cells do not display a two-

    16    stage mortality process.  So to distinguish more

    17    clearly between senescence and crisis, some groups

    18    have started to use the word "M1" for mortality stage

    19    one, and "M2" for mortality stage two.

    20                Senescence is different from crisis. 

    21    These are not just the same proliferative decline. 

    22    The main distinction is that cells in senescence or M1

    23    are truly non-dividing.  Whereas in crisis cultures,

    24    the apparent absence of proliferation on the

    25    macroscopic scale is actually the result of ongoing

                                                                        186

     1    cell division combined with ongoing cell death.

     2                This is an experiment that was performed

     3    in my lab.  What we show here is that elimination of

     4    the CDK inhibitor P21 in a pre-senescent normal human

     5    fibroblast causes an apparent extension of lifespan

     6    that is equivalent in magnitude to that elicited by

     7    SV40 large T antigen.  So also in human cells now we

     8    have been able to do ablative intervention.  That is

     9    eliminate the activity of certain negatively acting

    10    affecters and cause an apparent extension of lifespan.

    11                In terms of cell substrate design or the

    12    technology that would go into doing this, this was

    13    really strictly an aside, we have now developed

    14    methods -- these are really based on gene knockouts,

    15    homologies reculmination gene targeting, that can be

    16    used to delete entire genes, multiple genes in human

    17    cells, including normal human cells.

    18                So let me now turn to my last topic, which

    19    is the molecular clock of aging.  I think probably

    20    this is where I am going to overlap with what Jim has

    21    already said.  As I told you, there are some older

    22    observations that correlated entry into senescence

    23    with the lap cell division as opposed to chronological

    24    time.  Quite a few years ago, this has led to the

    25    proposal for the existence of some sort of a molecular

                                                                        187

     1    clock.  Then one envisioned that the running down of

     2    this clock would generate a signal that triggered the

     3    senescence program.

     4                Then the expression, for example, SV40

     5    large T could either prevent senescence by overriding

     6    a signal from this clock or by what I think is more

     7    likely now in light of new evidence, actually

     8    interfering with the senescence machinery itself.

     9                So as you know, the currently prevailing

    10    hypothesis is that the nature of the molecular clock

    11    is the attrition of telomeres.  This is a slide by one

    12    of my dear friends, Chris Counter, who has fancifully

    13    imagined H-TERP, which is the catalytic sub-unit of

    14    human telomerase sitting here at the end of a

    15    chromosome end.  So this is a telomere here.  Then

    16    catalyzing the addition of the telomere heximer.  You

    17    can see the telomerase RNA that acts as a template for

    18    that process right there.

    19                Germ cells and some key stem cells are

    20    known to express telomerase catalytic activity while

    21    the majority of somatic cells lack this activity.  The

    22    estimation of telomere shortening for one generation

    23    is in human cells between 50 to 100 bay spares.  So

    24    that's 50 to 100 bay spares per S phase.  This

    25    correlates reasonably well with the average telomere

                                                                        188

     1    length in a young human fibroblast of 18 to 20

     2    kilobases and the length of 8 to 10 kilobases in the

     3    senescent fibroblast.

     4                I think it's an important observation that

     5    senescent cells in fact contain appreciable

     6    telomerase.  So here we have a normal cell or a young

     7    cell.  We get attribution of telomerase.  At this

     8    point, the telomerase are maybe 8 to 10 kilobases in

     9    length.  This generates a signal.  If the cell is now

    10    driven into the extended lifespan phase, these

    11    telomeres will continue to erode because telomerase is

    12    not expressed in that state.  Eventually one enters

    13    into a crisis which is caused by erosion at the end,

    14    genetic instability, et cetera, et cetera.

    15                It is really the nature of this signal

    16    that I think is one of the enduring mysteries of the

    17    field.  One can really now beginning -- we can start

    18    to see the process as being composed of a clock, a

    19    signal, and then the senescence machinery itself which

    20    is most likely composed of the same players, CDK

    21    cyclin inhibitors, et cetera, et cetera, that are used

    22    in other types of responses such as differentiation

    23    and quiescence.

    24                The linguistic definition of senescence is

    25    the state of being or the process of becoming old. 

                                                                        189

     1    This term has therefore been used to describe

     2    essentially any sort of age-related irreversible

     3    proliferative decline.  In light of these new

     4    molecular insights, I prefer to use senescence in the

     5    more restrictive mechanistic sense to designate the

     6    response triggered in normal cells.  I really believe

     7    that senescence is an active genetically programmed

     8    process that responds to an inductive signal.  Perhaps

     9    telomere shortening, but that is not 100 percent

    10    clear.

    11                How the signal is generated is not really

    12    well understood.  One can argue that the ensuing

    13    growth arrest has the obvious advantage of preventing

    14    the cell from becoming grossly genetically unstable. 

    15    In contrast then, I think of crisis as an

    16    unphysiological state.  You have to do something to

    17    the cell to drive it to this point, and that it leads

    18    eventually to the catastrophic breakdown of chromosome

    19    stability, which is caused by critical telomere

    20    shortening on many chromosome ends.

    21                So now this is really just a restatement

    22    of the two-stage mortality process.  What I have added

    23    here now is telomere length in kilobases on the Y

    24    axis, the replicative age on the X axis.  So here we

    25    have a cell in the beginning.  If this happens to be

                                                                        190

     1    a germ cell or a stem cell, it will maintain telomeres

     2    because it will express telomerase activity.

     3                Most somatic cells will start down the

     4    slippery slope of telomere attrition, eventually

     5    entering into a physiological state of growth arrest,

     6    through which they can be driven by either the

     7    expression of certain viral oncogene or the eblation

     8    of certain inhibitory pathways that are intrinsic to

     9    those cells.

    10                The cells then enter into extended

    11    lifespan.  They continue to erode telomeres.  They

    12    enter into a state of crisis, which is characterized

    13    by genomic instability.  Finally, at this point, one

    14    can attain a truly immortalized derivative in the key

    15    step here, is the expression of telomerase catalytic

    16    activity.

    17                I should also point out that telomerase

    18    need not be expressed at the final step.  It has been

    19    shown experimentally that telomerase can be

    20    artificially or experimentally activated anywhere

    21    along this line, and that that will lead in some cell

    22    types, not necessarily all cell types, to

    23    immortalization.  However, I think the large body of

    24    evidence suggests that at least in vivo, and by this

    25    I mean during the natural development of malignancy, 

                                                                        191

     1    the activation of telomerase activity is a relatively

     2    late step.

     3                So if crisis doesn't exist in rodent

     4    cells, and bypass of senescence is sufficient for

     5    immortalization, how does telomerase become expressed

     6    in somatic cells, rodent somatic cells?  The bottom

     7    line here seems to be that telomerase is not very

     8    strictly regulated in rodent cells and tissue.  A

     9    variety of rodent tissues have been shown to express

    10    telomerase activity.  Telomerase negative primary

    11    cultures often become telomerase positive over time

    12    even prior to reaching senescence.

    13                In contrast, telomerase appears to be

    14    regulated very stringently in human cells.  Therefore,

    15    telomerase activation could occur in rodent cells that

    16    are undergoing immortalization either prior to or

    17    after the senescence bypassing event, and could easily

    18    occur in the subtle and gradual fashion so that no

    19    clearly apparent downturn in proliferative capacity of

    20    the ball culture would be observed.

    21                In other words, one step immortalization

    22    that one sees so often in rodent cells may in fact

    23    require two steps, the obvious step of senescence

    24    bypass and very likely a second step that may be very

    25    subtle, at least in rodent cultures.  That is, of

                                                                        192

     1    activating telomerase catalytic activity.

     2                So I think that is about as good a summary

     3    as I can think of in 20 minutes.  I will be glad to

     4    entertain questions.

     5                (Applause.)

     6                AUDIENCE MEMBER:  Bill Egan, from the FDA. 

     7    When you immortalize cells, you know, after they go

     8    into crisis or whatever, what becomes the length of

     9    the telomere?  Does it go back up to 20 kilobases? 

    10    What maintains the length of that telomere at a fixed

    11    --

    12                DR. SEDIVY:  That's a very good question.

    13                AUDIENCE MEMBER:  Why doesn't it become 30

    14    or 40 kilobases.

    15                DR. SEDIVY:  In fact, it seems that

    16    excessive telomere length is not good, at least in

    17    human cells.  It's been known for a long time that

    18    many spontaneously immortalized human cell lines which

    19    we love and honor like 293 and Hela, et cetera, et

    20    cetera, have very short telomeres.  These telomeres

    21    can be maintained at a length of one to two KB.  These

    22    cells seems to be perfectly happy with that.

    23                So I think it's more the maintenance of

    24    the telomere length rather than the absolute length of

    25    the telomere.

                                                                        193

     1                If you artificially introduce telomerase

     2    catalytic subunits into fibroblasts, what one

     3    typically sees is that the best clones are ones that

     4    build up telomere length to about 8 to 10, 12 KB and

     5    then maintain it at that level.  It seems to be a

     6    function of the expression level of the H-TERP gene,

     7    because if one does this experiment, you see cultures

     8    that very slowly erode their telomeres.  They will

     9    eventually senesce.

    10                You see cultures that build up telomeres

    11    to maybe 20, 30 kilobase in length.  That doesn't seem

    12    to be good for them because the rate of growth goes

    13    down.  So really the best cultures are the ones that

    14    maintain at least in fibroblasts.  So I think it's the

    15    maintenance rather than the absolute length.

    16                DR. HUGHES:  Would you please comment on

    17    Carol Greider's knockout mice?

    18                DR. SEDIVY:  Well, yes.  I didn't get into

    19    that at all because that's at least for the time being

    20    -- there are some paradoxes here.  Okay?  The obvious

    21    paradox is that mice have extremely long telomeres, 60

    22    KB on average.  This is the laboratory mouse. 

    23    Muskulorattus has perfectly normal telomeres, for

    24    example.

    25                In fact, this is really the other way

                                                                        194

     1    around because these cells live for a very short time

     2    in tissue culture.  When they undergo senescence, they

     3    undergo senescence with telomeres that are 50 KB in

     4    size as opposed to 60 KB in size.

     5                So there are really two answers to that. 

     6    One answer is that if you look at individual telomere

     7    ends, you will see that there's a certain degree of

     8    heterogeneity in that in fact in all cells, including

     9    mouse cells, you always see a minority fraction of

    10    chromosomes that have very short telomeres.  If the

    11    signal is caused by a perfectly short telomere that

    12    gives, for example, DNA damage-like signal, then you

    13    only need one per cell to give the senescence signal. 

    14    So that's one explanation.  I don't really know

    15    whether it's correct.

    16                The other explanation is that mouse cells

    17    don't senesce.  They neither undergo crisis or they

    18    undergo senescence.  In fact, that plateau that we are

    19    seeing during the immortalization is not senescence. 

