UNITED STATES OF AMERICA
	FOOD AND DRUG ADMINISTRATION
	CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
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	VACCINES AND RELATED BIOLOGICAL
	PRODUCTS ADVISORY COMMITTEE
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	MEETING
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	WEDNESDAY,
	MAY 16, 2001
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		The Advisory Committee met in the Grand Ballroom, Holiday Inn Gaithersburg, 2 Montgomery Village Avenue, Gaithersburg, Maryland, at 9:00 a.m., Dr. Robert S. Daum, Acting Chair, presiding.
PRESENT:
		ROBERT S. DAUM, M.D., Acting Chair
		C. ESTUARDO AGUILAR-CORDOVA, M.D., Ph.D.
		DONALD BLAIR, Ph.D.
		JOHN COFFIN, Ph.D.
		JAMES COOK, M.D.
		MICHAEL DECKER, M.D.
		PAMELA S. DIAZ, M.D., Member
PRESENT (Continued):
		ALEX J. VAN DER EB, Ph.D.
		WALTER L. FAGGETT, M.D., Member
		BARbARA LOE FISHER, Member
		JUDITH D. GOLDBERG, Sc.D., member
		DIANE E. GRIFFIN, M.D., Ph.D., Member
		STEPHEN HUGHES, Ph.D.
		SAMUEL L. KATZ, M.D., Member
		KWANG SIK KIM, M.D., Member
		STEVE KOHL, M.MD., Member
		PAMELA McINNES, D.D.S., Msc.(Dent.)
		PHILIP MINOR, Ph.D.
		LAWRENCE MOULTON, Ph.D.
		MARTIN MYERS, M.D.
		SUZETTE PRIOLA, Ph.D.
		DAVID S. STEPHENS, M.D., Member
		SIDNEY WOLFE, M.D.
		NANCY CHERRY, Executive Secretary

	C-O-N-T-E-N-T-S

Conflict of Interest Statement 	4
Introductions 	4
Introduction to the Session on Designer Cell
	Substrate, Dr. Andrew Lewis 	16

Designer Cell Substrates for Vaccine
	Development:  Concepts and Issues,
	Dr. Steve Hughes 	35

Adenovirus Biology as Related to Development
	and Use of Adenovirus Vectors, Dr.
	Estuardo Aguilar-Cordova 	54

Adenovirus Transformation of Human Cells and the
	Development of 293 and PER.C6 for the
	Manufacture of Defective Adenovirus Vectors,
	Dr. Alex van der Eb 	77

Adenovirus Transformed Cell Tumorigenicity and
	Transformed Cell-Host Interaction that
	Determine their Tumor Forming Capability,
	Dr. James Cook 	100

Quantitative Assessment of the Risks of
	Residual DNA, Dr. Keith Peden 	131

Introduction to Adventitious Agent Issues,
	Dr. Philip Krause 	161

Transmissible Spongiform Encephalopathy Agents
	as an Issue in the Use of Neoplastic Cell
	Substrates, Dr. Sue Priola 	168

Adventitious Agent Testing of Neoplastic Cell
	Substrates, Dr. Philip Krause 	196

Review of OVRR-CBER Issues with the Use of
	Adenovirus-vectored Vaccines and their
	Complementing Designer Cell Substrates,
	Dr. Hana Golding	228

Committee Discussion 	239

	P-R-O-C-E-E-D-I-N-G-S
	(9:01 a.m.)
		ACTING CHAIRMAN DAUM:  Good morning.  We will begin our session with turning the floor over to Nancy Cherry, who will read the conflict of interest statement.
		MS. CHERRY:  First of all, I'd like to welcome you all to this meeting, and then I will read the statement.
		The following announcement addresses conflict of interest issues associated with this Session 2 of the Vaccines and Related Biological Products Advisory Committee meeting on May 16th, 2001.
		This open session is focused on discussion on adventitious agent testing, tumorigenicity testing, and issues related to residual cell substrate DNA of novel and neoplastic cell substrates used to manufacture viral vaccines.
		No temporary voting members have been appointed for this session.  
		To determine if any conflicts of interest existed, the agency reviewed the submitted agenda and all financial interests reported by the meeting participants.  As a result of this review, the following disclosures are being made regarding the discussion May 16th.
	Drs. Griffin, Aguilar-Cordova, and Ketner have each been granted a waiver in accordance with 18 USC 208(b)(3), which permits them to participate fully in the discussions.  
		Also, in accordance with Section 2635.502 of the Standards of Conduct, Drs. Coffin and Moulton have been granted appearance determinations which permit them to participate fully in the discussions.
		Drs. Daum, Goldberg, Griffin, Kim, Stephens, Blair, Priola, Hughes, Cook, McInnes, and Minor have associations with firms that could be or appear to be affected by the Committee discussions.  However, in accordance with 18 USC 208 and with the section I referenced above of the Standards of Conduct, it has been determined that none of these associations is sufficient to warrant the need for a waiver, for a written appearance determination or for exclusion.
		The agency has determined that the services of Dr. van der Eb as a non-voting guest are essential.  Dr. van der Eb has reported that he received a consulting fee for scientific advice on Crucell's human cell line.
		In addition, the agency has determined that the services of Dr. Michael Decker as a non-voting guest from industry are also essential.  Dr. Decker is employed by Aventis Pasteur as the Vice President of Medical and Scientific Affairs.  He reported a financial interest in a firm that could be affected by the committee discussion.
		In addition, Dr. Decker's employer has associations with university researchers and with major vaccine manufacturers.
		In the event that the discussions involve specific products or firms not on the agenda and for which FDA's participants have a financial interest, the participants are reminded of the need to exclude themselves from the discussions.  Their recusals will be noted for the public record.
		With respect to all other meeting participants, we ask in the interest of fairness that you state your name and affiliation and any current or previous financial involvement with any firm whose products you wish to comment on.
		Copies of all waivers and appearance determinations addressed in this announcement are available by written request under the Freedom of Information Act.
		And I do have one other announcement.  The Committee management specialists that did so much work to put this meeting together are, I guess, both sitting out at the front desk now.  Denise Royster is being assisted today by Rosanna Harvey, and if you have any problems, please see them.
		ACTING CHAIRMAN DAUM:  Thank you very much, Nancy.
		There's a peculiar microphone feedback in the room that seems to be resonating around when anyone is speaking.  It sounds like someone whispering, and I realize after a while that it's me and it's my echo going around.  We had it when Dr. Patriarca was speaking last time also.
		Can you give it a thought?  Maybe I'm just sitting at the funnel here.
		PARTICIPANT:  Are you hearing it now?
		ACTING CHAIRMAN DAUM:  When I speak I am.
		Also, cell phones, beepers, all the things you can't use on airplanes, please don't use them here either.  Different reason.  They really distract the tone of the discussion and the Committee deliberations, and I'd very much be grateful if everybody now thought about whether they have a beeper or cell phone that could ring and disrupt the Committee.
		I would like to take a few minutes to go around the table and have people introduce themselves this morning, and I would like to ask that there be a slight discrimination in the process, unless the way we usually do it, and that is we'll start with Dr. Griffin and come down as far as Ms. Fisher, which are the standard Committee members, and then I'm going to ask everybody else, starting with Dr. Myers and working our way around, to not only say who they are and what their affiliation is, but sort of explain how that affiliation gets them here in one sentence or two.  Why are they consulting to our Committee for in general or for this particular issue.
		I think that would be helpful in terms of orienting everyone toward the discussion.  So, Dr. Griffin, would you start us off, please?
		DR. GRIFFIN:  So I am Diane Griffin from Johns Hopkins.  I'm the chair of the Molecular Microbiology and Immunology Department in the School of Public Health, and I'm going to explain a little bit about myself.
		I'm interested in the pathogenesis of viral infections.
		ACTING CHAIRMAN DAUM:  Perfect.
		DR. STEPHENS:  I'm David Stephens from Emory University, Director of the Division of Infectious Diseases.  I'm a bacteriologist, not a virologist.  So I'll pass to the next person.
		ACTING CHAIRMAN DAUM:  Committee members need to be less explicit in this regard.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  This is not a total expose, but rather an opportunity for the Committee to understand why the consultants that are here today, in fact, are.
		Dr. Goldberg.
		DR. GOLDBERG:  Hi.  Judy Goldberg.  I'm the Director of Biostatistics at New York University, School of Medicine.
		DR. KATZ:  I'm Sam Katz, a pediatric infectious disease person from Duke who's spent most of his career studying vaccines.
		DR. DIAZ:  I'm Pamela Diaz, pediatric infectious disease person and the Director of Infectious Diseases for the Chicago Department of Health.
		DR. KOHL:  I'm Steve Kohl, pediatric infectious diseases and at the Argonne Health Science University, with an expertise in viral immunology.
		DR. KIM:  I'm Kwang Sik Kim.  I'm head of pediatric infectious diseases at Johns Hopkins School.  My work has been primarily on the pathogenesis of infectious diseases, primarily on bacterial infections in pediatrics.
		MS. FISHER:  Barbara Loe Fisher, President of the National Vaccine Information Center, a nonprofit organization that's concerned about vaccine safety.
		DR. MYERS:  I'm Martin Myers.  I'm the Director of the National Vaccine Program Office.  Background:  pediatrician in infectious diseases interested in pathophysiology, particularly animal models of Herpes viral infections; former Chairman of Pediatrics.
		MS. McINNES:  I'm Pamela McInnes, Deputy Director, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases.  NIAID is, of course, an important funder through public money, expenditure on basic, applied, and clinical research in infectious diseases.
		DR. VAN DER EB:  I am Alex van der Eb, emeritus professor at the University of Leiden, with expertise in viral transformation and cancer in general.  I'm still active in the lab and scientific advisor to Crucell, a member of the Scientific Advisory Committee.
		DR. DECKER:  I'm Dr. Michael Decker.  I'm a member of the Departments of Preventive Medicine and Infectious Diseases at Vanderbilt University, where for, oh, ten or 15 years I've been actually involved in clinical research and vaccines.  Recently I've joined Aventis Pasteur as Vice President for Scientific and Medical Affairs, and I'm here because through a typical federal process, I am the vaccine industry representative to VerPAC.
		DR. AGUILAR-CORDOVA:  I'm Estuardo Aguilar.  I'm with the Harvard Gene Therapy Initiative, and I've been asked to come here primarily because of my work in antiviral vectors and their use in gene therapy applications.
		DR. COFFIN:  John Coffin.  I'm a professor in the Department of Molecular Biology and Microbiology at Tufts University and also part-time Director of the  NCI's HIV Drug Resistance Program and also part-time cranberry grower.  And I'm here, I guess, because my research over quite a number of years has been engaged in understanding how retroviruses work and how they transform cells and issues related to that.
		DR. COOK:  I'm Jim  Cook.  I'm Chief of Infectious Disease at the University of Illinois, and my research interest is adenoviral early gene expression, especially E1A and how it affects the cell's response to the inflammatory response in host..
		DR. BLAIR:  I'm Don Blair.  I'm Chief of the Oncogene Mechanism Section of the Center for Cancer Research at the NCI and have a long history of interest in DNA biological activity and tumorigenesis.
		DR. MOULTON:  Larry Moulton.  I'm a biostatistician at Johns Hopkins University, and I spend the majority of my time working on vaccine safety and vaccine efficacy studies.
		DR. KETNER:  I'm Gary Ketner from the Department of Molecular Microbiology at the Johns Hopkins University now Bloomberg School of Public Health, and I'm an adenovirus geneticist.
		DR. MINOR:  I'm Philip Minor.  I'm from the National Institute of Biological Standards and Control in the United Kingdom.  We're concerned with quality control and quality issues and regulation of viral vaccines, and we also get involved in viral contamination, issues of biological products.
		DR. WOLFE:  I'm Sid Wolfe.  I'm a general internist by clinical training, and since leaving NIH 30 years ago, I've spent most of my time at the Public Citizens Health Research Group in activities that relate to the FDA, drugs, biologics, and I think I'm here because we've worked closely, sometimes in an antagonist way, but closely with the FDA for 30 years to try and sort through problems.
		This is certainly one of the most interesting and important issues that's come at least to my attention, and I'm glad to be asked to participate.
		DR. PRIOLA:  I'm Sue Priola from the Rocky Mountain Laboratories, which is an off, off, off campus branch of National Institutes of Health, and I'm here to provide information about infectivity TSE infection, and tissue culture cells and the risks involved.
		DR. HUGHES:  I'm Steve Hughes.  I'm from the HIV Drug Resistance Program of the NCI, and I have a longstanding interest in retroviruses and retroviral vectors.
		ACTING CHAIRMAN DAUM:  And I'm Robert Daum.  I'm from -- I'm with parainfluenza virus infection.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  I'm from the University of Chicago.  I'm head of the Section of Pediatric Infectious Diseases there.  My interests include antimicrobially induced stress in Gram positive bacteria, and that's my day job, and my closet research concerns clinical evaluation of vaccines and strategies for improving immunization rates in inner city children.
		And so with that, I welcome everybody, members and guests, to our meeting.  We have obviously a very distinguished panel of consultants today to help us with these important issues.
		And at this point I'd like to move on with the body of the meeting and call on Dr. Andrew Lewis from the FDA, who will introduce us to this session on so-called designer cell substrates.
		While Dr. Lewis is walking up to the podium, could the FDA folks tell us who they are also and just in the same kind of brief, USA Today format?
		DR. PEDEN:  Yes, my name is Keith Peden.  I'm in the Division of Viral Products in the Office of Vaccines at CBER.  We're involved in the regulation of vaccines, and as a nighttime job we do some research on HIV.
		DR. KRAUSE:  Phil Krause in the Laboratory of DNA Viruses.  I'm interested in viral latency and in viral detection.
		DR. GOLDING:  I'm Hana Golding.  I'm the Chief of the Laboratory of Retrovirus Research in Division of Viral Product.  I'm very much involved in regulation of HIV vaccine, and my scientific  world has been focused on HIV cell entry and HIV vaccine development.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		DR. GRIFFIN:  I am Diane Griffin from Johns Hopkins.
		DR. STEPHENS:  I'm David Stephens from Emory University.
		DR. GOLDBERG:  Judy Goldberg from New York University.
		DR. KATZ:  Sam Katz from Duke University.
		DR. DIAZ:  Pamela Diaz, Chicago Department of Health.
		DR. KOHL:  Steve Kohl, Argonne Health Science University.
		DR. KIM:  Kwang Sik Kim, Johns Hopkins School of Medicine.
		MS. FISHER:  Barbara Loe Fisher, National Vaccine Information Center.
		DR. MYERS:  Martin Myers, National Vaccine Program Office.
		DR. COFFIN:  John Coffin, Tufts University and sometimes NCI.
		DR. COOK:  Jim  Cook, University of Illinois.
		DR. BLAIR:  Don Blair, NCI.
		DR. MOULTON:  Larry Moulton, Johns Hopkins University.
		DR. KETNER:  Gary Ketner, Johns Hopkins.
		DR. MINOR:  Philip Minor from the National Institute of Biological Standards in the U.K.
		DR. WOLFE:  Sid Wolfe, Public Citizens Health Research Group.
		DR. HUGHES:  Steve Hughes, NCI.
		ACTING CHAIRMAN DAUM:  And I'm Robert Daum from the University of Chicago.
		DR. LEWIS:  And by way of introduction, I'm Andrew Lewis, as it says on this slide.  Maybe we need to cut the lights down a bit.  Can people see this better now?
		I'm the Chief of the Laboratory of DNA Viruses, Division of Viral Products.  I came to the FDA about a little over five years ago, having spent basically a 30-year career at the National Institutes of Health studying adenoviruses and adenovirus transformed cells.
		My role in introducing today's session is twofold.  The first is to review the status of the Office of Vaccines' approach to the use of neoplastic cell substrates for viral vaccine development and, second, to introduce the topic of designer cell substrates and the issues associated with their use for vaccine manufacture.
		Is this better?  Keith, could you see about focusing this slide?  Is that better?
		Okay.  Thank you.
		Several of the topics for discussion today have evolved from studies of viral oncology, using in vitro tissue culture systems in studies of neoplastic development in vivo using animal models.
		To understand the terminology that's evolved from these fields that will be used by some of the speakers today, I've defined in this slide what we mean when we say we need neoplastic cells, cell transformation, cell line tumorigenicity and viral oncogenicity.
		Neoplastic cells is, for our discussion today, used in its broadest sense to include spontaneously transformed cells, virus transformed cells or other types of immortalized cell lines that may be either tumorigenic or non-tumorigenic.
		Transformation is a process by which normal cells are changed by viral or cellular oncogenes or spontaneous events to become immortal neoplastic cells.
		Tumorigenicity if the ability of neoplastic cells growing in tissue culture to multiply and develop into tumors when injected into animals, and oncogenicity is the ability of a virus or viral or cellular genes to convert the cells of an injected animal into tumor cells.
		Now, the use of neoplastic cells for vaccine manufacture has been discouraged since 1954.  A number of factors are contributing to the need to reconsider neoplastic cell substrates for vaccine development, and those factors that are related to the discussion today are presented in this slide.
		First, cell lines capable of complimenting the growth of defective viral vectors used as antigen delivery systems and hence of vaccines.
		Second is the development of virtual vectored HIV vaccines.
		Finally, progress in understanding carcinogenesis and detecting adventitious agents, and the successful experience with highly purified biologicals that are actually derives from tumor cells.
		Discussions regarding issues associated with the use of neoplastic cell substrates were begun in the Office of Vaccines in 1996.  The outcome of these initial discussions was the development of a systematic approach to consider and evaluate these issues.
		This approach consisted of the five steps, which include identifying the issues, developing appropriate models to evaluate each issue, developing the necessary data to establish the validity of the models used to issue your evaluation, developing criteria to consider levels of risk, and discussing the approaches or this approach in public forums and meetings.
		In the initial stages of implementing this approach, six issues were identified.  These issues and the concerns they generated are presented in this slide.  The issues were discussed in detail before the committee in 1998 and again in May of 2000.
		Of the six issues that we identified originally, only Issues 2, 3, and 5 will be the focal points for today's discussion.  
		Issue 2 includes adventitious agent contamination with the possible transfer of known or unknown viruses.  For purposes of today's discussion, we will include agents of transmissible spongiform encephalopathy under the category of adventitious agents.
		Issue 3 includes residual cell substrate DNA contamination with the possible transfer of activated oncogenic and/or infectious genetic information.
		And Issue 5 includes viral-viral and viral-cellular interactions with the possibility of transfer of novel or recombinant viruses, and for the issues that we will be dealing with today, this includes replication competent adenoviruses.
		Now, to manage the model and risk assessment aspects of the Office of Vaccines' approach, what we're calling a defined risk evaluation was developed.  The basic aspect of this evaluation includes assessing quantitative where possible the risk posed by the issues, establishing the probability of a worst case scenario for plausible issues, using available data to evaluate plausible risk individually and cumulatively, and using cumulative data to assess the relative risk of the product.
		The concept and implementation of the defined risk evaluation will be presented in more detail by Drs. Peden and Krause when they discuss residual substrate DNA and with adventitious agent issues later this morning and this afternoon.
		To implement the public discussion stage of the CBER approach, our plan was presented to the Advisory Committee in November of 1998.  During this meeting, the Committee recommended that we develop the plan into a draft document and present the plan for discussion at an international workshop on cell substrates.
		This recommendation was implemented over the next nine months and culminated in a workshop on neoplastic cell substrates that was held in Rockville, Maryland, in September of 1999.
		Additional discussions at the Office of Vaccine followed this meeting and the public discussion of neoplastic cell substrates was continued at the May Advisory Committee last year.
		Now, to briefly summarize the substance of the Office of Vaccine's presentations at the May 2000 Advisory Committee meeting, neoplastic cell substrates were divided into five categories.  Category 1 included human cells used for vaccine manufacture that are transformed by known mechanisms.  Since there are no cell lines like this, hypothetical examples include the diploid WI-38 and MRC-5 cell strains that are immortalized by human telomerase gene.
		Category 2 includes early passage human diploid cells transformed by known mechanisms.  Examples include the  293 cells and PER.C6 cells that are going to be the focal point of our discussion today.
		Category 3 through 5 represent non-human primate cells transformed spontaneously.  These include VERO cells, CV-1 cells and BSC-1 cells.  All cell lines that are derived from tumors of any species, and those cells lines that are not covered by Categories 1 through 4.
		Examples of these types of cells in Categories 3 through 5 include HeLa cells and the HUT-78 cells, which is used to propagate HIV virus.
		Now, these categories were developed based on estimations of the difficulties in managing the regulatory issues associated with different types of cells.  Possible management approaches were presented for each category.
		However, today time doesn't permit me to review the variety of issues and approaches that were raised by cells in each of these categories.  This information is available in the transcripts of the May 2000 meeting, which are present on the CBER Web site.
		Of these five categories, only Categories 1 and 2 as examples of designer cell substrates are going to be discussed today.
		And as I mentioned, the subject of today's meeting is to consider issues associated with designer cell substrates which fall into Categories 1 and 2, as you just saw.  For today's discussions, we're defining designer cell substrates as normal human cells.  They're neoplastically transformed by a known viral or cellular oncogenes or by immortalizing cellular genes.
		Because it's now possible to engineer or design all types of mammalian cells to express desired traits, this definition may need to be altered in the future.  In the next talk, Dr. Steve Hughes will present in more detail the development of designer cell substrates and the issues associated with their use.
		Like the factors that are stimulating the need to use all types of neoplastic cells and substrates for vaccine development, there are a number of factors behind the need to develop and use designer cell substrates for vaccine development.  These factors include the development of cells to complement the replication of bioengineered viral vectors, increasing experience with viral vectors in gene therapy and the production of biologically active proteins, and hence the development of vaccines and the development of HIV vaccines.
		I should like to point out the development and use of bioengineered defective viral vectors to serve as vaccines by delivering immunizing antigens requires the use of cells containing the missing copies of the defective viral genes to assist the growth of the defective vector.
		In the third talk this morning, Dr. Aguilar will have much more to say about viral vectors and especially adenovirus vectors as vaccine delivery systems.
		The designer cell substrates we'll be considering today include 293 cells, which are human embryonic kidney cells transformed by restriction enzyme flea fragment of the Adenovirus 5 genome.  Frank Graham described this cell line in 1977.
		PER.C6 cells, which are human embryonic retinal cells that are transformed by a clone fragment of the Adenovirus 5 genome, these cells were described by Frits Fallaux in 1998.
		Because there's been very little published on PER.C6 cells and a considerable amount of information has accumulated on 293 cells since they became available in 1977, much of our discussion today will focus on 293 cells.
		The talk by Dr. Alex van der Eb later this morning will discuss the origins and the characteristics of these cell lines.
		The regulatory issues associated with the use of designer cell substrates are similar to the issues associated with the use of other types of neoplastic cell substrates.  These issues include tumorigenicity and the ability of cells on tumors in animals, residual cell substrate DNA contamination, and the possible contamination with adventitious agents.
		And in contrast, the cells are transformed spontaneously, are derived from mammalian tumors that arise in animals or humans.  Designer cells have the perceived advantage of starting with cells that are known to be normal and are neoplastically transformed by a known mechanism.
		From a regulatory perspective, this type of information provides an additional level of assurance that unknown factors which might be present in the cell substrate of less certain origin are not available to enhance any risk to vaccine recipients.
		The issue that tops the list of concerns with the use of designer cell substrates and neoplastic cell substrates, in particular, is their tumorigenicity, which is their potential to grow into tumors when injected into rodents.
		For many years assays of tumorigenicity have been used to discriminate between cells that are suitable for vaccine development and those that are not.
		The risk believed to be associated with the capacity to produce tumors in animals are noted in this slide.  Tumorigenicity has been perceived to be a trait associated with high risk, and due to the possibility of transferring cell components, either DNA or proteins or possibly viruses, with oncogenic activity to vaccine recipients.
		However, proteins from tumor cells are unable to sustain neoplastic development, and they're unable to transform cells.  This leaves cell DNA and oncogenic viruses as the risk factors associated with cell substrates that are tumorigenic.
		In order for the Committee to appreciate what we mean when we talk about tumorigenicity of adenovirus transformed human cells, studies on the tumorigenicity of 293 cells are presented in the next slide, and they're compared with A-549 cells, a cell line that was established from a human lung tumor.
		I have to apologize for the transfer of our information by computer to the people making the slides because I became Lew is rather than Lewis, and the mouse obviously suffered a discrepancy as well.
		But in this slide, what we're looking at are a series of tumorigenicity assays, one done by Frank Graham and two done by myself.  In the original description of the 293 cell line, Graham reported that the cells weakly tumorigenic, and they produced tumors in only three of 20 animals inoculated with -- and I think this may be hard to see -- but that's ten million cells per mouse.
		We repeated this experiment ten years later, did a little more detailed inoculations and the animals inoculated with 100 million cells per mouse, ten million cells per mouse, and a million cells per mouse, and we basically discovered or found, got the same results that Frank Graham got in that of the number of cells required to produce tumors in mice was somewhere in the range of ten million cells.
		The way that these data are reported is in terms of the  TPD-50 value, which is tumor producing dose at a 50 percent endpoint.  That's the number of cells that's required to produce tumors in 50 percent of the mice, and these numbers are basically comparable.
		However, when you compare these to A-549 cells, which is the cells derived from human tumors, it only takes about 1,000 cells to produce tumors for 50 percent of the mice.
		Therefore, the A-549 cells are about 1,000 to 10,000-fold more efficient in inducing tumors in animals than are the 293 cells.
		Dr. Jim Cook is going to have a lot more to say about tumorigenicity of adenovirus transformed cells later this morning.
