FOOD AND DRUG ADMINISTRATION

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CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

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VACCINES AND RELATED BIOLOGICAL PRODUCTS

ADVISORY COMMITTEE

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OPEN SESSION

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TELECONFERENCE MEETING

ON CBER LABORATORY SITE VISIT

LABORATORY OF ENTERIC AND SEXUALLY

TRANSMITTED DISEASES

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This transcript has not been edited or corrected, but

appears as received from the commercial transcribing service.

Accordingly the Food and Drug Administration makes no

representation as to its accuracy.

CONFERENCE ROOM C

BUILDING 29B

NATIONAL INSTITUTES OF HEALTH

BETHESDA, MARYLAND

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MONDAY, NOVEMBER 18, 2002

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**ADVISORY COMMITTEE**: (Telephonically)

ROBERT S. DAUM, M.D.

Professor of Pediatrics

University of Chicago Children\'s

Hospital M/C 6054 Chairman

WALTER L. FAGGETT, M.D.

Medical Director

Medical Assistance Administration

D.C. Department of Health

BARBARA LOE FISHER

C-Founder and President

National Vaccine Information Center

JUDITH D. GOLDBERG, Sc.D.

Director

New York University School of Medicine

Division of Biostatistics

DIANE E. GRIFFIN, M.D., Ph.D.

Professor and Chair

Molecular Microbiology & Immunology

Johns Hopkins University School of Medicine

SAMUEL L. KATZ, M.D.

Wilburt C. Davidson Professor and Chairman Emeritus

Department of Pediatrics

Duke University Medical Center

DAVID M. MARKOVITZ, M.D.

Professor

Division of Infectious Diseases

Department of Internal Medicine

University of Michigan Medical Center

AUDREY F. MANLEY, M.D., M.P.H.

President Emeritus

Spelman College

Rear Admiral (Ret)

U.S. Public Health Service

GARY D. OVERTURF, M.D.

Professor of Pediatrics

Department of Pediatrics

University of New Mexico School of Medicine

Ambulatory Care Center

**ADVISORY COMMITTEE**: (Continued)

PETER PALESE, Ph.D.

Chairman and Professor

Department of Microbiology

Mt. Sinai School of Medicine of New York City

JULIE PARSONNET, M.D.

Associate Professor of Medicine and Health Research and Policy

Stanford University

DAVID S. STEPHENS, M.D.

Professor of Medicine

Division of Infectious Diseases Division

Department of Medicine

Emory University School of Medicine

RICHARD J. WHITLEY, M.D.

Professor

Pediatrics, Microbiology and Medicine

Department of Pediatrics and Microbiology

University of Alabama at Birmingham

**CONSULTANT**:

ESTUARDO AGUILAR-CORDOVA, Ph.D., MA.D.

Director

Advantagene,Inc.

**EXECUTIVE SECRETARY**:

JODY SACHS, D.P.M.

Scientific Advisors & Consultants Staff

Center for Biologics Evaluation and Research, FDA

**COMMITTEE MANAGEMENT SPECIALIST**:

DENISE ROYSTER

Scientific Advisors & Consultants Staff

Center for Biologics Evaluation and Research, FDA

DR. BILL FREAS

Director

Scientific Advisors & Consultants Staff

Center for Biologics Evaluation and Research

**FDA PARTICIPANTS**:

NEIL GOLDMAN, Ph.D.

Associate Director for Research

Office of the Director, CBER

KAREN MIDTHUN, M.D.

Director

Office of Vaccines Research and Review, CBER

RICHARD WALKER, Ph.D.

Division Director

Division of Bacterial, Parasitic & Allergenic Products

OVRR/CBER

DENNIS KOPECKO, Ph.D.

Laboratory Chief

Laboratory of Enteric and Sexually

Transmitted Diseases

DBPAP/OVRR/CBER

SCOTT STIBITZ, Ph.D.

Principal Investigator

Laboratory of Enteric and Sexually

Transmitted Diseases

DBPAP/OVRR/CBER (Telephonically)

PHILIP BOUCHER, Ph.D.

Staff Fellow

Laboratory of Enteric and Sexually

Transmitted Diseases

DBPAP/OVRR/CBER

**[I N D E X]{.underline}**

**[PAGE]{.underline}**

Call to order

\- Robert Daum, M.D.

VRBPAC Chair 6

Announcements

\- JODY SACHS, D.P.M.

VRBPAC Executive Secretary, FDA 7

Open Session:

Overview of Division of Bacterial,

Parasitic & Allergenic Products

\- RICHARD WALKER, Ph.D.

Division Director 9

Overview of Laboratory of Enteric and

Sexually Transmitted Diseases (LESTD)

\- DENNIS KOPECKO, Ph.D.

Laboratory Chief, FDA 15

Overview of Research Activities

\- Scott Stibitz, Ph.D.

LESTD, FDA (Telephonically) 24

Overview of Research Activities

\- Philip Boucher, Ph.D.

LESTD, FDA 37

Open Public Hearing 44

Adjournment 34

**[P R O C E E D I N G S]{.underline}**

(1:05 p.m.)

DR. DAUM: I\'d like to begin by calling the meeting to order. Jody,
would you like to go through another roll call first, or are you
satisfied with that?

DR. SACHS: No, I\'m happy. There\'s maybe two other people \-- three
other people \-- we\'ll just notify everybody.

DR. DAUM: Everyone is on except Drs. Griffin, Palese \--

DR. PALESE: I\'m here.