    20    It's a differentiation-like process.

    21                There are people, including myself, that

    22    given this kind of loose distinction between

    23    quiescence, differentiation, and senescence, would

    24    prefer to define senescence now as a process that is

    25    triggered by telomere erosion.  Obviously when a mouse

                                                                        195

     1    primary fibroblast culture undergoes senescence, it's

     2    not doing it, probably not doing it because it's

     3    receiving a telomere signal.

     4                I don't know if that is -- does that make

     5    sense?

     6                DR. HUGHES:  I had hoped you would comment

     7    on the mice themselves.

     8                DR. SEDIVY:  The mice themselves?  What do

     9    you want to know about the mice themselves?  They are

    10    alive.

    11                DR. HUGHES:  Yes, I know.  But in the

    12    Greider experiment with telomerase knockout.

    13                DR. SEDIVY:  If you knock out telomerase

    14    in mice, it takes six organismal generations to

    15    observe lethality.  Okay?  What you see at each

    16    generation is that the average telomere length.  So

    17    generation one, it's 50.  Generation two, it's 40. 

    18    Generation three, it's 30.

    19                If you take mouse and real fibroblasts at

    20    any one of these generations, they senesce in vitro on

    21    schedule.  Is that what you wanted?

    22                DR. HUGHES:  (Inaudible.)

    23                DR. SEDIVY:  Well, I think I just offered

    24    you one explanation for that.  That is that what we

    25    are calling senescence is not senescence.  It's

                                                                        196

     1    something caused by some insufficiency in the median

     2    that is in fact triggering a differentiation event. 

     3    Actually, Jim McDougall and I also don't quite agree

     4    about what's happening in his keratinocyte cultures

     5    because what he is calling senescence, some

     6    keratinocyte biologists would prefer to call

     7    differentiation.

     8                DR. NABEL:  Okay.  If we could move the

     9    questions along, maybe brief answers.  Then we'll move

    10    onto the next speaker.

    11                AUDIENCE MEMBER:  Alex van der Eb, Leiden. 

    12    You just already answered, I think, my question, which

    13    was why do mice cells, mouse cells enter senescence

    14    while they have such long telomeres?  In fact, you

    15    answered already part of that question.

    16                Do these cells that enter a so-called

    17    senescence have high levels of P21 or P16 or something

    18    like that?

    19                DR. SEDIVY:  Yes, they do.  Yes, they do.

    20                AUDIENCE MEMBER:  So there is a signal

    21    then.

    22                DR. SEDIVY:  If you take a knockout mouse

    23    for P21 that doesn't undergo senescence.  It just

    24    keeps going.  But you know, that's what I was trying

    25    to say.  That is that op regulation of P21 is not a

                                                                        197

     1    molecular market for senescence.  P21 is op regular

     2    because of oxidated stress, osmotic stress,

     3    differentiation signals.  This is a very general

     4    machinery that is used to establish cell cycleresce.

     5                I, in fact, don't know of any molecular

     6    marker that is specific for senescence.  This includes

     7    the famous senescence-specific betagalactocytis

     8    activity.  You know, you see a lot of people staining

     9    cells, and they turn blue and they say it's

    10    senescence.  Everybody knows if you put hydrogen

    11    peroxide on your cells, they turn blue as well.

    12                AUDIENCE MEMBER:  Just a brief comment for

    13    those people who might be setting up assays that would

    14    be monitoring P21 sip.  We, as I showed, found that

    15    P21 is elevated in a subset of the HPB infected cells. 

    16    We did three other related assays.  One was to look

    17    for P21 MRNA.  It turns out it's abundant in all

    18    differentiated cells.  But there is a post-

    19    translational control on the accumulation of P21.

    20                It turns out what happens is that if there

    21    is not a signal that unscheduled in a synthesis is

    22    underway, namely, abundant cyclin E, then proteosomes

    23    rapidly degrade the P21 that's translated.  When we

    24    put in proteosome inhibitors, P21 piled up in all

    25    cells and all replication was blocked.

                                                                        198

     1                We went on to ask one additional question. 

     2    That is, how does P21 actually block S phase or DNA

     3    synthesis.  Unexpectantly, it had nothing to do with

     4    blocking cyclin E activity.  It turns out cyclin A,

     5    CDK-2 or cyclin A CDC-2, can phosphorylate DNA

     6    preliminary cell and all these other subunits I

     7    showed.

     8                The one thing cyclin A can't do is bind to

     9    PCNA.  But when the P21 sip piles up in these cells

    10    that have excessive cyclin E, the way the P21 is

    11    actually functioning is by binding to the PCNA and

    12    blocking elongation, not initiation.

    13                DR. LEWIS:  This may be a naive question. 

    14    Is there any change in the activity of endogenous

    15    oncogenes in cells at about the time they are entering

    16    into senescence, especially rodent cells?

    17                DR. SEDIVY:  By activity, you mean level

    18    of expression?  I am not aware of that.

    19                AUDIENCE MEMBER:  What happens in

    20    spragues?  I mean are they different than

    21    muskulorattus?  Do they have shorter telomeres that

    22    they go through?

    23                DR. SEDIVY:  What happens in spragues is

    24    exactly the same that happens in muskulor.

    25                AUDIENCE MEMBER:  So even though one has

                                                                        199

     1    60 KB and one has 2 KB?

     2                DR. SEDIVY:  I mean, you know, this kind

     3    of all argues that the length of telomeres has nothing

     4    to do with this plateau in mouse cells that we define

     5    as senescence.  Okay?  I think there's a result that's

     6    kind of floating around, which is also consistent with

     7    that.  That is, we all know the wonderful experiment

     8    of expressing telomerase in human fibroblasts which

     9    causes immortalization.  It doesn't do that in mouse

    10    cells, which also argues that the length of telomeres

    11    in mouse cells is not what is triggering this growth

    12                DR. NABEL:  John, I am going to just end

    13    with one last question.  You referred to the notion of

    14    program of senescence and it being dominant.  I am

    15    just wondering, has anyone ever done a cell fusion

    16    experiment where you have taken cells approaching

    17    their limit and then fused to neo-natal cells.  Is it

    18    in fact dominant?

    19                DR. SEDIVY:  Yes.  Those are very old and

    20    classical experiments.  In general of course,

    21    senescence is a dominant state.

    22                DR. NABEL:  Thanks.  The last talk in this

    23    session is from Frits Fallaux.  The title of his talk

    24    is using defined adenoviral genes and primary human

    25    cells for the generation of immortalized cell

                                                                        200

     1    substrates.

     2                DR. FALLAUX:  First of all, I would like

     3    to thank your organization for inviting me here.  The

     4    subject of my talk will be on the generation and

     5    characterization of new helper cell lines for the 

     6    construction, provocation, and protection of

     7    recominance replication effective adenoviral vectors.

     8                In the past few years, the interest in

     9    vectors derived from human viruses.  This is caused by

    10    the fact that from the many years of intensive

    11    fundamental research on human adenoviruses, it has

    12    been found that adenoviruses have several favorable

    13    characteristics, including high stability of variance. 

    14    The variance is very easy to grow into pure with very

    15    high fibers.  It has a very broad host range. 

    16    Importantly, it has the capacity to transduce non

    17    milotic cells.  This makes adenovirus a very potent

    18    gene therapy.

    19                It is known that it has very low kinisity,

    20    and there is there ample experience with adenoviruses

    21    as vaccines.

    22                This slide shows a schematic

    23    representation of the adenoviral genome.  It is a

    24    double stranded linear DNA molecular of approximately

    25    36 KD, carrying several genes, flanked by inverted

                                                                        201

     1    herminal repeats.  The genes are sub-divided in so-

     2    called early genes and in late genes, depending

     3    whether they are stressed early or late during the

     4    lytic infection.

     5                This slide shows you a scheme of the

     6    classical methods to construct the common

     7    adenoviruses.  All currently used adenoviruses carry

     8    a deletion in E-1.  This renders the virus replication

     9    effective, and it also provides space to insert

    10    therapeutic genes.

    11                Now in the old days, we used to isolate

    12    the DNA from wild type adenoviruses at 5 or S-2. In

    13    purified DNA, and I just -- the restriction enzyme

    14    cla-1, which puts ones in area region one.  We then

    15    purify the large fragment.

    16                In addition, it also needs the

    17    construction of an adaptor plasmid which carries the

    18    transcriptase unit, including geno-fenchfras, but also

    19    the left inverted herminal repeat and a part of the

    20    adenovirus sequence which is also present in the large

    21    fragment.  Pro-construction of these two moleculars in

    22    so-called helper cells, and the helper cell is the 293

    23    cell made by Frank Reim.  Upon close inspection,

    24    another mination occurs, creating now the recombinant

    25    adenovirus, carrying the gene of interest at the

                                                                        202

     1    position that we want.

     2                You can proficate these elongated viruses

     3    due to the fact that the helper cells complement the

     4    missing elong function.

     5                Now despite the encouraging results of

     6    things so far with the use of recombinant

     7    adenoviruses, there are also several problems

     8    associated with the use of such vectors.  These

     9    problems include the growth infectivity range.  That

    10    is, you do not only infect the target cells, but also

    11    non-target cells.  This may cause pathogenicity.

    12                Also, the viruses are rather immunogenic. 

    13    We only leave off E-1, and all the other viral genes

    14    are still present and can be expressed to low levels,

    15    resulting in numerous responses by the host, both

    16    humoral responses, antibodies, and cellular responses

    17    against new cells.  The cells are killed and the

    18    therapeutic effect is lost within several weeks.

    19                Another issue is the occurrence of

    20    replication competent adenovirus, abbreviated RCA.  I

    21    will focus on this topic.  There are various sources

    22    of replication competent adenoviruses.  In a sort of

    23    infection, during the production of viral, or as an

    24    earlier stage, or during the construction of the

    25    recombinant factor, especially when you use classical

                                                                        203

     1    methods, if you use the large clavon fragments.  If

     2    the digestion if not complete, you have RCA, namely

     3    the wild virus.

     4                It has also been shown recently that you

     5    can generate RCA by homologous recrimination because

     6    the factor and the helper cell carry adenovirus

     7    sequences that overlap.  As a result, by homologous

     8    recrimination, you can get RCA.  I will focus on this

     9    source of RCA.

    10                Well, how does it work, homologous

    11    recrimination resulting in RCA?  This is a scheme of

    12    a typical elongated factor.  This is a scheme of the

    13    integrated adenovirus sequences in the helper cells.

    14                The helper cell line is 293, and more

    15    recently, we made alternative cell line 911.  Both

    16    helper cells carry the evon A, evon B in chorion

    17    regions.  But in addition, they also carry sequences

    18    that enclose structural protein lines, downstream of

    19    evon B, and upstream of evon A, be left for determinal

    20    repeat.