		The potential risk associated with residual cell substrate DNA in vaccines prepared in designer cells represents another concern.  DNA from neoplastic cells can contain activated oncogenes, viral oncogenes, the genomes of oncogenic viruses, latent viruses, as well as retrovirus proviruses.
		Clone cellular oncogenes can induce tumors in rodents, and DNA from oncogenic viruses and cloned viral oncogenes can also induce tumors in rodents.  Latent viral genomes in retrovirus proviruses sequestered in cell DNA can be infectious.
		Due to these observations, the possibility must be considered that residual DNA from designer cell substrates could transfer either neoplastic activity or infectious virus genomes to vaccine recipients.
		The talk by Dr. Peden later this morning is going to cover in detail the issues associated with the use of residual DNA.
		The third concern associated with the use of designer cell substrates is the possibility of adventitious agent contamination.  All cell substrates are subjected to possible contamination with adventitious agents.  Due to their laboratory origins, the designer cell substrates might represent a risk of adventitious agent contamination because they're neoplastically transformed and may be tumorigenic.  Designer cell substrates might represent a risk of contamination with unknown, possibly latent oncogenic agents.
		Dr. Krause in his talk this afternoon will address the issues specifically associated with evaluating designer cell substrates for adventitious agents.
		I'd like to conclude my talk by saying that today we are facing a transition.  By considering the issues associated with the use of Adenovirus 5 transformed cells, such as 293 cells, we're confronted with the first of the truly novel neoplastic cell substrates that we've discussed with the Committee over the past three years.
		As these cells fall into the category of being tumorigenic, they represent a transition from the previous way of thinking about cell substrates that goes back over four decades to future ways of thinking about cell substrates.
		As with most of these types of situations, this transition presents risks that must be confronted.  However, this transition also presents the possibility of future rewards.  Those rewards will come from the ability to maximize the benefits that can be obtained by the application of molecular technology to the development of safe and effective vaccines.
		The challenge facing us today is to objectively review the data that's available on these types of cells, determine what these data tell us about their potential to produce safe and effective vaccines.
		I think that's the end of the slides.  To assist CBER and the Committee in this review, we've invited those individuals who have introduced themselves to you whose work qualifies them as experts with sufficient experience with defective viral complementing cell systems and the issues they raise to review the relevant data before the Committee, to answer Committee questions, and to offer their opinions regarding the issues that need to be addressed.
		Before they begin to speak, I'd like to just take this opportunity to thank them for the time that they have used to assist the Office of Vaccines, the Committee, and the public in these discussions.
		This concludes my talk.  I'd be happy to try to answer any questions.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Lewis.  That provides a useful setting for us to continue hearing about this issue.
		It also reminds some of us that it's time for our annual visual screening test.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  We do have the opportunity run behind a little bit here in terms of scheduling if there are Committee questions.  Alternatively, we can get some more information on the table and then initiate discussion.
		Is there Committee input?  Dr. Goldberg, and then Dr. Griffin.
		DR. GOLDBERG: Yeah, just on your table of tumorigenicity where you show the rates of tumorigenicity in the 293 cells in the nude mice, can you give me some feel for how you feel that you can distinguish these levels?
		For example, you have -- I can't see.  I'm sorry -- you don't observe any tumors in four nude mice at ten to the sixth in one experiment and in another experiment you observe four of four.
		And you know, any calculations I do would suggest that you really with four animals can't distinguish.
		So can you give me some feel for what other information you're bringing to bear on this to make the distinctions about what the TPD-50 is?
		DR. LEWIS:  I guess I'm having a little bit of a hard time hearing what you were saying.  You're trying to understand how we calculate the TPD-50?
		DR. GOLDBERG: No, I think I do know how you do that, but my concern or my question really is:  how do you feel that based on these experiments with four mice at each of these dose levels that you can really estimate the TPD-50 with any certainty to make a distinction between --
		DR. LEWIS:  Okay.
		DR. GOLDBERG: -- ten to the three and ten to the sixth, for example?
		DR. LEWIS:  Basically, the data that was used to do this came from a series of titrations that we did on Adenovirus 12 transformed mouse cells.  These assays were repeated ten times, and each time they were done in four mice, but nude mice are expensive, and each time they were done the standard deviation of those assays was about plus or minus .6 of a log.  Okay?
		So based on the information that we obtained with that, we are reasonably confident that this represents an accurate way of reflecting this type of information.
		The data on the 549 cells and many of the 293 cells were repeated at least twice, and the numbers are basically the same.
		ACTING CHAIRMAN DAUM:  Dr. Griffin.
		DR. GRIFFIN:  Well, I guess I was being puzzled by the same table.  And maybe I just missed this, and I got a little clued in what you just said.
		The difference between the 293 cells and the A-549 cells is one is Ad. 5 transformed and the other is Ad. 12 transformed?
		DR. LEWIS:  No.
		DR. GRIFFIN:  And they have different --
		DR. LEWIS:  No.  A-549 cells are a cell line was established from a human.  I believe it's an oat cell (phonetic) carcinoma.  Okay?  And they were established directly from the human tumor in the tissue culture.  They are not virus transformed.  They are a cell line that developed from a human tumor that developed in nature, a spontaneous tumor in the human.
		DR. GRIFFIN:  So the point to be made from this is that cells differ in how likely they are --
		DR. LEWIS:  Well, yes, that's one point.  The second point is that it takes a large number of Adenovirus 5 transformed cells to produce tumors.  This is true both in adenovirus transformed mouse cells, Adenovirus 5 transformed hamster cells, as well as adenovirus transformed human cells.
		They fall into a category that most people would define as weakly tumorigenic, and this is a characteristic of Adenovirus 5 transformed cells.
		DR. GRIFFIN:  Thank you.
		ACTING CHAIRMAN DAUM:  Okay.  Thank you.
		I'd like to move on then at this point -- thank you very much, Dr. Lewis -- to Dr. Steve Hughes' presentation, entitled "Designer Cell Substrates for Vaccine Development:  Concepts and Issues."
		Dr. Hughes.
		DR. HUGHES:  This is somewhat smudged.  I may challenge people's optical state once again.
		Thank you.
		Since this subject has been so ably introduced by Andy, I'll try and go through this quickly.
		Basically the question to consider, of course, is how designer cell substrates, in fact, differ from other permanent cell lines  with transformed cells, and basically in the past, spontaneous transformation has been used to establish cell lines, and that simply means you take cells from an animal usually or an embryo and passage them in culture, and it's a particular characteristic of rodent cells that after some period of passage the cells undergo some sort of change, which we still don't understand clearly, that alters both their ability to grow permanently in culture and alters some of their physical and biological properties.
		The other way that cells have been immortalized or immortal cells have been derived is as was just mentioned, from tumors taken from either humans or animals, and in some cases these can be established directly in culture, and in some cases these tumor cells are then serially passaged in animals, and both of these methods have been used to establish a number of types of cells lines that basic researchers, such as myself, use routinely in the laboratory.
		And it's very convenient, but it has a particular disadvantage, and that is that in neither case do we have any clear notion of what changes have taken place in these cells, what it is about these cells that differentiates them from the normal cells that don't have the properties of either being transformed or growing forever in culture.
		And so one of the things that I think makes everyone a little nervous about these types of cells is not necessarily that they have something specific wrong with them, but, in fact, the very fact that we don't know what it is that has changed them.  We don't know how they differ from the normal cells that everyone feels reasonably comfortable with.
		And as an alternative to that kind of idea, what's meant, as Andy has just told you, by a designer cell substrate that differentiates it from these two types of cells is that one now can take specific -- which I have not spelled correctly -- specific DNA segments either derived from virus, derived from cells, that can change the growth properties of normal cells, and in so doing, we now have something in which we understand what agent it is that is causing the cells to behave differently.
		And that gives us some particular handle and some particular feeling that we have at least some idea of what's going on.
		This does not, of course, eliminate all the worries that one might have.  There are issues.   One of the issues is that there is the question of whether this specific DNA that when it's added actually has some sort of risk associated with it.
		Of course if this DNA segment is capable of causing the cells to grow forever in culture, it may have oncogenic potential, and in fact, as you've just heard discussed by Andy, there is some reason to think that in the case of the adeno early region that there is some oncogenic potential of that.
		So you really would worry about carrying the DNA if you're using a vaccine preparation with the vaccine material that you're going to use.
		So there is actually still a question of the degree to which this is a serious concern, and you'll hear more about that later today.  I would also be pleased to tell you one of the things that was discussed the last time this group met was that there should be in a sense a collaboration between the NCI and the FDA to try and get a more quantitative assessment of what the risk is in terms of using defined amounts of defined oncogenic DNA segments.
		I'm pleased to say that that interaction has reached the point where it's funded and that there will be some quantitative studies to try and establish exactly what the risk is at least from some defined DNA segments.
		As Andy has also mentioned, there is the issue of adventitious agents, that is to say, that any cell, whether it's a cell that is permanent in culture, whether it's a normal diploid fibroblast can be infected with virus, can have other agents associated with it.
		And in both of these cases, as I just tried to allude in the DNA, in part the question here is both understanding what sorts of things pose risks and, secondly, trying to understand how it is we can determine what agents, in particular for adventitious agents, what adventitious agents might be present.
		And so what I've tried to say is that the issue, I think, from the DNA is at least in part one of risk assessment, and I think that there actually are reasonable ways of defining what the risks are.
		One can take, particularly if one knows what DNA segments one is dealing with; one can take those DNA segments; one can take defined  amounts of those DNA segments; one can inject them into animal models, and one can define the oncogenicity, and based on that, one can get some reasonable measure, some idea of what it is that we're facing in terms of the risk.
		And in the case of adventitious agents, one of the nice things about modern molecular biology and biotechnology is that we now have much better ways of looking for at least nucleic acid bearing agents, and you'll hear, I think, a little later today in considerably more detail than I intend to discuss the sorts of things that are under consideration as ways of doing this.
		So the question then becomes given that we have these tools and given that we have these problems, what sorts of things should we do.  How should we go about trying to be as safe as possible?
		And I think one of the things, and I think it's going to come up in considerably more detail, is culture history.  You would like to know where the cells have come from.  You would like to know where they have spent time.  It's sort of like worrying about where your teenage children go at night.
		And there are a couple of things that I think are worth discussing, although probably only briefly, that might not be sort of immediately obvious if one just thinks about passage or culture history, and it's been alluded to, I believe, earlier that, in fact, the source of the serum and what might be in the serum turns out to be a substantial consideration.
		And this is true for both agents like BSE and, of course, for viruses as well.  And I think there's another issue that I don't believe has been discussed in any particular detail, but actually I think does matter not even so much for designer cell substrates, but for substrates that are derived in a sense directly from tumor material, and that's the idea that it's one of the traditional methods for deriving particularly cell lines from human tumors, is to passage the cells in mice.
		And there's a particular consideration which is one which makes for one of John Coffin's favorite stories, that suggests that there is an element of risk here that people don't always consider. 
		Mice, of course, contain several families of endogenous retroviruses, and some of these endogenous retroviruses preferentially replicate in cells derived from mice, and some actually replicate preferentially in non-rodent cells.
		And one of the things that happens when, for example, human cells are passaged through nude mice is that that provides a wonderful opportunity actually for xenotropic viruses -- these are the viruses that like to replicate in non-rodent cells -- to actually infect the human cells.
		And it's quite possible actually in this kind of culture history to add an adventitious agent that one would really not normally think would be one you'd have to look for in a cell derived from a human or a primate.
		So these sorts of considerations, I think, are very important and certainly I think we have to give substantial consideration to having a defined culture history not so much because it will necessarily rule out all possibility of adventitious agents, but we can understand if we know the culture history what sorts of adventitious agents we should look for.
		And one of the problems in searching for adventitious agents with, for example, the nucleic acid technology that will be used for many viruses is you only find the things you look for, and if you know what to look for, it makes your job much easier.
		And the final thing that I think is perhaps the most challenging part of the problem, the part of the problem that I'm not sure I have a particularly good idea yet how to resolve, is the issue of the stability of the genotype or the phenotype of the cells.
		And the reason this is a consideration actually goes back to the idea that I introduced the talk with that, in fact, you can derive cells, cell lines by simply passage in culture; that, in fact, there is such a thing as spontaneous transformation.
		And of course, not only is there spontaneous transformation, but upon passage the properties of the cells in culture upon prolonged passage can change.  They don't have to change, but change can occur.
		Now, that means that, in fact, the phenotype, for sure, and probably the underlying genotype has altered during the passage of the cells.
		Cell lines do change upon prolonged passage in cell culture, and so the question then arises if that is true, how do we gain confidence that the properties of the cells after some period in culture, in fact, match the properties of the cells with which we began.  That is to say if we're quite confident that we've made a designer cell line that has the desirable properties and has only the changes we put in and then we passage it for a long time, given that the cell lines can change, how do we know that the cell line hasn't changed?
		And that seems to me to be one of the substantial requests that we need to consider, and of course, one of the old standards of tissue culture people is simply to use cells that have been passaged a relatively small number of times, and that, of course, because these changes appear to be spontaneous, some sort of genetic accident, by using low passage cells, the chances that some change has taken place seems to be better.  The possibility that there's a change seems to be less.
		But the final thing that I think we ought to at least begin to think about is the idea that we might at least in some cases give consideration to using some sort of regulatable system to drive the expression of the gene that causes the cell to change its properties.
		And if we imagine, for example, that we have some sort of promoter that has a switch on it that we can turn on and off so that we can turn on and off the gene that we're interested in that is causing the cells to be transformed, then if that is the cell -- I'm sorry -- that is, if the gene we've added is, in fact, the agent that changes the properties of the cell, if we switch that gene off, then the cells' properties ought to fall back to that of the starting cell, which was not permanent or transformed.
		And I mean, it may be that I'm throwing this out as an idea, not as a solution.  You may not want to use necessarily an inducible promoter, but the idea that I think is central here is somehow to find a way to regulate the expression of the gene you're interested in, whether it's some sort of dominant negative effect either at the protein level, at the nucleic acid level or the inducible promoter.
		The idea that we want to, I think, think about is can we validate, can we determine after some passage that the agent that we think is changing the properties of the cell is, in fact, the responsible agent -- I'm sorry -- the gene, the designer gene that we've added, or have there been some additional changes in the genotype and phenotype that are influencing the behavior of the cell.
		And I think giving some consideration to that idea will be important, and I think at that point I'll stop, and if there are questions I'd be happy to answer them.
		ACTING CHAIRMAN DAUM:  Okay.  I'd like the questions at this time to be focused mainly on Dr. Hughes' presentation.  There will be plenty of time for more general discussion later.
		DR. COFFIN:  Steve, there's an issue you didn't raise actually that comes up particularly when one is considering these cells for growth of viral vaccines, and that is the potential of the cells to actually contribute genes to the vaccine virus itself by some sort of recombination and the consequences of that, and I think that's an issue that could arise, particularly if the retroviruses of a cell line has picked up an endogenous xenotropic virus or with the early genes of adenovirus.
		DR. HUGHES:  I deliberately, as i'm sure you're aware, avoided that issue both because I think there will be consideration of the recombination issue by others later and because I believe that the issues are somewhat different for adenovirus, which I think we're supposed to be focused on today, and retroviruses.
		And I think the issue of recombination and the mechanics of recombination particularly as they pertain to retroviruses are a bit beyond the scope of the discussion we have here.  So that was a deliberate omission.
		But I certainly think that as Dr. Coffin points out that the issue that he raises is a real one, and that we should give very careful consideration to issues of not only what the viruses can do to cells but, in fact, in some more complicated sense what the cells or things in the cells can do to the viruses.
		ACTING CHAIRMAN DAUM:  Thank you, Dr. Coffin and Dr. Hughes.
		Dr. Kim.
		DR. KIM:  Are there any designer cell substrates on the horizon or on the radar screen that are shown not to be oncogenic or less likely to be oncogenic?
		DR. HUGHES:  I'm not qualified to answer that.
		ACTING CHAIRMAN DAUM:  Would you like to try, in looking for answers to this question?
		(Laughter.)
		ACTING CHAIRMAN DAUM:  Sorry, but I do recognize you are number two in line.
		Dr. Aguilar-Cordova.
		DR. AGUILAR-CORDOVA:  Yes.  You talk about the transformation of cells and for a cell to become tumorigenic there's some old data showing a series of events.  So if one has only one agent, wouldn't that just make ten minus one  and the oncogenic event may still be there on that genotype?
		DR. HUGHES:  I'm not quite sure I precisely understand your question, but it certainly is the case that we now believe that for most tumors multiple genetic changes are needed, but many of the things that we regard as tumorigenic, whether they're chemical agents or viral agents, and these studies have been confirmed by genetic manipulation of mice, that anything we do that moves us one step closer to the required number, be it two, three, five, whatever it is, if we add any one thing to the list of changes, if we make any of the changes, that by doing that, by making the change, you do bring the cell closer to a transformed phenotype, and that you can show, for example, in mice by the p53 knockout mice, which have only a single change, get spontaneous tumors at a very high rate because one layer of safety has been removed.
		And I think that's the sort of thing that we would be nervous about, and of course, some of the cells may have more than one change so that you could actually from the DNA, depending on what the cell substrate was, actually deliver more than one of the things necessary to drive a cell in an animal or a human towards the transformed phenotype.
		So the single one is not good, and some of the cells may have had more than one.  So I think it makes us feel better that there are more than one, but I don't think it means that things were perfectly safe.
		ACTING CHAIRMAN DAUM:  Dr. Minor, please.  Then Dr. Kohl.
		DR. MINOR:  The tumorigenicity assays done in rodents for very, very good technical reasons clearly, but is it possible that there are actually species effects; that if you took the immune response out of things, that you would find a different tumorigenicity ranking in a different species?
		I mean, how relevant are the rodents do you think to a human situation?
		DR. HUGHES:  I think the answer is -- and this is by definition a speculation because the experiments, in general, can only be done in rodents.  So you have to sort of extrapolate.
		But there is enough good data, I think, from chemical carcinogenesis to make one believe that, in fact, there are very strong species effects in some cases.
		And I think that's a concern, but I think the choice in some sense experimentally is between doing the experiments in rodents, in which you have the worry that it may not perfectly reflect what happens in humans, and not doing the experiment at all.
		And while I have some reservations of the exact sort you mention, in terms of worrying about doing the experiments in rodents and applying it to humans, I certainly would rather have rodent data and try and worry about the extrapolation than have no data at all.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Kohl and then Dr. Myers.
		DR. KOHL:  That was my question.
		ACTING CHAIRMAN DAUM:  Dr. Myers, please.
		DR. MYERS:  I guess I have two questions.  On the confidence of the stability of the genome, would you be more confident if the designer segment were excised and the tumorigenicity was lost?
		And the second question is related to that, and that is could you tell us about the relevance of the tumorigenicity limited to nude-nude mice?
		DR. HUGHES:  I don't think it's probably technically feasible to excise the segment, but I think there are ways of setting up the experiment so that you can interfere with the expression.
		DR. MYERS:  Knock it out?
		DR. HUGHES:  Knockout technology is probably not the easiest, but the point I'm really trying to get at is what I think you want to look at is not necessarily the precise technology or necessarily even to limit yourself to a precise technology, but to be able to somehow develop either a technology or technologies that will allow you to ask the question that, in a sense, you're posing.
		If you then interfere with or obliterate the expression of the thing you think is driving the cells towards this permanent tumorigenic phenotype, does that, in fact, change the behavior of the cells as you would expect?
		And if you can do that, I think you're quite confident that there's nothing else going on or nothing substantial going on.  I don't know that I -- I think actually in terms of responding to the nude mouse question I'd feel more comfortable probably deferring to my colleague Don Blair, who I think is much more of an expert on tumorigenicity in nude mice than I am.
		DR. BLAIR:  Well, I guess the question is, if I understand it, is in an immunocompromised animal does the fact that the cell is tumorigenic have any relevance to normal situations, and I guess, you know, the argument would be that at least by demonstrating the tumorigenicity in the nude, you've shown the potential, and the failure to be tumorigenic in an immunocompetent system presumably arises from the immune response which could at some stage, at some mechanism be lost or be modified.
		So I think, you know, the demonstration of the tumorigenicity in a immunocompromised system is important because it does demonstrate that there is that potential, as opposed to no potential or what presumably is no potential at all.
		ACTING CHAIRMAN DAUM:  Thank you for clarifying, Dr. Blair.
		Dr. Lewis, then Dr. Aguilar-Cordova.
		DR. LEWIS:  Just in response to Dr. Kim's question about immortalized cells that are not tumorigenic, the only system that we're aware of in which that occurs by immortalizing cells with the human telomerase gene data suggests that it takes three different genes, telomerase, ras, and SV40 to transform a normal cell to a cell that is, in fact, tumorigenic.
		You can immortalize cells with hTERT with a telomerase gene, and those cells, as far as I understand right now from what we're aware of in the literature, are not tumorigenic.
		But so far nobody has proposed one of those as a designer cell substrate for our attention.
		ACTING CHAIRMAN DAUM:  Thank you, Dr. Lewis.
		DR. AGUILAR-CORDOVA:  My question actually follows very well on that, and it's a follow-up on what I started to say.
		So if it's a series of events, would a particular event, whether it be the telomerase, the SV40 T antigen or myc, be oncogenic depending on the background of the cell that it hits so that they're complementing oncogenes?
		And I guess that begs the question to whether any cell, tumorigenic or normal, is any safer depending on what the target cell is.  It would appear normal if it only has an activated end myc, for example.  It wouldn't appear tumorigenic, nor immortalized perhaps.
		DR. HUGHES:  Those of us who have spent many, many hours, days peering through microscopes at cells that had single oncogenes delivered by, for example, retroviruses that give very high levels of expression could in many cases see the effects either by change in morphology, change in growth pattern, change in behavior of adding a single gene.
		So I think there are certainly reasons to believe that even if it takes multiple changes to produce the Frank phenotypic tumorigenic phenotype in vivo, that the adding of individual oncogenes one at a time or in some cases removing or ablating tumor suppressor genes actually does substantially change the properties of the cells, even if it's not sufficient necessarily to drive the cell to its full Frank transformed phenotype.
		So I actually believe it is important to in some sense keep track, and I also think, as I tried to say earlier, that because these things are, in fact, additive as far as we know in humans, that providing one or two steps in the direction of the oncogenicity is something we'd like to avoid, I think.
		One could argue in many cases that chemical carcinogenesis provides exactly one or two of the changes and the long duration that you see to the development of the tumor after the initial exposure to the chemical  insult actually represents the fact that the chemical may have changed only one or two things, and the rest must occur spontaneously later.
		But that still enhances the risk substantially, and I think those are things that we need to worry about.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Hughes.
		I think we'll move on, if you would.  That was a very helpful presentation, to Dr. Aguilar-Cordova -- I hope I'm not butchering your name -- who will tell us about adenovirus biology as related to development and use of adenovirus vectors.
		DR. AGUILAR-CORDOVA:  Can you hear me?
		Yeah.  So I'm just going to give a general, generic background on the adenoviruses so that we can use this for further discussion and their use as vectors.
		So adenoviruses were identified in the early '50s from an adenoid tumor, from an adenoid tissue, thus the name adenoviruses, and it's associated with some fairly common illnesses in the lay group referred as the "common cold," some eye inflammations, et cetera.
		It's composed of a linear, double stranded DNA encapsidated in a protein shell; has no envelope, and there are many different types of adenoviruses in nature.
		They are primarily classified based on the organism of origin, and so there are two major groups:  Mastadenoviruses and the Aviadenoviruses, those that come from mammals and those that come from birds.
		Hopefully nobody else has slides.
		(Laughter.)
		DR. AGUILAR-CORDOVA:  The further characterization is in the antigenicity of the terminal knob in the fiber protein and hexon epitopes and, thus, the serotype, and you'll hear about Serotype 2, Serotype 5, the most commonly used, and there are many other different serotypes, and also by hemagglutination, binding of the fiber protein to red blood cells.
		And it turns out that some groups of adenoviruses have more tumorigenic ability in rodent cells.  None have been shown, as you will hear probably later on, to be tumorigenic in humans.
		The virus is icosahedral.  It has 240 hexons, 12 for each of 20 triangular phases and 12 pentons, and as you can see, it has these little fibers.  The fibers and the pentons and the hexons are what constitute the serotype of the virus.
		Inside that icosahedral protein base there is a double stranded genome flanked by two terminal repeats and some proteins that go with that genome, and here's the list of them.
		Primarily the terminal protein is important to keep the stability of the genome and to condense it.  
		The gene structure is linear as well, but it does express from both strands.  What you've heard here is E1A and E1B.  The E1 region, these are critical for expression of other genes of the virus, and thus the majority of the vectors that are used in adenoviruses are vectors in which these two genes have been deleted and replaced by the gene of interest.
		There are two origins of replication.  In the ITRs, inverted terminal repeats, transcription units include five early genes, the E1A and E1B that I just mentioned, the E2 region, the E4 region, and the E3 region.  I'll talk a little bit more about these.  There are two delayed early and one major late transcript that includes late one through five.
		The E1A, there are two proteins, and this is host activation, and what this really does is activate the transcription and induce hosts to enter S phase, and that activates a transcription of most of the other adenoviral genes.
		E1B is also two proteins, and it induces cell growth, and we know no what E1A and E1B do this in part by binding to some of the cellular genes that regulate cell cycle and expansion, like p53 and retinal blastomagy (phonetic).
		E2 has three proteins.  It's involved in virus reproduction, DNA replication, in particular.  E3, there are four proteins.  It's believed that this is protection from viral infection, and thus it down regulates the ability of the cell that has the virus to be immunogenic.
		One of the down functions is that it down regulates the expression of MHC Class 1, and thus the host can't recognize the other proteins going on.