DR. DAUM: Hello, Dr. Palese. Everyone is on except Drs. Griffin and
Whitley at this point. Dr. Diaz we know wasn\'t coming, and Dr. Gellin
is still a question mark.

ARBITRATOR HOCKENBERRY: Estuardo Aguilar-Cordova is about to join us on
the call.

DR. AGUILAR-CORDOVA: I\'m here.

DR. SACHS: Great.

DR. DAUM: Okay. Thank you. You\'re not on my voting list, D.
Aguilar-Cordova, so I made a mistake. I\'m sorry.

So, with that attendance information, Jody, why don\'t we turn the floor
over to you for announcements.

DR. SACHS: Okay. I just have a little bit of a meeting statement to
read. Good afternoon. I\'m Jody Sachs, the Executive Secretary for
today\'s meeting of the Vaccines and Related Biological Products
Advisory Committee. I\'d like to welcome all of you to the 92nd meeting
of the Advisory Committee. There is a speaker phone for public
participation located here in Conference Room C of Building 29B, on the
NIH Campus.

This afternoon\'s session will consist of a presentation and committee
discussion that are open to the public. We will then go into a closed
session until the meeting is adjourned, as described in the Federal
Register Notice of October 30, 2002.

Should a committee member get dropped from the teleconference, simply
call back at the 888-324-0789 number and give the pass code as VRBPAC.
The operator is under instructions only to connect the committee members
to this line. Again, you have the number so, if you get disconnected,
please call back. If you have any problems while we\'re on the call, you
can press \"\*0\" and the operator will help you.

We ask that you do not place us on hold because many clinical centers
have background music, and it can be very distracting to those remaining
on the teleconference line. However, I strongly urge everyone to use
\"\*6\" or a mute button \-- it is the same as a mute button \-- since
there are many lines connected, the background sound will be decreased
and the quality of sound for everybody would improve if you just use
mute.

I just want to let everybody know who is in the room wiht me, in front
of me. Dr. Karen Midthun is here. She\'s the Director of the Office of
Vaccines Research and Review; Dr. Neil Goldman, Associate Director for
Research, Office of the Center Director in CBER is here. Dr. Richard
Walker, Director of Division of Bacterial, Parasitic & Allergenic
Products. And we have two of the people in the Laboratory of Enteric and
Sexually Transmitted Disease Labs, Dr. Dennis Kopecko, who is the
Laboratory Chief, and Dr. Philip Boucher. Also on the call is Dr. Sam
Katz, who is the Site Visit Team Chair.

I would now like to read a public statement for the record. The
following announcement addresses conflict of interest issues associated
with today\'s meeting of the Vaccine and Related Biological Products
Advisory Committee on November 18th, related to the review and
discussion of intramural research programs of the Laboratory of Enteric
and Sexually Transmitted Diseases.

Based on the agenda made available, it has been determined that the
committee discussions present no potential for conflict of interest.

At this point, I\'d like to turn the meeting over to you, Dr. Daum.

DR. DAUM: Thank you very much. We\'re now going to go to the open
session portion of this afternoon\'s meeting. The meeting, of course, as
Jody mentioned, is devoted entirely to the findings of the CBER
laboratory site visit of the Laboratory of Enteric and Sexually
Transmitted Diseases.

So, we are first going to hear from Dr. Walker regarding the overview of
the Division of Bacterial, Parasitic and Allergenic Products. Dr.
Walker.

DR. WALKER: Thank you. Good afternoon. I\'d like to take just a few
minutes to, as Dr. Daum said, give you a quick overview of what goes on
in the Division of Bacterial, Parasitic and Allergenic Products.

Basically, the division is focused on assuring safe and effective
products for immunological control of bacterial, parasitic and
allergenic agents affecting human health. It\'s important to keep in
mind that the scientists in this division perform both research and
review functions. In some cases for our investigators, this review
function could be 30 to 50 percent of their time.

Another point I\'d like to make focuses in on the review functions that
these people do. I\'d like to emphasize that their review work is a
cradle-to-grave type operation because it begins with pre-IND meetings
with sponsors to help provide guidance, and then through the IND
process, continuing meetings and review, license actions, and then it\'s
very important to keep in mind that even post-licensure of a product
that the task of this division goes on because there\'s review of
biological deviation reports, there\'s inspections, lot release issues,
and so forth. So, these people have a lot of work in the review are as
well as in the scientific area.

I\'d like to make another point about \-- that focuses in on the
scientific area that these people in LESTD and other parts of the
division deal with is in there are quite variety of products. When you
consider the products that are existing now or that are possible in the
foreseeable future, I think of respiratory pathogens, from Streptococcus
to Moraxella, sexually transmitted diseases, pathogens that might be
encountered by penetrating inoculation, like Malaria and Borrelia, and
so forth; special pathogens which has become a big issue in recent
years, like Bacillus anthracis, Clostridium, Franciscella and Yersinia;
diarrhea-causing pathogens which will be discussed a bit today, and so
forth, allergenic products whether it\'s latex or cockroach antigens and
skin test antigens. So there\'s quite a variety of things that the
people in this division have to cover.

And so the division, to do this, is made up of about 100 people
organized into eight laboratories. There\'s the Laboratory of Methods
Development Quality Control, which seeks to develop and standardize and
evaluate quality control methods for bacterial vaccines, and also
develop, evaluate and apply serological methods to measure immune
responses in vaccine trials. And also this group more recently is
working to coordinate quality assurance activities within the division
and provide leadership and the initiative to accredit CBER quality
control testing laboratories.