    21                Those sequences are also present in the

    22    factors.  So that a sequence overlap 5 prime and 3

    23    prime of the therapeutic gene.  As a result, you can

    24    get homologous recrimination by which the recominance

    25    virus now trades its therapeutic gene for region E-1,

                                                                        204

     1    and becomes replication competent.  Now what you can

     2    do about this is to avoid the sequence overlap.  We'll

     3    come back to that later.

     4                Well there are only a few helper cell

     5    lines available when you work with recombinant

     6    adenoviruses.  We are fortunately in our lab to have

     7    three of them, including the two in our free cell

     8    line, recently an iomosa line, and even more recently,

     9    the PER cell line.  All three cell lines are obviously

    10    of human origin.  They are all derived from primary

    11    diploid embryonic cells.  293 is derived from kidney

    12    cells, 911 from retinoblasts, and PER C-6 as well.

    13                Now when I started to work with

    14    recombinant adenoviruses in our lab, which is the lab

    15    of Professor van der Eb at the Leiden University, I

    16    used obviously 293 cells, and I met with some

    17    technical difficulties.  Since we had a panel of adeno

    18    virus transformed human cells, including cells of

    19    kidney, lung, and retinoblasts, I decided to screen a

    20    panel of cells in order to find an alternative for

    21    293.

    22                From this panel of cells, I selected one

    23    particular retinal cell line.  We named it cell line

    24    911.  The reason for this name was to get the

    25    attention of our colleagues in the U.S.

                                                                        205

     1                (Laughter.)

     2                Well, the construct we used to make the

     3    911 cell line is shown over here.  It carries the

     4    adenovirus sera type 5 nucleotides 87 to 5,788,

     5    including evon A and evon B.

     6                Now I want to be short on the 911 cell

     7    line.  The most important findings were that they

     8    performed very good in virus titrations.  We also

     9    found that the virus use of 911 are up to three times

    10    higher with various viruses, also recombinant viruses,

    11    three times higher than obtained from 293.

    12                Some other characteristics of this cell

    13    line are that they express very high levels of evon A

    14    and evon B, are highly transfectable, which is

    15    important when you want to construct recombinant

    16    viruses at the classical method.  The use of the

    17    viruses are very high, as told, and they perform very

    18    well in titration assays.  So we concluded that 911 is

    19    a good alterative for 293.

    20                However, I have shown you the construct we

    21    used to generate the 911 cell line.  We now have a

    22    situation which is similar to 293.  Namely, and also

    23    in 911 cells, besides evon A and evon B encoding

    24    sequences, also sequences of the left inverted termin

    25    are repeat, and sequences in part encoding protein 9

                                                                        206

     1    are present.  So again, there is overlap and you can

     2    create RCA.

     3                So what we decided to do is start all over

     4    again and make now the cell line in combination with

     5    a so-called matched vector, now sequence overlap. 

     6    What we did was to make a so-called packaging

     7    construct carrying only the evon A and evon B encoding

     8    sequences in which evon A is driven by PGK, a

     9    heterologous promoter, and a heterologous poly and

    10    signal, and lay matched vectors that are deleted of

    11    exactly that elong region which is present in the

    12    packaging construct.  Thus affording sequence overlap

    13    and thus eliminating homologous recrimination as a

    14    source of RCA.

    15                This shows you one of the packaging

    16    constructs we constructed.  Present are adenovirus

    17    sera 5, sequences four, five line to 3,511.  Those are

    18    only the evon A and evon B encoding sequences.  Evon

    19    A is driven by the human PGK promoter.  Evon B is

    20    under its natural promoter, and directly flanking the

    21    evon B stop codon as the polyadenylation signal

    22    derived from hepatitis B virus.

    23                Now before we decided to transfect in this

    24    vection construct into our retinoblasts, we decided to

    25    do some functional assays with this construct first. 

                                                                        207

     1    We did this because first we only had a few frozen

     2    ampoules of the retinoblasts.  Second, the packaging

     3    construct contains several PCR fragments.

     4                This slide shows you some of the functions

     5    or features of adenovirus evon A.  The features are

     6    that domains 1 and 2 are involved in the regulation of

     7    expression of genes.  Evon A is known to associate at

     8    the protein level with cellular proteins, P-105 RB,

     9    cyclin A, P-300.  I think the list is growing.  All

    10    these different features result in the transformation

    11    and immortalization.

    12                Not shown is the feature or function of

    13    evon B.  Evon B prevents the cells from growing into

    14    apoptosis as a result of the activities of evon A.

    15                Now this is the actual functional assay we

    16    performed.  At the left is shown the constructs we

    17    tested.  At the right is shown the number of colonies

    18    we obtained upon transfection of one or five microprin

    19    of these plasmids into baby rat kidney cells.  When we

    20    transfected in a construct expressing only evon A, we

    21    only found on average one focus or colony upon

    22    transfection of one microgram.  This is very low. 

    23    This is caused by the fact that expression of evon A

    24    in primary cells, in the absence of evon B, is toxic,

    25    causing apyltosis.

                                                                        208

     1                You can see this in the second line. 

     2    Transfected this plasmid to come in plasmid in evon B,

     3    and we did obtain reasonable amounts of foci.

     4                Obviously also the construct we used to

     5    generate 911 resulted in focus formation. 

     6    Unfortunately, also when you are packaging construct,

     7    which we used to make the PER cells gave foci, thus

     8    indicating that the packaging construct allowed the

     9    functional expression of early region non-probenes.

    10                So then we went to the actual experiment,

    11    transfected the packaging construct into primary human

    12    diploid retinalblasts.  We could establish seven

    13    clonal cell lines.  We tested these clones for first,

    14    expression of evon A and evon B proteins.

    15                Now we found that all clones expressed

    16    very high levels of evon A in both 55 and 21 K evon B,

    17    when compared to 293 and 911.  We also looked at

    18    vector use.  We looked at three clones, clone 3, 5,

    19    and 6.  As you can see, we found that the three PER

    20    clones tested exhibited similar use of recombinant

    21    viruses, compared to 293 and 911.  Since PER C6 played

    22    the highest use, we decided to analyze this clone in

    23    further detail.

    24                Now of course the major issue for us was

    25    to test whether or not our approach to use the PER

                                                                        209

     1    cells in combination with matched adenovirus vectors

     2    would reduce or even eliminate the generation of RCA. 

     3    This testing has been performed at the enzyme.

     4                What I did was to amplify an RCA free

     5    master stock of a typical adenovirus vector and

     6    amplify it to 293 or PER C6.  What I found, I can

     7    summarize it for you, is that amplification of 293

     8    resulted in RCA positive vector batches in

     9    approximately 50 percent of the places.

    10                Now for a clinical setting, that means

    11    that you might consider to throw away half of what you

    12    had made.  In the case of PER C6, fortunately in none

    13    of the batches amplified on PER C6, we were able to

    14    detect RCA, not even in a large scale production

    15    setting.

    16                So we concluded that our strategy to make

    17    a PER cell in combination with new matched vectors

    18    severely reduced, maybe even eliminated the RCA

    19    problem, at least by homologous recrimination.

    20                The next two slides summarize some of the

    21    other features of PER C6 cells that contain three to

    22    five copies of the packaging construct, very high

    23    levels of evon A and evon B, comparable to 293 and

    24    911.  Good use of the different vectors, also similar

    25    to the other two producer cells.

                                                                        210

     1                The cell line was very stable.  We have

     2    now come over passage 250 actually.  So far we have

     3    not detected RCA, and the list of productions with

     4    different vectors is still increasing.

     5                We have a master cell bank available for

     6    PER C6, also a working cell bank.  Importantly, the 

     7    PER cells were made on a GLP conditions, using

     8    certified U.S. bovine serum and trypsin.  Currently,

     9    InterGene is doing all kinds of tests which were

    10    necessary for the use in the chemical setting,

    11    including mycoplasma and sterility testing.  In the

    12    academic lab, you can simply draw the cells to

    13    standard medium.

    14                Finally, I would like to thank all the

    15    people that are involved in this project.  Number one,

    16    the PER cells were generated in the lab of Professor

    17    van der Eb at Leiden University, in the Applied

    18    Virology Group, supervised by Dr. Gugen.  The

    19    packaging constructs were made by Edie von Frel of

    20    InterGene, and all the downstream processing, a lot of

    21    work is currently being performed by InterGene,

    22    supervised by Valeria in the Adenovirus group,

    23    supervised by Dr. von Laud.  As I told, all the RCA

    24    testing was performed at Genzyme by Kathy Hay here. 

    25    Thank you.

                                                                        211

     1                (Applause.)

     2                DR. NABEL:  Thank you.  We'll take

     3    questions.

     4                DR. FRIED:  Mike Fried.  Do these cells

     5    form tumor in nude mice?

     6                DR. FALLAUX:  Actually we did not test

     7    that yet.  However, we did test this for the 911 cell

     8    line, the weakly tumorogen in nude mice.  So you might

     9    expect the PER cells would exhibit the same feature

    10    with respect to that.

    11                DR. FRIED:  What is weakly tumorogen?

    12                DR. FALLAUX:  Compared to some of the

    13    let's say adenovirus sera type 12 transformed rodent

    14    cells, they are much less tumorigenic.

    15                DR. SHEETS:  Becky Sheets, FDA.  I had a

    16    similar question, but I have a couple of other.  Does

    17    the PER cell stay diploid or is it aneuploid?  Also,

    18    the individual from whom you obtained the retina, did

    19    they have wild type RB genes or were they -- you know,

    20    was this someone that died of retinal blastoma?

    21                DR. FALLAUX:  For your first question,

    22    they are no longer diploid.  I don't know all the

    23    details, but we do find chromosome duplications I

    24    guess in PER C6 because we have tested recently.

    25                Your second question.  This may sound

                                                                        212

     1    silly, but we are currently trying to find out the

     2    information of the donor.  The cells were isolated in

     3    the early 1980s, and we're now working backwards to

     4    find out those details.

     5                DR. NABEL:  John?

     6                AUDIENCE MEMBER:  293 cells have become

     7    quite popular in the laboratory for reasons that have

     8    little to do with their ability to support adenovirus

     9    vector replication.  Have you checked these other

    10    cells, for example, high levels of transvectibility in

    11    being a good host for other kinds of viruses and that

    12    kind of thing that make 293 cells so beloved by many

    13    virologists?

    14                DR. FALLAUX:  In fact, all adenovirus

    15    transformed cells are as highly transvectible as 293

    16    cells.

    17                DR. NABEL:  Okay.  If there are no further

    18    questions, then I think we can just proceed onto the

    19    panel discussion.  John Coffin will chair that.  If

    20    the panel members want to come forward and get

    21    started.