		And the E4, there are at least four proteins, and it has miscellaneous activities, such as regulation of transcription, MRNA transport, and DNA replication of the virus.
		The late genes, there are five of them.  They're mostly involved in the structure of the virus itself and the stabilization of that core.
		The viral life cycle and replication is really two faces.  There are early events, which occur in the first six hours after infection, and that includes absorption, penetration of the virus into the cell, disassembly of the virion core, and transcription and translation of the early genes.
		One that begins, the late events by definition start, and that's in the next 18 hours or so, and that is when there is the construction of new virions.
		And we have approximately ten to 50,000.  Traditionally it was said there's about 10,000 virions per cell.  We know now that there are -- when we produce them in the laboratory, we can produce a lot more than that.  So wild type probably also does more than that.
		Now, the next step, of course, is that's the virus.  How does one use the virus to optimize gene transmission and, therefore, its potential use in gene therapy?
		And there are two key factors of adenoviruses that actually have allowed us to use this variance as good, effective vectors.  One is that we can package up to 105 percent of its capacity.  So when we take out the E1A or the E1B genes, that gives a little bit of space, and then you have actually a five percent wiggle room there.
		And the other very important issue is that one can manipulate the virus in a circular form.  That is, the ITRs can be circularized in a plasmid-like structure, and one can clone and change contents that way.
		So given those two factors, we can then manipulate the virus easily and make it an efficient vector. 
		This obviously you will not be able to read, but it is to show that there are many different viral vector vehicles that one can use.  The most common ones are retroviruses, adenoviruses, and associated viruses and Herpes viruses, and they all have pluses and minuses depending on the use that one will have for them.
		Retroviruses are often used as an advantage, which is that it will enter the cell efficient, integrate so there will be stable expression for long terms, and that one has no viral genes in the most common of these vectors.
		And the disadvantages is that they're hard to produce.  They have a limited insert size, and that they may integrate randomly and thus cause mutation.
		On the flip side for adenoviruses, the advantages often listed are that it enters cells sufficiently, produces high expression of therapeutic gene, the transgene of interest; does not integrate into the host chromosome, and thus the disadvantages that are often related that it's not a long-term expresser; that the viral genes are often in the vector so that it's immunogenic, and it gets disposed of by the host fairly quickly.
		In the case of vaccines, this may be a very useful disadvantage.  So it may not be a disadvantage.
		The same as for the other vector types, there are advantages and disadvantages depending on what one needs to use them for.
		So typically on first generation vectors what one does is put the gene of interest instead of the E1A/E1B region.  The E3 region is often deleted to create a little bit more space.  The E3 region is totally for all apparent reasons irrelevant for in vitro expansion.
		But as you can see, there would be a lot more of the viral genes that are still within the vector, and even though there is no E1A, there is some leakage and some expression of those viral genes after transduction.
		So typically then one would create this in the laboratory, clone whatever gene of interest one wants, use that plasma to transfect a packaging cell.  In this case we're talking about 293 cells or PER.C6 cells.  These are cells that express the E1A/E1B constitutively.  So they can entrance, complement the deficiency of this vector, and then one can produce a lot of those virions in the laboratory.
		And theoretically once that virion then gets used to infect the target cell, it will not make anymore virions because it will not have this E1A/E1B region.
		In that type of vector, the virus has approximately 8 kb of space for foreign DNA, as I mentioned, its replication deficiency.  One can product them in very high titers in the laboratory, often close to ten to the 13th viral particles per milliliter.  It affects a variety of tissues.  It goes through a receptacle CAR for coxsackievirua adenovirus receptor, and it's fairly prevalent throughout nature, and then it also uses some integrants.
		And it can get into nonreplicating tissues, such as antigen presenting cells, and it can have high levels of transgene expression.
		Now, there have been many different evolutions of the types of adenoviral vectors that are currently in use.  What I've described to you so far is what is referred to as the first generation.  That's E1 minus, and it can be E3 positive or minus.
		The second generation vectors or so-called second generation is E1 minus as well, and then they had an additional mutation either in the E2 gene or E4 gene, and again, E3 positive or negative.
		They haven't been quite referred to as any generation.  I just called it generation X here.  They are the ones that are E1A positive, E1B minus, and then the E3 region that's again positive or minus, and various farther generations like X.1 here is E1A and E1B with a conditional promoter so that they replicate only specific tissue types in which that promoter is active.
		And the final generation at least so far is X.2, which are helper dependent, and these are closer to what a retroviral vector would be like in as far as viral gene content.  Everything has been deleted from the vector backbone, except the ITRs in the packaging sequence, and it has been replaced by some DNA content.
		It does require at least 28 kb of DNA.  So often these vectors come with stuffer DNA of some origin.
		I want to give you a couple of examples though of how this generation of changes may not be that critical in the development of vectors for their use.  For example, this is data from O'Neill, et al., in which they were using a second generation and comparing it to a first generation vector.
		Here the dark bars are a vector that has the E1 and the E2 region deleted, deletions in the E1 and the E2 region, and the light bars are just an E1 vector.  At one time it was ten to the -- these are dose per kilogram -- one times ten to the 12th, three times ten to the 12th, and one times ten to the 13th, and this is the platelet count of the animals or mice.
		As one can see, the toxicity was perhaps slightly different.  One times ten to the 12th dose, but it equilibrates very quickly at three times ten to the 12th and one times ten to the 13th.
		So the thrombocytopenia that is often caused by this virus was no different in a first generation than the second generation.
		And here is again using liver enzymes, which is another known toxicity of this adenoviral vectors, and you can see that one times ten to the 11th, one times ten to the 12th, and then only a three times ten to the 12th is there a difference, and again, at one times ten to the 13th there's no difference.
		So maybe the changes from first to second generation might give a slight window of difference in as far as the toxicity, but the profile seems to be the same.
		Now, this was not the case when we got to the generation X.2, the gutless or helper dependent adenoviral vectors, and here what we see is a first generation vector with alpha-1 anti-trypsin, and this is a gutless vector with the same insert.
		This is the level of expression through time.  These are weeks, and you can see with the first generation it's a peak of expression within the first few days, leading to baseline, no expression later.  These are just different doses of the vector ranging from 3.2 times ten to the 11th to 1.2 times ten to the 10th.
		And you can see  that with the gutless vector, expression is much prolonged.  Putatively, this may be due to the fact that there are genes, viral genes in the first generation that are still being expressed and that are still being immunogenicity against that content of genes.
		And here is the toxicity profile.  This is the liver enzyme toxicity profile, and you can see a peak.  That is very clear in the first generation vectors, and it does not occur at any of the doses tested here with the gutless vector.
		But it is not only the adenoviral genes that can induce the immune response.  In fact, it is also the trans-gene that can induce the response.  This is some data from Morall, et al., in PNAS in 1999, and what we see is in the little light blue color is a gutless vector, and it's human alpha-1 anti-trypsin again, and in the multi-color lines is a first generation vector.  This is in baboons.
		And what we see is that two of the three animals that received the gutless vector have a very long term expression.  This is out 100 weeks here, and all of the first generation vector animals had lost all detectable expression by 20 weeks.  Most of it was lost by ten weeks.
		But there was one animal with a gutless that actually lost all expression by ten weeks, and that animal, in fact, had developed an immune response to the alpha-1 anti-trypsin.
		So these vectors can be used to generate an immune response to the coded gene.  Of course, in addition to the variables that I just spoke about, there's variables for the analyses of the vectors, and I will just go very, very brief couple of slides on this mostly to give you a sense of what is not known rather than what is known.  I will not go through all of the safety testing of quality control that's normally done for all viral agents.
		And in order to do that, I want to just give you an idea of how these vectors are typically quantified, and so we have the quantity of particles that we can detect easily, but then we also need to know of those particles how many of them are functional and can transduce the gene of interest into the target cells.
		Often what we do is we just layer a soup fully of particles on top of some target cells.  The problem with this is that these particles, depending on how deep this soup is, how long the assay gets allowed to progress, and many other variables, most of these particles will never reach one of the target cells.  So may not be detected, and whether they get detected one time or another is quite variable.
		So much so that we sent out an identical preparation to six laboratories, and we had a two log differential in the determined titer from those laboratories, all experienced with adenoviruses and also how they get handled may be very specific.  This is just a bit of data from a paper by Neiber Hoffman, et al., and you see here you can't tell the distance, but there's a seven log differential on the vector that was shipped just across town into a clinical setting as an experiment for shipment only from a vector that was shipped on dry ice and a vector that was not shipped on dry ice.
		So it turns out in that particular case it was the CO2 that was seeping in, dropping the pH, and there was a seven log differential that could be lost in a very short period of time.
		So in conclusion, adenoviruses, I think, I've shown that due to their biology they can be converted into efficient gene transfer vehicles.  They're not inherently dangerous, even as wild type viruses.
		Not all adenoviral vectors have equivalent toxicity profiles.  Additional safety of the second generation vectors may be transient and only in a small, temporal stage.
		And this is actually very clear because standardization of the dose specifications is necessary, and I'm told there is a very standard way of determining the potency and the quantity of these entities.  It is very difficult to analyze the data as a whole, and thus, assurance of clinical potency.
		And just as a last mention, there is currently a working group that is developing a standard of wild type adenoviruses so that all of these things can be compared and quantified.
		That's all I have.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		That was extremely helpful.  It raises many questions for us to consider.
		Dr. Coffin and then Dr. Kohl and then Dr. Stephens, Dr. Katz.
		DR. COFFIN:  You said something early on regarding the difference between adenoviral vectors and retroviral vectors that I think has something of the status of an urban legend without ever actually having been subject to a real test, and that is the idea that retrovirus vectors might have some greater danger because of the property of integrating their DNA into a cell, in the cell DNA, whereas adenoviruses don't do that.
		That's actually not true, of course.  Adenoviruses do, of course, -- adenovirus DNA does get integrated after infection at some low event, and I believe that the -- I don't know exactly what the numbers are and I don't know if anybody knows exactly what the numbers are, but it's quite likely that you make up that entire difference in efficiency in integration by the difference in the doses of adenoviral vectors versus retroviral vectors that you give, and in fact, the probability of the integration of a fragment of adenovirus DNA may be equally high as a probability of retrovirus DNA integration after administration of these as gene therapy or vaccine vehicles.
		Can you comment on that?
		DR. AGUILAR-CORDOVA:  The fact is that they do integrate occasionally.  It's not their standard method of operation, but they do integrate occasionally.  I didn't mean to imply that they were more dangerous or not.  I was just reading off a list of often stated events.
		The one difference would be that they do no carry a promoter enhancer in there, in the LTR.  So at least the promotion enhancement effect of the integration would be different.
		DR. COFFIN:  But there must be one somewhere in the vector or it wouldn't be any good as a vector, and often more than one, of course, if --
		DR. AGUILAR-CORDOVA:  Often, yes, and so how they compare as far as integration I can't comment.  I don't know that there is any data.  There is data showing that they do integrate on occasion, yes, and they persist.
		ACTING CHAIRMAN DAUM:  Dr. Kohl, please.
		DR. KOHL:  Two questions.
		Thanks for your talk.  It was enjoyable and elucidating.
		You mentioned the concept of leakage.  Could you elaborate that a little further, and can you get enough leakage from a gene that supposedly is deleted that you can get a competent virus injected into the host?
		DR. AGUILAR-CORDOVA:  So the leakage that I was speaking about was from the genes that were not deleted.  Obviously the one that's deleted can't leak on there.
		However, there is the potential for recombination with the gene inside the packaging cell and thus leading to a replication competent virus.  I believe that that will be spoken to as far as why the PER.C6 cells were developed, and the difference between them and the 293 cells.
		DR. KOHL:  Can you elaborate a little bit more on the toxicities of the adenoviral vector?  What causes that?  What's the mechanism of the toxicity?  The platelets, the liver function transmission and other toxicities as well?
		DR. AGUILAR-CORDOVA:  Yes.  So what we know are what the toxicities are, and particularly, of course, as you've heard, there was an incident in the University of Pennsylvania where a young man died due to a large dose of an adenoviral vector directly injected into their hepatic artery.
		In that case what was seen was a DIC like syndrome with upper respiratory distress, and what we have seen in many animal models is an elevated liver enzyme content, often transient and recoverable, and not just animal models.  Many Phase 1 studies have seen the same thing.
		Thrombocytopenia, believed but not shown to be caused due to endothelial cell damage and leakage because it is consumptive thrombocytopenia.
		DR. KOHL:  Who does the mechanism?
		DR. AGUILAR-CORDOVA:  The mechanism is probably cytopathic effects of the vector and potentially an immunological response to the original infection.
		ACTING CHAIRMAN DAUM:  I have Dr. Stephens, Dr. Katz, Dr. van der Eb.
		DR. STEPHENS:  In this discussion, there's some at least in my mind confusion about the issues of gene therapy vectors versus vaccine vectors, and I'd like you to kind of clarify that issue for us, if you would.
		More specifically, the question relates to the E3 sequence and whether you think that should be in or out of the vaccine delivery vector.
		DR. AGUILAR-CORDOVA:  Sure.  So not just in this discussion is there a confusion of vaccine or gene therapy.  I think as a member of the RAC we've had that discussion a lot, too, what should be accepted or not, and many of the gene therapy applications especially in cancer are, in fact, vaccines.  We're trying to vaccinate against cancer.
		And Dr. Ginsberg started working with an E3 region in early '80s, I believe, or before, and he's  big proponent of leaving the E3 region in when one wants to create an immune response because in that situation, it is possible that the adenovirus itself is an adjuvant to whatever one wants to create an immune response against.
		The flip side of that is that in some studies in my laboratory and others, when transducing a gene that one wants to create a CTL response against, for example, with an adenoviral vector, we tend to get an awful lot of CTL against adenovirus and very little to none against the gene of interest.
		So it could go either way.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Katz, please.
		DR. KATZ:  What is the receptor for adenovirus and what cells express the receptor?
		DR. AGUILAR-CORDOVA:  The known receptor, and there are probably others, the known receptor is a module called CAR, coxsackie adenovirus receptor, and its distribution is fairly ubiquitous.  Epithelial cells are especially high expressers, and it also uses some integrants as co-receptors.
		ACTING CHAIRMAN DAUM:  Dr. van der Eb.
		DR. VAN DER EB:  I'd like to come back to the issue of leakage, leakage that you mentioned.  Even a deleted or undeleted vectors are supposed not to express the rest of the viral genome that is still present in the vector.  That is because the E1A gene is the master switch that directs the expression of the rest of the viral genome.
		Now, it's known already for a rather long time that leakage may occur and expression of the rest of the viral genome occurs when very high multiplicities of infection are used.
		For reason then this creates a kind of E1A-like activity in the cell and leads to expression of the rest of the viral genome with consequent reaction of the host cell immunological reaction of the host.
		DR. AGUILAR-CORDOVA:  Right.  So in order for efficient transcription of the other genes, the E1 region is necessary.  However, it seems like there is some gene expression, although maybe not at sufficient levels to produce virions and to create the E1.
		But given the data, there seems to be some expression of the other viral genes even in its absence.
		DR. VAN DER EB:  Is the data of Tom Shenk from long ago?
		DR. AGUILAR-CORDOVA:  Right.
		ACTING CHAIRMAN DAUM:  Two last questions, please.
		DR. COOK:  I'd just like to respond to the question about E3 in or out.  E3's job from the virus' point of view is to shut down Class 1 expression or travel to the surface through Golgi mechanisms.  So theoretically if that were uniformly true and you were trying to make a vaccine and it required expression of that peptide in the cell in which E3 was co-expressed, it would be a good idea maybe not to have E3 present.
		The truth is when you infect with adenovirus in normal human cells or cells that don't express E1A, that phenomenon is very late in infection.  It doesn't happen until probably 48 to 72 hours after infection with that virus.
		So chances are the peptide expression could occur, depending on what the kinetics are.  If the cell co-expresses E1A, the E3 effect is much greater.  So it might depend on how you rate the system, but theoretically, at least, one would have to consider whether E3 is downregulating Class 1 expression on the surface and whether that alters antigen presentation or peptide expression on the surface of the gene of interest.
		ACTING CHAIRMAN DAUM:  Thank you, Dr. Cook.
		Dr. Blair.
		DR. BLAIR:  Yeah.  This comes out of my retroviral background, but is there any evidence that there's an encapsidation of cell DNA/RNA protein in the virus as it's assembled?
		DR. AGUILAR-CORDOVA:  Not that I know of.  Not at the level that we've seen in retroviruses certainly with the RNAs, especially of viral-like proteins or viral-like particles and so on.
		But certainly there's the possibility of that, and certainly there's the possibility of recombination events, non-specific recombination events that would package random pieces of DNA.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Aguilar-Cordova.
		And we will shorten the 20-minute scheduled break to a 15-minute break.  I have 10:40.  We'll reassemble at 10:55 and continue with Dr. van der Eb.
		(Whereupon, the foregoing matter went off the record at 10:39 a.m. and went back on the record at 10:58 a.m.)
		ACTING CHAIRMAN DAUM:  Would everybody please settle down as quickly as they can?
		ACTING CHAIRMAN DAUM:  We're ready to continue with the open session.
		We will now call on Dr. Alex van der Eb to tell us about adenovirus transformation of human cells and the development of 293 and PER.C6 cells for the manufacture of defective adenovirus vaccines.
		Welcome, Dr. van der Eb.
		DR. VAN DER EB:  Thank you.
		So what I would like to do is to describe to you how and why we have made two different cell lines, adenovirus transformed human embryo cell lines which are called 293 and PER.C6.  Both cell lines were made in my lab, and also the cells, the starting material, was prepared by myself at the University of Leiden.  The 293 cell was made by Frank Graham in 1973 from human embryonic kidney cells that were made from fetal tissue one year ago by myself one year before that, so that was in 19 -- probably in 1972, whereas the PER.C6 cell was made by Ron Bout and Frits Fallaux in 1995 from an embryonic  retina cultures that were made from fetal tissue by me ten years before that, in 1985.
		This just shows you again the adenovirus genome and you have seen it already.  The interest in this virus was due to the fact that the viruses can transform cells in tissue culture.  In fact, all human adenoviruses or almost all can transform cells in tissue culture, and also that certain types of adenoviruses can induce tumors in experimental animals.
		The transforming region -- oh, I hope it survived -- the transforming region is associated with the left-most about ten percent of the genome that harbors the E1 region.
		We became interested in transforming -- in the question whether human cells could be transformed, and therefore, I will tell you how we got the transformed human cells, and it all started actually in 1972 when Frank Graham in my lab developed the calcium phosphate DNA transfection technique, which made it possible in the first place to make infectious virus with intact viral DNA.
		If you transfect the intact viral DNA of Adenovirus Type 5 into permissive human cells you get infectious virus, but it also turned out -- do you have a pointer here?  There is no pointer?  Okay.  Thank you.
		And it turns out that not only it was possible to get infectious virus by transfecting human cells with the intact viral DNA, but also purified DNA proved capable of transforming cultured rodent cells, but human cells could not be transformed.
		And the reason is that these human cells got destroyed by the viralytic (phonetic) reaction.  If the DNA was sheared, however, up to 3 mega delta Daltons, up to three million Daltons, it turned out that  the transforming potential of rodent cells still remained intact, indicating that only a portion of the viral genome, a rather small proportion of the viral genome is necessary for transformation.
		As I said, purified Adenovirus 5 DNA transfected into permissive human cells yields effective virus, but human cells could not be transformed, everywhere interested in transforming human cells by adenoviruses just in order to find out whether that is possible.
		But we found some evidence that permissive human cells could be perhaps transformed from the fact that semi-permissive rodent cell cultures could be transformed if the DNA of adenovirus was sheared into smaller pieces, and these were Syrian hamster kidney cells.
		The transforming activity, and this was done with detailed shearing studies at that time; there were no restriction enzymes.  There was no DNA cloning at that time; that the transforming activity associated with the 11 left percent, left-most 11 percent of the adenovirus genome, and this all affect the basis of the transformation by adenovirus of the human cells with fragments of adenoviruses.
		So the reason why we wanted to transform human cells is just to answer the question whether human cells can be transformed at all by adenoviruses, human adenovirus, and if so, which part of the adenovirus DNA is required to transform cells?  Is that the same area that is also needed for transformation of rodent cell or is it less or is it more?
		And then can we simply develop a model to study transformation of human cells?  And that was at that time important because although there was no evidence that human adenoviruses have anything to do with cancer in humans, it was still an open issue, an open question, and in fact, it is at this moment still, although clearly there is no evidence that human adenoviruses have anything to do with cancer in men.
		So the method that we followed was take human embryonic kidney cultures.  Why kidney cultures?  And that is mainly because of the fact that the rodent system, the rodent model that we used were always baby rat kidney or baby mouse kidney or baby hamster kidney.  The kidney cells were very suitable for these transformation studies with sheared adenovirus DNA.
		When we transfected these human embryonic kidney cultures with sheared purified Adenovirus 5 DNA with the calcium phosphate technique  using carrier DNA of salmon sperms.  So this was not restriction enzyme fragments.  They were just not yet usable.
		One year later we had resection enzyme available to make pure DNA, but the first and also the 293 cell was made with sheared Adenovirus 5 DNA, then simply scored for transformed colonies as we did with the rodent cultures.
		So the kidney material, the fetal kidney material was as follows.  The kidney of the fetus was, with an unknown family history, was obtained in 1972 probably.  The precise date is not known anymore.
		The fetus, as far as I can remember was completely normal.  Nothing was wrong.  The reasons for the abortion were unknown to me.  I probably knew it at that time, but it got lost, all this information.
		The kidneys of the fetus were then isolated and the kidney cells were isolated in the so-called still air cabinet.  There were no laminar flow hoods at that time, and this is simply a still air cabinet that was also used all over for tissue culture and worked quite well.  There was UV lights in it just to sterilize it, and that was all.
		So as we did also for the rat kidney cells, the surrounding membranes were removed as completely as possible, and the kidneys were then minced with scissors, trypsinized, and the cells that were recovered after removing the trypsin were cultured in medium containing bovine serum, calf serum.  That is what we know.
		And this calf serum was obtained not from a commercial source.  We either got it from somebody else, from another lab, or we made it ourselves from blood, calif blood.
		Rodent, monkey, and other human cell cultures took place in the same general area at that time.  So there was one cell culture room, and there all of the experiments, all the cell culture work was being done.
		There was also experiments with viruses, but that was in a separate virus cultured unit, and we used in addition to Adenovirus 5 whole viruses, also the oncogenic Adenovirus 12, as well as SV40 and possibly also already Herpes virus, but maybe Herpes virus was not yet used at that time.
		So the method was DNA from wild type Adenovirus 5, was isolated from virions.  So we had to prepare the DNA by first growing and purifying the virions, and the DNA was then fragmented by shearing in this case through a 22 gauge needle up to about eight million Daltons.  There was no cloning strategy at that time, and the DNA fragments were transfected as I already indicated with salmon sperm DNA with the calcium technique.
		The results were rather disappointing.  In the first experiment of quite a number of dishes there were not a single transformed colony.  So we repeated it.  Again, no transformed colony.
		However, after many other experiments, we found finally one transformed colony which was visible in the cultures, and that colony appeared 33 days after transfection was seen, 33 days after transfection.
		This colony, this single colony was picked and established and became the 293 cell.
		There were two colonies here mentioned, and that is because one, the second colony, was only seen after the cells at the end of the experiment, the cultures were fixed and stained, and one other colony was seen at the edge of a dish which we had missed.
		So the single transformed colony which would give rise to 293 was very difficult to expand.  The cells hardly grew at all, and after five months in culture, it was possible to freeze down the number of ampules.  Only three ampules, passage four was that.
		And at that time the cells started to grow faster, but still relatively poorly and a doubling time was at least a week or more than a week.
		So it appeared from these experiments that human cells are resistant to transformation by Adenovirus 5.  Although these cells replicate extremely well in replicating Adenovirus 5, they cannot be transformed with the same DNA that also transforms rodent cells quite efficiently.
		So up to this moment, it's still unclear why human cells are resistant to transformation by adenoviruses.  One possibility is that the 293 cell came out of a cell line that had some kind of a mutation so that it became permissive to transformation.
		Another possibility is that since this is primary human embryonic kidney which consists, of course, of many different cell types, that there is one -- that there are very few cells in the whole culture which are permissive to transformation, and that this particular cell came from one of those transformation prone cells.
		We will never know probably, but one possibility is that the 293 cell is actually a neural cell that was present in that culture.  We don't know, and this is something that occurred to me when I was traveling here to Gaithersburg, and so that is a possibility that can probably be tested because neural cells appear to be more prone to transformation by adenovirus.
		We also tried human diploid skin fibroblasts for transformation, never any positive result.  We also have tried human embryonic lung cells.  No positive transformation.
		Anyway, around page 13, the cells went into crisis, the same type of crisis that is also seen when SP40 transformation is followed in human diploid fibroblasts, for example.  They always go in crisis.
		This crisis lasted nearly three months.  During that time, the cells remained on the dish or gradually started to die, some of them at least.  So you have to defeat the cultures for a long time.  Nothing happens.  There is no cell division.
		And then the culture started to recover for some reason not in the same way as usually in the case of SV40, but apparently cells all over the plate begin to grow, whatever that means.  We don't know what happens in this crisis base.
		After crisis, when telomerase is activated, apparently when the cells start recovering, the cells were subcultured and the growth rate increased significantly.  Several ambules were shipped then by Frank Graham to McMaster in Canada, where he went to Anestilles (phonetic) in 1974, and the data have been published in several papers.
		I would like to show here also the part of the adenovirus genome present in the 293 cells.  It is not completely sharp, but it doesn't really matter, and this is the left-most 4,041 nucleotides.  There is also some E4 region present in these cells, which is not expressed, however.  So this is in the 293 cells.