Another laboratory, the Laboratory of Bacterial Polysaccharides is
involved in characterization of immune responses to polysaccharide and
conjugate vaccines, in standardization of methods for relevant clinical
application. They also have been instrumental in developing novel
physical and chemical methods for improved evaluation of licensed and
experimental vaccines involving polysaccharides.

A third laboratory is the Laboratory of Biophysics, which use high-end
instrumentation to help characterize biopolymers such as
polysaccharides, DNA, proteins and macromolecular assemblies such as
vaccine/adjuvant complexes. And the high-end equipment like applying NMR
technology to vaccine characterization.

Another laboratory in the division is the Laboratory of Respiratory and
Special Pathogens, which deals with Bordetella pertussis, as well as
more recently with Bacillus anthracis and Yersinia species. And the work
that these people are doing is to characterize the virulence factors and
studies on mechanism of actions of these factors, and also the
regulation of gene expression of these factors.

Another laboratory is the Laboratory of Bacterial Toxins, which deals
with neurotoxins as well as iron-regulated virulence factors in
Corynebacterium diphtheriae, and also in Bacillus anthracis.

There are two other laboratories before the laboratory that we\'re going
to talk about today. The Laboratory of Mycobacterial Diseases and
Cellular Immunology, which evaluates protective innate and adaptive
immune responses to intracellular bacteria, and they are presently
involved in assessment of DNA vaccination strategies against
tuberculosis, as well as identifying new antigens that might be useful
in anti-tuberculosis vaccines.

The seventh of the eight laboratories that I want to talk about today is
the Laboratory of Immunobiochemistry, which deals with allergenic
products. They are concerned with allergen structure and function, and
immunomodulation of the allergic responses, and the study of chamokines
and receptors in the modulation of immune responses to allergenic
agents.

Then the eighth laboratory, which Dr. Kopecko will be talking much more
about this afternoon, is the Laboratory of Enteric and Sexually
Transmitted Diseases, which of course is dealing with enteric pathogens,
but also in more recent times is dealing with several bioterrorism
agents, and I\'m sure Dr. Kopecko will mention that a little bit more.

I\'d like to conclude by just bringing your attention to some of the
realities facing our Researcher/Reviewers in the division. Some of the
things they have to deal with are, as you would expect in a large
organization, are bureaucratic hurdles, like personnel and other things
that sometimes make work challenging, even though we\'ve got very good
people working with us. Another issue these Research/Reviewers have by
working in the Federal Government, they have the issue of funding levels
are uncertain from year to year, and dependent upon the appropriation
process.

But the most unique thing, I think, about the FDA Researcher/Reviewers
as opposed to people in other government agencies like CDC or NIH is
that since these people are reviewers, the timing of their workload is
determined by the sponsor submissions and not by CBER. And so they have
to carry out the research work and also meet deadlines that are part of
the review function, and that is one of the challenges that our
investigators are meeting.

And, finally, when we had the actual site visit, I asked the Site Visit
Committee to not only help us by reviewing the individuals and reviewing
the programs that they are directing, but also comment on the current
and future directions of the research.

So, hopefully that gives you a very brief overview of our
Researcher/Reviewers and sort of the nature of the work that goes on
within this division, and I\'ll be glad to answer any questions that you
might have.

DR. DAUM: Thank you very much, Dr. Walker. The floor is open for
committee question or comment.

DR. KATZ: This is Sam Katz. I guess I\'ll reserve mine until after we go
through the report.

DR. DAUM: That sounds fine, your choice. Any others?

(No response.)

Then why don\'t we move on to the next item, which is we\'re going to
hear from Dr. Kopecko. Welcome, Dr. Kopecko, and we will have an
overview of the Laboratory of Enteric and Sexually Transmitted Diseases.

DR. KOPECKO: Thank you very much. Good afternoon to everyone. It\'s a
great pleasure to have the opportunity to introduce the lab to you. In
ten minutes, I\'ll try to give you a little bit of a flavor for who we
are and what we do both regulatory and research-wise, and mention a few
of our future directions.

If you have the Site Visit Notebook in from of you, you can see from
Section 2 that the laboratory is comprised of three sections \-- the
Pathogenesis Section, the Gene Regulation Section, and Dr. Stibitz and
Dr. Boucher will be reviewing research in that area for the review
period. And the third is a new section, the Immune Mechanisms Section,
that Dr. Walker is heading up, that\'s just begun, and there wasn\'t any
significant amount of research carried out during the review period, but
as we move more into anthrax and Shigella vaccine development, that
Immune Mechanism Section carries great importance.

The lab also is evolving from five personnel to 14 personnel, so we\'re
undergoing some significant changes space-wise and personnel-wise. We
currently have 13 people onboard, with one more hopefully to join us
within the next couple of months.

If you can see from Section 3, our lab was started in 1994, replaced the
former Micoplasma Lab. It was initiated to handle a large number of INDs
and regulatory work in enteric diseases, and also to cover sexually
transmitted diseases which, in fact, were thought that there would be an
onslaught of STD applications, which has not actually come yet, but
there has been a tremendous increase of enteric disease applications.

The lab, as Dr. Walker mentioned, has a bifunction, as all labs here at
CBER. We are tasked with carrying out regulatory review in our area of
specialty, as well as to conduct research which enhances our knowledge
base, which allows us to increase both the quality and speed of the
regulatory review of various products. So, our research is aimed at our
area of specialization.