    22                DR. COFFIN:  By my calculation, we are

    23    running almost exactly an hour late.  A check with the

    24    board outside reveals that we really probably can not

    25    go much past 3:00 before we start to lose people quite

                                                                        213

     1    seriously.  So we probably should shoot for an hour in

     2    which we either have a lot or a little to do,

     3    depending on the will of the crowd and our host.

     4                This panel discussion actually as two

     5    functions.  One is a discussion of the last topic

     6    covered.  That is the designer cell substrates, two

     7    talks we heard today and the one yesterday.  Then

     8    secondly, where we really earn the generous honoraria

     9    that FDA is paying us, where we try to summarize and

    10    hopefully answer some questions that might be useful

    11    to our host in terms of development of policy, ideas

    12    for further meetings, experimentation and so on.

    13                Tentatively we'll plan to sort of split

    14    the discussion half and half between these issues, but

    15    I think we can play that by ear as we go along. 

    16    Again, I expect widespread audience participation,

    17    particularly since these are topics that I myself am

    18    not really actively working in and am familiar with.

    19                The questions that on the first part, on

    20    the designer cell substrates, that we were charged to

    21    address are summarized on this overhead.  Before I

    22    turn it on, I want to apologize in advance for two

    23    things.  One is my handwriting is very bad, so you are

    24    going to be subjected to that for a while.  Secondly,

    25    I was given a rather blunt instrument to write with. 

                                                                        214

     1    Thirdly, of course I'm not well enough organized like

     2    some of the previous chairs who have prepared these

     3    ahead of time.

     4                So this is a paraphrase, I hope an

     5    accurate one, of the issues that were raised, that are

     6    raised in the points to consider.  The first regarding

     7    designer cell substrates is the issue of whether cells

     8    that are derived by the kinds of defined means that we

     9    have seen, and we have seen the example of

    10    introduction of telomerase plus or minus oncogenes,

    11    viral oncogenes, or viral oncogenes alone whose

    12    function among other things was to stimulate or

    13    inactivate genes that are involved in senescence.

    14                Whether cells that are created in this way

    15    in fact offer significant safety, create safety issues

    16    relative to other cell lines, whether they offer

    17    advantages or disadvantages, whether we can go through

    18    the sort of defined risk algorithm that was given to

    19    us at the beginning of the meeting, to address these

    20    and anything else.

    21                So if we could get onto the first point

    22    here.  Are there significant safety issues relative to

    23    tumor or neoplastic cell lines?  In other words,

    24    uncharacterized, what we should call it perhaps,

    25    uncharacterized cell lines.  Cell lines that have just

                                                                        215

     1    been handed to us either in tumors or that have arisen

     2    by means we don't really understand very much about in

     3    culture.

     4                Would anybody on the panel care to --

     5                DR. SEDIVY:  Yes.  I would just like to

     6    make a very brief comment about designer cells.  I was

     7    asked to talk about the history of immortalization. 

     8    In fact, what my lab works on is more related to

     9    interventions, genetic interventions in human cells. 

    10    So you know, obviously we have talked about putting an

    11    H tert and putting in various viral oncogenes.  So

    12    really here the issue is can we make a cell line that

    13    is immortal and it has a particular spectrum of

    14    phenotypes that we want by absolutely defined genetic

    15    interventions.  I think the answer now is yes.  We can

    16    do a lot better than putting in SV-40 large T or E-1A

    17    or E-7, because as have heard even today, we don't

    18    really know exactly down to the last T what these

    19    viral proteins are doing to the cell.

    20                So in fact, what we can do is we can

    21    delete cellular genes using gene knockouts, and

    22    produce very much the same effect.  That is, we can

    23    really now contemplate really designing cells without

    24    the use of viral oncogenes.  I think that putting an

    25    H tert is obviously a necessary step, but this is a

                                                                        216

     1    cellular gene.  So I think that's probably okay.

     2                DR. ONIONS:  It's really a question of

     3    clarification from my point of view.  But it seems to

     4    me that one of the advantages of cell lines that come

     5    from potential oncogenic background is they have some

     6    of the features from mass culture that industrity. 

     7    That is, they can be grown in an anchorage-independent

     8    way.  They can be grown in high density in fermenters. 

     9    What's the position with telomerase immortalized?

    10                I assume actually they are mimicking much

    11    more the kinds of cell type that Dr. Hayflick would

    12    define as -- I forgot what he used, apologies -- the

    13    first stage of cell strains.  That probably do not

    14    have those particular phenotypic properties.  Do you

    15    know what the stage of those cells are?

    16                DR. SEDIVY:  Well, you know, we have a

    17    really limited experience.  This game has only been

    18    played for a few months, maybe a year in some

    19    privileged labs.  I think what we're really talking

    20    about here is proof of principle.  In my lab, we're

    21    not interested in growing cells in fermenters.  We are

    22    interested in cell cycle progression.  But I think if

    23    somebody wanted to make a cell line that grows well in

    24    fermenters, I think it may be a good idea to

    25    contemplate some of these new approaches.

                                                                        217

     1                DR. ONIONS:  Yes.  The point I was trying

     2    to make was that if you start down the road of trying

     3    to produce new cells and immortalize them, and you try

     4    and use procedures that you think are of in a sense,

     5    the safest, whatever that means, those might not be

     6    the steps that you actually need to actually produce

     7    an industrially useable cell line regardless of the

     8    importance of the science.  The practical end may not

     9    be what you want.

    10                DR. FRIED:  What else besides, you said

    11    you would knock out genes?  I mean would this be in

    12    human cell lines or instead of using the viral

    13    proteins?

    14                DR. SEDIVY:  I think in general, the first

    15    thing you have to do is you have to immortalize the

    16    cell.  For that reason, we put an HH tert.  That is a

    17    technical feature that we need because to do the

    18    knockouts, we have -- you know, there's limited time

    19    to do a knockout.  We can do two knockouts before a

    20    human fibroblast undergoes senescence.  But if we want

    21    to do more, and we're obviously interested in doing a

    22    lot more, it is very convenient to put tert in there

    23    at that point.

    24                If you don't want tert, you know, tert now

    25    comes flanked with lock sites, so that you can take it

                                                                        218

     1    out later on if you are interested in that.

     2                DR. COFFIN:  Can you be more specific

     3    about what you would knock out?

     4                DR. SEDIVY:  Pardon me?

     5                DR. COFFIN:  Want you to be more specific

     6    about what you are knocking out.  Which genes have you

     7    knocked out?

     8                DR. SEDIVY:  Well what we are interested

     9    in doing is we are interested in dissecting the

    10    machinery that establishes senescence.  So not in a

    11    single cell line, but at this point, we have P-21, cip

    12    1, RB, B-53, and P-16 ink 4A knockouts in various

    13    combinations.  So you know, the vectors are available.

    14                One interesting point about human cells

    15    that is interesting to the technocrats I think is that

    16    until this time, we do not see the requirement for

    17    isogeneic DNA.  So in fact, these vectors that we have

    18    made, and some other labs have contributed to this,

    19    can be used and essentially -- and have been used in

    20    any human cell with equivalent frequency of

    21    recombination.

    22                DR. HAYFLICK:  Yes.  We have a lot of

    23    information about the biological properties of H tert

    24    transformed normal human cells now.  There are the

    25    ones with which Choma is familiar, are now as I

                                                                        219

     1    indicated several days ago, approaching, and in one or

     2    two cases beyond 400 PDLs.  Many of them are beginning

     3    to show some signs of aneuploidy.  They are still

     4    anchorage dependent.  Their virus spectrum seems to be

     5    identical to that with which we are familiar prior to

     6    H tert transformation.  Finally, the studies that have

     7    been done on animal inoculation of these cells

     8    indicates that they are not neoplastic.  So that we do

     9    know that much.

    10                However, we are not talking about two

    11    other classes of cell populations that are important

    12    candidates to consider.  I mentioned these in my

    13    opening remarks on Tuesday night.  Namely, normal

    14    human diploid cells that can be immortalized using

    15    repeated treatments of exposure to chemical

    16    carcinogens.  We published on this on a population

    17    that that explanation defines about 25 years ago. 

    18    It's called SUSM-1.  It's freely available.  It is

    19    from a normal human diploid fibroblast transformed by

    20    multiple exposures to MMNG.

    21                A second class of cells that also falls

    22    into this category of normal human cell populations

    23    transformed with something other than a virus, by way

    24    of example, is KMSD-6 transformed by a former student

    25    of mine, using multiple exposures to cobalt 60

                                                                        220

     1    radiation.  That cell population was also described

     2    and in fact is used commercially today.  It was

     3    described about 25 years ago.

     4                I think it is very important to realize

     5    that it's possible to immortalize well-characterized

     6    normal human diploid cells like WI38 and MRC5 and

     7    perhaps others, utilizing non-viral means.

     8                I would also like to repeat what I

     9    mentioned the other day because it's been restated

    10    again, and it's not accurate.  That is, that there are

    11    spontaneously transformed normal human cell

    12    populations.  I will be happy to provide the

    13    references to whoever would like to have them.

    14                DR. COFFIN:  Any response to that?  Do we

    15    feel in terms of this first issue that there are

    16    significant safety, differential safety issues of

    17    these kind of cells relative to the relatively

    18    uncharacterized lines?

    19                DR. MINOR:  From the point of view of the

    20    infectious agent side of things, I mean I don't see

    21    much difference between, you know, a brand new

    22    tumorigenic cell line that appears in your hand and

    23    one that's actually being designed to actually appear

    24    like that.  It seems to me they are both

    25    uncharacterized and you would have to look at both of

                                                                        221

     1    them very carefully.  I'm not sure that you have new

     2    infectious issues simply because you designed it to be

     3    transformed.

     4                DR. FRIED:  Something came up the first

     5    day.  Would it be worth getting new cells that really

     6    grow well with defined media so we can avoid serum and

     7    any problems that come with that as something one

     8    might think about?

     9                DR. COFFIN:  If you avoid serum, I think

    10    would be a highly desirable trait in vaccine

    11    production if one could engineer that.  Obviously

    12    there are BSE issues, and that sort of raises what

    13    might be the tip of an iceberg.  Is that a practical

    14    goal for production?

    15                DR. FALLAUX:  Actually, for the

    16    production, we now can grow PER C6 in serum-free media

    17    in suspension.

    18                MR. LEWIS:  Lewis, FDA.  Are there any

    19    restrictions to the telomerase immortalization?  In

    20    other words, there are some populations that you can

    21    immortalize and others that you can't, or is this a

    22    universal way of immortalizing all types of human

    23    cells?

    24                DR. SEDIVY:  You know, I am by no means

    25    expert on this, so I can, you know, basically restate

                                                                        222

     1    what I think is already out in the literature.  That

     2    is that you can definitely immortalize human

     3    fibroblasts, pre-senescence fibroblasts by putting H

     4    tert in.  That's been shown now in a number of

     5    laboratories.