		Now, in addition to basic research, adenovirus DNA also became interesting.  Adenoviruses became interesting as factors for gene therapy.  So this occurred in the '80s when people started to think about gene therapy, introducing genes into cells, first retroviral vectors, and also later adenoviral vectors.
		And adenoviral vectors, in contrast to retroviral vectors, in fact, have as have been already said today a deletion in the E1 gene and in the place of the E1 gene, you can clone the gene of interest, and the reason that E1 deleted adenovirus was chosen was, in fact, the present or the availability of the 293 cells, which turned out to be very suitable packaging cell line for these first generation viral vectors because they expressed the  E1 genes.
		So E1 deleted recombinant adenovirus vectors were being used more and more for gene transfer purposes.  Adenovirus vectors, you've heard it, are quite suitable for similar reasons.  They have certain disadvantages also compared to retroviral vectors, for example, but they certainly have a number of important advantages.
		The cells to grow the replication deficient E1 deleted adenovirus vectors were also available, and those were the 293 cells, and in fact, already in 1994 the first clinical study with an E1 deleted adenovirus vector was done, was made by Crystal in '94, which probably was the, if I remember correctly, the CFTR gene.  That was the first clinical trial gene in an adenovirus vector.
		So there was a new use now for the 293 cells.  Two, nine, three for quite some period of time was the only packaging cell line available for growth of adenovirus vectors.
		E1 deleted vectors for gene transfer became common practice.  More and more groups started to use adenoviruses as a factor, but there were also limitations to the available packaging cell line, 293.  It became apparent at that time, and that is that due to recombination between E1 sequences from the 293 cells into the E1 deleted vector could occur, and this gave rise to formation of replication competent adenovirus, RCA, and it turned out to be very difficult to produce large batches of RCA free vector, and of course, the RCA is almost identical to the vector with the gene of interest, and therefore cannot be physically separated from the vector.
		So it was clear that 293 was not really the ideal vector for gene therapy used, and therefore, we decided around 19 -- oh, this first, just to show you here, here is the recombinant vector in which the E1 gene is deleted, and instead of it, the gene of interest can be inserted here.
		And here are the 293 cells with a proportion of the adenovirus genome integrated with the E1 gene and P9, protein 9, and there turns out to be quite a considerable overlap at both sides between the recombinant adenovirus vector, first generation vectors of both sides of the E1 region.
		And be recombination you can get back RCA, which is essentially, again, the wild type adenovirus.
		So the combination of the E1 gene in the vector yields wild type virus, which is capable of replicating.  This could cause toxicity, high concentrations perhaps of virus in places where you don't want it.  It could give rise to uncontrolled dissemination not only of the wild type virus, but also of the recombinant factor that replicates together with it in the same cell.
		It could theoretically also yield new virus strains in the case of capsid modified vector where the capsid is modified in such a way, for example, that it can attach to other receptors and other cells.  So in that case when that becomes replication competent you can say that this is a partially new virus that you have created.
		Also, it could yield, give rise to replication deficient E1 containing viruses in the case of multiply deleted vectors, for example, vectors that in addition to deletion of E1 also deleted in E2, E2A, for example, when E1 is reinserted into the vector, it will still be replication deficient because the E2A gene is absent, but this vector, in theory, is immortalizing or transformation competent virus that you have created.
		So in 1995, Brahm Bout (phonetic), Frits Fallaux, Brahm Bout from IntroGene and Frits Fallaux from our university Gene Therapy Group, decided that we should try and make a new helper cell line and matching factor in such a way that there is no sequence overlap between the factor and the advanced sequences in the cell line.
		And indeed, in order to make a new system that allowed pharmaceutical production of adenovirus vectors, three of RCA.  It should also meet pharmaceutical standards.  If you start all over again you can just as well try to do that, and it could be the basis for the manufacture of multiply deleted Adenovirus 5 vectors also.
		So we choose the human embryonic retina cells at that time.  Why not kidney cells?  Simply because these cells were so resistant to adenovirus transformation that we didn't think it would be worthwhile to try it all.
		Human embryonic retina was chosen because Gallimore had shown not long before that that human embryonic retina was permissive to transformation, could be transformed by adenoviruses, Adenovirus 5 and 12, and that was, again, based on some other studies in animals, and therefore, we decided to take human embryonic retina cells.
		So they can be transformed by Adenovirus 5, and also at least in some of the cases that we have studied, there is no real crisis.  So the cells become transformed and then go on to become immortal without a real crisis in which the whole culture stops the fighting.
		Transformation is still a rather low efficiency, but anyway, there is transformation, and it is reproducible.
		So I isolated retina from a fetus, from a healthy fetus as far as could be seen, of 18 weeks old.  There was nothing special with a family history or the pregnancy was completely normal up to the 18 weeks, and it turned out to be a socially indicated abortus, abortus provocatus, and that was simply because the woman wanted to get rid of the fetus.
		We got this.  There was permission, et cetera, and that was, however, was in 1985, ten years before this.
		This shows that the cells were isolated in October '85, Leiden University in my lab.  They were then isolated in separate cell culture area, which contained a laminar air flow cabinet, and that was we did it in the cell culture area of the three different cell culture rooms that we had available at that time.  That was only devoted to diploid cell cultures, human cell cultures.
		The cell culture media were, of course, from certified supplies.  At that time already '85, I should say, the cells were frozen, stored in liquid nitrogen, and in 1995 one of these files was thawed for the generation of the PER.C6 cells.
		We used defined E1, identifiers E1 DNA construct, in order to eliminate sequence homology between the cells and the vectors, and that would allow RCA contamination free E1 deleted vector production.
		The E1 genes in the PER.C6 cells were regulated not by the E1A promoter, but by the PGK promoter, and the whole thing was all sequenced and functionally characterized.  I can show you if you are interested the data on expression of the viral genes.
		Transfection was carried out at Leiden University.  There was no carrier DNA used.  In '95 was this.  The transfection yielded a number of different colonies after about 18 days, and one of several of those were isolated.  One of them, Clone 6, finally was established and gave rise to PER.C6, and Clone 6 was chosen because it gave the highest yield of viruses and also had rather high expression of E1A and E1B gene products.
		These cells did not go through crisis, but it is possible that in some case a crisis appears, as I remember from experiments of Phil Gallimore, that sometimes a short crisis may be observed.
		So after the transformation event at the university in Leiden and after the colonies were picked, everything was transferred to IntroGene, which was close by also in Leiden, in fact, in the same building, and the whole documentation control was done by them.
		In the dedicated cell culture area defined materials were used, of course.  Cell banks were laid down at IntroGene, passage 29, 33, and 36.
		This shows you the information.  The recombinant identifiers itself it shown here.  The P9 gene is still present at the right hand and, in fact, of E1, and here is the area where E1 is deleted and the gene of interest is inserted.
		Then the PER.C6 cell was transformed by an E1 gene construct with a PGK promoter and a polyacid of Hepatitis B virus, I believe.
		And here is no sequence homology and real RCA has never been observed in many, many different experiments.
		And this is the final slide just showing you some comparisons between 293 and PER.C6.  Again, I remind you that both cell lines were made in my lab for different reasons.
		The objective, as I indicated, is for 293 -- was basic research, and we have done many different transformation studies after that, not transformation studies, but gene expression studies with human embryonic kidney cells in the years following that up to now, I would say.
		PER.C6 was made just for pharmaceutical manufacturing of adenovirus vectors.  As to RCA free, PER.C6 is RCA free.  Two, nine, three is not.
		The history documentation of the cell line has been carried out completely for PER.C6 and was not done at that time for 293.  We had no donor information on 293 or what was available got lost, and this is available for PER.C6.  Containment at that time was a little primitive perhaps and was now done in a laminar flow cabinet.
		The serum sources were of noncommercial use.  Probably I have made it myself.  Certified samples -- supplies were now used for serum and medium, et cetera. 
		Crisis free history just means that 293 had a crisis, was not crisis free at the long crisis, and these cells, PER.C6 had no crisis for some reason.
		And then pharmaceutical industry standard.  I realize that this sounds a bit commercial, but PER.C6 were made for that particular purpose.  Also, as far as I know, more than 50 different companies have taken license for PER.C6.
		Two, nine, three was not in the same way characterized, is in the public domain, whereas PER.C6 is licensed.
		So I think I'm at the end if somebody wants to see the data again of virus production and so on, but I don't think that's very important.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. van der Eb.
		We'll take a couple of questions.  Dr. Decker, then Ms. Fisher.  Dr. Kohl.
		DR. DECKER:  Did you say that adenovirus is not capable human diploid cell transformer because of cytolysis?  The human cell --
		DR. VAN DER EB:  Yeah, yeah.  Well, yes.  What I said is that you -- if you take intact DNA or a virus and you put it on human diploid cells, the types that we have used is the embryonic kidney, the skin, embryonic lung.  Then you see lytic reaction, and that will just wipe out a whole culture area.
		DR. DECKER:  Does that imply that an attenuated and human diploid cell adapted might then adapting it that way so you didn't get the cytolysis might unmask a transformation capability?
		DR. VAN DER EB:  Theoretically that is a possibility.  I don't believe it because the three different types of diploid human cells that we have tested were so resistent to transformation just by DNA, also by fragments of the DNA, resection fragments of the DNA.  We did it later also, but I don't believe that that is a big issue.
		What I think is that there might be other tissues, cells and tissues in the human body that can be transformed, for example, retina cells.  It could be that neural cells are also transformed.
		It is known that Adenovirus Type 12 is more clearly -- much more efficient in transforming neural cells of neural origin than Adenovirus 5, but this is five that we are talking about here.
		ACTING CHAIRMAN DAUM:  Thank you.
		Ms. Fisher, then Dr. Kohl and Dr. Minor.
		MS. FISHER:  From your chart you said that there was no cell crisis with the --
		DR. VAN DER EB:  No.
		MS. FISHER:  -- use of PER.C6, but before that you said that there was a short crisis observed.
		DR. VAN DER EB:  There have been, I believe, a short type of crisis observed by Gallimore in some cases, but he just described that the cells slowed down a little bit, and during one or two weeks did not seem to grow and then took off again.
		So you can perhaps not say that that is a crisis.  I don't know.  That could be a crisis, but the whole culture just took off again and continued.
		ACTING CHAIRMAN DAUM:  Dr. Kohl, then Dr. Minor, please.
		DR. KOHL:  Regarding the possibility of prion transmittable diseases, can you tell us more specifically about the fetal calf history of PER.C6, especially back in '85?
		You said it was from certified sources, but I'd like --
		DR. VAN DER EB:  Yeah.
		DR. KOHL:  -- to know more about that, number one.
		DR. VAN DER EB:  Yeah.
		DR. KOHL:  And, number two, can you tell us about the neurological history of the mother and the father of the fetus?
		DR. VAN DER EB:  I can, yes.  As to the source of the serum, we were able to  trace back that -- let me see where I have it -- that the serum was obtained in August of 1985 from -- it was Flow, Flow serum, and it was not exactly stated where the serum came from in this particular case, but the Flow serum samples that we got in the years before and afterwards were all from North American sources at that time.
		Also we had sometimes GIBCO, also North American sources.  It was certainly not European source.
		Yes, we got these cells.  These serum samples were selected by the University of Rotterdam for growth of diploid cells very carefully, and they usually get something like seven different samples either from Flow or GIBCO or both, and they test which one is the best for cloning of human diploid cells.
		And if they select one, the batch is large enough so that they can have enough for half a year, and we had the other half.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Minor, please.
		DR. VAN DER EB:  Oh, prion.  No, you had another question?
		DR. KOHL:  The neurological histories of the mother and the father.
		DR. VAN DER EB:  Both the mother and the father.  The mother was completely normal.  That I know and had -- there was nothing wrong with the mother.  She had at least two children afterwards in the same hospital in Leiden, which were completely healthy.
		The father was not known, not to the hospital anymore, what was written down, and unknown father, and that was, in fact, the reason why the abortion was requested.
		ACTING CHAIRMAN DAUM:  Dr. Minor.
		DR. MINOR:  You may have said this and I missed it.  Is there anything know about the copy number of E1 in the PER.C6 and the site of integration of the DNA?  In other words, is E1 really all there is to it?
		DR. VAN DER EB:  Yeah.
		DR. MINOR:  Or is it where it's actually put in the --
		DR. VAN DER EB:  No, E1 is the only thing that's present that are in PER.C6.  I can't exactly remember.  Maybe somebody in the audience can correct me.  I think about six or seven copies in it, which are all located close to each other.  So they may be in that kind of tandem repeat that you often see after transfection with calcium phosphate, and it's only -- that is, I think, only one side on one chromosome is integrated, nothing else.
		ACTING CHAIRMAN DAUM:  Thank you.
		I think at this point we're going to thank Dr. van der Eb very much for another informative presentation, and ask Dr. Cook to tell us about adenovirus transformed cell tumorigenicity and transformed cell host interactions that determine their tumor forming capacity.
		DR. COOK:  So what I'd like to do is focus on tumor development per se to start with in experimental tumorigenicity models and how that might relate to the question at hand and then talk a little bit about the ability of the E1A gene of this E1 region of Adeno. 5 to sensitize cells in which it's expressed to immunological injury.  That's been our area of interest and I think it to some extent explains the lack of tumorigenicity of these cells in immunocompetent animals.
		Let's see.  Is that number one?  Could you go back one just to make sure I didn't hold it down 12 times?  Yeah, okay.
		So what I'd like to do initially is talk about this basic premise about tumorigenicity as it relates to cell substrates.  I think that's an area  of concern when I read over the documentation.  The potential risk statement that Dr. Lewis has provided:  what are the determinants of experimental tumor formation that are being used to characterize these tissue culture cells used for vaccine production?
		And then some general observations thinking about experimental tumorigenicity as the data that we understand from that relate to potential risk, and then, as I mentioned, tell you something about E1A induced sensitivity to apoptotic injury and E1A induced rejection of cells expressing these proteins, and then just a very little bit at the end about E1A in humans.
		So the focus here is on the E1A as the immortalizing, enabling oncogene in the cells that are being created, and I guess theoretically its potential risk as a contaminating bit of DNA that would go across in the vaccine.
		So the potential risk statement cut down a bit says something like tumorigenic cells are more risky than non-tumorigenic cells as vaccine substrates, and that seems to be a focus of -- an appropriate focus of concern, and taking that on directly, I think, is an interesting thing to do, and this comes, as Dr. Lewis mentioned, from this Armed Forces Experimental Board in 1954 where there was a preference for normal cells over human tumor cells, not cells like 293 or PER.C6.
		And so I think the question at least as I think of it is the concern about the use of human tumor cells, like HeLa or something of that sort, the same thing as the concern about using human cells that form tumors.  Those things may seem to be similar, but I think they're different, and that is form tumors in nude mice.
		So what I'm going to try to convince you is that I think experimental tumorigenicity as we measure it is not the same thing as primary tumor development because I think this is an important distinction.  We're talking about the tumorigenicity of E1 expressing cells or E1 immortalized cells, and I don't think that's the same thing as tumor formation from a single immortalized or mutated cell in vivo that goes on to successfully form a tumor in us or in a mouse.
		And it's important, I think, not to confused these things.
		So the variables that can be affected, that can affect experimental tumor formation, that is, the ability to form tumor in an animal like a nude mouse with a set of tissue culture cells, the first thing is the host that you select.  That makes a big difference.
		And this shows you something about some experiments we've done where you can see a huge host difference in the apparent tumorigenicity of the cells.
		Let me crank this up.
		So these are three sets of types of mice.  These are normal C57 Black 6 adult mice.  These are C57 Black 6 mice that are nude and, therefore, lack of functional thymus and lack of functional T cells that have an intact natural killer cell response, a different kind of host defense.
		And these are CD3-epsilon transgenic mice, and it's not interesting to know what they are, except for the fact that they lack both T cell and NK cell responses.
		And then what's been done here is to take a mouse sarcoma that is a relatively non-immunogenic mouse sarcoma, and this is collaborative study done with Jack Routes, and these cells were either tested in the three types of mice, either in their E1A negative parental state or in the state that they would be after being transfected with the E1A gene alone, no E1B, not the whole E1 region, just the E1A gene.
		And the question was:  did E1A expression affect the tumorigenicity, and if so, did that relate to the type of animal that you use to test tumor development?
		And the answer is obvious.  All of the red lines are the control cells not expressing E1A, and all of the green lines are the efficiency of tumor formation using the parameter that Dr. Lewis mentioned, the TPD-50, the efficiency of tumor formation by the cells in the different animals.
		And the lower the TPD-50, the more efficient tumor formation occurred.  So you can ignore all of the red lines because they're essentially all the same, and that is the non-E1A expressing sarcoma cells were very efficient at forming tumors in all three types of animals.  The E1A expressing cells were essentially unable to form tumors in normal mice, formed tumors sort of in a medium fashion reasonably well in nude mice, those that lacked a T cell response, and formed tumors with equal efficiency as the E1A negative cells in the animals that lacked both types of cellular immune defenses.
		And so the type of host you pick determines a lot about the type of answer you get.  You can say that this cell is, quotes, non-tumorigenic, whereas this cell is highly tumorigenic, but it really just depends on the animal chosen.  So I think that's clearly an important parameter to consider.
		The next thing that's important is the threshold effect.  Dr. Lewis mentioned numbers of cells used to challenge animals, and so the cell dose you pick determines the result you get as well.  So, again, you could create a non-tumorigenic cell by just picking too low a dose.
		And here is an experiment from a study that Dr. Lewis did with these Adeno. 12 transformed mouse cells.  These were Balb/c cells, and they were inoculated into adult immunocompetent animals at various doses over time.  This is the number of cells injected.  This is the tumor incidence of these animals.
		And what you can see is there are essentially no tumors up to 100,000 cells.  So if you happen to get unlucky and pick 100,000 or ten to the fifth cells as your number, you'd call these non-tumorigenic cells.
		On the other hand, if you picked 100 million or 10 million cells as your dose, you'd say they're highly tumorigenic.  So dose matters.  Host matters, dose matters.
		The next thing is the rout of inoculation.  This is something that's not as well quantitated, but there's no question in my mind that the route that is used for a given challenge makes a difference, and in our experience intraperitoneal inoculation results in an apparently more efficient tumor positive result than subcutaneous inoculation, and there are other kinds of things you could do, like inoculate into the immunologically privileged sites.
		Another result is to think about the observation period.  How long do you wait before you score tumor development versus non-tumor development when you're doing an experimental tumor formation assay?
		This in our laboratory is a notorious experiment because we did a collaboration with another investigator.  He had published a paper saying that human breast cancer cells expressing the E1A gene were non-tumorigenic and E1A had, in fact, converted these cells into non-tumorigenic cells.  It had reversed their tumorigenicity.
		Well, the thing was he looked at these cells only after about 14 to 18 days.  We repeated the experiment, and so what's shown here is the percentage of animals that are tumor free over time after a challenge, with the breast cancer cell itself.  These are all nude mice.  So they lack T cell responses, have some NK cell defenses.  So this is the E1A negative breast cancer cell.  This is a ductile epithelial cell carcinoma.
		There was the E1A expressing cells, and
sure enough, if you look at about two to two and a half weeks, you get zero tumor formation or 100 percent tumor free  animals, whereas all the animals that had been inoculated -- that's not true -- some of the animals that had been inoculated with the E1A negative cells developed tumors, and that tumor incidence continued to increase over time or the tumor free number decreased.
		But if you waited a bit longer, then the E1A positive cells begin to make tumors in up to 20 percent of the animals over time.  So if you get unlucky and pick a very short observation period, you might come up with the wrong conclusion and call a cell nontumorigenic.
		So the host matters.  The cell dose matters, and the route of inoculation matters, and how long you wait to call the endpoint matters.
		Well, then the last thing, I don't have any data to show on this, but there are several other things when you look at the literature that can make a difference in the methodology chosen to determine whether cells make tumors or don't.
		One thing that I find interesting because almost all of these experiments are done by sticking a needle into a mouse or a hamster or a rat, injecting some cells into that needle track, and then asking whether they form tumors or not.  That's not normal.  That's not what happens after a UV irradiation or exposure to a carcinogen.  This causes some kind of trauma at which site the cells now have a chance to grow.
		And so I think there may be a wound effect, and we were talking earlier about some of the observations that have been made in Rous sarcoma virus where tumors form at the site of wounds.
		That is an artifact that can affect tumor formation, I think.  Foreign bodies can affect tumor development as well.  For example, you can attach cells to plastic plates and put them into animals and get tumors.  These cells wouldn't form tumors at all through any route of inoculation like 3T3 cells if you didn't attach them onto these little disks, and so the foreign body can make a difference in your tumor assay.
		So you have to be careful about how you interpret those, and the other thing that can happen is if you just provide fibroblast as a mixed culture in with your putative tumor cells, you can get tumor forming efficiency that is much greater.  So there is some feeding effect or something that goes on when you can do certain kinds of mixed culture inoculations.
		So my point is that tumorigenicity is not all the same.  It depends on how you rig the system, how you set it up.  All of these experiments can be used quite effectively to ask questions about one cell versus the other, but I don't think they are anything like at least in my mind what goes on when you get a spontaneous single transformation event and ask that cell to grow into a tumor, especially when you think about cell dose.
		There are very few spontaneous transformation events that result in the immediate generation of 100 million cells or ten million cells.
		So what I'd like you to think about as a possibility is the fact that tumor formation is not the result of a single activity at least as we look at it in experimental tumorigenicity, but it's really an orchestra of events, and if all of these things aren't working right, you don't get a very good outcome or in our case you don't get a tumor.
		You need to have in most cases that we know about continuous oncogene expression or continuous in our case E1A expression.  That probably isn't always true, but almost always true.
		That gene has to successfully cause cell cycle disregulation, and it probably has to hit to Achilles heel in the cell cycle that relates to what some people call the p53 Rb pathway where something has to occur to cause that to go out of whack.  Otherwise you don't get immortalization.
		The cells gave to survive this, and it's important to think about the fact that when you try to over express E1A in normal mammalian cells it usually kills them.  So it is a proapoptotic event itself.  So it's not easy if you're trying to transfer a lot of E1A and some contaminating DNA to get those cells to survive because they would tend to die as a result of E1A expression itself.
		Then you have to have this magical threshold cell number.  I don't think single E1A positive cells probably ever form tumors.  I was taught early in medicine never to say "never" or "ever," but I think it's probably true that it's probably true that it's very hard to get a tumor with a single E1A positive cell.  You probably need millions at least.
		There probably would have to also be local tissue factors.  Steve Frisch from California has shown that if you plate E1A positive cells on collagen matrices as opposed to plating them on plastic, they tend to do poorly.  So you could argue that maybe the reason that E1A positive cells grow poorly in nude mice compared to normal mice is that they don't like being on biometrices and that tends to be inhibitory to their growth.  Most of us grow them on plastic dishes and they look fine.
		And then they also have to escape from this cellular immune response that I described in this three-mouse experiment.  If they don't escape from that, then the cells are likely to be killed, even if they make it through all of these other hurdles.
		So I think it's very unlikely that this combination of events would occur in a well orchestrated fashion with transmitted DNA without a lot of help.
		I'm sorry.  Would you go back one?
		This is just a summary of a lot of work done on trying to characterize how E1A sensitizes cells to a variety of proapoptotic injuries.  We've mostly been interested in immunological injuries since they relate more to whether a cell will form a tumor or won't form a tumor in vivo, but a number of other injuries do the same thing, and that tells us something about the mechanism, I think.
		So if you look at a variety of types of things, chemotherapeutic drugs, hydrogen peroxide, potassium ionophores, irradiation; this is a protein synthesis inhibitor; where the things we're more interested in like natural killer cells, cytolytic T cells, TNF alpha, or the TNF receptor apoptosis inducing ligand trail, all of these things can selectively induce apoptosis in an E1A expressing cell.
		So there were a lot of things going against E1A positive cells in trying to survive in vivo or trying to survive when exposed to a variety of types of injuries.  
		This may say something about the pathway through which E1A sensitizes, but what it does say is that an E1A positive cell doesn't have an easy time of it trying to survive in vivo, especially when you consider these kinds of defenses.
		It's also important that these events, this sensitization of cells to immunological mediated proapoptotic outcomes are not necessarily dependent on normal cell genetics.  So p53 minus cells are cells that lack expression of the Rb gene, are also sensitized by E1A just like normal cells are.
		So if you wanted to postulate the worst scenario, you'd say, well, maybe E1A would be transmitted, and it would hit a cell that already had a mutation and that would give a chance for a bad outcome.
		The fact is the  most common mutations in human cells that lead to neoplasia are in this pathway, E1A sensitized cells expressing mutations in that pathway to immunological injury.  So they're not going to be protected by that second mutation.
		This just shows you something about the range of experiments that have been done with human cells.  This is just an experiment with natural killer cells to show you that human cells are very much like other cells.  These are mouse 3T3.  This is a continuous rat line that was immortalized with ras.  This is BHK21, a spontaneously transformed hamster cell line.  THE BHK21 is highly tumorigenic, and its tumorigenicity is eliminated by expressing E1A.
		These other lines, all when expressing E1A become more sensitive to natural killer lymphocyte induced lysis, and this is just a human fibrosarcoma cell line, stably expressing E1A, and it does the same thing.
		If you generalize this to other types of things that we know about, a variety of types cells from different tissue origins from humans are sensitized to various apoptotic injuries.  Epithelial cells, notable among those 293.  We've also looked at this breast cancer cell I told you about; fibroblastic cells.  This is a fibrosarcoma line.