Within LESTD, we conduct basic research into the genetic and molecular
bases of disease pathogenesis and the host response to infection. And
the current events that have been analyzed during the last review period
involve both prokaryotic and eukaryotic mechanisms involved in bacterial
invasion of mucosal epithelial cells \-- that is, induced endocytosis
and also induced exocytosis \-- as well as the global regulation of
expression of bacterial virulence components, which Drs. Stibitz and
Boucher will address.

Our regulatory responsibilities, you can tell from the title, cover both
enteric and sexually transmitted diseases, but I wanted to summarize
some of the more common and some of the less common types of agents that
we cover.

This includes, obviously, cholera, Typhoid Fever, Shigellosis, as you
might expect, but also enterotoxigenic E.coli and other pathogenic
E.coli such as Campylobacter, LOB-attenuated bacterial vector based
vaccine development for multiantigen use. These include both modified
Salmonella strains, Shigella strains and cholera strains. We also cover
Helicobacter pylori, the use of live enteric bacteria such as attenuated
Salmonella typhinerium given IV to treat metastatic cancer, various UTI
pathogens, proteus, E.coli, intestinal strain replacement for probiotic
therapy, the use of bacterially derived enzymes for treatment such as
alosperigenase for acute lymphocytic leukemia, bacterial phase for
therapeutic antimicrobial use, bovine or chicken immunoglobulin
concentrates from specifically immunized animals for human use, and
genetic harbored vegetables, to mention some of the products that we
cover.

These encompass products that are administered by oral, intranasal,
parenteral, intrarectoral, or transcutaneous routes, and they also cover
evaluation of new adjutants for orally administered products.

Currently within the lab we cover about 110 different IND products. We
are receiving about 12 to 14 new IND applications per year, so you can
see how that list is growing. We probably drop about five to seven per
year that become inactive. We also are reviewing one full biological
license application on Cholera currently, and we have several BLA
supplements under review.

So, that covers the general lab and our regulatory responsibilities.
I\'d like to take a couple of minutes and go back to Section 9 and just
quickly summarize some of the research carried out in Molecular
Pathogenesis Section.

Our main focus was to look at the cell biology of invasion of intestinal
epithelial cells by Campylobacter jejuni, which I\'ll get to summarize
in a minute, but we also carried out a number of more minor projects
which all turned out to result in publications, a couple of which I want
to point out because I think they have enhanced significance.

One involves Nanobacteria, which was reported by a Alevi Pejander
(phonetic) in Finland, to be a new species of bacteria that were very
small, filterable through .1 micron-filters, potentially important
adventitious agent biological products because they are found in more
than 80 percent of bovine and human serum, thought to be the main cause
for human kidney stone formation, and more recently touted to be the
main cause of atherosclerotic plaque, still being pushed by Dr.
Pejander.

We followed up his results, which were mostly phenomenological studies
with the heaviest piece of data being PCR 16Srod assembled DNA. We were
able to obtain growth of these same coccoid like structures that had a
calcified outer shell. They looked like coccoid bacteria. However, when
we got into molecular evidence, we found by PCR we obtained the same
sequence that Pejander obtained, but in going to gene bank, we found
that he had misinterpreted the data and, in fact, this was the sequence
from phylo bacterium, which is a common water-borne contaminant that\'s
found in PCR reactions that are carried out for a high number of cycles.
Also, the growth of these structures could not be inhibited in azides,
which all respiration-dependent organisms would be inhibited by. So, the
upshot of these studies that we published in P&AS was that there was no
credible molecular data to support the existence of Nanobacteria. We
feel that these structures are actually hydroxy-appetite crystals that
are triggered into initiation by various macromolecules in blood and in
saliva as part of a natural process. And, in fact, as Pejander reported,
you can destroy these macromolecules by heavy gamma-irradiation. So,
there is a way to knock them out, but we didn\'t feel that there was any
strong evidence to support the existence of this new group of organisms,
removing it at least for the current time as an important potential
adventitious agent.

The second project that I want to mention quickly involved Shigella
sonnei. This was a project that we started some 20 years ago, trying to
make a modified hybrid vaccine in salmonella that would protect against
typhoid and Shigella, and this involved moving the LPS genes from
Shigella sonnei in Salmonella typhi, Ty21a. The difficulty is this block
of genes is very large, and more recently we were able to clone downsize
to about 12kb the essential genes. We found that they are stably
expressed in Salmonella off of an illegal copied plasmid versus the
typical multi-copied plasmid that most cloners use. We also found that
there is an adjacent insertion sequence, Is91, that causes instability
of this. Once that\'s deleted, it stabilizes the clone region for
production.

And we also found that this form one polysaccharide is expressed in two
forms in Shigella and in E.coli, both as a lipoaliga saccharide attached
to core as normally, but also as a capsule where it\'s lipid-bound and
stuck in the outer surface of the cell. And we were able to transfer
this into Salmonella and make a candidate vaccine.

These studies were published recently in Infection and Immunity,
patented, and we had one firm who\'s carrying out commercially the
development of this into a vaccine. We are extending these studies to
include Shigella dysenteria and a couple of other Shigella serotypes for
future vaccine development, together with development of a live oral
vaccine against anthrax.