     6                You can also immortalize retinal pigmented

     7    epithelial cells as reported by the Texas group.  I am

     8    not sure whether it's been published yet, but I have

     9    heard that you can immortalize T cells, CD8 positive

    10    peripheral lymphocytes.

    11                Jim McDougall says that you can not

    12    immortalize keratinocytes unless he said something

    13    different yesterday, in that you need to interfere

    14    with the RB pathway in addition to putting H tert. 

    15    There's also some indication that breast epithelial

    16    cells may need an additional step to become

    17    immortalized.  That's all I know at this time.

    18                DR. LEWIS:  Since you can immortalize T

    19    cells and you suspect that those would grow in

    20    suspension like normal T cells, and you could grow

    21    them in any large suspension culture, that you would 

    22    need to.

    23                DR. SEDIVY:  I don't think this work has

    24    been published, so I think I'm just telling you

    25    something that I heard at another meeting.  So maybe

                                                                        223

     1    we should just cool it.  But I would presume that if

     2    they grew in suspension before tert, they would grow

     3    afterwards as well.

     4                DR. COFFIN:  Obviously these immortalized

     5    T cells would be of great interest to people who are

     6    interested in growing attenuated HIV vaccine.

     7                DR. FRIED:  But I mean once you have

     8    something that's immortalized, you could always select

     9    for something that grows in suspension, or you could

    10    try anyway.

    11                DR. COFFIN:  But when doing that, of

    12    course as soon as you start selecting for these

    13    additional characteristics, when you are introducing

    14    new and uncharacterized genetic changes.

    15                DR. FRIED:  Right.

    16                DR. COFFIN:  That these are less important

    17    than safety issues, than changes that might have led

    18    to immortalization in the first place.

    19                DR. SEDIVY:  You know, I think I would

    20    also like to second the point that was brought up

    21    earlier.  That is that sure, we can always make the

    22    claim that we know exactly what we did to the cells

    23    ourselves, but we don't know what the cells have done

    24    on their own during those zillions of passages that

    25    they are growing in my lab.  It's definitely being

                                                                        224

     1    documented that H tert immortalized fibroblasts are

     2    karyotypically very stable.  But if you passage them

     3    for long periods of time, you will find anemploy

     4    these.  So I don't think that that's any different

     5    from any other established cell lines.

     6                So it's really, you know what you did, but

     7    --

     8                DR. COFFIN:  But you don't know what

     9    happened.  To bring a point to this, if one is

    10    concerned about issues of what might happen with DNA

    11    from the cells that was carried along, then it sounds

    12    like, it sounds from what I'm hearing like there may

    13    not be a great deal of difference between using these

    14    cells and using these kinds of cells as compared to

    15    using relatively uncharacterized cells.

    16                Although there are very good reasons for

    17    making such cell lines, that this particular issue may

    18    not be the most important one.

    19                DR. SEDIVY:  You know, you could do some

    20    pretty neat things that under certain circumstances

    21    may be very advantageous.  For example, if somebody

    22    wanted to knock out the endogenous PR gene, that could

    23    be done.  You could make a cell line like that.

    24                DR. COFFIN:  You can also use factors that

    25    make these cell lines highly susceptible to viruses

                                                                        225

     1    you might want to be growing on them, and things like

     2    that.

     3                AUDIENCE MEMBER:  I'll just make a comment

     4    on one of the papilloma transform cell lines that

     5    Margaret Stanley initially isolated.  It harbors -- it

     6    was from a cervical dysplasia and harbors episomal

     7    HPV-16 from which E-6 and E-7 are expressed.

     8                Paul Lambert sub-cloned an isolate that

     9    carried generous, approximately a thousand copies of

    10    this episome in a fairly homogenous state.  The

    11    problem with it, and it sounds very appealing to have

    12    episomal maintenance of your E-7 gene.  The problem is

    13    it's dreadfully unstable, and nobody has been able to

    14    keep it with the episome.  It tends to integrate and

    15    completely rearrange, and has gone aneuploid.

    16                So any efforts toward trying to utilize an

    17    episome are probably doomed to a degree of failure

    18    because of the risk of -- well, you have to maintain

    19    episomal replication in addition to your chromosomal

    20    replication.  It just doesn't seem to work.

    21                DR. COFFIN:  And this of course, this kind

    22    of issue gets far more amplified when we're talking

    23    about growing up 10 to the 13th.

    24                AUDIENCE MEMBER:  Yes, absolutely.  We

    25    can't keep this thing going for three months.

                                                                        226

     1                DR. FRIED:  Also, every time you knock out

     2    an allele, you have to lock out the other alleles to

     3    get them both.  So that means more passages, and they

     4    get away.  I don't know whether that's good or bad. 

     5    I mean if you finally end up with the cell type that

     6    has a lot of positive features, it may not really

     7    matter whether they are instability of chromosomal,

     8    and stable or rearranged.

     9                DR. ONIONS:  Just as a general principle

    10    about whether it's useful or not to engineer cells

    11    rather than go out and select a transformed cell, a

    12    pre-existing transformed cell from a tumor, it does

    13    strike me that again, that it's under control and that

    14    you have a number of choices.

    15                The kinds of studies that PER C-6 has been

    16    involved in give you a very precise engineered system

    17    that's absolutely ideal for vector production.  But it

    18    perhaps also highlights with respect to the mistake

    19    that was made.  That is, that you had another

    20    possibility here where you could actually choose the

    21    cell.  You could validate its origin.  You could check

    22    the person.  That is the other advantage of you being

    23    able to engineer materials, that you can actually pre-

    24    select the actual source of the material that you

    25    start with.  That would have been an advantage that

                                                                        227

     1    was unfortunately missed in this particular case. 

     2    That's not to undervalue the value of these cells, but

     3    it does seem to me that that's what engineering cells

     4    can give you.  It gives you control at each stage of

     5    the process.

     6                DR. FALLAUX:  Can I mention that it's

     7    nowadays rather difficult to take primary human,

     8    especially immuno material.

     9                DR. ONIONS:  It was -- I understand only

    10    too well.  It's not at the end of the day a criticism

    11    of PER C6 success, which I think are excellent.  But

    12    really just that where possible, that that should be

    13    done.

    14                DR. COFFIN:  Okay.  So I think we have a

    15    consensus here that there's lots of useful things

    16    about such cells, but that we really don't' know

    17    whether they enhance any particular safety issue or

    18    not.  I think that's a sort of at least some sort of

    19    closure on that particular point.

    20                The points we were asked to address also

    21    included the use of the defined risk algorithm that

    22    was mentioned at the beginning of the meeting, to

    23    evaluate these kinds of things.  Andy Lewis had that

    24    on his slide, which I have asked him to put back on.

    25                This will also, I mean with this, we will

                                                                        228

     1    sort of segway into the general discussion as well I

     2    think, because these are the issues.

     3                So the question is, can we go through and

     4    do this, and is it possible in this particular case,

     5    just using this as an example of this kind of

     6    procedure, to assess the level of risk posed by these

     7    issues, infectivity, infectious and so on,

     8    quantitatively.  My own feeling right now is that

     9    we're no where near a position to do this, certainly

    10    for DNA issues.  We might be able to put some sort of

    11    numbers on viral issues.  It's a little hard to see

    12    exactly how because there's so many different ones,

    13    which could have a different contribution.  But maybe

    14    we can get some further comments on these sort of

    15    issues from the panel.

    16                DR. ONIONS:  My only comment, and I

    17    understand why a defined risk approach was used, and

    18    it certainly makes you think.  I mean that's one of

    19    its great virtues.  I think one of the real intrinsic

    20    problems, that if you applied, you can give yourself

    21    a false sense of security.  It would strike me that

    22    Phil's story about SV40, if it turns out that is the

    23    origin, SV40 in people, would have given you such a

    24    false sense of security, I think, because you might

    25    have come through that exercise in the 1960s.  I'm not

                                                                        229

     1    sure how you would have predicted that that agent was

     2    there a priori.

     3                So I'm not sure that you can give

     4    guarantees that 1 and 10 to the 6th dose is one-half

     5    X, if you don't know what X you are looking for.

     6                DR. SEDIVY:  Yes.  I mean I broadly agree

     7    with that.  I think it's worth trying to do some sort

     8    of numerical calculations, so long as you don't

     9    believe the numbers that you get out at the end of it.

    10                (Laughter.)

    11                Because I think one thing it will do is it

    12    will tell you where you think you are confident, on

    13    what stage of the process you are actually confident. 

    14    Then you can actually question whether your confidence

    15    is misplaced or not.  But I think if you come out with

    16    a number, I think you are asking for trouble.

    17                DR. LEWIS:  Yes.  I don't think that we

    18    discounted that.  I think in sort of going through

    19    this thing, what we were trying to do was to figure

    20    out exactly where we can be reasonably confident of

    21    what we're doing and where we can not be confident of

    22    what we're doing.  But we always recognize that when

    23    push comes down to shove, the bottom line is that

    24    we'll always -- we can never be sure.

    25                So at some point in time, it requires a

                                                                        230

     1    leap of faith to say this product or this cell line,

     2    or whatever, can in fact be used.  I think what we're

     3    trying to figure out a way to do is to make sure that

     4    when we get to the point or we have to make that leap

     5    of faith, that it's better to find than it would be if

     6    we did nothing at all.

     7                So the attempt here is to develop sort of

     8    a way of thinking about narrowing that margin of

     9    error, or at least to develop a margin of error that

    10    is better than it would be if we're just doing it on

    11    intuition.

    12                DR. ONIONS:  I think that's absolutely

    13    right.  I was taken by Neil Cashman's risk assessment,

    14    quantitative risk assessment today.  I think again,

    15    what it did, although I think he himself didn't

    16    believe certain the numbers at the end of the day, it

    17    makes you think about the process.  I think that's

    18    fine and I think I would agree with that.

    19                DR. HUGHES:  One of the things that's true

    20    about the numbers that I've seen is no one has

    21    attempted to put what I guess I would call a

    22    confidence interval on the numbers.  One of the things

    23    that makes me uncomfortable is that I think in some

    24    cases the uncertainty is as large as the number.  I

    25    would feel a bit more comfortable with a calculation

                                                                        231

     1    with which I'm fundamentally uncomfortable, if I had

     2    a better notion of how uncertain people were about the

     3    assumptions they were making in generating the numbers

     4    in the first place.

     5                DR. ONIONS:  I think that's what we're all

     6    saying, is I think I started it off by criticizing the

     7    whole approach.  I think what Phil said is what I

     8    think.  I think Dr. Lewis said the same thing.  That

     9    is, don't believe the numbers.  All it is is gives you

    10    a manner of approaching what are the issues, really. 

    11    I think that's the way it should be treated.

    12                I agree.  I don't think anyone should

    13    believe the numbers at the end of the day.