		Hematopoietic cells, K562 has been shown by Stiewe in a publication in 2000 to become sensitized to reactive oxygen intermediates when expressing E1A, but not when not expressing E1A, and ACLS2, which is an interesting cell line because it doesn't express either p53 or Rb, when it expresses E1A becomes much more sensitive to natural killer cell injury, and I've already told you about some of the injuries.  These are the ones that have been tested against human cells expressing E1A.
		So this is like the atomic chart or the intermediary metabolism pathway.  It's one of these things that we never remember, and I just show it for the purpose of saying that E1A probably hits multiple targets in the apoptosis cascade.  This is an incomplete representation of what goes on during apoptosis, but there are preliminary data.  This isn't well worked out, but there are data that E1A causes over expression of p53, which can drive apoptosis.  E1A positive cells -- this was all done in 293 cells ‑- express some kind of what's called an oncogene associated factor that is probably like a mitochondrial-like factor that can be involved in apoptotic activity.
		E1A can probably facilitate the conversion of Procas Base 8 to Cas Base 8 (phonetic).  In certain cell types, it can increase cell surface depth receptor expression, and we've done several studies now, and I've just submitted a publication about E1A repression of the cellular NF kappa B (phonetic) defense against apoptosis.
		So probably the bottom line is that there are multiple sites in cells that are targets for E1A rendering cells sensitive to a variety of apoptotic injuries, especially immunological ones.
		So the last point is E1A in humans, and I guess to try to put the idea of contaminating E1A in perspective, we should think about what goes on elsewise (phonetic), and that is there are studies.  This is a study by Jimmy Hogg from Canada, from his laboratory, that shows that E1A actually may persist in normal human tissues after infection. 
		In these studies what they did is they took a lot of people that had lung disease, and they asked whether those that progressed to emphysema or chronic bronchitis had something unusual about them, and I think the truth be told, he had somebody who had done a post doc in Alex's laboratory or somebody's who had learned how to look for E1A, and they looked for E1A in these lung tissues, and what they found was a lot of E1A sequences in both normal people and those that progressed to emphysema based on PCR technology.
		So it's not easy to do, but when people have looked, they have found E1A, at least in this one laboratory, have found E1A in normal tissues, and that may speak to the persistence of adenoviral infection.  It may say that there is integration that goes on that we aren't very good at detecting, but it's probably not true that E1A is never around and these viral oncogenes are never expressed in humans anyway, even vaccine recipients.
		And then there are two clinical trials that are going on now and fairly extensive studies in which E1A is actually being used therapeutically.  It's being put into people that have cancer to ask whether it can help reject tumor cells.  One of those is in a company that has the name of the virus that's called ONYX.  It's really an old Arnie Levine that has a mutation that allows it to selectively kill p53 minus cells, which many human tumors are.
		So this virus infection is given to people who have p53 minus tumors, and that virus preferentially replicates in those tumors and destroys the tumor tissue preferentially to normal tissue.
		It's not that simply, but that's the idea.
		There is also an M.D. Anderson study in which E1A is being used in gene therapy to inject in liposome vectors into people that have, for example, metastatic ovarian or peritoneal metastasis with ovarian carcinomas, and it's also being considered for breast carcinomas.
		And here the idea is that E1A represses expression of a cell surface receptor that's necessary for growth factor stimulation of the tumor cells.  So not only do we probably have E1A already in our tissues.  If we were to look carefully and certainly can support persistent infection with adenoviruses, but E1A is being used therapeutically in humans, and so far now admittedly these aren't long-term vaccinations of babies, but so far there's no evidence from the safety testing that this has caused an adverse outcome in these patients.
		So my conclusions are that experimental tumor formation in nude mice does not predict tumorigenicity of an isolated oncogene, in this case E1A.  Even if the E1A oncogene were transmitted in a contaminating DNA, it's very unlikely that it would become stably expressed in recipient cells because of transfection inefficiency.  Dr. van der Eb just told you how hard it is to transfect human cells with sheared adenovirus DNA or certainly with E1A itself it's actually even harder.
		Direct apoptosis that occurs during attempted establishment of these cells would probably kill many of the cells in which the DNA would try to get itself inserted.
		Even if E1A did become stably expressed in recipient cells, it's very unlikely that such cells would provide a tumorigenic risk because there wouldn't be a threshold cell dose.  You wouldn't have the millions of cells up front that you'd need to get over the hurdle of getting tumor formation initiated.
		Now, the viability of the E1A positive cells on biometrics, Steve Frisch's data, suggesting that these cells do relatively poorly when cultured on things like we might have in our soft tissues, and the susceptibility of these cells even if they did get established to immune mediated apoptosis, I think, would rule against their survival.
		And then also we have to think about the fact that expression of E1A in humans might be normal, and when therapeutically used in humans, it can actually be directly apoptotic and can reduce expression of critical growth factor receptors.
		So I think E1A expression as a contaminating force from the vaccine would probably not likely be a major problem.
		I'd be glad to answer questions.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Cook, for an enlightening presentation.
		We'll take a few questions.  Dr. Griffin, then Dr. Stephens, please, and then Dr. Faggett.
		DR. GRIFFIN:  It's my understanding that the cells we're talking about express both E1A and E1B.
		DR. COOK:  Right.
		DR. GRIFFIN:  And that E1B is a counter force, an anti-apoptotic factor.  So how does that fit in with all of your conclusions that you're making about the safety of E1A when you're really talking about giving both?
		DR. COOK:  Right.  I specifically didn't talk about E1B.  There's no evidence that E1B expressed alone does anything in terms of its ability to immortalize cells. 
		They're looking at both.  The only reason that E1B is interesting in the contest of E1A is it allows E1A -- it prevents E1A from destroying cells.  So without E1B what you get is a tremendously inefficient immortalization event, very few colonies formed.  So E1B really has to be around to prevent the apoptotic response and to bind p53, the two proteins that E1B makes.
		So I don't think that E1B is a factor.  All I can tell you is when it gets down to what I think is the key event at least from our perspective is that if you look at the immunological injuries and ask whether they still happen, from a killer cell point of view, a natural killer cell, a cytolytic lymphocyte or an activated macrophage still kills cells expressing E1B perfectly well, even though it does it in an E1A specific manner.
		The only thing that E1B can do in human cells when co-expressed with E1A is to repress the TNF induced apoptotic response.  That's a unique thing that Linda Gooding showed years ago.
		So I don't think E1B really adds a whole lot to the mix, other than allowing the cells to be immortalized in the first place.
		Then the other thing that I would have to think about is how do you think about transmitting this DNA when these are two separate genes under the control of two different promoters, and now what you have to do is get the orchestra to work even harder.  You've got to put the two genes together again to get them expressed in the same cell if you're going to try to transmit this as contaminating DNA.
		So now the odds even go up higher.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Stephens and then Dr. Faggett.
		DR. STEPHENS:  I was struck by one of your papers talking about this family of oncogenes, and you compare the HPV oncogene, which has very different properties than EA-1, and I just wondered if you could comment on the structural relationships and possibility of mutation of EA-1 to produced an HPV-like oncogene.
		DR. COOK:  Well, E1A and E7 have some sequence similarities.  You can actually do with an analogous gene SV40, you can do mix and match experiments where you can cut out a bit one, stick into the other and get a competent gene.
		So they have conserved sequences.  I think the reason for this is that all of these genes need to do the same thing when they get into the cell, and that is to regulate the cell cycle, and they do that by binding Rb and by binding p300 and Kreb binding proteins.
		So there are reasons that they have co-evolved these.  They aren't the same virus.  As far as anybody know, they didn't evolve together, and they aren't just recombinations.
		The data you're referring to are that E1A does all of the things I'm talking about, and so far as we can tell, when E7 is expressed in cells with the exception of one or two reports, it doesn't sensitize cells to some of these other kinds of injuries like TRAIL or to killer cell injury, and so E7 is a different beast, even though it shares some of these same cell cycle regulatory bits.
		The ability to convert E1A into E7, other than making a chimeric molecule, which can work -- Jack Routes has done that.  He's made an E1A E7 chimera.  What happens is the E1A phenotype is dominant, and so E7 sort of lacks what E1A has.  It doesn't look like it has any evil force that's trying to outdo E1A.
		There are no mutations so far that we have been able to make in E1A that's eliminated these activities, although if you eliminate enough of the genome, if you take enough of E1A out, you can reduce its ability to sensitize things, but it's not just a simple mutation.  It's a deletion.
		So I don't know that we could, other than chimera formation between E1A and E7 just making single based changes or some kind of frame shift or something in E1A create an E7-like gene product.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Faggett, please.
		DR. FAGGETT:  Thank you very much.
		It was a very clear presentation of a very complex topic.
		You did mention that you were comfortable in terms of safety issues with the A-1.  What was the level of sensitivity and types of tests used to bring you to that level of comfort in terms of safety?
		DR. COOK:  Well, what I'm saying is that I think the -- I'm talking about odds.  I suppose we could talk to statisticians, which I'm not, but the odds of getting multiple unlikely events that occur at a rate of maybe one in a million to occur simultaneously to get the event that you want to see happen, which is the transfer of an E1A oncogene into a vaccine recipient at the level of DNA contamination you're talking about, and have that gene by itself now be able to immortalize the cell, have that cell survive itself, and then have it survive an immune response.  I think the odds could be -- probably can't be calculated because you don't know all of the numbers, but I think they're very low.
		DR. FAGGETT:  It's a question of ten nanograms of DNA fragments estimated to be the smallness to get some effect.  Is that a level?
		DR. COOK:  Ten nanograms per vaccine dose is what I think I heard earlier.
		DR. FAGGETT:  Okay.  So how do you --
		DR. COOK:  I've have to try to do some math.  I think it would be mental gymnastics, but it probably could be done.
		DR. FAGGETT:  Thank you.
		ACTING CHAIRMAN DAUM:  We will return to this very subject in our discussions later.
		Dr. Diaz.
		DR. DIAZ:  You mentioned that there was a lot of E1A just naturally occurring in human cells, different types of human tissue.  Could you expound on that a little bit in terms of what's known?  Is it expressed?  Does it perform similar immunologic regulatory functions in humans?
		And, secondly, is there any known E1B-like genes in human tissue also?
		DR. COOK:  Yeah, it's important to put it into perspective so that I don't overstate this.  So there is a laboratory in Canada in which they have found evidence based on PCR analysis of E1A sequences in lung tissue.  It was in a lot of different lung specimens that they sampled.
		As far as I know, there aren't other laboratories that have done the same thing.
		There's a laboratory in Australia where they've found E1A-like activity in mammalian cells, including human cells, but haven't convinced anybody that it's E1A yet.
		So there's a different in finding E1A, as Hogg's lab did, and finding E1A-like activity, as many other people have.  So there probably are E1A-like functions in normal cells, and that's probably why E1A decided to ape this during evolution anyway, because it helps regulate cell cycle.
		A far as E1B expression in normal human cells outside of initial infection or persistent infection, I'm not aware of any data that E1B independently has been detected in human tissues unrelated to infection.
		ACTING CHAIRMAN DAUM:  There are four more hands.  We'll take these four questions, and then we're going to move on.  Dr. Minor, then Dr. Ketner, Dr. Aguilar-Cordova.
		DR. MINOR:  I'm interested in your threshold description.  If you had a PD-50 of 1,000 and you give it to ten mice, five of them will go down.
		If you had 100 cells, for example, went into 100 mice, are you saying that none of them will go down?
		DR. COOK:  I think that's true, actually, yeah.
		DR. MINOR:  Has anybody ever done to see--
		DR. COOK:  Andy, you've given a lot of -- you know the answer.
		DR. LEWIS:  Yes, we did that very experiment with the Adeno. 12 because we found that the TPD-50 for the Adeno. 12 transformed Balb/c mice embryo cells was a million cells, and so we asked the question:  what would happen if you put 100,000 cells into 100 mice?
		And we did that experiment, and we got no tumors.  So it looks like that threshold is real.
		We did several other experiments to address that question the same way, like cloning and doing this, that, and the other, and the TPD-50 on sub-clones of the population were again a million cells.
		So nothing we could do would alter that threshold.  I can't say that there's thing that couldn't be done to alter the threshold, but we were unable to do it considering that as a criticism for the proposal we were making.
		ACTING CHAIRMAN DAUM:  Can I ask a follow-up and this be -- save one of the four questions?
		Is this threshold phenomenon in your belief a probability event where if you just give enough cells, one will do it, or is this a quorum sensing kind of thing where if you give enough cells they will start doing something?
		DR. LEWIS:  Yes.  Well, I think what we ruled out by the experiments we did was the possibility that one cell out of a million cells was doing the job.  All the data that we have suggested it took the collective action of million cells to overcome whatever it was that was prohibiting the cells to form a tumor mass.
		Now, exactly what it's overcoming I don't know.  I think the question was as to whether this was -- and these were adult Balb/c mice, by the way.  They were not newborns or immunoincompetent animals.  These were adult animals.
		So the question I think we raised but were never able to answer was the possibility that it was something about the immune system that it took a million cells to overcome.  In other words, that was the level of competence at which that animal could function immunologically with all of his anti-tumor defenses intact.
		And if you challenged him with enough, he could no longer respond.  Jim may have some other ideas about that, but that's as far as we were able to take the logic.  We were never able to test that.
		ACTING CHAIRMAN DAUM:  It may be fun to return to that this afternoon as an issue.  I'd like to get the focus back to your presentation.
		Dr. Ketner is next, and then Dr. Aguilar-Cordova.
		DR. KETNER:  Yeah, this is just a comment on the persistence of E1A sequences in human lung samples.  It turns out it was shown a long time ago, 20 years or so, that human peripheral blood contains, after an adenovirus infection has come and gone, contains residual, intact viral genomes.  They're probably replicating slowly in peripheral blood cells, and so those E1A sequences that were found, these are detectable by relatively insensitive techniques like Southern blotting.
		So those E1A sequences that were detected by PCR may well just have been intact replicating genomes in the blood in those tissues.  So I think it overstates it to say that there are cells in which there are, you know, E1A persistent sequences.
		ACTING CHAIRMAN DAUM:  Thank you for a clarifying comment.
		Dr. Aguilar-Cordova.
		DR. AGUILAR-CORDOVA:  Yeah, I was wondering on the cell thing you were mentioning that had E1A-like activities if these cells could complement the E1A minus vectors, and what other known genes, such as E7 or other viral genes, may have that function of complementing and then producing the replication competent activity.
		DR. COOK:  The clearest experiments have been done with mouse embryonal carcinoma cells, the cell line F9, and I think some of the break-away studies from Australia may be similar, but in F9 cells what happens is these are interesting cells because they can be differentiated in vitro with retinoic acid into terminally differentiated cells.  Without retinoic acid, they're undifferentiated embryonal carcinoma cells.
		The undifferentiated cells will complement a virus called DL312, which has no E1A and grows very inefficiently in normal cells.  When these cells are differentiated, that complementation goes away.
		So this is the basis upon which it has been concluded there is some E1A-like activity in normal cells.  This is a mouse embryonal carcinoma cell.
		The question about whether other human persistent viruses or like HPV where E7 is expressed in an epithelial carcinoma or something like that, I don't know of any evidence that E7 can be expressed in the cell and complement a defective adenovirus.  I don't think -- at least as far as I know, that's not the case, but I don't know the data.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Cook.
		We're going to move on now to our final morning, so to speak, presentation.  We'll hear from Dr. Peden at the FDA regarding quantitative assessment of the risks of residual DNA, and after which we'll take a break for lunch.
		DR. PEDEN:  Thank you.
		ACTING CHAIRMAN DAUM:  While setting up, I'd like to congratulate Dr. Peden for providing us with a handout with his slides on it.  It makes it much easier to follow the talk.
		DR. PEDEN:  Thank you.
		I was going to say good morning, but it is, in fact, good afternoon.
		This presentation will discuss issues of whether residual cell substrate DNA in vaccine manufactured in neoplastic cells poses a risk to vaccine recipients.
		At the outset, I should like to say that while these views expressed are largely my own, I would like to acknowledge the sage and helpful discussions of many colleagues, particularly Andy Lewis, Phil Krause, and Becky Sheets.
		I would discuss what the perceived risks are, the proposed mechanisms whereby there could be a risk.  I will review the relevant in vivo data that do exist and present some calculations and assumptions upon which they are based.
		The overall conclusion I want to leave you with is that the small amounts of residual cell substrate DNA, and by this I mean ten nanograms, poses an acceptably small risk to vaccinees, and this value of ten micrograms did not come from my whim.
		Oops, can I go back one more?
		It has been discussed for many years, and at the WHO expert commission in biological standardization in 1998, the residual DNA from continuous cell lines, ten nanograms or less, was proposed as a reasonable level, raising it from 100 picograms, .1 nanograms or less, that was reached in 1986.
		So what types of risks are there?  There's the perceived risks associated with residual cell substrate DNA, has generally considered to be oncogenic risk, but there is another risk which I'm going to discuss, too, the infectivity risk.
		So the proposed mechanisms whereby an oncogenic risk by cell substrate DNA comes for several ways.  One is with the introduction of a dominant activated oncogene.  For example, an activated rat oncogene or in the issues we're discussing today, the dominant activated oncogenes could be E1A or E1B.
		Insertional mutagenesis is another mechanism whereby DNA could be oncogenic.  While this mechanism has been found in certain cancers of animals, such as the disruption of cellular tumor suppressor gene or activation of a cellular dominant oncogene, by promotion of insertion mechanisms, calculations have been done by many people, and the risk associated with a single integration event by this type of -- to disrupt the cellular tumor suppressor gene or dominant oncogene is considered by people like Reinhart Court (phonetic) to be one in ten to the minus 18 or one in ten to the 18.
		So this is a very low probability event, and it comes about -- this has been seen in animals were activation has occurred from an integrating, replicating system, such as a retrovirus, and a single insertion event from residual DNA is unlikely, very unlikely to pose this.
		Another mechanism that's been pioneered by Walter Doerfler is that DNA methylation pattern changes following integration.  While this occurs in system trends, the lack of a phenotypic consequence of this makes this very difficult to study.
		The other risk of integrating -- of cell substrate DNA is the one I talked about, cell substrate DNA they incurred in infectious genome.  The DNA virus, which is polyoma virus, Herpes virus, and papilloma virus exist as integrated into cells.
		Well, in addition, of course, retroviruses have a provirus state, such as HIV-1 and HTLV and human retroviruses.  So by infectious DNA what we mean is that if this DNA is introduced into the appropriate system, then infectious particles are produced, and an infection can be established.
		So one other thing that influences the nature of the inoculum.  For example, we concern linear versus circular DNA as not being studied greatly.  The single stranded versus double stranded, where the state of the DNA is free versus the chromatin associated.  In most lysed cells, of course, the DNA will not be free, but be chromatin associated.  This has not been assessed in any study except one, that chromatin was injected into animals.
		The size distribution of the DNA is obviously very important.  If the DNA is sheared to a size that's below the size of a normal gene, such as normal genes excluding neutrons are about 3,000 base pairs.  Then clearly this DNA will not be able to produce a product.
		Now, the route of inoculation can be important as you heard from Dr. Cook.  Most of the studies, various animal studies were done with intramuscular, intradermal, subcutaneous, intranasal and oral route, and  also by intraperitoneal route.   However, an application in Phase 1 would be considered unfavorably, I would imagine, with this route.
		Assumptions.  Before we can go into the calculations, we need to make certain assumptions here, and I hope you can read that, but what I wanted to do is to go through these assumptions.  I'll read them slowly, and I hope you have the handout.
		For a given DNA, the level of response of the cell to that DNA is proportional to the amount of that DNA.  That makes somewhat sense.  The activity of the gene integrated in the chromosomal DNA was part of a plasmid or phage vector is equivalent or lower.
		Now, we have to make this assumption because we have no other way of doing this.  However, it's likely that the DNA integrated into chromosomal DNA being controlled by its own promoter, requiring certain perhaps tissue specific transcription factors, may not, in fact, be active at all, whereas we put a gene on a plasmid or phage vector and we've designed it such that it expresses very well.
		So if anything, this is going to over estimate the risk rather than underestimating the risk, but that's the only thing we can do.  So we're making this assumption.
		And the amount of uptake of a given gene by a cell and the expression of this gene in the cell is related to the concentration of the gene in the DNA.
		Now, because a single copy is represented at approximately one-millionth of the haploid mammalian genome, the amount of DNA corresponding to a single copy gene is a million-fold less abundant for equivalent amounts of cellular DNA compared with the plasma DNA with the same gene.
		Now, as I say, the genome is of nine -- the haploid genome is three billion base pairs, where as I say, the normal gene is maybe 1,000 base pairs.  So there's a million-fold difference in the size of the genomes.
		So, for example, that is, for the uptake and expression of a gene in mammalian genomic DNA equivalent to one microgram of plasma DNA, one million micrograms or one gram of mammalian DNA is to elicit an equivalent biological effect, giving the assumptions on the previous slide.
		Conversely, the single oncogene is present at one microgram of mammalian DNA, the equivalent amount of the same oncogene if cloned in the plasma is one times ten to the minus six micrograms or one times ten to the minus three nanograms or picogram.
		So there are the assumptions on which we've made some calculations.
		So now I want to go through some of the data that has been presented in the literature about oncogenicity, and this is the work done by Hsien-jen Kung, and colleagues, in 1983, where they're using a cloned src gene, two micrograms induced tumors in seven out of ten chickens inoculated subcutaneously in the wing web.
		If you inoculated a cloned Rous sarcoma genome, the entire genome, this two micrograms induced tumors in six and six.  Presumably this comes about because the Rous sarcoma virus DNA initiated an infection.
		So two micrograms was found to induce tumors in about 70 percent of the animals.  A study in 1990 by Halpern and colleagues got essentially the same or similar results using in this case 20 micrograms of DNA by the same route, and inoculated intravenously.  Also it gave tumors.
		So we can conclude that two micrograms of DNA, which is equivalent to about two and a half times ten to the 11 molecules of cloned v-src oncogene is oncogenic in chickens.  
		The study that was done by Burns and colleagues inducted the oncogenicity of ras, of the activated ras gene in mice.  Activated ras from the T-24 gene, ten micrograms inoculated by scarification in the mouse skin.
		Lymph anginose sarcomas developed in 33 out of 34 animals within 12 months, usually within 12 weeks.  However, the normal ras gene failed to induce tumors.  So at least in this route, ten micrograms, which is equivalent of about one times ten to the 12 molecules of an activated ras gene is oncogenic in adult mice.
		If we looked at the oncogenicity of polyoma virus DNA, polyoma virus DNA, of course, is infectious in mice, but in hamsters it is not infectious.  It causes tumors by interperitoneal, subcutaneous routes, using either super clonal DNA or linear DNA.  As you can see, it's tumorigenic.
		This is the viral DNA isolated from the virion.  If you clone the polyoma virus DNA and inject in newborn hamsters, it is also tumorigenic.
		Now, the minimum amount of DNA they found activity was .2 micrograms of the linear DNA, and gave it to 22 percent of the animals.  So if we use that figure, it can conclude that .2 micrograms, which is about two times ten to the ten molecules of polyoma virus DNA is oncogenic in newborn hamsters.
		So then we're going to move on to what data are available on the infectivity.  Now, I'm going to talk about several studies on retroviruses, and in the first ones I'm going to talk about is work done by three groups over the years, using Simian immunodeficiency virus through various routes and establishing infection in various animals, and the number of genomes, for infection.
		In the bovine leukemia virus using ID route with either a facilitated Achiheinig (phonetic) lipid facilitator DOTAP in the Caprine (phonetic) arthritis encephalitis virus in goats, again, and feline leukemia virus and feline immunodeficiency virus.
		Now, the only reason for presenting this slide is, although you probably can't see it, is that the number of genomes for infection with these disparate systems and different routes of inoculation is remarkably similar.
		The highest number of genomes required is about two times ten to the 13, and the lowest is one times ten to the 12 in this system.  So it's somewhat remarkable.  Even though a dose response study was not generally done, with the possible exception of the Purcell group, it is quite remarkable the number of genomes or molecules of these proviral clones.
		And yet to establish an infection, we're very similar within about a 20-fold range.  The dose response study was done by Portis and colleagues with a murine retrovirus using 19, 3.8, .38, and .038 micrograms intraperitoneally and found that 3.8 micrograms intraperitoneally was the last of the level to establish an infection.
		Interesting, they also found that super core DNA was unable to establish an infection, whereas linear DNA established an infection very well.
		That result disagrees somewhat from other people's, but probably reflects the system as well as the clones.
		So the summary then of the infectivity with cloned viral genomes, viral DNA coming from the retroviruses, 15 to 500 micrograms intramuscular injection, and so that if you calculate the number of genomes, it's about one times ten to the 12 to 2.3 times ten to the 13 genomes required for infection.
		Polyoma virus, in contrast, from five time ten to the minus five micrograms or one microgram variously, and the minimum estimate then is 1.3 times ten to the seven genomes.  So if we can conclude from that the infectivity of different retroviral DNAs is quite similar, quite surprisingly perhaps is similar, and depending on the route of inoculation, 15 micrograms can be infectious for retroviruses, but the infectivity of polyoma virus DNA is higher than approximately 50 picograms or .05 nanograms is sufficient to establish an infection.
		So it may be informative to compare the oncogenicity and infectivity where it's possible.  So for polyoma virus DNA, that's the only one where we can, in fact, compare directly the oncogenicity.  Point, zero, two micrograms is the minimum amount of DNA that was tested that was oncogenic, and that's about 3.6 times ten to the ten genomes infectivity, however.  The infectious dose 50 is 1.3 times ten to the minus four micrograms.  So this is about 2.3 times ten to seven genomes.  
		So there's about three orders of magnitude difference with the infectivity of polyoma versus the oncogenicity, and SV40 is about an oncogenicity of a microgram.  Retrovirus, the infectivity, again, is 15 to 30 micrograms.  It's about one to two times ten to the 12 genomes.  V-src was two micrograms at about two, two and a half times ten to the 11 molecules.  Inactivated ras, ten micrograms, is nine times ten to the 11 molecules.