Now, the last thing I want to mention is our work on Campylobacter
jejuni. We carried out a number of in vitro tissue culture invasion
assays into various cell lines, using a variety of different inhibitors.
And to understand the cell biology of entry, these studies have been
summarized in 2001 Trends in Microbiology Summary, for the most part. We
found that the organism recognizes a receptor that\'s probably in the
junctional space, the receptor is not yet characterized, and it\'s
carried within cuveale interactions of the bacteria with this receptor
trigger, we think, secretion of invasion effectors \-- this is work by
Mike Comp (phonetic), not ours \-- these invasion effectors, we found,
trigger an up-regulation of PI3 kinase, a release of intracellular
calcium from intracellular stores, activation of calmodulin and protein
CON-AC, which cause a structural rearrangement \-- and this is a
microtubule dependent up-take mechanisms as opposed to a microfilament
dependent that most other bacteria use \-- the cytoskeleton is
reorganized, the bacteria enter through a microtubular like extension,
they are carried in an endosomal vacuole along microtubules via the
molecular motor dinaene to raise the lateral surface where exocytosis
occurs. So, that\'s the current state of understanding for Campylobacter
jejuni entry. And if there aren\'t any questions, I\'ll turn things over
to Dr. Stibitz.

DR. DAUM: Dr. Kopecko\'s very interesting presentation is open for
questions and discussion.

(No response.)

Well, I guess there aren\'t any questions or discussion, Dr. Kopecko,
which means you were crystal clear. Thank you very much.

We will call on Dr. Stibitz for an overview of research activities in
his area.

DR. STIBITZ: Thank you. And thank you for letting me phone in. I\'m
actually in southern Illinois, in my ancestral home, and I\'m willing to
bet I\'m the only person who literally dialed-up to the conference call.

The section of LESTD that I\'m concerned with is interested primarily in
the study of one particular aspect of bacterial pathogenesis, and that
is the regulation of virulence factor expression. The model system that
we use is that of Bordetella pertussis, which has a very well-developed
regulon governed by the BvgAS two-component system. Now, BvgAS, like
other two-component systems, contains an environmental sensor protein
which is localized in the inner membrane, BvgS in this case, and this
protein communicates with the cytoplasmic response regulator protein,
which is BvgA in this case. These two proteins communicate by the kinase
activity of the BvgS sensor protein, and when BvgA is phosphorylated, it
binds to the promoter regions of virulence.

Now, in pertussis, these include two which I\'ll mention, the fha which
encodes the filamentous hemagglutinin, an important adhesion, and ptx
which encodes the pertussis toxin responsible for some of the hallmark
symptoms of whooping cough.

Now, we know from in vitro transcription studies that BvgA phosphate is
both necessary and sufficient to activate transcription of virulence
seen. However, there are interesting and significant differences in how
this occurs, and a classic experiment which demonstrates this is that if
one grows pertussis under conditions where the virulence regulon is
turned off and then shifts it to permissive conditions, one sees that
fha transcription is initiated within minutes while that of ptx takes
several hours. And it has been suggested to represent a temporal pattern
of gene expression upon infection.

Now, this difference in one of the aspects of virulence gene regulation
that we\'re most interested in, and our ultimate goal is the complete
understanding at a molecular level of the interactions of BvgA with
promoter DNA and with RNA polymerase in the process of transcriptional
activation. And to accomplish this, we\'ve taken a combined biochemical
and genetic approach.

Now, until now, our efforts have concentrated on the fha promoter. This
is one of the simplest, and actually the simplest, BvgA regulated
promoter described. And it was our hope that we could best learn the
details of BvgA action in this system and then broaden our sights to
include other promoters. And, in fact, that\'s exactly the juncture that
we now find ourselves at, and it has been quite satisfying.

Now, Dr. Philip Boucher, who will be speaking in a few moments, has been
the driving force behind the biochemical analysis, and he\'ll be telling
you more about that, but I just wanted to mention that that work has
recently been accepted for publication in Molecular Cell.

I have been primarily involved in the genetic analysis, and I\'d just
like to go over that briefly. Past work includes both forward and
reverse genetics. In terms of forward genetics \-- in other words,
screening for mutant phenotypes and then determining the site and nature
of mutations involved \-- we have isolated mutants which are affected in
the differential regulation \-- in other words, ones which can express
fha but not ptx, and a reverse of that, those which are hyperactivated
for ptx expression and down for fha expression.

In terms of reverse genetics \-- in other words, introducing specific
mutations like bacterialistic mutagenesis and then determining their
phenotypic effect \-- we have confirmed to role of the amino acid D54,
we have made short C-terminal deletions of BvgA which had an interesting
phenotype in that they were lethal to the cell and were suppressed by
mutations in the alpha subunit of RNA polymerase and, more recently,
we\'ve made a number of 16 substitution mutants at different positions,
and many of these had interesting phenotypes as well either in terms of
differential regulation or, in one case, we believe the mutation which
affects oligomerization of BvgA.

Now, my future genetic analysis of BvgA involved a full-scale genetic
assault on BvgA and rests on some genetic screens which we\'ve
developed, which will allow us to identify mutations that specifically
affect the ability of BvgA to interact with RNA polymerase or to
interact with itself in the process of dimerization or oligomerization.
And I won\'t go into those, but input into these will be BvgA in which
we\'ve done either intense in vivo mutagenesis or, in fact, saturation
mutagenesis by alynase scanning. And what we hope to identify are
specific messages in BvgA which are involved in interacting with RNA
polymerase or with itself in the process of oligomerization and
dimerization.

