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

 

                                              PAGE

 

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

                                            (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.)