		So we went in the literature to find out what evidence, what data are available for intranasal inoculation.  Unfortunately there is almost none in terms of quantitative estimates.  So the only one we could find was looking at this study by Timer and colleagues in Japan several years ago by using DNA vaccine administered intranasally.
		So this was a clone gene of the hemagglutinin gene from influenza.  It was inoculated into mice via the nasal route.  Different amounts of DNA were administered in PBS, and after four weeks, a second intranasal inoculation of 1.5 micrograms was given as a good.
		So down here we can zero micrograms, 0.2 micrograms, .4 micrograms, and .6 micrograms, and they measured two responses, hemagglutinin inhibition assay and mucosal IgA to the vaccine, and after the primary inoculation, nothing was found except for the six micrograms.  There was a weak response.
		However, after the second inoculation, there was nothing.  No priming inoculation was given demonstrating that this 1.5 microgram secondary -- the boost was not immunogenic.  However, for all the levels of DNA, there was a substantial boost.
		So what this means to us is that even 0.2 micrograms of DNA, in other words, 20 nanograms, administered intranasally elicits immune response since the secondary inoculation boosts this response.
		Therefore, we're concluding that 20 nanograms of DNA can be biologically active when administered intranasally.
		So the other route that we're considering is the oral route.  Now, there aren't that many studies of this either, but Malcolm Martin and Mark Israel's group fed mice polyoma virus DNA between one or 0.5 micrograms, and zero out of 25 became infected with one microgram and zero out of 30 became infected with 0.5 micrograms.
		However, if you stick a gastric tube through, you can get some infectivity, but how this is doing this is through damage of the gastric tube in mice is not clear, but the important point is though that it's unlikely that small amounts of DNA will survive passage through the stomach intact.
		Now, the next study that looked at oral routes is a study by Duffler (phonetic) and colleagues, and they fed a phage DNA -- it's a 7.25 kb phage DNA -- large quantity DNA and followed its fate to see whether anything did escape through the stomach, and they did find DNA in feces, blood, small intestine, and large intestine in the leukocyte population.
		But the size, as you notice, is 100 base pairs to 1,700 base pairs, whereas the original genome was 7,000 base pairs.  So it's clearly getting degraded during passage through the stomach.
		They did find some in the blood, 194 base pairs to 976 base pairs, and perhaps interestingly, they also found about a 700 base pair fragments in blood, and they also found it integrated in 0.1 percent of the cells.
		However, these cells did not survive, and these cells came from the peyer's patches and from the spleen.
		So conclusions that small amounts of DNA can, in fact, pass through the stomach, but no full length DNA was detected, and DNA can be found integrated into the mouse chromosome in a few percent of the cells.  So you have to feed large quantities of DNA.
		So now we can go on to try to get some estimate of risks.  We'll first consider the oncogenicity.
		The cloned activated oncogene, for a single dominant activated oncogene per cell, one microgram of DNA has 152,000 oncogenes, and this figure comes from the DNA in 152,000 cells that's one microgram or one microgram carries 152,000 cell equivalents.  So that's where that figure comes from.
		We know from our previous studies of the literature and for polyoma virus DNA about 3.6 times ten to the seven genomes or molecules are required for oncogenicity, and the activated ras DNA, 9.1 times ten to the 11 molecules are required for oncogenicity.
		Therefore, the number of tumors expected from one microgram of residual DNA with a single dominate oncogene per cell is between 152,000 divided by this.  It gives you a value of 4.2 times ten to the minus six for polyoma, and a value of 1.7 times ten to the minus seven for activated ras, and if you take that the other way, therefore, for ten nanograms of DNA, not one microgram, the probability of an oncogenic event is between one and two times ten the five or 200,000 for polyoma virus, and one in ten -- six times six million for the inactivated ras.  So that's where the calculation comes from.
		For infectivity, we've looked at polio virus DNA, and viral genomes required for infectivity is ten to the seven.  So it's ten million.
		The probability of an infectious event using ten nanograms of mammalian DNA with a single copy of polyoma virus DNA is one in 7,000 events, one in 7,000 events.  This is rather high.
		This also points to the fact that it is very important to be able to determine that an adventitious agent, such as polyoma, not just polyoma; other adventitious agents are not present in the cell substrate since infectivity is a very easy -- much easier than oncogenicity.
		For the retrovirus cloned proviral DNA, the number of viral genomes we've determined is about four times ten to the 11, as a minimum, and the probability of an infectious event for ten nanograms of mammalian DNA with a single provirus is about one in three times ten to the eight, one in 300 million.
		So it's much more difficult for this.  OF course, these values all change if there are more than one copy of the oncogenic agent or of the retroviral provirus.
		So we can draw some conclusions from this.  Infectious risk of DNA can be more important than oncogenic risk, and as I said, therefore, it's very important to determine what level of adventitious agents are present.
		For the IM, intramuscular and subcutaneous routes, ten nanograms of DNA provides an estimated risk for the polyoma virus DNA, one in about 200,000 for an oncogenic event and about one in 7,000 for an infectious event.
		For the more likely events of cellular DNA now, cellular activated ras, there's one in six times ten to the sixth for an oncogenic event, and for proviral DNA for an infectious event, there's one in 300 million, three times ten to the eight.
		For the IN route, the intranasal route, ten nanograms of DNA as I've shown you has an estimated safety margin of one in ten to the sixth for what I'm quoting an expression event.  This is because you can detect the activity of that DNA.  However, that is not an unoncogenic event.  That's just what we can detect by immunological means.  So that's the best one we have.
		And for an oral route, one micrograms of polyoma DNA administered orally is not infectious.  So, therefore, ten nanograms by arithmetic calculation with one copy of polyoma DNA, the safety margin is at least one in ten to the eighth.  
		However, it's not all bad for the risk.  There are some mitigating factors for DNA.  As you probably all know, the uptake, expression, integration are inefficient processes.  Integration requires cell division, and so therefore, not all cells in the body, of course, are dividing.  In intramuscular routes, the myocytes, and muscle cells have a very low division rate, and integration is required for maintenance of DNA in the absence of the replicating system.
		The degradation of DNA vaccine manufacturer procedure usually requires degradation of the DNA, and of course, DNA is degraded in vivo.  It doesn't exist for very long in vivo.
		So if the DNA is degraded below the size of a gene, then that increases its safety.
		There's a host immune response to transfected cells, as Dr. Cook just mentioned.  So even if the DNA does get in and express these cells in an immunocompetent individual, it will likely be removed.
		What also Dr. Cook talked about eloquently is the multi-step nature of carcinogenesis.  We now know that it's not just a single event.  So even if an activated ras did get into that cell, that cell would not be established as a tumor right there.  There are many more steps that have to proceed before carcinogenesis can occur.
		And, of course, we all know that transmission of human cells in culture is much more difficult than transmission of rodent cells, and Dr. Alex van der Eb discussed some of these issues earlier on.
		So what are we doing at CBER to try to help to address this?  Well, Dr. Hughes mentioned that we have now initiated a study.  We have finally got some funding from the FDA, and in a collaborative study with CBER and the National Cancer Institute and the Center for Drugs, we're going to study in a systematic way to develop numbers that have been alluding everybody for 40 years.
		These issues come up every five to ten years, and still nothing is being done.
		So we're trying to develop sensitive animal models to detect oncogenic activity of DNA.  The models that have been heretofore used have not been systematically developed and are not very sensitive.  So what we're going to try to do is test newborn NIH Swiss and C57 Black 6 mice, the athymic nude mice, and K6/ODC mice, which is a constitutively promotive mouse line, and we're going to use them making the activated ras genes.
		And our colleagues, and we're very grateful to have John Coffin and Don Blair and Steve Hughes from NCI and Frank Sistair (phonetic) from CDER to help and advise us on these studies.
		The other assay we're trying to do with CDER is to get some quantitative estimate of DNA infectivity, and we want to develop quantitative in vitro assays first, of course, to affect the infectivity of proviral DNA in retroviruses.  It is not known what level of proviral -- levels of provirus that is in mammalian DNA are infectious.
		Howard Tammin (phonetic) has shown that in mammalian systems, the integrated provirus is, in fact, infectious in vitro, but no quantitative estimates of that were attempted.
		In addition, we'd like to go on and discuss the integrated and extrachromosomal genomes from DNA viruses.
		And I think that's it.  Thank you.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Peden.
		We will take a few questions if there are before adjourning.  Dr. Griffin.
		DR. GRIFFIN:  It seems to me that the quantitative studies you talked about are just crucial in order to really be able to tackle some of these issues, and I don't know anything about the K6/ODC mouse.  Is that one that -- I was thinking of using mice that already have the first hit, so to speak, and therefore, measuring oncogenicity, you know, you might get a readout that's higher or more likely to see tours.
		Because I guess one of the concerns for a vaccine oriented use is that you will provide the first hit, and you won't get tumors due to that, but then that the individual would be more susceptible to the second and third and fourth hits at subsequent times.
		If you could go into a system where some of those things are already present, if you would -- I don't know -- give us a better readout.
		DR. PEDEN:  Yes, you're right.   I didn't mention it, but, in fact, the idea of a first hit and then you give to a vaccinee and then subsequently you get additional hits is a worrying issue.
		We don't know how stable those initial hits are.  If you give it to a cell, where that cell eventually gets lost, you don't retain that so-called activated state in the cell.  We don't know that.
		As for the models, the ODC mouse is a carotin 6 promotor that drives olefin de-Kalb oxylase (phonetic), and that's been used in carcinogenesis activities because it's constitutively what they call promoted.  So if you add an initiator such as ras, it's a very sensitive model on the skin, of course, because --
		DR. GRIFFIN:  So it does provide a more sensitive --
		DR. PEDEN:  So it does provide that.  We discussed with Steve Hughes and John Coffin and Don Blair about which mice to use.  We're thinking about using the heterozygous p53 mouse, and perhaps Steve or the others could comment on that, but we decided in the first run through to do these mice.  I think the homozygous p53 knockout was generally felt, I think, by Steve just to have too much background.
		And so we're trying to balance it.  I'm glad you do point out the importance of doing these quantitative studies.  I don't want to lessen what's done in the past, but they were never intended for that role, and of course, the infectivity in monkeys is an incredibly expensive thing.  So there isn't any information on that.
		So I don't know whether Steve wants to comment on the particular animal models or Don or John.
		DR. BLAIR:  No, I think we chose, you know, sort of a reasonable spread of different strains of mice which might show up some strain differences.  The nudes as an immunosuppressed, perhaps more sensitive mechanism, and as you say, the problem with some of the p53 knockouts and things is as you increase the sensitivity, you increase the background, and you may obscure things.  So we went with something where we felt the background wasn't going to be insurmountable and we should see positive.
		DR. HUGHES:  I also would add that I don't think that the studies that have been outlined so far will necessarily be the only things that will ever be done, and I think that based on what we would like to do is to have what we hope is a reasonable preliminary survey and see, in fact, the degree to which we get response with relatively lower amounts of DNA.
		And depending on the outcome of these experiments, I think we would be better prepared to go back and actually plan and propose a second round in which, depending on what the outcome is, something like a p53 heterozygous animal might be employed.
		But because they're actually, as you point out and Dr. Peden has pointed out, there's relatively little really quantitative assessment, we thought we'd make what we intend to be the first attempt to do that, and then based on what we learn from that, try and go ahead in a reasonable fashion and maybe expand this to other model systems.
		ACTING CHAIRMAN DAUM:  This is, again, an issue that I think we should encourage everybody to raise in this afternoon's session when we're talking about the big picture issues with respect to this approach.
		I'd like to focus now on Dr. Peden's presentation though, and Dr. Aguilar-Cordova is next and then Dr. Faggett and Dr. Moulton.
		DR. AGUILAR-CORDOVA:  Yes.  Given the numbers that you presented and there may be various oncogenes in any one cell or activated genes or whichever, in your interpretation  would it make a significant difference whether it was a spontaneous tumorigenic cell line versus a designer cell line in which one of the many events might be known?
		DR. PEDEN:  So now are you saying the oncogenes from a spontaneously transformed cell is studied?  I mean, these are cloned oncogenes injected.
		DR. AGUILAR-CORDOVA:  This is like the worst case scenario.  What I'm saying is based on your statistical analysis that you presented --
		DR. PEDEN:  Well, I wouldn't grace it with statistical analysis, but --
		(Laughter.)
		DR. AGUILAR-CORDOVA:  Or statistical deductions.  I guess the general question would be would, say, in the presentation we heard earlier from Dr. Lewis, do you think that based on that would A549 residual DNA pose any significant different risk than 293?
		DR. PEDEN:  I think there's not universal agreement, I think, among us about that, but I think the general feeling is yes.  The main reason, you don't know what the oncogenes were that brought about the A549 phenotype.  So that's one thing.
		And also the other issue about A549 is you don't know what's in there.  Is there an oncogenic agent that brought about that?
		So I think in general terms there is a big difference between a cell that you know hits passage history and you transform it by another mechanism.  I think that's why we are calling these designer cell substrates and we call them at low risk, such as A549.
		So there's two reasons for that.  The both I've just said to you.
		I think for the study what we want to do is, in fact, determine ultimately the most sensible animal model where we can look at genomic DNA, DNA from A549, from PER.C6, to see where that can induce an oncogenic event.  So that's really where we're going at, although I didn't, in fact, say that.  It's to see whether you can, in fact, detect an oncogenic event using DNA from such things as A549 cells.
		That's where we want to go, but we need to develop these animal models, sensitive animal models for that reason.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Faggett, and then Dr. Moulton, and then we will adjourn for lunch.
		DR. FAGGETT:  Now, what prompted the change from the WHO requirements for residual cell substrate DNA levels from 0.1 nanograms in 1986 to the ten nanogram level in 1999?  Was there some --
		DR. PEDEN:  I think as we get more and more information, we do these experiments.  See, more information came, more experiments were done.  You may think they were deficient, and we may agree with you, but we have more knowledge and more data and also more clinical experience with some of these cells, such as Vero cells.
		So I think it's a combination of all things, but I wasn't at that meeting.  So maybe Phil and Andrew were at the meeting.  I don't know what specifically lowered except for these numbers that I just presented to you.
		But, Phil, do you want to say anything?
		DR. KRAUSE:  Yeah, maybe I could just comment.  I think an important thing to remember is that these meetings that have gone on in the past, in general, have been talking about the older kind of cell substrates, and here we're talking about newer kinds of cell substrates.
		And so I don't think that the idea of using an A549 cell, for instance, was specifically considered as one of the things that was going into raising that limit from 100 picograms to ten nanograms at that meeting, but instead the idea was to consider the kinds of cells with which there was much more substantial experience in vaccine and other biological production.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Moulton.
		DR. MOULTON:  Yeah.  This is a great start, I think, towards the full risk analysis.  I think a full risk analysis would have probability distributions associated with every one of the assumptions there.  There's a lot of assumptions we're making.  For example, the potential mitigating factors are going to reduce the risk.  There's other assumptions that could increase it, and a couple of people have already mentioned the problem of the low dose extrapolation, which could affect things by a couple of orders of magnitude.
		And I was wondering if there's a plan even before we get the result of these future experiments to try and put some probability distributions on there, every one of those assessments, and also a more general question of how does this risk analysis fit into the general risk management program at FDA.
		It's one thing to estimate or calculate a risk, estimate that point estimate, and if you're lucky get some kind of tolerance interval around it.  It's another to actually manage the risk once it's been estimated.
		DR. PEDEN:  Well, the way we manage the DNA risk is to require that the levels are low.  I mean that's the simple way to do that.
		As for the probability, many people have tried to make estimates, probability estimates at all of these steps, and i can give you lots of references where that was done.  I don't find that that's satisfactory nor useful because there isn't -- I mean, the probability of integration event, the probability of out-take of DNA, that's all those events you're talking about, and that's the evidence.
		The consideration of those is all done from in vitro studies.  They've been transfecting DNA in culture and measuring events, and so to me that's not very satisfactory to do that, to extrapolate from what goes on in vitro to what goes on in vivo.
		That's why I think we need the in vivo data to try to get real numbers as opposed to these estimates.
		ACTING CHAIRMAN DAUM:  This is a topic that I think must be also returned to this afternoon.  So perhaps, Dr. Moulton, you will remind us that we need to address this and bring it up again.
		I'd like to thank Dr. Peden at this point very much for another fine presentation that helped us think.  Dare I say you provided us a great deal of thought for food.
		We will now break for lunch.  It's 12:45 here in the Eastern time zone, and we will reconvene at 1:45 exactly.
		Thank you.
		(Whereupon, at 12:45 p.m., the meeting was recessed for lunch, to reconvene at 1:45 p.m., the same day.)

	A-F-T-E-R-N-O-O-N  S-E-S-S-I-O-N
	(1:49 a.m.)
		ACTING CHAIRMAN DAUM:  Okay.  We are in afternoon session.  
		Welcome back from lunch, and we'll begin with the adventitious agent issue this afternoon.  We have three or four presentations on that, and we'll begin with Dr. Philip Krause from FDA who will introduce us to the topic.
		DR. KRAUSE:  Vaccines are among the most effective public health interventions ever devised.  Vaccines, however, are very dependent on public confidence in their safety, and that's really for several reasons, but one of the most important is that vaccines are generally given to healthy people, many of whom may never be exposed to disease which one is trying to prevent, and another reason, of course, is the need in many cases for many vaccines to generate high enough immunization rates to get some level of herd immunity, without which one doesn't get maximal vaccine benefit.
		And of course, since many vaccines are given to healthy children, these issues are even more important.
		An important component of public confidence in vaccine safety, of course, is their freedom from adventitious agents.  So what is an adventitious agent?
		Well, for the purposes of this discussion at least we're defining an adventitious agent as an infectious agent that is extraneous to the product, and so a vaccine strain obviously is not an adventitious agent, but something that's carried in from the outside or carried in with the cell substrate, is potentially an adventitious agent, and an obvious goal then is to insure that final products don't contain adventitious agents.
		Now, there are two additional points I want to make, and just because I say final products here doesn't mean that intermediate products should contain adventitious agents.  In fact, all through the production process it's important to keep adventitious agents out of vaccine manufacture, and the other thing which we've spent some time talking about already is trying to define in some quantitative way the terminology of "should not contain."
		So any negative result obviously needs some quantitation associated with the sensitivity of the testing, which allows one to exclude that something is there.
		Now, in general the OVRR approach to adventitious agent issues has included identifying potential issues, including theoretical ones, discussing these issues in public, and Dr. Lewis described the two previous Advisory Committee meetings at which this came up, as well as the public international meeting on cell substrates, and of course, this meeting is another component of that, making decisions based on the best available science and, in addition to that, insuring that any potential issues are known and understood by research subjects and investigators.
		So today we're going to be talking about two potential adventitious agents or types of potential adventitious agents.  One is transmissible spongiform encephalopathy agents, and the other is viruses.
		Directly following this brief introduction, Dr. Sue Priola will talk in some more detail about TSE agents, and then after that, I will provide some general principles of adventitious agent testing, as well as a more detailed discussion of virus as potential adventitious agents.
		So to introduce Dr. Priola's talk, I'm just going to go over a few things related to our consideration of transmissible spongiform encephalopathy agents and cell substrates.  And the kinds of issues that can come up that might increase the risk that a cell substrate could harbor such an agent.
		And I guess one possibility is that because some transmissible spongiform encephalopathy diseases, such as Creutzfeldt-Jakob disease and Gerstmann-Straussler syndrome are at least in part genetically determined.  The question arises of whether a cell substrate which contains mutations that are associated with these types of diseases might then harbor a greater risk of either having TSE agents in it or, if exposed to TSE agents causing infection or being infected with them and thereby transmitting them to a vaccine recipient.
		The second question, which is a general one which comes up and which this committee has addressed before to some degree at least, is the consequence of potential exposure to serum from countries where bovine spongiform encephalopathy or the risk of BSE exists.
		And this is an issue which has come up in the context, in particular, of cells which have been in laboratories in the early to mid-1980s at a time before this risk was as widely appreciated as it is now.
		And then, of course, there are other factors that could theoretically increase the risk of TSE infection of cell substrates.  One of these is PrP expression levels.  It's been shown at least in some systems that cells that express higher levels of the PRNP gene that encodes PrP, that the risk of those cells being able to propagate TSE agents is higher, and then also there's the question of whether cells of neuronal or retinal origin might also have greater risk of at least propagating TSE agents if exposed or of containing them in the first place.
		Now, there are a couple of neoplastic cell specific notions that are also TSE related.  One of them is that because neoplastic cells often have some associated genomic instability, whether, in fact, that genomic instability might itself induce mutations in the PRNP gene, which might then have some consequence on the ability of those cells to harbor TSE agents.
		And then also, it appears that at least in some cells the ability of a cell to undergo apoptosis helps to prevent infection with TSE agents in them, and because neoplastic cells have in most cases at least some apoptosis pathways abrogated, the question then is whether a neoplastic cell might a priori be a little bit more susceptible to infection with TSE agents.
		So at this time, we're following the approach to TSE issues that's outlined on this slide, and that is where possible to determine the family and medical history of the cell donor with respect to TSE risk factors; to ask that the PRNP gene be sequenced; to ask that Western blots be performed to look for the presence of protease resistant  PRP; and to insist that they be negative, of course; and to determine if exposure to fetal bovine serum from countries with BSE or with the risk of BSE could have occurred, and then if some possible exposure to questionable fetal bovine serum has occurred, to perform a risk assessment based on the dilution factor from the time of that exposure, based on the assumption then that the cells cannot support replication of a BSE agent.
		There are several evolving ideas which we don't think can be applied right now, but which may be applicable in the future.  One of them is if possible exposure to fetal bovine serum of unknown origin has occurred, to document that the cells cannot support replication of the bovine spongiform encephalopathy agent.
		And this time I don't believe that there are any cells in tissue culture that have been demonstrated to replicate the BSE agent.
		To evaluate the level of PrP expression, which although it is generally accepted, I think that cells that express higher levels of PrP might have a greater risk of propagating TSE agents if exposed.  The question then also comes down to defining what exactly would constitute a safe level and understanding in a way that can be applied quantitatively what kind of controls would be necessary to use such an assay in a regulatory setting.
		To evaluate for the presence of TSE agents by animal inoculation, and as many of you know, the monkey, the primate models take a very long time to carry out, but there are promising transgenic mouse models which could potentially be used after they are appropriately validated to get more rapid answers to these kinds of questions in an animal model, and of course, the general principle that once new assays for detection of TSE agents become available, to introduce them for cell substrate testing as soon as is feasible.
		So with that, I would like to stop here.  I'm happy to take any questions, but if there aren't any, then we'll go on to Dr. Priola's talk on TSE agents as an issue in the use of neoplastic cell substrates, and I think she'll concentrate, in particular, on issues associated with TSE agents in cell culture.
		ACTING CHAIRMAN DAUM:  I think we'll go right on if that's okay because we'll have a change to ask questions.
		DR. KRAUSE:  You'll get another crack at me.
		ACTING CHAIRMAN DAUM:  That's right.  Thank you.
		Dr. Priola here?  There she is.
		DR. PRIOLA:  I would just like to thank -- can everybody hear me? -- to thank Dr. Krause for giving such a terrific introduction.  He left me with a few things to talk about, but he hit really on every major point that is going to be an issue in what I go over.
		What I want to do is basically give a very brief introduction, basic introduction to TSE diseases, and then describe using experimental examples what we know about the difficulties associated with passaging TSE infectivity into tissue culture.
		So do you have the presentation up?  There you go.
		I think as we all know that TSE diseases are long term transmissible, degenerative diseases.
		Have you frozen up there?  It's coming on.  It's the good at explanation point.  Oh, no, there it goes.
		(Laughter.)
		DR. PRIOLA:  I want to wait until this comes up.  Okay.  There we go.  Thank you.
		All right.  So the TSEs are slow, fadable, and transmissible brain diseases that affect a variety of mammals, including, of course, humans.  Creutzfeldt-Jakob disease in humans is the prototypic, one of the prototypic TSEs, scrapie in sheep, and of course, BSE in cattle.
		One of the things about the TSE diseases is there's this phenomenon called the species barrier to infection, and it's this that has caused all of the concern when it was realized that SE from cattle apparently has crossed over and caused a new form of CJD in people in the U.K.
		And usually the species barrier is quite strong in these diseases, and so it was unexpected that cattle BSE would necessarily pass into people.
		And, of course, it's this concern that now that we have BSE in cattle populations that contamination of bovine products used in tissue culture could then passage that TSE infectivity into tissue cultures using those products.
		Experimentally, the primary systems are TSE infections of mice and hamsters, and most of what I'm going to talk about today deals with that.
		It's important to remember that within the TSE there are different strains of TSE agents.  So there has to be some caution in interpreting experiments using one strain of agent and one species of animal and extrapolating those data to other species and other strains.  I'd like you to keep that in mind.
		Infection, of course, is believed to be primarily from ingestion or inoculation.  It's an extremely long disease course.  It can take from months to decades to appear.  It is always fatal.  There are no preclinical diagnostic tests available.  There's no effective post or preclinical treatments available.
		If that weren't bad enough, it turns out that we're still not entirely clear what the exact composition of the infectious agent is, but the fact that it's unusually hard to kill and that there's no viral or bacterial association with TSE infectivity -- good Lord.  Can I go back?  Good luck.  Okay -- led to the hypothesis that, in fact, it was an infectious self-replicating protein that was responsible for these diseases.