Now, recently two other people have joined the laboratory, and I\'ll
just briefly describe their projects. Dr. Wendy Veal Carr is an ERDA
Fellow who came to us from Bill Schaeffer\'s lab at Emory University,
and Wendy is embarking on a project which, if successful, will allow us
to zoom out from our detailed mechanistic analyses of BvgA to an
organismal or host pathogen interaction level. And she\'s adapting
RIVET, which stands for recombinase-based in vivo expression technology.
This system was developed by Andy Kanuli, who is not at Tufts, and it
was to examine in vivo gene expression oligliocollar. The system uses a
DNA recombinase as a reporter gene, and the readout is the degree of
recombination of a construct in the chromosome which, upon resolution,
leads to a block with antibiotic resistance. And that\'s after
appropriate strain construction one is able to infect an animal \-- in
our case, a mouse \-- and then recover the bacteria at different time
points and, by affecting the degree of recombination, affect the level
of expression that that particular gene \-- in our case, we\'ll expand
an fha and ptx \-- has experienced.

And we plan to use this to determine if the differential regulation seen
in vitro with fha and ptx develop in a manner accepted in vivo and,
secondly, to determine the effect of mutation \-- for example, which
knock out important adhesions such as fha \-- on the expression of
pertussis toxin.

And then, finally, Dr. Brian James has just very recently joined us,
actually, since the site visit, and he joins us from Dr. Robert
Bender\'s lab at University of Michigan. And Brian is responsible for
getting our anthrax project up and running, and in the initial phase
he\'s developing new genetic tools specifically for powerful molelic
exchange vectors and an enhanced efficiency of DNA transfer, and then we
plan to use those tools to study genetic regulation of virulence in
anthrax. And I\'ll stop there.

DR. DAUM: Thank you very much, Dr. Stibitz. Dr. Stibitz\' interesting
presentation is now open for committee discussion and comment.

(No response.)

I was wondering if you could possibly say a couple of sentences about
the big-picture role of fha and the pathogenesis of pertussis \-- why is
this of particular importance? Of the vaccine antigens that we talk
about and bandy around their importance of the acellular vaccines, why
this particular antigen important to the pathogenesis?

DR. STIBITZ: Well, I guess the most sort of convincing evidence of that
is that the mutants of pertussis which lack expression of fha are
decreased \-- they have decreased ability to adhere to host cells, and
in some models have decreased virulence.

DR. KATZ: If you ask John Robbins, he says it doesn\'t do anything.

DR. STIBITZ: Fair enough. I think it is safe to say that the animal
models that we use may not be the best animal models for assessing the
effect of fha, but at least, you know, one aspect of \-- I think whether
or not fha is absolutely important in infection in a mouse model, we can
still assess the degree of in vivo regulation using the system that
we\'re going to use.

DR. KATZ: I guess what I would like you to comment on \-- I was one of
the visitors, so I heard this, but I don\'t know if the committee
members did \-- what is the relevance of Bordetella research to a
laboratory of enteric and sexually transmitted diseases?

DR. STIBITZ: Yes, I get this question every time.

DR. DAUM: I imagine you do.

DR. STIBITZ: There are two answers to that. One is sort of the
historical story of how I came to be where I am, and I don\'t think
that\'s particularly relevant. I think what\'s more relevant is the fact
that the type of approach that we take, until recently, we were probably
the only group within the division taking a very genetic approach. That
set of knowledge has turned out to be very useful for the type of
products which we particularly find in the Enterics Lab. For example,
rationally attenuated live vaccine which use the exchange methods that
we use often in pertussis and actually developed for use in pertussis,
and then also issues generally relating to mutation rate, population
genetics, and so forth, that impact upon genetic stability, and these
also impact upon live vaccine preparation, as well as issues that relate
to products we see, such as the replacement flouro-type of approach that
Dennis mentioned, as well as bacteria therapy, which is also something
that\'s coming to the fore again.

So, I think pertussis itself is not relevant. I think the system that we
study, the regulatory system, is widely relevant to bacterial
pathogenesis as a whole, including enterics, and then our specific
method technology base help us greatly in the reviews that we do.

DR. DAUM: Thank you.

MS. FISHER: Dr. Daum?

DR. DAUM: Yes.

MS. FISHER: Barbara Fisher. May I ask a question?

DR. DAUM: Please.

MS. FISHER: Is any of your work going to lead us to genetic screening
techniques for those who would be at high risk of potentially reacting,
say, to pertussis vaccine?

DR. STIBITZ: I don\'t believe so. In other words, that would be
primarily a host characteristic, and our techniques are not designed to
assess that aspect of the host/parasite relationship.

DR. DAUM: Thank you, Dr. Stibitz. Are there other committee questions or
comments?

DR. PALESE: Yes. Peter Palese, please. Can I ask a question about the
anthrax?

DR. DAUM: Certainly.

DR. PALESE: A new person who is coming, we work on real characteristics
of the anthraces. Now, are there facilities available at your
institution, or how are you going to test for A-virulence factors?

DR. STIBITZ: Well, initially, we\'re trying to develop new genetic
tools, and that may take some time. When we do get to the point where
we\'re working directly with anthrax, much of our work can be done with
the A-virulent strain. At this point, we\'re seeing a need for doing
actual work with fully virulent anthrax, although, if that happens, we
do have a petri facility. In fact, we\'re actually going one better and
we\'re doing much of the initial work in Bacillicereous, which is a very
close relative of anthrax, but much, much less virulent. Does that
answer your question?

DR. PALESE: Yes. Thank you very much.

DR. AGUILAR-CORDOVA: This is Estuardo Aguilar. I wonder, Dr. Stibitz, if
you could comment \-- I was also on the review team \-- but if you could
comment on the independence of Dr. Boucher within the work that you are
doing and the collaboration that involved.