    14                DR. HUGHES:  I think it might help if when

    15    someone put down a number, they at least put down a

    16    range of numbers, and that would generate a range of

    17    confidence at the end.

    18                I think what people will see when they do

    19    that, is that the ability to define the confidence

    20    interval is going to expand when you multiply the

    21    numbers together.  I think that act may in a sense

    22    help define how uncertain the number actually is.

    23                DR. COFFIN:  I think from a regulatory

    24    standpoint, what often happens is that the far end of

    25    the confidence interval on the worst possible side is

                                                                        232

     1    taken, and then that's propagated through.  You never

     2    see the other side.

     3                DR. ONIONS:  I don't want to just go into

     4    an academic discussion about risk assessment because

     5    I'm not really interested in it, in that formal sense,

     6    but there are two other approaches that are used.  The

     7    engineering industry uses a form of analysis that

     8    doesn't do risk assessment like that.  It actually

     9    looks for holes.  It looks for what could go wrong. 

    10    In a sense, that's really probably what we ought to be

    11    doing.  Then there are four mechanisms of that kind of

    12    analysis.

    13                The other form of analysis is the one that

    14    has become fashionable in the U.K., which is this

    15    concept of the precautionary principle, which

    16    ultimately, it seems to me, means you don't ever do

    17    anything because you never know what might happen,

    18    which seems to me completely dumb.

    19                DR. COFFIN:  So we're voting against the

    20    precautionary principle.  You can't be sure of

    21    anything, but you can be sure of that.

    22                Are there any other points anybody would

    23    like to make about this?  One could say that this is

    24    a useful way to organize your thoughts on this

    25    subject, but shouldn't be taken as giving you either

                                                                        233

     1    additional grounds or comfort or discouragement,

     2    unless you actually had a situation where you had

     3    measurable quantities.

     4                Are there any other issues or questions

     5    regarding the designer cell substrate issue that

     6    anybody wishes to raise?

     7                DR. SHEETS:  Hi.  Becky Sheets, FDA.  I am

     8    going to ask the whole panel what I tried to ask Dr.

     9    Hughes earlier.  That is, the kinds of questions that

    10    sponsors ask us.  One question I would ask is we've

    11    heard a lot of people in this meeting say that the

    12    oncogenic DNA issue has been put to bed.  Has it?

    13                DR. COFFIN:  That segways us into the

    14    next, into the general discussion, which is fine.

    15                Before we go into that, can we see if

    16    there are any other specific issues regarding cell

    17    substrates?

    18                DR. SHEETS:  Any questions about the

    19    quantitative?

    20                DR. COFFIN:  If we do that, we can turn

    21    off that slide, put up my next one, and then you can

    22    ask the question.

    23                DR. SHEETS:  There's one question.

    24                AUDIENCE MEMBER:  Jerry Sato from Merck. 

    25    I understand the reluctance to put a number on

                                                                        234

     1    something when you have such degree of lack of

     2    confidence and the assumptions that are going into it. 

     3    But I do think that getting an order of magnitude of

     4    where we are is actually helpful in our thinking about

     5    what we feel comfortable going forward with or not.

     6                When you have a lack of confidence in each

     7    of those areas, you also have to ask the question,

     8    what are the chances that all of your assumptions are

     9    wrong?  In other words, are two of them wrong, three

    10    of them wrong, five of them wrong, seven of them

    11    wrong?  Because you have to put a degree of that's not

    12    likely to happen.  So if you multiply the lower end of

    13    the confidence interval for all those things, then you

    14    will never do anything.  But that's not the way it

    15    works in reality.

    16                So I think it would be useful for somebody

    17    from the engineering community, where they design

    18    bridges that aren't supposed to fall down and other

    19    things, to try and put a bit little more

    20    sophistication into this analysis, because in the end,

    21    somebody is going to ask our community, which is the

    22    regulatory community, the academic community, and the

    23    industrial community, for the number or at least what

    24    they thought the number was when they went forward

    25    with their act of faith.  Because there is a certain

                                                                        235

     1    amount of common sense that goes into it, which is the

     2    basis of the act of faith.  Then there is whatever

     3    kind of quantitation we can put into it.  It's the

     4    combination of those two things that I think we are

     5    going to have to reassure the general public about.

     6                DR. HUGHES:  I would recommend to you the

     7    book Strategy and Conscience by Anatol Rappaport,

     8    which attempts to deal with the issues having to do

     9    with what was called strategic thinking, in which you

    10    calculate, for example, the probability of some

    11    unlikely event, such as thermonuclear war.  Mr.

    12    Rappaport does a very good job of making clear why

    13    doing the calculations when you don't have the proper

    14    data is in fact a very risky and misleading

    15    proposition.

    16                AUDIENCE MEMBER:  I guess it might be

    17    worth pointing out that there are, however, some cases

    18    where you clearly do have the proper data.  Right? 

    19    You know the sensitivity of a specific assay for a

    20    specific adventitious agent, if in fact you choose to

    21    figure out what it is.

    22                So you can actually answer, based on those

    23    kinds of questions, and based on that kind of data,

    24    the specific question of how sure are you that

    25    something isn't there.  If a better assay comes along,

                                                                        236

     1    and it's still negative, then you can say by how much

     2    more certain you are.

     3                So just because you can not come up with

     4    good estimates for some of the numbers, seems to me it

     5    would be crazy to throw the baby out with the bath

     6    water and claim then that you shouldn't attempt to

     7    come up with good numbers for those things that you

     8    can.

     9                DR. RABINOVICH:  Gina Rabinovich, NIH.  A

    10    non-regulator asking a question from experience

    11    learned this summer, in which we have been dealing

    12    with using a quantitative number, i.e. the numbers

    13    that the Federal agencies and the global agencies has

    14    set for acceptable limits of mercury, i.e. methyl

    15    mercury, and then trying to attempt to understand what

    16    those uncertainty factors mean for thimerosal and

    17    vaccine exposure im.

    18                The concern I have, and I think it has

    19    been heard, is that these numbers take on a life of

    20    their own.  They become the standard against which

    21    things are measured.  So that that kind of concern

    22    needs to be entered into, attempting to use the data

    23    when those data do exist, but understanding the limits

    24    to it.

    25                DR. COFFIN:  That's inherent in

                                                                        237

     1    regulation, that things become standard.

     2                DR. LEWIS:  Lewis, FDA.  To follow up a

     3    little bit on what Phil was saying.  I think one of

     4    the areas that we could approach with some confidence

     5    is the ones who saw Keith Peden's data last night

     6    using the tac man assay to detect JC, BK, and SV50 in

     7    human tissues.

     8                Now if someone comes in with substrates

     9    they derive from a neuroblastoma, which we learned at

    10    the DNA tumor virus meeting a year or so ago, is it's

    11    usually contaminated with BK virus.  We wanted to be

    12    sure that there was no BK virus in that substrate. 

    13    Then we could apply this assay with a fairly

    14    sufficient level of sophistication and say with some

    15    certainty, based on the volumes and things that were

    16    tested, the level at which that particular genome or

    17    that particular virus is absent.  So I think that we

    18    sort of view that as a possible starting point for a

    19    quantitative approach.

    20                Now obviously you can't do that unless you

    21    know exactly the probes and things that you are

    22    working with, and you define the limits of their

    23    ability to detect things.  But I think this is one of

    24    the sort of examples that was going through our mind

    25    when we were thinking about how to do this.

                                                                        238

     1                So you start at the place where you might

     2    be able to generate some relevant data that's

     3    meaningful.  The other stuff will fall into place as

     4    we get better.

     5                DR. ONIONS:  I mean I think that's exactly

     6    right.  I think more and more that we move to assays,

     7    that we get good quantification on, we can validate

     8    them, and it's the sensitivity and limits of

     9    detection.  I think all of that is absolutely

    10    essential.  I mean I absolutely 100 percent concur

    11    with that.  I think it does add confidence to those

    12    specific questions.

    13                I think when you are asking specific

    14    questions, then I 100 percent agree.  I think my

    15    concern is perhaps that where you try and make

    16    assumptions, for instance, of residual DNA.  I mean I

    17    was the one who said I thought it should be put to

    18    bed.  That is because all the evidence that I had

    19    heard didn't convince me that there was a risk.

    20                On the other hand, I actually believe that

    21    no one has done the right experiment to actually

    22    convince me of that, in a formal scientific sense.  So

    23    we're then dealing with the area of conjecture.  That

    24    conjecture is based on non-quantitative data.

    25                DR. COFFIN:  We're leading into you,

                                                                        239

     1    Becky.

     2                DR. SHEETS:  I'm patient.

     3                DR. COOK:  I'm sorry.  Jim Cook.  As I was

     4    sitting there thinking about how you would describe

     5    these issues to a patient or to a group who is asking

     6    you about the wisdom of using a vaccine, it seems like

     7    in addition to trying to generate some logic about

     8    calculations of numbers and risks, that every

     9    opportunity that you have, it would be worthwhile

    10    going back to history and saying well, we have done

    11    virtually something like this along the way ever since

    12    vaccines have been developed, and the experience with

    13    this approach has been the following.

    14                So maybe there could be some real numbers

    15    in a historical sense, used to color or give some more

    16    real meaning to these, what are otherwise theoretical

    17    things, to help communicate this to the public, as

    18    well as to provide some, a little bit more logic than

    19    just phenomenology to the calculations that are being

    20    made now.  So if history is used to color the

    21    estimations, that might be of some use.

    22                DR. COFFIN:  How comforting is it to tell

    23    --

    24                DR. COOK:  Say it again?

    25                DR. COFFIN:  How comforting is it to tell

                                                                        240

     1    patients that there are three or four cases of

     2    paralytic polio?

     3                DR. COOK:  Well, I think you have to be

     4    honest with them.  You say look, you are one of 250

     5    million people.  The odds for your child is the

     6    following, and I think it's a very good idea to use

     7    this vaccine.

     8                It is those kinds of conversations that

     9    eventually lead adolescents into getting hepatitis B

    10    vaccine.  We're doing a miserable job of this, by the

    11    way.  I think as a culture, you know, if we bat 50

    12    percent, we're lucky.

    13                DR. COFFIN:  That's clear in light of

    14    things in the movies, because lately we're doing a

    15    terrible job where these people show up with these

    16    anecdotal cases of somebody's child gets vaccinated,

    17    and then two months later is diagnosed with autism. 

    18    It's automatically due to the vaccine.

    19                DR. ONIONS:  But I think history can also

    20    be a dangerous thing.  I mean the British government

    21    has been criticized, partly justifiably, but I think

    22    partly unfairly for the problems of the way BSE

    23    problem goes up.

    24                But the issues concerning public health

    25    were based on people were asked what is the risk, what

                                                                        241

     1    is the risk of the human population of the BSE

     2    outbreak, when we had a few hundred cases of BSE in

     3    cattle.  Well, there were only a few hundred cases. 