		In the early '80s, a protein was found that was very closely associated with TSE infectivity in Stan Prusiner's lab, and that protein was called prion protein, or PrP, and I think as most of us are aware here, PrP is, in fact, a normal host cellular protein.  It's something that's expressed in all mammals, and it's expressed almost ubiquitously.  It's very difficult to find a tissue or cell that does not express PRP.
		In its normal form it's sensitive to digestion with cellular proteases, and for that reason I'm going to call it PrP-sen for protease sensitive.  As I said, it's almost ubiquitously expressed, and it's soluble.
		During TSE disease, the normal form of PrP, which is shown here -- it's a cell surface glycoprotein -- is converted to an abnormal form that is now partially resistant to proteinase K, and the partial proteinase resistance comes about because when you expose this molecule to proteases, what happens is instead of digesting everything away as it would if it were just PrP-sen, it basically can only clip away part of the end terminus, and you see this very characteristic side shift down like this, and that is what we define as PrP-res or protease resistant PrP.
		This form is now insoluble.  It's heavily aggregated, and it is completely TSE specific.  It's the marker that everybody looks for.  If you find PrP-res, by definition you have a TSE disease.
		It's found primarily at high levels in the CNS in some animal models it's found in the lymphoreticular system as well.
		It has a different confirmational structure than normal PrP.  The normal PrP is primarily optihelical.  When it gets converted to the abnormal form, it becomes beta sheeted structure, and it's believed that it's this difference, it's this change in confirmation that accounts for the different properties of these molecules.
		Now, while there's still controversy within the field about the role of whether or not PrP-res is the infectious agent, the role of PrP in TSE diseases is absolutely undeniable.  You need normal PrP for infection.  Mice that don't express this gene do not get sick following exposure to TSE agents.
		Mutations in this molecule can strongly influence disease susceptibility and species barriers to infection.  PrP-res, the abnormal form is associated with toxic events in the brain and is, of course, always associated with infectivity.
		So in terms of human TSE diseases and the dangers involved, not dangers, the possibility involved with deriving neoplastic tissue culture from human cells, there are three groups of human TSE diseases.  
		There is the sporadic form known as Creutzfeldt-Jakob disease or CJD.  There's no known exposure to TSE infectivity.  This form of the disease is not associated with mutations in PrP.  It accounts for the vast majority of TSE cases.  It's extremely rare.  It happens annually worldwide at an incidence of about one case per million people.
		There's also familial forms of the human TSEs.  These include familial CJD, Gerstmann-Straussler-Scheinker syndrome.  These forms are also not associated with any known exposure to infectivity.  They are, however, associated with mutations in the PrP molecule.  They are even more rare than this radic (phonetic) disease with about one to ten cases per 100 million people.  There are very few families in the world that have been identified that carry these mutations.
		Finally, I have lumped together here under the term "infectious TSE" infections which are a result of exposure, a known exposure or presumed exposure to infectivity, and these include iatrogenic CJD cases that arise following exposure to contaminated medical instruments, and of course, variant CJD in Great Britain.
		Is there a problem?  You can't see the slides?  I'm not speaking clearly enough for you to see the slides?  Sorry.  I'm glad you told me.
		All right.  So where in the heck was I?  Okay.  So in terms of it being an issue in cell culture, the problems are can you infect tissue culture cells easily with TSE agents.
		You're going to have to wait a minute.  I've got to reorganize myself.
		And as alluded to by Phil, there are three basic issues.  First of all, development of new cell lines from CNS tissue of patients who are potentially contaminated or who are potentially infected with Creutzfeldt Jakob disease or a derivation of cell lines from an individual carrying familial PrP mutations, and I'll address both of these issues.
		There's also what has become a concern particularly in the last couple of years whether or not tissue culture cells exposed to bovine derived products could become TSE infected, and I'll address this and the susceptibility factors that might be involved.
		And finally, given these two possibilities, what is our ability to detect TSE infectivity in these cells?
		All right.  So the first thing I want to address is is it, in fact, possible to derive persistently infected TSE infected cultures from immortalized cell lines derived from human cells, and these experiments were all done many years ago, 20 to 30, almost 40 years ago now.
		And in these experiments essentially tissue was taken from the brain or spinal cord of CJD or Kuru patients who had died of CJD or -- this thing is extremely sensitive.  I've got it.  It's okay -- had died from CJD or Kuru, were maintained in tissue culture for over 300 or up to 300 days, and then the presence of infectivity assayed for by bioassay in primates, and they could always detect infectivity.
		Similar experiments were done in mouse and sheep models of scrapie.  The point I want to make here is that even though you could maintain infectivity if you just kept the cells alive, the minute you start passing them, you lose infectivity.
		Okay.  So after one or two passes in the human system, you lose infectivity, and you can't recover it upon further passage.  You can, however, derive immortalized cell lines infected with TSE agents and other systems because this approach did work in both the mouse and sheep system.
		So even though it didn't work for human, theoretically it's been shown to work in at least two other system.  So the possibility exists.
		What about familial TSE diseases?  There are a wide variety, up to 20 mutations in the PrP gene that have been associated with familial TSE disease, and they're spread throughout the molecule, and we don't know exactly how they lead to disease, but the idea based on the protein only theory of the TSEs is that these mutations lead to altered biochemical properties of the normal PrP molecule.  It then spontaneously converts over to the abnormal form, accumulates, and causes disease.
		So what is the evidence that this actually happens?  Because, of course, this is the concern if you should immortalized either cell line derived from an individual carrying a familial mutation.  Does this actually happen?
		And in my laboratory over the last few years, we've been working with PrP mutants that involve insertion of extra copies of an octapeptide repeated motif, and when you look at these mutations, so what we've done is basically taken a PrP molecule and just added more and more numbers of these repeats and asked about the properties.
		I hope you can see this.  What you find is that if you look at two of the cardinal properties of PrP-res, the abnormal form, protease K resistance and aggregation, you can see the red line here.  As you increase the number of repeats, you do, indeed, increase the relative protease resistance of PrP.
		To put it into perspective, the green line represents the abnormal form PrP-res.  At the level of PK where all of the protein is destroyed, PrP-res hasn't even been touched.  So this molecule is not PrP-res by this definition.
		Similarly, when you look at the amount of it aggregated, yes, as you increase, as you have these mutations, the molecule begins to aggregate more.  It still does not do so to the same extent as PrP-res.
		So while the biochemical properties of these molecules change and they begin toe acquire some properties reminiscent or res, they are not PrP-res, and believe me, we've tried.  We cannot get them to spontaneously form PrP-res.
		All right.  So this is all in tissue culture.  What about in vivo?  If you take these cells and stick them into animals, you also don't get TSE disease.  Okay?  So just sticking in the cells.
		If you make transgenic mice over expressing these mutations, so try to mimic what happens in people, what do you get?
		And what I've done here is really very briefly summarized experiments done by several labs where they basically inserted human associated familial TSE mutations in to the PrP gene, overexpress them in mice, and then assayed them for disease, and a couple of points I want to make here.
		Number one, you need overexpression to get any sort of neurological disease.  You don't need the mutation necessarily because without the mutation and over expression, you still get neurological disease.  Okay.  So overexpression itself is sufficient to cause symptoms in the animal.
		For these top two models when they looked at these mice, they did show signs of neurological dysfunction, no TSE disease, and they certainly didn't passage infectivity.  There's this one GSS associated mutation where this was overexpressed in animals, had the neurological disease, and there may have been some transmission into limited -- in a limited way into other mice.
		So this is the one instance where there may be some evidence that this mutation could induce disease.  When you look, however, at this same mutation in a context where it is not overexpressed, which is much more analogous to the human system, all right, so this is an instance where mouse was made where the mutation was put into the PrP gene and that gene put right back into the correct spot in the chromosome at the correct copy number.  You don't get neurological disease.  You don't get TSE disease, and you don't get infectivity, and the laboratory that did this the last I heard had looked at 800 and some mice.  So they're looking very carefully.
		So there's no really strong, convincing evidence that mutations, familial TSE mutations will generate spontaneous TSE disease in tissue culture or in transgenic mouse models.
		Now, what about infection of tissue culture cells, particularly in regard to exposure to bovine derived tissue culture products?
		What are the susceptibility factors?  How much infectivity do you need?  How is easy is it to get an acute versus persistent infection?
		And what I've tried to do on this next slide is summarize all of those points in a little model, and what I've shown here is each of these black boxes represents what I think is a major block to persistent TSE infection of tissue culture cells.
		First of all, you need extremely high -- you need high multiplicity of infection.  So routinely what we used to do is brain material, high titered infectious brain material.  This automatically calls into question whether or not the very low amounts of TSE infectivity that have been hypothesized to be carried around in blood would be sufficient to overcome this barrier where you need a whole lot of agent to get a little bit in.
		The second thing, even if some of this agent gets in that has to be considered is, first of all, does the cell express PrP-sen theoretically any cell that expresses normal PrP is susceptible.  So if it expresses PrP-sen, it's got the right sequence.  So say this is bovine, BSE, PrP-res, and this is human PrP-sen.  Right away you've got a problem.  The sequences of these genes do not match, and that will be an issue in terms of the abnormal form reacting with the normal form and causing propagation of the abnormal form.
		Okay.  So let's say all of this is okay.  This matches that.  There's enough PrP there.  The sequence is the same.  It's folded appropriately.  This process occurs.  You've passed the second barrier.
		Then comes the third one, and this is a big one, and that is that as you start to passage these cells and dilute out whatever cells might have become infected, you lose infectivity.  
		Even if you get an infected monolayer, in many cases there are as few as one percent of these cells infected.  All right?  And the consequence of that is that if you look at an infected cell layer like this for PrP-res, by current techniques you're probably not going to find it.  Okay?
		So while the presence of PrP-res is a very good indicator that the cell is infected, the absence does not mean it is uninfected, and that's what I want to show here.  This is an experiment I did years ago where we tried to infect cell lines that express relatively low levels of PrP-sen.  We assayed at various times after exposure to an infectious brain homogenate.  
		We looked both for PrP-res, which we could not find, and we looked for infectivity in mouse bioassay, and we easily found infectivity, and at every pass and at equivalent levels.
		Okay.  When we did this experiment in cells that had been maintained in tissue culture a bit longer, so that these were relatively new cells; these had been maintained a couple of months longer; all of a sudden we couldn't infect them, and this gets back to the point that Dr. Hughes made earlier this morning that cells change over time in culture, and in this instance, it changed in a good way for you guys and a bad way for me because, you know, I want infected cells.
		The other point I want to make, and Dr. Krause referred to this a couple of times in his talk, is this issue that if you overexpress PrP-sen, and this again is probably not something that should be a concern with the cell lines that are being used here, if you artificially overexpress PrP-sen, now you get a tissue culture line that is more susceptible to infection, and the idea is with more PrP-sen, you can accumulate more PrP-res, which you can then detect more easily, and you can infect more easily.
		And our experience has been this is simply not true.  It's completely unpredictable, and this is a piece of data that hasn't been published yet that just shows in neuronal and non-neuronal cells exposed to four different strains in this instance of mouse scrapie.  So eight combinations.  In only one case is there very clear evidence of formation of the abnormal form, and that's this one right here.  All the others are negative.
		And I could not have predicted.  I mean I could have thrown a dart at this thing and predicted it just as easily as the result I got.  So even overexpression, while it could be considered a susceptibility factor, you can't use it to predict susceptibility.
		And just to summarize, all the different cell types that have been looked for susceptibility to TSE infection, there have been both neuronal and non-neuronal cells described that are susceptible to mouse and this bottom one here, the kidney epithelial, to sheep scrapie if they expressed the sheep PrP gene.  All of these cells, including a couple of cell lines that were referred to earlier this morning by Dr. Lewis, they're using vaccine production.  These guys have not been shown to maintain human TSE infectivity.
		So there are a lot of nonsusceptible cell lines, and you can see a lot of these are human derived.
		Okay.  So finally, to briefly mention what our ability to detect TSE infectivity in the absence of detectable PrP, in the absence of bioassay, there are now three approved and marketed tests for checking for PrP-res in infected animals, and these are being used extensively in Europe at slaughterhouse levels to look at cattle, potentially BSE infected cattle.
		These tests are all maximized for high titer tissue, such as brain or spinal cord, and they're only sensitive to PrP-res levels that you detect shortly before or at clinical signs.  So this is where you have a lot of infectivity present in the animal.
		This is not like tissue culture cells where you have very little infectivity present.  Okay?
		There is another test that's coming through in the approval process.  This is the Adelphia test, which may be a bit more sensitive than these three.  There are a couple of future tests that have not really been -- in particular, the capillary immunoelectrophoresis have not been rigorously tested, but have been proposed to be able to detect extremely low levels of PrP-res in tissues such as blood.
		So the difficulties with all of these tests, the postmortem test, they're based only on res detection.  They're maximized to high titer tissue, and we haven't actually tested them against our tissue culture cells.
		Their sensitivity, again, is not terribly -- not as good essentially as bioassay in either the same species or in appropriate transgenic mice.  So bioassay remains the gold standard test for detection of TSE infection, and as Dr. Krause referred to, the time required for this assay often makes it impractical, but it's very important.
		So given the data that we currently have in tissue culture, it's, you know, unlikely that human neoplastic cell substrates will be TSE infected or that exposure to potentially BSE contaminated bovine tissue culture products could lead to persistent TSE infection, but of course, as we're all well aware, you cannot guarantee zero risk.
		And in the absence of that, what can you do to at least assess it as carefully as possible, the presence of TSE infectivity in these cultures?  And this is the answer I came up with, and after finishing it, I realized it's basically what we do in the lab to assess whether our tissue cultures contain infection.
		There are now commercially available tests that can be used to do this, and as, again, Dr. Krause mentioned, you assay for PrP-sen, its expression level, determine its sequence.  If it's a familial mutation, throw the cell line out.
		Assay for PrP-res, and this would best be done in multiple cell sub-clones from the passage, as well as periodically at different cell culture passes because of the potential instability of the cell.
		Bioassay for infectivity if all of this is negative.  If any of these are positive, you know, again, throw out the cell line.  If these are all negative or if this is negative, bioassay for TSE infectivity would have to be done, I should think.
		And in terms of looking for human TSE infectivity or BSE, perhaps the best bet is to use human PrP or bovine PrP expressing transgenic mice.  Keep those animals for up to two years, watch for clinical signs, and at the end of the day, look in the brains for pathology and/or PrP-res, and to do this, you know, relatively routinely if you're worried.
		So I'll stop there pretty much on time and take any questions.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Priola.
		And we'll have this presentation open for discussion.  We'll begin with Dr. Decker.
		DR. DECKER:  Just a simple clarifying question to see if I've absorbed your implicit definitions correctly.  When you and Dr. Krause used the term "infected," you're referring to a cell line that actively is producing the abnormal prion protein, and if we're just talking about pouring in some preformed abnormal prion protein, you're going to use the word "contaminated" or something like that?
		DR. PRIOLA:  Yeah, that's correct.  When I use "infected," I mean persistently infected.
		DR. DECKER: So we've got a cell there that's actually --
		DR. PRIOLA:  Producing infectivity.
		DR. DECKER: -- producing.
		DR. PRIOLA:  Yeah.  It's making PrP-res, it's accumulating PrP-res, and it's making new infectivity.
		DR. DECKER: Rather than simply pouring contaminated --
		DR. PRIOLA:  Exactly.
		DR. DECKER: -- bovine serum in there which eventually gets diluted out and doesn't reproduce.
		DR. PRIOLA:  Right, which is what's suggested by those immortalization experiments, yeah.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Coffin, please.
		DR. COFFIN:  It's technically feasible, although probably not very pleasant, to take cell lines that one imagines one might use for vaccine production purposes and actually knock out both copies of PrP.  From what I understand, such cells would be expected to be perfectly viable and you wouldn't notice the difference.  The whole animals are perfectly viable without --
		DR. PRIOLA:  Yes.
		DR. COFFIN:  -- PrP.  Do you think that would be an advisable thing to do?
		DR. PRIOLA:  Well, if you took away the PrP, then I think you'd have a cell line that could be very strongly argued would be completely resistant to TSE infection, and I have never made knockout cells myself, except from knockout mice, for example, PrP knockout mice, but if it could be done, and it was not technically too difficult, then, yeah, I think it would be -- it would take away an element.  You know, what little risk there is, it would take that away because PrP knockout mice are resistant completely to infection.
		ACTING CHAIRMAN DAUM:  There was one more hand here.  Was it Dr. Myers?
		DR. MYERS:  When you're growing, in effect, cell in culture and it loses the infectivity, talking to people about this, the implication for me is that if you're growing the cell fast enough, the cell outgrows the PrP-res.  Is that not an appropriate way of looking at it?
		DR. PRIOLA:  Well --
		DR. MYERS:  Because an awful lot of your infected cell lines are very slow growing things, and you mentioned this business about the TSE from the infected brains and so on.  Is that a misconception?
		DR. PRIOLA:  You know, I don't think it's a hard and fast rule.  The neural 2A cells, which are mouse neural blastoma cells, we have troubles with losing infectivity in these cells if we don't use very specific types of medium.
		There's another persistently infected cell line called an SMB cell that is perfectly stable.  It is slow growing.  These fibroblast cells I showed you where it appears we got infectivity in there based on the res signal, we have to split those if we're not careful every two to three days.  They are not slow growing, and they are very reproducibly infectable based on PrP-res with this one strain.  So I don't think it's a hard and fast rule.
		ACTING CHAIRMAN DAUM:  Thank you. 
		Dr. Katz.
		DR. KATZ:  I have a naive mechanical question.  When you talk about passages and failure or success at pass, you're talking about passing suspensions of cells, not just tissue culture fluid; is that correct?
		DR. PRIOLA:  That's correct.  These are passage of TAP cells, yeah.
		DR. KATZ:  Thank you.
		ACTING CHAIRMAN DAUM:  Dr. Aguilar-Cordova and then Dr. Stephens.
		DR. AGUILAR-CORDOVA:  I was wondering.  When you talk about some people being contaminated with hospital instruments, would that be expected to be a large inoculum, and then from your experiments in vitro, it seems like you really need a huge inoculum to get anything going.  So could that be a big differential between in vivo and in vitro?
		DR. PRIOLA:  Yeah.  I mean, in vitro you definitely need much more infectivity to get a productive infection of tissue culture cells.  As to how much infectivity is actually on a contaminated surgical instrument that passes this, I don't know, but I do know that when it happens, the people who have -- what essentially happens is an operation is done on a subclinical CJD patient, and those instruments are used in other patients before it's realized, and those other patients do get CJD.
		So it's pretty effective, and with presumably, you know -- well, I don't know the dose.  I don't know the dose, but it may be very low.
		ACTING CHAIRMAN DAUM:  Dr. Stephens, and then Dr. Coffin.
		DR. STEPHENS:  Just a follow-up on the PrP knockout question.  What does PrP do in normal cells?
		DR. PRIOLA:  That's a really good question.  It's not precisely known, but there was a recent really nice paper in science that suggested that if you cross-link it in differentiated neurons, it activates the Finnkinees (phonetic) pathway.
		DR. STEPHENS:  And as a follow-up, was there any evidence, is there any evidence of an inoculum effect in new variant CJD in terms of BSE exposure?
		DR. PRIOLA:  You mean inoculum effect, how much you need?
		DR. STEPHENS:  Yeah, how much?  I mean, is there a relationship in any of the epidemiological studies to beef exposure?
		DR. PRIOLA:  Yeah.  Not being an epidemiologist, I don't know the precise answer, but I do know that the current thinking right now is it's inefficient because -- it's inefficient.  You're crossing a species barrier.  You're going orally, both of which are bad things.  So you presumably need more, a pretty good bolus.
		DR. STEPHENS:  I appreciate that, but is there data that supports that?
		DR. PRIOLA:  Not that I'm clear on.
		ACTING CHAIRMAN DAUM:  Michael Decker might have data about that, an answer for that.
		DR. DECKER:  I don't know if there's the type of hard data we'd like, but an illustrative anecdote is the town in England where the butcher shop that served the local community was using centuries old techniques that heavily contaminated the resulting meat with neural tissue, and there was a very high attack rate, a cluster of human variant CJD in that town.
		So they found both an unusual preponderance of disease and an unusual practice that would give them a high load.
		DR. PRIOLA:  Yeah, I'm not sure what the ‑- I know there was a cluster of five patients, but I'm not clear what the total number of people were that were exposed, and so what the exact attack rate was.  Do you?
		DR. DECKER:  No, but five out of the 90 in England is a pretty high proportion.
		DR. PRIOLA:  Oh, for sure.  Oh, yeah.
		DR. DECKER:  The proportion of that town's population to the total English population.
		DR. PRIOLA:  Definitely, yeah, yeah.
		ACTING CHAIRMAN DAUM:  Dr. Coffin and then I'd like to move on.
		DR. COFFIN:  So what do we know about infectivity in serum of affected animals and their offspring?
		DR. PRIOLA:  In terms of cattle?
		DR. COFFIN:  Yeah.
		DR. PRIOLA:  In cattle?
		DR. COFFIN:  In cattle, yeah.
		DR. PRIOLA:  There are only two reports that I know of that have suggested there might be very low levels of infectivity in blood of cattle, one of which is in an experiment where they experimentally inoculated cattle and assayed tissue over the entire course of disease.  There was a point at which they detected infectivity in bone marrow cells from a pool of three samples.
		And there was a recent report last year where it's a bit more indirect, where sheep infected with BSE -- a bunch of sheep were infected with BSE, and at various time points blood was transfused from those sheep into naive sheep, and one of those sheep got sick, the implication being that infectivity got out into the blood.
		DR. COFFIN:  But have there been serious attempts made to look into --
		DR. PRIOLA:  There have.
		DR. COFFIN:  -- serum products and other blood products?
		DR. PRIOLA:  Commercial serum products?  Not that I --
		DR. COFFIN:  Or in serum that's obtained, you know, large amounts of serum from large numbers of significant --
		DR. PRIOLA:  Yeah, and in terms of hamsters, yes, there was.  Bob Rohwer did a really heroic effort to do that, and pure concentrated hundreds of mLs of hamster blood and was able to detect using lots of animals that there's less than 1 LD-50 out of all that material.
		DR. COFFIN:  So it seems --
		DR. PRIOLA:  It's extremely low.
		DR. COFFIN:  -- at the moment, based on our current thinking, it seems improbable --
		DR. PRIOLA:  Extremely.
		DR. COFFIN:  -- that there were these significant risks from serum, to begin with.
		DR. PRIOLA:  Based on our current knowledge.
		DR. COFFIN:  Based on our current knowledge.
		DR. PRIOLA:  Absolutely.
		ACTING CHAIRMAN DAUM:  A burning final comment.
		DR. MINOR:  I mean, while the bone marrow result is out there, okay, I think it was regarded as slightly questionable, and I think also that the efforts that people have made to find infectivity in cow blood as opposed to bone marrow have uniformly been negative, and that's even going cow to cow, as I understand it.
		DR. PRIOLA:  Yeah.
		DR. MINOR:  And I think there's also an issue of the species that you're looking at will give you a different level of infectivity in the blood perhaps.
		DR. PRIOLA:  Sure, absolutely.
		ACTING CHAIRMAN DAUM:  Thank you for that clarification.
		I'd like to move on to Dr. Krause, who promised we would have our opportunity to have at him again, and here it is.  Dr. Krause will talk about adventitious agent testing of neoplastic cell substrates.
		DR. KRAUSE:  I'm just going to dive right in and view this as a continuation of where I left off.  This slide shows a number of different episodes throughout the history of the world and the U.S. of contamination of biological products with adventitious agents, and I don't want to go into this in very much detail, but I do want to focus on the contamination of polio and adenovirus vaccines in the late 1950s and early 1960s with SV40 as an example of something we really want to avoid repeating.
		And as many of you know, millions of people received SV40 contaminated polio and adventitious vaccines into the late 1950s and early 1960s, and these vaccines were produced in primary Rhesus monkey kidney cells.
		SV40 was identified as a virus; it was discovered after this, and the basis for discovering SV40 was the fact that supernatants of primary Rhesus monkey kidney cells cause tumors in laboratory animals and also were shown to cause cytopathic effect in primary Circa Pithacus (phonetic) monkey kidney cells, or different species of monkey, which then led to the discovery by Dr. Hillaman (phonetic) at Merck of SV40.
		The vaccine seeds were treated with anti-SV40 neutralizing antibodies in the early 1960s to rid them of SV40.  Epidemiological studies suggest luckily that there was no adverse sequelae to the vaccinated children, and however, recently SV40 DNA has been detected in some human malignancies by PCR, and this highlights a significant problem that one has with adventitious agents, which is that sometimes the concerns and the potential effect might not become apparent for a substantial period of time, until a substantial period of time after the contamination event has occurred.
		So a couple of lessons learned here are obviously the value of insuring that the products are free of adventitious agents, and also the particular importance of insuring the freedom of products from oncogenic agents or potentially oncogenic agents, especially for vaccines that are given to children.
		So I've listed here a few basic principles of adventitious agent testing.  We've already talked about how this kind of testing should consider quantitative issues and how important it is to understand the sensitivity of one's assays and what one precisely is tested for and what the meaning of a negative result actually is.
		It's important to consider issues that are specific to the material in question, and I would argue that also where possible one should use tests that have the potential to detect unsuspected or even undiscovered agents.
		Now, to talk about adventitious agents, I think it's useful to think a little bit about how viral vaccines are produced.  The center of viral vaccine production is the cell substrate, and of course, for live viral vaccines or inactivated viral vaccines, some cell substrate is required to grow the virus.
		You add to that a vaccine seed and various other raw materials, which can include serum, medium, stabilizers or things like that, which then together in the production process after usually some kind of purification or inactivation lead to the final product.