DR. STIBITZ: The word you use is the word I would use to describe it.
Basically, Phil brings a set of knowledge and tools to the problems that
I don\'t possess, and he\'s developed all the biochemistry. He\'s done
that essentially on his own. I work in some of the mutant construct, and
so on and so forth, but Dr. Boucher has done all that biochemistry from
the ground up, and it\'s a project that he initiated and it\'s really a
collaboration. I did the genetics and he does the biochemistry. If you
have more specific questions, I could elaborate, but the degree of
independence is very high.

DR. DAUM: Dr. Palese, a followup?

(No response.)

All right. Thank you. Okay. Other committee questions, comments?

DR. SACHS: I just have a very quick comment. This is Jody Sachs. I just
wanted to remind everybody that a Transcriber is recording this telecon,
and it\'s easier for the Transcriber if everybody repeats their name
before speaking when they comment. Thank you.

DR. DAUM: Thank you.

DR. AGUILAR-CORDOVA: This is Estuardo Aguilar one more time.

DR. DAUM: Please.

DR. AGUILAR-CORDOVA: Just another quick question for either Dr. Stibitz
or Dr. Kopecko. Both of you have mentioned an increased number of
people. Is there also an increase in space that\'s being considered?

DR. STIBITZ: Do you want to field that one, Dennis?

DR. KOPECKO: Yes, I will. This is Dennis Kopecko. Yes, we do have an
increase in space. We still would like to get more space because even
with renovations that are ongoing, we are still going to be fairly
cramped. But within the limitations of the division and the Center, we
can\'t ask for anymore, really, currently. But, yes, there is some
additional space, and certainly some additional funding for per capita
research.

DR. DAUM: Thank you.

DR. KATZ: Refreshing refrain. This is Dr. Katz. You said you were going
from five to 14 people, which is what you said in your introductory
remarks. Those additional nine people, at what level are they
scientifically? Are these doctoral, post-doctoral, what level people are
these?

DR. KOPECKO: Good question. Let\'s see here. If we go back to Section 2,
to the organizational chart, you\'ll see that in the Molecular
Pathogenesis Section, there are two Post-Doctoral level individuals
joining. Dr. DeQi Xu, who just recently joined within the past week, who
is going to be working on Shigella vaccines, and a second Post-Doctoral
Fellow to work on Campylobacter pathogenesis. Dr. Stibitz mentioned two
additional individuals, Dr. Wendy Veal Carr and Dr. Brian James, who
joined at the Post-Doctoral level. Dr. Manuel Osorio is a Staff Fellow
Ph.D. Cellular Immunologist, who joined us back in July, in the Immune
Mechanisms Section, and then we have an additional beginning research
technician, and that sort of fills out the field. So, mostly
professional level, on one technical level.

DR. KATZ: Thank you.

DR. DAUM: Other comments, questions?

DR. FAGGETT: This is Dr. Faggett. One question relative to constant
research versus regulatory. What is the breakout in terms of how much
time \-- sounds like the new Fellows will be doing primarily the
research \-- but in terms of how much time just on a regulatory?

DR. KOPECKO: This is Dr. Kopecko. That\'s a very good question. In order
to carry out competitive research, you obviously have to have some
people in the lab that spend the majority of time there. And what Scott
and I have sort of evolved into is a situation where he and I take the
majority workload for regulatory, so we\'re splitting up around 40 to 45
IND applications each, and then the remaining 20 to 30 are parceled out
with other members of the laboratory, that way trying to keep most of
the other individuals in the lab most of the time, but they are all
involved to some extent with regulatory, other than a couple of the
Post-Doctoral Fellows who are not FDA personnel, but they are actually
individual contractors.

DR. KATZ: This is Dr. Katz. I think when we made our review, if I
remember correctly, Dr. Kopecko, you told me 50 percent of your time is
spent on regulatory, is that correct?

DR. KOPECKO: That\'s correct.

DR. KATZ: So, I think that gives you a little idea, Walter.

DR. FAGGETT: Thanks, Sam.

DR. DAUM: Other input?

(No response.)

Good. thank you, everybody, for your comments. And let\'s move on to
hear from Dr. Boucher, who will give an overview of his research
activity. Dr. Boucher, welcome.

DR. BOUCHER: Thank you. I thought I would very briefly summarize the
work I presented to the Site Visit Team a couple of months ago, then
discuss a current project, and then finish off with what is planned for
the future.

My interest in this lab since my arrival here in 1994 has been on a
biochemical approach to an understanding of how a bacterial response
regulator effects control of expression of virulence factors. This work
has evolved from early investigations describing how the phosphorylated
BvgA regulator of Bordetella pertussis binds to simple promoters to more
recent experiments that have global implications for molecular genetics
in bacteria.

Initial studies employing DNA binding and both in vivo and in vitro
transcription assays formed the basis of our understanding as to how
BvgA activated transcription at the fha and ptx promoters that have
already been discussed by Scott. For example, it was discovered that
activation of both promoters involves multiple dimers organized in such
a way as to suggest an interaction with the C-terminal domain of the
alpha subunit, one of the polymerase subunits.

Again, as Scott mentioned, the most recent work presented a couple of
months ago will be coming out in the January issue of Molecular Cell, so
I\'ll be going through it very briefly here. Using a technique termed
affinity cleavage of DNA, we have developed a structural model of how
both the BvgA regulator and RNA polymerase interact at a virulence
factor promoter, specifically the fha promoter.

The technique is based on the ability of a chemical nuclease to cleave
DNA primarily at sites dictated by the binding specificity of the
DNA-binding protein to which it is bound. Analysis of cleavage patterns
generated by such modified proteins bound to promoter DNA allows the
development of structural models capable of addressing certain critical
questions such as how many molecules of a given regulator bind a
particular promoter and how are the different proteins in a
transcription initiation complex oriented relative to each other.