     4    The general assumption was, and it was a widely shared

     5    general assumption by those who were informing the

     6    area, the people who worked on scrapie and so on,

     7    well, scrapie has no evidence whatsoever of scrapie

     8    transmission to man.  We have been eating scrapie-

     9    infected sheep for generations and it doesn't seem to

    10    have been transmitted to people.  There is no evidence

    11    of that whatsoever.

    12                The probable likelihood that BSE is of

    13    scrapie origin that's gone through the rendering

    14    process, because we have never seen a spontaneous

    15    spongiform encephalopathy in cattle, so that's

    16    probable origin ipso facto, you know, there's no

    17    problem.

    18                It wasn't quite as glib as that because

    19    actually within a year, all the controls on human food

    20    were in place and so on and so forth.  They were badly

    21    conducted, but they were theoretically in place.

    22                So I think history can also be dangerous. 

    23    I'm not sure that we can always learn the right

    24    lesson.

    25                AUDIENCE MEMBER:  I get your point, but I

                                                                        242

     1    think there is a difference between an ongoing

     2    epidemic that's yet resolved, and the experience of X

     3    number of hundreds of millions of doses of polio

     4    vaccine.

     5                So what I am saying is, if you are going

     6    to make a calculation about the likelihood of one in

     7    a million or one in 10 or 100 million happening with

     8    a vero cell or with a primary cell, you can say well,

     9    we've got experience making polio vaccine in vero

    10    cells, and there have been the following hundreds of

    11    million doses given, and the likelihood is that when

    12    the incidence of the real disease gets so low that you

    13    are finally going to see some background.  That's the

    14    real --

    15                DR. ONIONS:  I'm sorry.  You

    16    misunderstand.  What you are saying, I absolutely

    17    agree.

    18                DR. COFFIN:  Do you want to continue this

    19    discussion or do you want to break into --

    20                AUDIENCE MEMBER:  Very similar aspect,

    21    although a little bit less scientific.  As I perceive

    22    the discussion, of course there's this highly

    23    sophisticated, highly conscientious scientific

    24    community, and there's the general public on the other

    25    side.

                                                                        243

     1                The general public, to my perception,

     2    consists of at least two sub-groups.  One group that

     3    is sort of generally benevolent and would believe

     4    scientists.  But there is a very strong group that is

     5    not believing scientists.  They are sort of using that

     6    as a political tool to attract attention.  In our

     7    country, we have had this experience with the Green

     8    Party, that has become very influential in the

     9    European Union and maybe in other countries as well.

    10                Now one thing I also, since we're ending

    11    and coming to the end of this meeting, would like to

    12    raise, isn't it the responsibility of scientists also

    13    to do something to better educate the public?  I know

    14    this is a utopian goal, but at least if we could

    15    increase enough of people in the general public who

    16    are educated or better educated in science and biology

    17    and biomedic issues, we would at least have a

    18    political community that might support scientific

    19    issues more valuable than we have had it so far.

    20                You know, in our country at least, every

    21    time when the Greens demanded to stop all biomedical,

    22    all gene technology research, and it course never

    23    happened.  After they come to power, maybe they have

    24    different outlooks on life.  But I think as a

    25    scientific community, unless we do something at least

                                                                        244

     1    for the future, we might be in a very difficult

     2    situation to defend certain issues.

     3                If I confronted some of the violent

     4    ideologically pure Greens in our country, because the

     5    trick is, we have been discussing here, I'm sure they

     6    would say "Shut it down because this is unsafe,

     7    totally unsafe."

     8                So what I am trying to recommend is we

     9    have to do something to have more people in the

    10    general public who can appraise and can assess the

    11    difficulty and the uncertainty in any biological

    12    research.  We can never get down this figure to 10 to

    13    the minus 80.  So we have to raise understanding on

    14    the other side.

    15                DR. SHEETS:  So have we put oncogenic DNA

    16    to bed?

    17                DR. COFFIN:  No.  I would like to use the

    18    sort of summary -- I think that's in a sense, an

    19    overhanging issue.  We have talked about infectious

    20    risks and measurements and so on considerably today

    21    and in the past few days.  I think an overhanging

    22    issue is this oncogenic DNA issue regarding the

    23    specific charge of the meeting, which is the use of

    24    tumor versus other kinds of cells, tumor and

    25    neoplastic whatever, transformed cells versus other

                                                                        245

     1    kinds of cells as substrates for vaccine production.

     2                Although I think many of us here, perhaps

     3    all that's here, feel this is not a risk to be really

     4    concerned about in a scientific sense, I think many of

     5    us here might agree that the issue is not completely

     6    put to bed in the sense that we can't put any real

     7    good numbers on it.

     8                So now if you ask your question.

     9                DR. SHEETS:  Okay.  Has oncogenic DNA been

    10    put to bed?

    11                DR. HUGHES:  I will answer in two

    12    different ways.  I will give you my opinion, personal

    13    opinion, and then I will tell you what I think should

    14    be done, which is slightly different.

    15                Personally, and this would apply if you

    16    approached me to do something to myself, I am not

    17    concerned.  However, it is my view that the data that

    18    we have, particularly for the consequences of putting

    19    DNA into animals, is not sufficient to satisfy me as

    20    a scientist.  I am going to try to help my colleague,

    21    Dr. Coffin, and some of my colleagues at the FDA to

    22    try and organize a simple study that would be more

    23    satisfying to me.

    24                I think I would feel more comfortable if

    25    we had more data that was of the experiments done on

                                                                        246

     1    a larger scale under more controlled conditions.  I

     2    think that would give me a greater degree of comfort.

     3                DR. ONIONS:  I think I almost entirely

     4    concur with that comment actually.  I did say a

     5    comment, and said it partly to be provocative, but on

     6    the other hand, I think I share that opinion.

     7                I have seen nothing that would convince me

     8    at the moment on the data, which there's a singular

     9    lack of, or just from I suppose theoretical reasoning,

    10    to suggest that this would really be a significant

    11    danger.  But on the other hand, there are the tools

    12    now, and I say yet again, but I think some of the

    13    transgenic models offer that possibility for testing,

    14    whether or not DNA is a risk.  It can be done in a

    15    series of graded experiments from taking the worst

    16    case examples of actually simply just repeating -- of

    17    injecting oncogenes at various titrations into animals

    18    that are already primed with oncogenes as a

    19    transgenic, down to taking tumor DNA down to taking

    20    normal DNA.

    21                I mean those experiments, they are pretty

    22    straight forward to do.  I mean the interpretation

    23    might be a bit more complex, but they can be done.  I

    24    think they are worth doing.  It might help you to put

    25    some limits, broad limits on the thing we're all just

                                                                        247

     1    making conjectures about.

     2                DR. MINOR:  I don't think it's been put to

     3    bed either.

     4                DR. COFFIN:  For the same general reason?

     5                DR. MINOR:  Broadly speaking.  I mean I

     6    think it's clearly a very, very complex issue about

     7    how you actually induce a tumor.  So you do your 3T3

     8    assays and you pick up H ras.  Okay?  I mean it's an

     9    artifact of 3T3s or was that just a question of how

    10    common H ras is.

    11                If you go and put your DNA in

    12    intravenously, is that the same as putting it in

    13    subcutaneously, for example?  I mean if you put it in

    14    because it's been picked up by an envelope virus, is

    15    that going to make any -- will it be picked up by an

    16    envelope virus?  Will that make any difference?

    17                I mean it seems to me that there are so

    18    many sort of loose ends to it that I don't think while

    19    there is no evidence that DNA is tumorigenic, and I

    20    buy that 100 percent, it doesn't seem to me that it's

    21    necessarily been dealt with properly.  That is why I

    22    guess I am agreeing with what the previous two

    23    speakers said.

    24                DR. LOEWER:  So as I already have said, I

    25    personally believe that there's not a real big risk

                                                                        248

     1    with purely oncogenated DNA.  Purely oncogenated means

     2    three or five or six.  But I realized that there is

     3    still, I believe, lack of experimental data.  This was

     4    already mentioned by John Coffin, in saying that since

     5    18 years, this question is on the table.  Since 18

     6    years, no additional experiments have been performed.

     7                I would like to propose that regulatory

     8    authorities, which are involved in regulation of these

     9    biologicals, the major ones, that we should sit

    10    together and to join the efforts and maybe decide on

    11    experiments which can be done in the foreseeable

    12    timeframe.

    13                But I look forward to see what types of

    14    experiments John may recommend.

    15                DR. FRIED:  I think most of the evidence

    16    we have so far, which is limited, says at least

    17    putting DNA into animals, we haven't seen anything

    18    happen.

    19                We have only seen in NIH3T3 cells, and we

    20    now know that there is a defense mechanism in the cell

    21    when it sees an oncogene.  That is this P, this arf,

    22    which is the alternative reading frame of P-16.  So

    23    it's like an immune system of the cell, specifically

    24    for oncogenes.  The radiation activation of P-53 is a

    25    different pathway.  This turns on, and arf activates

                                                                        249

     1    P-53, and P-53 then closes itself to go through

     2    apoptosis.

     3                The only positive things are in NIH3T3

     4    cells.  They are the classic cell where the arf gene

     5    is inactivated.  Probably that's why people have been

     6    using that for years.  They are very easy to transfect

     7    because maybe even transfection kills the cells in

     8    terms of P-53.

     9                But that said, I would like to see a lot

    10    more injection of DNA from different tumor lines into

    11    animals, and to really put it to bed.

    12                DR. COFFIN:  It's striking to me that one

    13    of the very few, if only successful experiments with

    14    injected activated oncogene DNA in chickens is the one

    15    that Hsing-Jien Kung did that was off-site, or in any

    16    animal, that was Hsing-Jien Kung.  It was actually

    17    fairly efficient sort of transient transformation of

    18    cells, but transformation of cells is always

    19    transient.  It's virtually impossible to immortalize

    20    them.  They are much harder then human cells to

    21    immortalize.  It's been done once, to my knowledge.

    22                So there may be something very

    23    fundamentally different going on in that model,

    24    because there may be some fundamental difference in

    25    chickens as compared to mice, as compared to humans,

                                                                        250

     1    which already have important --

     2                DR. EGAN:  Bill Egan from FDA.  I would

     3    certainly like to work with you and other regulatory

     4    authorities to try and design and our colleagues in

     5    PHS, to try and design these experiments and do these

     6    experiments, and get the data, and get away from the

     7    remark I quoted from Maurice Hilleman from 30 some-odd

     8    years ago about this debate being a philosophical or

     9    ecclesiastic debate because we simply don't have the

    10    data.  Here we are 30 some-odd years later, you know,

    11    with the same question, with the same debate.  It's

    12    still opinion.

    13                I must say I also feel that personally,

    14    myself, I don't think there is a large risk from the

    15    DNA.  But then again, the kinds of risks that we're

    16    talking about are very small risks, very, very rare

    17    events.  Things like one in a million are not

    18    acceptable or in many cases are not acceptable.  Those

    19    are hard data to get.

    20                While I may not feel there is a risk to

    21    me, the bottom line basically for the approval of

    22    almost any of these vaccines, is would I put this into

    23    my children.  There it becomes a much more

    24    conservative process.  If putting it into my children

    25    is putting it into other people's children, it's the

                                                                        251

     1    same thing.

     2                DR. HUGHES:  If you want one more piece of

     3    data that should give you some comfort, it is the

     4    experience attempting to make antibody to the oncogene

     5    sarc, which involved putting an avian virus into a

     6    number of mammalian species under circumstances in

     7    which the virus absolutely does not replicate.

     8                In adult animals, to my knowledge, no cell

     9    growth was ever seen.  The only experiments that

    10    succeeded, to my knowledge, were those initiated by

    11    John Burge, in which he put enormous amounts of ras

    12    sarcoma virus of a subgroup that would infect

    13    mammalian cells.  We are talking sort of 10 to the

    14    10th infectious units.  Into immunologically naive

    15    baby bunnies.  In those animals very transiently,

    16    there were small nodules which regressed.

    17                In those animals, you did see antibody to

    18    the oncogene sarc, implying that there was transient

    19    uptake of the DNA, at least probably permanent uptake

    20    of the DNA.  But even under those circumstances in

    21    which the delivery of the DNA is extremely efficient

    22    and every copy of the DNA carries a known potent viral

    23    oncogene under circumstances where the DNA will not

    24    replicate but will insert, do you see any permanent

    25    transformation?  Again, I'm not suggesting that this

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     1    is sufficient.  I am one of the advocates of more

     2    experimentation.  But the data that we have suggests

     3    that this is not a simple process.

     4                DR. EGAN:  No, but I mean these are the

     5    kind of data that start to put brackets around the

     6    numbers for the levels of risk.

     7                DR. COFFIN:  Of course you have enormous

     8    problems, including the fact that sarc is never seen. 

     9    It's a human oncogene.  For many years, the most

    10    popular viral model.

    11                AUDIENCE MEMBER:  I had a question.  If

    12    given the unknowns, and given the data that was

    13    presented about hit and run DNA modification

    14    potentials, would the panel in the context of this

    15    type of vaccine development, and given the unknowns,

    16    give the vaccine to someone with a strong family

    17    history of malignancy or who was a cancer survivor who

    18    we know is at increased risk for a second cancer, if

    19    that was you or your family member?

    20                DR. COFFIN:  The question, to sort of

    21    focus that a little bit, the question is whether we

    22    would consider there to be a greater risk in certain

    23    sub-populations who might have sort of pre-activated

    24    oncogenes or some other fact of predisposing.

    25                DR. ONIONS:  I understand the question,

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     1    but it sort of arose --  I will go backwards, because

     2    one of the things that used to concern me was when

     3    people were doing clinical trials with rusvel vectors,

     4    and they were using marker studies.  That sort of

     5    study did worry me, because actually what you were

     6    there doing was putting something, inserting something

     7    into somebody who probably already had a preexisting

     8    oncogenic hit.  That struck me as being dubious,

     9    mildly.

    10                In this situation, I would have thought

    11    that unless you have got somebody with a Li-Fraumeni

    12    syndrome or something, that you are dealing with

    13    changes that are somatic changes in a few cells, even

    14    if they are going to risk of a second cancer.

    15                So the likelihood that you hit the right

    16    cell is pretty low.  So I wouldn't have thought it up

    17    the risk -- I'm sure the risk has increased, but I

    18    wouldn't have thought that risk has increased

    19    significantly, unless of that sort where you've got

    20    mutation.

    21                DR. MINOR:  It would also depend on what

    22    you are trying to protect them against too, wouldn't

    23    it?

    24                DR. SHEETS:  Before we lose our entire

    25    audience, I wanted to -- I think we have gotten a

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     1    pretty clear answer on the oncogenic DNA issue.  I

     2    wanted to ask another kind of question that FDA has

     3    asked, before we lose our entire panel.  That is, are

     4    there risks, additional risks that one perceives in

     5    using a continuous cell line such as vero cells,

     6    particularly vero cells, which is immortal but not

     7    tumorigenic at the levels that vaccines are made, past

     8    the level that vaccines are made.  Is that worse in

     9    any way than diploid cells, for a live viral vaccine?

    10                DR. MINOR:  This is apart from the DNA

    11    issue?

    12                DR. SHEETS:  Well, in a continuous cell

    13    line, certainly there are -- it may be aneuploid, but

    14    it's not tumorigenic in animals.  So you can comment

    15    if you'd like about whether you think the DNA is

    16    oncogenic.

    17                DR. MINOR:  I would say that the DNA from

    18    vero is as questionable as the DNA from anything else. 

    19    I mean John, with whom we discussed these matters,

    20    more or less said the same thing.  I think that it

    21    doesn't matter how malignant it is.  Maybe it depends

    22    on how many oncogenes you put in there.  So I would

    23    have thought that a vero is as questionable as

    24    anything else, or is not as questionable as anything

    25    else.

                                                                        255

     1                DR. SHEETS:  So you wouldn't suggest to

     2    make a live viral vaccine in vero cells?

     3                DR. MINOR:  I think it would depend on the

     4    live viral vaccine.  I mean I think OPV clearly has

     5    been made in vero cells.  You can scrub it clean.  I

     6    think you can more or less destroy anything that's

     7    actually hanging on the end.

     8                I have more serious thoughts perhaps about

     9    things like a paramixo virus vaccine, because you

    10    couldn't clean it up so much perhaps.

    11                DR. SHEETS:  What about the sort of crude,

    12    less purified live viral vaccines, not the purified

    13    vaccines like we heard about last night with OPV, but

    14    the things that are just filtered cell culture

    15    supernatant?

    16                DR. MINOR:  Right.  They might be figit. 

    17    I'll tell you that.  Which is not to say there's any

    18    good reason for me to be uncomfortable with them. 

    19    It's just that they make me feel uncomfortable.

    20                DR. COFFIN:  We are very fast losing our

    21    audience, so I think --

    22                DR. HUGHES:  Isn't it partly the question

    23    do you know the life history of your cells as opposed

    24    to the state of the cells at the end?

    25                DR. SHEETS:  Vero cells are a bank that is

                                                                        256

     1    well characterized.  The reason for the question is

     2    that we have numerous live viral vaccines of the sort

     3    I described that are being proposed to be made in vero

     4    cells.  Manufacturers prefer vero cells because one,

     5    they can be characterized.  Two, you get a high yield. 

     6    Three, they can be grown in the sorts of fermenter

     7    culture that you heard about.

     8                DR. HUGHES:  I'm not particularly bothered

     9    as long as I know that the sort of life history of the

    10    cell.  But I think the question is, if you have a cell

    11    that's been in culture for a long time and has had a

    12    complicated culture history, do you know that history?

    13                AUDIENCE MEMBER:  I asked the last panel

    14    the same question.  It comes down to the question

    15    really is the adventitious agent issue put to bed as

    16    well.  Do we now have the assays in place that can

    17    easily be applied to validate the freedom from

    18    adventitious agents of these kinds of new cell lines?

    19                The answer that Dr. Broker gave in the

    20    last panel suggested that one could attempt to use DNA

    21    chips and things like that, which to my knowledge

    22    aren't assays that at least tomorrow I could go out

    23    and do on a cell line and give me some confidence.

    24                So my question to you is, sort of using

    25    the standard assays that you are all aware of, without

                                                                        257

     1    developing further assays for this specific purpose,

     2    do we have enough information to be sure that these

     3    kinds of new cell lines are safe from the adventitious

     4    agent perspective?

     5                DR. ONIONS:  That's another unanswerable

     6    question, isn't it.  I would just make the point that

     7    I think you have to adopt somewhere between good

     8    science and pragmatism.  I mean you could

     9    theoretically go and do representation difference

    10    analysis on all these cell lines.  Actually, I don't

    11    think it's possible because you don't usually have the

    12    partner.  But theoretically you could do that.  That's

    13    not really a practical solution.

    14                It does seem to me that we do know virus

    15    types that tend to be latent in cells, and that it's

    16    sensible to perhaps think of strategies of widening

    17    the brief of detecting those agents, because I'm not

    18    convinced that the kind of routine types of infecting

    19    -- infectability assays when they work are as

    20    sensitive as PCR, as just Phil pointed out.  But I am

    21    not convinced that always the right infectability

    22    assay is present to actually detect certain agents. 

    23    So that you are probably relying on a combination of

    24    things.  Perhaps we do need to look at redundant PCR

    25    for certain agents.

                                                                        258

     1                DR. COFFIN:  I would think the producers

     2    would have a big attraction, is set up the same assay

     3    and use it for everything.

     4                DR. MINOR:  I mean I think you could also

     5    argue that you have used these assays for looking at

     6    human diploid cells and primary cultures, and all that

     7    sort of stuff.  Right?  What's the difference in

     8    principle in terms of adventitious agent contamination

     9    between those and the cells you are looking at here? 

    10    I am not sure there's much difference.  But are the

    11    concerns as big or as little.

    12                AUDIENCE MEMBER:  For one, I would like to

    13    thank Dr. Andy Lewis for bringing us together.  This

    14    has been a very stimulating week.  Last night Dr. Vyas

    15    showed us a picture of the thinker.  But that actually

    16    prompted me to remember that Rodin placed that

    17    gentleman directly above the gates of hell.

    18                (Laughter.)

    19                The question I would like to pose right

    20    now is whether we're walking in through the gates or

    21    out.

    22                DR. COFFIN:  I would like to also second

    23    the thanks to the organizers for setting this up and

    24    bringing us here.

    25                DR. LEWIS:  Yes.  On behalf of the

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     1    sponsors and those of us at CBER who worked on this,

     2    we really appreciate the effort that the session

     3    chairs, the panel chairs, and the speakers have put

     4    into this meeting.  When you attempt to put something

     5    like this together, there's always a question of how

     6    it's going to turn out.  I think the success that we

     7    have enjoyed here the past three days is a tribute to

     8    the work, an incredible amount of work, that has gone

     9    on on a very short period of time.

    10                I think that I was very concerned when we

    11    were trying to contact folks in May to do this by

    12    September.  For those of you who rose to the

    13    challenge, I can't thank you enough on behalf of the

    14    sponsors.

    15                With that in mind, I hope everybody has a

    16    great trip home.  Get your papers in whenever you can. 

    17    Thank you.

    18                (Whereupon, at 2:57 p.m., the proceedings

    19    were concluded.)