Using this technology, we described a novel architecture in which
multiple dimers of BvgA bind along one fact of fha promoter DNA while a
part of the polymerase complex, the alpha C-terminal domain, binds to
the opposing face along the same linear stretch of DNA.

In addition, modeling studies revealed that the orientation of BvgA
monomers within the dimers is the same as that recently demonstrated by
x-ray crystallographic methods for the C-terminal domain of the related
response regulator called NarL bound to its DNA-binding site.

Since these experiments were conducted, we have extended our analysis of
transcription initiation complexes to other virulence factor promoters
of Bordetella pertussis. Previous studies using relatively low
resolution analyses made certain predictions whose validity, in light of
our current understanding, were now being brought into the question. In
the pertussis toxin promoter, for example, stoichiometry of BvgA binding
and novel orientations for how BvgA maneuvers itself onto a very unusual
binding site had previously been suggested.

Using our affinity cleavage assay, we recently determined that the
previous studies had underestimated the number of BvgA dimers bound to
the toxin promoter and that the unusual BvgA binding site within this
promoter bound 2 dimers of BvgA in a very usual and unconstrained
manner.

At another promoter, the bipA promoter, we and others have shown that
activation at this promoter requires a concentration of BvgA that is
intermediate between that required to activate the fha promoter and that
required to activate the toxin promoter. Indeed, at the relatively high
in vitro concentrations needed for toxin activation, the bipA promoter
appears to be repressed. Our in vitro affinity cleavage assay has aided
us in distinguishing sites that are likely involved in activation from
sites that are likely involved in repression. Studies of other critical
virulence factor promoters are currently ongoing.

Conclusions from the work just described also suggested other
interactions between BvgA and the RNA polymerase. For example, our data
suggests that BvgA may interact with one or both of the following: The
C-terminal domain of the sigma subunit of RNA polymerase and the
N-terminal domain for the alpha subunit of RNA polymerase.

Experiments utilizing both an in vitro biochemical approach and an in
vivo 2-hybrid protein-protein interaction assay are underway to address
these possibilities. As there are only two copies of the alpha subunit
per polymerase complex, it is also important to understand whether both
copies are required for full BvgA-dependent promoter activity. In vitro
transcription experiments are also underway to address this question.

And, lastly, another project is in the proof of concept phase of
investigation. How exactly phosphorylation of BvgA engenders an active
complex is not fully understood. Based on structural data of
unphosphorylated forms of homologous proteins, it has been suggested
that phosphorylation results in the unmasking of the DNA Binding Domain.

Perhaps due to the instability of the phosphorylated form of response
regulators, it has thus far not been possible to crystalize this form.
In an attempt to circumvent these limitations, we are opting for the use
of a physical method that would structurally compare the two forms of
BvgA. This technology termed FRET, for florescence resonance energy
transfer, is based on the ability of an activated flourophore to
transfer its resonance energy to a neighboring flourophore.

As the probability of this transfer is dependent on the distance
separating the two flourophores, molecular distances can be calculated
if energy transfer is monitored for a series of flourophore pairs
distributed within a single biomolecule. If measurements are made for
each pair in both the phosphorylated and unphosphorylated forms of BvgA,
a model can be derived that describes the motion of one domain \-- for
example, the C-terminal DNA binding domain \-- relative to another \--
for example, the N-terminal masking domain. The construction of
preliminary BvgA mutants that will eventually harbor flourophore pairs
is currently underway.

That about concludes my brief summary of my lab activities, and I can
entertain any questions.

DR. DAUM: Dr. Boucher\'s presentation is open for committee comment and
questions.

(No response.)

I would like to ask the same question I asked of Dr. Stibitz, if you
wouldn\'t mind, and that is, can you tell us a little bit about how fha,
which is good after some impressive regulation, factors into pertussis
status physiology, and how important it is to the conduct of the
organism or the ability of the organism to go about its business?

DR. BOUCHER: Certainly. First of all, it is clear that fha, the
expression of fha in Bordetella plays an integral role in its
pathogenesis. Without the fha, it really has no clear way of adhering to
epithelial cells. However, with the work that I am conducting, fha
really here is looked upon more as a readout of the activity of the
two-component systems that we\'re studying, and less really as an
antigen for any possible vaccine.

DR. DAUM: I understand that, but it\'s still an interesting issue for
me, so I have to ask it anyway.

Other committee comments? Questions?

(No response.)

Input?

(No response.)

Dr. Boucher, thank you.

DR. BOUCHER: Thank you.

Jody, I think it\'s that bewitching time for the open public hearing. Do
we know if there\'s anybody interested in addressing the committee?

DR. SACHS: No, but I\'ll just read a statement.

As part of the FDA Advisory Committee meeting, we are required to hold
an open public hearing for those members of the public who are not on
the agenda, who would like to make a statement concerning matters
pending before the committee.

I have not received any requests at this time. If there is anyone who
would like to speak \-- address the committee at this time, this is the
opportunity. And I look around the room where I am right now, and
there\'s no one from the public wishing to speak. So, I will close the
open public hearing and ask you, Dr. Daum, to continue the meeting.

DR. DAUM: That sounds fine. So, we\'re going to move into closed session
at this point, and I guess that\'s going to ask those individuals who
need to take their leave at this point, to please do so.

(Whereupon, at 2:00 p.m., the open portion of the meeting of the
Advisory Committee was concluded.)