		This entire thing takes place in a facility which hopefully is designed in such a way as to minimize the opportunity for contamination at any of these steps, although there is the risk, of course, that personnel who perform these steps might contaminate a vaccine.
		But a basic principle of producing these vaccines is that all of the materials that go into the vaccine, the vaccine seed, the raw materials and the cell substrate, need to be shown to be free of adventitious agents as they enter the process.
		Now, purification steps could potentially reduce adventitious agent burden if some were able to get into the production process, and there is a model for thinking about that that has been used in evaluating therapeutic products at CBER in which some cell substrates might contain viral particles, and the ability of a purification scheme then to remove those particles is then determined, and that's called investigation of viral clearance.
		And a general principle there as well then is that the purification scheme which is used needs to be able to remove no only the amount of material that you think could be present in the cell substrate, but also some additional amount which represents a safety factor.
		It's also worth noting that for live vaccines a purification process could actually potentially concentrate adventitious agents.
		Okay.  So as we look at adventitious testing then, I've talked about the importance of quantitation, but I just wanted to go through four individual points here because in policy making not only do we need to understand how sensitive an assay is, but it really does need to estimate the pretest probability of a problem because that will influence the kinds of assays that one thinks needs to be done.
		One needs to consider the number of doses or dose equivalents that can be tested. One needs to understand the sensitivity of the assays, and a very important component of that then is in performing assays to include the appropriate controls that allow one to define how sensitive any given assay was, and the need to consider safety margins in deciding what kind of assays need to be performed.
		So what are factors that could influence adventitious agent risk in a vaccine?  Well, if you are thinking about the cell substrate obviously, the species from which the cell substrate comes can have an influence on the kinds of agents you would look for.  You would look for different agents in non-human versus human cells, for example.
		The cell type or tissue of origin obviously makes a difference.  Under this I include three points.  Various previous exposures while the cell was still in its host could have an influence on the kinds of adventitious agents one might want to look for.
		For instance, if one were dealing with cells that were derived from fetal tissue, one would look for agents that are known to be able to cross the placenta, for instance, whereas if they are derived from an adult, one would look for appropriate viruses for that, and depending on the type of tissue, one might look for agents that are trophic for those kinds of tissues.
		Tumor association also could influence one's thinking about adventitious agent risk, and in particular, the knowledge of the transforming event could potentially mitigate that, and in the designer cell substrates that we're talking about today, if one does have definite knowledge of the transforming event, then perhaps one might be less worried about the tumorigenicity of those cells, although if a cell line were derived from an actual tumor, one might be more concerned, especially if one had no knowledge of the transforming event.
		And then the ability to bank the cells and to characterize them in some detail before they're actually used such that they can be used each time in the same way is also a very useful property in terms of reducing adventitious agent risk.
		And then whatever is known about the maintenance or passage history of the cell obviously also is an important component of thinking about what kinds of adventitious agents need to be looked for.
		So if we look at potential vaccine cell substrates and think about ones ability to characterize them for adventitious agent risk, well, whole animals, for instance, embryonated hen's eggs (phonetic) or even the mice which are used to make Japanese encephalitis virus vaccine may be less well characterized than some of the kinds of cells that can be studied in tissue culture before they're inoculated, although in the case of embryonated hen's eggs, one can use a specific pathogen free flocks, which can substantially mitigate one's risk.
		If one is using primary cells, and of course, the classic example of this or the primary monkey kidney cells were used in oral polio vaccines, one has some period of time prior to inoculation to characterize.  One can also maintain uninoculated control cells for longer periods of time as was done in the production of that vaccine.
		But it is, in general, less easy to characterize these cells than it is either diploid cell strains or neoplastic cell lines.  So diploid cell strains are cells like WI38 and MRC-5, which have been used for many years to produce vaccines line rubella and varicella, and because these cells can be banked, one can characterize the bank of cells and go back to it repeatedly and assume that it will be the same each time.
		Well, what about neoplastic cell lines?  They can be banked and, therefore, can be characterized, and they actually also have several other potential advantages which are worth considering today as we think about designer cell substrates.
		One of them is host range.  In many cases, there are viruses that can be grown in these cells that simply can't be grown in other cells.
		Another one is the ability to bank them.  Another one is that very often neoplastic cell substrates can be adapted to serum free growth, which is a property which is not as readily given to the other kinds of cell substrates we talked about.
		And they can be made, as in the case of designer cell substrates, to express complementing genes.
		So why then would someone be concerned about neoplastic cell substrates and adventitious agents, and in particular with oncogenic viruses?  Well, if there is a neoplastic cell line for which the mechanism of transformation is not completely understood, there is, of course, always the potential that an oncogenic virus was involved in the cell line's neoplastic transformation.
		And if that were the case, one would want to know that and be certain that that virus did not survive through to the cell substrate.
		Some of these cell lines, just by virtue of the fact that they grow very well and have been around for a very long time in many different laboratories are more likely to have uncertain histories, and that provides an opportunity for contamination of them.
		And of course, when one is talking about oncogenic viruses, oncogenic viruses have the potential to lead to very severe consequences that are quite difficult to evaluate in short-term clinical studies.
		So this slide shows the kinds of testing that are normally done on cell substrates for viral vaccines.  So the tests are listed here to the left, and I describe here in these two columns, one of them whether there's an amplification step involved in the test, which gives you some sense of the sensitivity of the test, and to the right the potential to detect the unsuspected, which then gives some sense of the ability of this test to find something that one doesn't know is there and to give some additional assurance on this point of unknown or undetected, unsuspected oncogenic viruses.
		So tissue culture assays, of course, have been used for many years in qualifying cell substrates.  If a virus grows in the specific cells which are being used in the tissue culture assay, there is an amplification step, and it can be fairly substantial.
		On the other hand, some viruses will not grow in tissue culture, and so although there's an amplification step and although there is the potential to detect the unsuspected, there is a hole in tissue culture testing.  We try to make up for this hole by animal studies, which include inoculation of eggs, inoculation of animals with either death or weight loss as an endpoint, and also animal antibody production assays, the so-called mouse antibody production, rat antibody production, and hamster antibody production assays, which also work to the degree that an agent will replicate in the systems which are being dealt with and have amplification steps associated with that, and in general have the potential to detect the unsuspected as well with the exception of the animal antibody production assays, which only detect the agents which the antibodies are shown to be directed at.
		And then there also are molecular assays, in particular, specific PCR, which is a very sensitive way of finding a given agent, but it doesn't help you at all in detecting the unsuspected, and then newer assays like the PCR based reverse transcriptase assay, which is very sensitive because it has a molecular amplification step.  It also has the potential to detect any unsuspected retrovirus.
		And then there's electron microscopy, which is a very insensitive test because it does not include any amplification step, but of these tests, perhaps there's the greatest potential to detect the unsuspected because it could potentially detect any virus if it were there in adequate quantity.
		I think it's worth saying a word about comparisons between PCR and biological assays in just thinking about testing of adventitious agents.  PCRs tend to be much more sensitive for small samples with low residual DNA, and the reason is because the PCR can detect a very small amount of nucleic acid, but it only can detect it in a fairly small sample.  So the amount of a product or of a cell substrate that can be evaluated by PCR is necessarily limited.
		On the other hand, biological assays are much more sensitive for large samples because you can put many doses either into an animal or onto a tissue culture flask.
		PCR assays are very specific, whereas biological assays have a greater potential to detect the unknown.  PCR assays will work independently of growth characteristics, and so even if a virus does not grow in a specific system that it's being tested in, one can detect it by PCR.
		Biological assays, on the other hand, require growth in a specific system.
		On the other hand that's also a flaw in PCR because a positive PCR result doesn't necessarily mean that there's a live virus.
		Biological assays, on the other hand, provide a more relevant endpoint.  For instance, it's only in a biological assay that one can detect oncogenicity.
		So if one's interested in  improving one's ability to detect oncogenic viruses, I guess one way to look at that is to think about the methods which have been used to discover oncogenic viruses or discover viruses in the past, and really four main methods that have been used are listed on this slide.
		These include animal inoculation and looking for some kind of an endpoint, and for oncogenic viruses, this has traditionally been the appearance of tumors, thereby verifying the oncogenicity of the virus.  A tissue culture, electron microscopy, and molecular methods.
		Now, tissue culture and electron microscopy are fairly well covered by the tests that are generally currently asked for with viral vaccines.  So I'm going to go into a little more detail on animal inoculation and molecular methods as potential ways of better understanding or better providing greater assurance that a cell substrate is free of an adventitious virus.
		Animal inoculation to look for a tumorigenicity endpoint or an oncogenicity endpoint is a method which has been used for many, many years, and really this recapitulates practically the entire history of virology.  Rous sarcoma virus, for example, was discovered in 1911 based on the ability of tumor extracts to cause tumors when inoculated into animals, and this method has been used to detect pox viruses, papova viruses, and adenoviruses up through and including the 1960s.
		And again, I point to SV40 as an example of a virus which was originally detected by this method, and it is the kind of thing that one would like to be able to avoid in dealing with cell substrates.
		If one looks at the ability of viruses to induce tumors in animal assays,  this slide shows some examples of what happens when you inoculate these viruses into either baby hamsters, mice, or rats, and a yellow, which actually is orange on my screen, but it looks sort of yellow from here, means that you do get an oncogenic endpoint.  So a tumor of some kind is formed.
		And what you can see is that if you use a combination of hamster assays and rat assays, one has a pretty good chance of detecting the viruses that are detectable by these means, which include retroviruses, polyoma viruses, and adenoviruses.  In general, human Herpes viruses and papilloma viruses though can't be detected by these kinds of methods.
		So what can we say about animal tests for oncogenic viruses?  Well, they could be used in cases where additional confidence that a product is free of adventitious oncogenic viruses is desired.  We know that many tumor viruses are cell associated, and so we would argue that inoculating two animal models of either rats and the hamsters with cell free lysates of cell substrates, where lysates would have a better chance of getting cell associated viruses, followed by fairly extensive observation would lead to the maximum sensitivity in these kinds of assays.
		So what can one say about the kinds of material that ought to be tested?  I already alluded to the fact that testing the cell substrate independently of just the final product would have some value, but I wanted to go into that in just a little bit more detail here as well.
		So testing a cell substrate has value for insuring product consistency because that way you know what the material is that you're dealing with each time you make the product.  If you're concerned about the potential for existence of an adventitious agent in a cell substrate, you certainly want to make sure it's not there in the cell substrate rather than just look at the final product because that's the only way you can be sure that there is -- or it's the best way to insure that there aren't going to be interactions between the vaccine strain and whatever adventitious agent is present.
		You have to look at the cell substrate.  You need to know how much is there or isn't there in order to apply the principles of viral clearance that I discussed, and I mentioned that we think that looking at the lysate probably makes a little bit more sense than looking at the supernatant because the lysate will also capture viruses that are cell associated, although one could potentially make an argument in favor of looking at supernatants as well.
		What about looking at the final product?  Well, one concern with looking at final products, especially of vaccines, of adenovirus vaccines is the potential for interference.  We heard that at very high titers there is some leakage with these vectors, and so there's the potential that cells that otherwise might show an oncogenic phenotype will simply be killed.  If one inoculates enough of this stuff, one will get a generalized inflammatory response, which then also might interfere with an oncogenic endpoint.
		And we know that E1 has an effect on apoptosis, and E4 also has a little bit of a pro apoptotic effect, which then might also lead to some potential interference of a final product then with whatever one might be trying to rule out in the presence of a cell substrate.
		On the other hand, testing of final product would give assurance that the vector itself is non-oncogenic, and so would potentially have some value in this kind of assay.
		It looks to me like a slide was skipped.  Can you back me up?
		So I just want to talk a little bit about where this is going and talk about molecular approaches to virus detection and how one might be able to use a newer, broadly specific approaches in finding adventitious viruses.
		And one approach which has been used to discover several different Herpes viruses over the last few years has been to use consensus PCR primers, which are directed to the DNA polymerase region to detect related viruses.  So generic primers or consensus primers, which detect Herpes virus DNA polyemerases are used, and then samples that are suspected to have new Herpes viruses in them can be evaluated using those primers, and new viruses have been discovered that way.
		This same general scheme also was used to identify the West Nile virus when it recently came to New York.
		Molecular subtraction assays also have been used to discover viruses.  These include methods like representational difference analysis.  Two viruses have been discovered over the last few years using this method.  One of them is Human Herpes Virus 8 and the other is TTV.
		And the problem with molecular subtraction assays is that you need something to subtract from your sample.  So if you have a cell substrate, unless you have something which is genomically identical, except for the potential adventitious agent, it becomes very difficult to interpret one's result.
		In our laboratory we've been trying a slightly different approach which we call nonspecifically amplifying viral nucleic acids, and the basic principal here is that instead of doing a molecular substraction, we're doing a physical subtraction in which we take a sample and attempt to get rid of all of the cellular nucleic acids in a way which preserves the viral nucleic acids and then use completely nonspecific PCR methods to amplify what's left over.
		And the kinds of techniques we used for that separation include ultra centrifugation and nuclease digestion.  We haven't investigated filtration to a great degree yet, but we will.
		And this next slide just shows one of our early experiments in which we took fairly small quantities of Varicella Zoster virus, spiked it into a million Vero cells and applied this method to ask whether we could find any VZV, and so we did these nonspecific PCRs on the nucleic acid that we extracted from this and ran them on these gels.  We then cut these bands out of the gel and cloned them, and both to our delight and our surprise, all of these bands turned out to have VZV sequences in them.
		We have since taken this general approach and have expanded it to a large number of other viruses, including RNA viruses by adding a reverse transcriptase step, single stranded DNA viruses, smaller DNA viruses, retroviruses, have also looked at cells that constitutively produce viruses in addition to cells into which viruses are spiked and have been successful in finding viruses under these circumstances.
		So we think that this kind of method has a lot of promise also for doing a better job at finding adventitious agents when one doesn't know exactly what it is that we might be looking for.
		Did I skip a slide here?  Maybe not.
		It's important in discussing any of the issues that we're talking about today to not only consider the theoretical issues that we're worried about and some of the risks from viral adventitious agents are theoretical, and these issues all have to be placed in the context of the entire product.
		And a very important component of that is potential benefit of the product.  And so it can be easy to look at these kinds of issues and to become discouraged by them, but these issues are only one side of the equation, and it's important to remember that.
		So how do we think that this entire way of thinking can be applied to Adenovirus Type 5 transformed human designer cell substrates for vaccine production?
		Well, if we think about TSE testing, as I said earlier, it's important to consider the cell type and potential exposures to BSE.  The tests that one can consider are a sequencing of the PRNP gene, Western blot, or the ELISAs that Dr. Priola talked about, and the idea of adding newer, more sensitive tests as they become available.
		Also we believe it's very important that research subjects and investigators and IRBs be well informed of these issues, and so the informed consent investigator brochure and other documents should reflect that need.
		One other point I wanted to make about TSE issues that I forgot to mention in my introduction is that this entire issue will be presented in a separate meeting of the TSE Advisory Committee in the near future, and so the reason we're discussing TSE issues so heavily at this meeting is really not for the purpose of coming to a final resolution of them, but instead for the purpose of having the Advisory Committee discuss our general approach and also for providing information to the Advisory Committee and to the public on that general approach.
		And then as this slide shows a potential approach to virus testing of Adenovirus Type 5 transformed human designer cell substrates for vaccine production, and that includes obviously to perform the standard testing as I've show it, including extensive tissue culture and electron microscopy.  One would want to insure that the test would detect any agent based on the fetal origins of the cells, the cell type and the cell history.
		And we would argue that although the mechanism of transformation of these cells is likely Adenovirus Type 5 genes, we would recommend doing extensive testing for potential oncogenic adventitious agents as well, which would include cell lysate oncogenicity testing and other tests as they become available.
		And as was the case on the other slide, we regard it as being very important that research subjects, investigators, and IRBs be well informed of the issues associated with moving into these new kinds of cell lines.
		So thank you very much.
		ACTING CHAIRMAN DAUM:  Thank you, Dr. Krause.  You touched on many issues that I hope the Committee will come back to during our discussion period this afternoon, but for now we'll see if there's questions specifically about the material that you've covered.
		Dr. Decker and then Dr. Goldberg.
		DR. DECKER:  It seems clear that the designer cell substrates pose issues with respect to the neoplastic line of tumorigenesis that are difference from those posed by currently used cell substrates, but it's not clear to me -- so if I'm missing it, point it out -- it's not clear to me that the designer cell substrates pose any new or different issues with respect to adventitious agents.
		DR. KRAUSE:  I think that one really has to look at each cell line by itself, and so there certainly is the potential for that to be the case.  If, for example, the history of 293 cells as it's gone through different laboratories is not as well known as one would like, then that may be a special adventitious agent related issue for 293 cells that one would want to consider.
		But I think that what you're really trying to get at though is the question of the designer cell substrates having a known mechanism of transformation, and many of the issues that I discussed with respect to neoplastic cells having to do with the notion that if one doesn't know the mechanism of transformation one might be more worried about certain kinds of adventitious agents.
		And I guess the argument that we would make is that although we're fairly certain that we understand the mechanism of transformation in these designer cell substrates, in order to provide the greatest possible public confidence as one moves into cell substrates that are tumorigenic, even if we believe them not to be oncogenic, that doing tests that provide additional levels of insurance on that point will bolster public confidence in these new cell substrates.
		DR. DECKER:  Well, I'm trying to separate the issues here, and I still haven't heard that the designer cell substrates with respect to adventitious agents pose any challenge that wouldn't be faced by any new, non-designer cell substrate line.
		In other words, if I was going to create MRC-5 cells now new, you would ask the same questions about adventitious agents and do you want me to do the same things.  The fact that these are transformed or designer really has nothing to do with what you want done for caution with respect to adventitious agents, right?
		ACTING CHAIRMAN DAUM:  If I can help maybe bridge the understanding here, I think he means with respect to adventitious agents.
		DR. DECKER:  Yeah, only with respect to adventitious agents, separating adventitious from oncogenic.  Okay?  Two separate questions.  Contamination with PRP protein is a question, and the ability to replicate that has nothing to do with whether or not they're designed agents.  It would be true of any novel cell substrate, wouldn't it, that we'd want to look at that?
		DR. KRAUSE:  One would want to look at it.  We might have greater concerns though about a cell that has retinal origin, as was mentioned.
		So since one of the designer cell substrates we're talk -- I'm not sure how you can.  I agree with you that in general if the only difference between a new diploid cell line and a designer cell substrate is the fact that the designer cell substrate has had some manipulation which has caused it to become immortal, and if one also stipulates that everything else about it is very well controlled and that one understands the milieux in which that occurred, and if one is absolutely certain that the intervention that you made to that diploid cell line to turn it into a designer cell substrate is, in fact, what caused it to become immortal, then I think you would be right.
		I think that one can make arguments in favor of doing the kinds of things that Dr. Hughes spoke about to provide additional assurance on those kinds of points.  So if one adds an immortalizing gene to a diploid cell strain and then demonstrates it by turning it off, then the cell line is no longer immortal, and that provides a very high degree of assurance that that immortalizing gene is, in fact, the only thing which caused the immortalization of that cell.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Goldberg, please.
		DR. GOLDBERG:  Yeah.  When you talked about the needing a quantitative framework for decision making, which goes to some of the questions that were asked this morning, and the first thing that you said was to estimate the pre-test probability of a problem.  I mean, have you any thoughts about what you mean by that or can you --
		(Laughter.)
		DR. GOLDBERG:  -- indicate to some of us?
		DR. KRAUSE:  Sure.
		DR. GOLDBERG:  To some of us who are living.
		DR. KRAUSE:  I think it's very difficult to do, but --
		DR. GOLDBERG:  I agree.
		DR. KRAUSE:  -- for example, in applying the principles of viral clearance to a therapeutic product, if you know by electron microscopy of a certain sensitivity that there are viral particles present which you believe not to be infectious, but you just want to be sure.  Then you start off with some pre-test probability based on a positive electron microscopy result if there was something there.
		And so if you figure, just to pick round numbers, if you could figure that the sensitivity of the EM test allows you to pick up ten to the sixth particles per cc, if you see something there that implies that there could be as much as ten to sixth, you want to have some safety factor built into that, and so you may then require 12 logs of clearance if you want to have a ten to the sixth safety factor.
		If, on the other hand, that test is negative, it's not clear to me that you would want to start off with the assumption that it would have been positive or that it would have been positive only at the level of its sensitivity.
		So if you believe the sensitivity of that test, you'd be ten to the sixth.  If that test is negative, it's not clear to me that you would start off with the assumption if there are 9.9 times ten to the fifth particles there.
		So I think that one has to look at each of these products individually, but I think that the idea of spending at least some time initially thinking about how likely you think a problem is and using that to guide the sensitivity of the assays that need to be done is an important component of thinking about doing this.
		DR. GOLDBERG:  Well, that gets back to the kind of questions that Dr. Moulton raised this morning, which really is what sorts of assumptions can you make and what kind of distributions can you put on the possible sensitivity or probability of detection by any of these assays, and you're really accumulating a battery of tests and so that you can model this and get some ideas.
		And the question is can you do this with any of the even in vitro data to see what you might be missing under various models and assumptions and has anyone done that sort of work?  
		I'm not saying necessarily you.
		ACTING CHAIRMAN DAUM:  Can I interrupt here for just a second?
		DR. GOLDBERG:  yes.
		ACTING CHAIRMAN DAUM:  I think this is a very, very important point, and I think it's the essence of what I would imagine our discussion is going to be when we finish the presentations, but what I'd like to just do is just make sure there's no comments or questions about these presentations because what you're hitting at, I think, is the essence of where we're going to go with this.
		You were first and then Dr. Katz and then Dr. Kohl, and then we're going to move on.
		DR. AGUILAR-CORDOVA:  I think I would like to follow up on the previous questions by Dr. Decker.  That is, if the designer classification just means that you know one of the events that occurred in immortalizing or transforming that cell line, and I'm a big concerned on this because if one detects any oncogenic transformation in any tumor cell line, then you know that one event as well, and it would just become the same as a designer cell, I would imagine.
		And then it's only the infectious or adventitious agents that we're talking about that you would be concerned with; is that correct?
		DR. KRAUSE:  I guess I'm not sure I understand.
		DR. AGUILAR-CORDOVA:  So as an example, somebody spoke about A549s and 293 cells earlier on, 293 cells, you know, the E1A and E1B section.  It's A549s you find out that have a mutation in p53.  Now you know one transforming event, and you can take that away or test for that.
		If you know that, then would they become designer cells?
		DR. KRAUSE:  No, I think the way we -- we've defined designer cells fairly narrowly as cells which have been immortalized by defined means where the immortalization is part of the design of the cell.  So those may well be cells that can be well characterized and  ultimately with enough information one could determine they're safe to use.
		But I don't think we would say that those cells have the same degree of information about them as a cell where one is starting with just defined information about the mechanism of transformation.
		ACTING CHAIRMAN DAUM:  Dr. Katz, is this about adventitious agent testing?
		DR. KATZ:  Absolutely.  Not testing; just an amplification or a clarification of those who are less than 60 years old.
		It was implicit in your statement, but I'm not sure everyone appreciates that SV40 contamination was not confined to live oral polio vaccine.  It was inactivated polio and inactivated adeno. because the formalin step that was sufficient to inactivate those viruses did not inactivate SV40.
		And an anecdote at least for a long-term observation, I can give you the names of three individuals who were injected repeatedly with SV40 inadvertently in trying to prepare polio skin test antigens, and none of us has a brain tumor, and we're all still alive.
		(Laughter.)
		DR. KRAUSE:  I'm pleased to know that.
		ACTING CHAIRMAN DAUM:  Thank you.  
		As we all are, Dr. Katz.
		PARTICIPANT:  But we're going to keep an eye on you.
		ACTING CHAIRMAN DAUM:  Dr. Kohl, please.
		DR. KRAUSE:  And, in fact, Dr. Katz is right.  I did not mean to imply that it was only oral polio vaccines. In fact, if you look at the people who seroconverted to SV40, you had an easier time finding seroconversion among people who received the inactivated vaccines for precisely the reason you say.
		DR. KATZ:  We had very high titers.
		DR. KRAUSE:  Right.
		ACTING CHAIRMAN DAUM:  Dr. Kohl, is this about adventitious agent testing?
		DR. KOHL:  Yes, adventitious agents only.
		Phil, I enjoyed your talk, and the thing that I'm sure causes you to lose sleep at night, and some of us as well, is the unknown adventitial agent that we, you know, at this moment can't even anticipate.
		And you mentioned some new molecular studies, the subtraction studies, et cetera.  Are those being recommended or suggested?  What's the status of those for new products?
		DR. KRAUSE:  I think these are tests which perhaps are not yet in a state where they can be recommended universally in a regulatory sense because they may not be well standardized enough or may not have a good enough sense of what the controls ought to be and so forth, but this is, I think, a direction in which we need to move.
		Certainly the kinds of primers which can detect families of viruses are becoming better known, and so if there are people in the audience who are involved in putting together these kinds of tests, I think it would be very valuable to do that.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		I think at this point we're going to move on to our final scheduled presentation.  Dr. Golding will review OVRR, CBER issues with the use of adenovirus vector vaccines and their complementing designer cell substrates.
		Following her presentation, we'll have an open public hearing, a brief break, and then we will begin getting at these issues that the Committee is chafing at the bit to begin discussing, which is a good thing.
		DR. GOLDING:  Okay.  So as many of you know, I'm Hana Golding, the Chief of the Laboratory of Retrovirus.
		And the task that I was given today is to try and put all of this very detailed and informative talks that you have heard today into some sort of perspective and to extract the essence of all of the talks and then translate them into the key issue that made our approach in trying to move forward into new cell substrates, and specifically designer cell substrate, into the arena of new vaccine development.
		And while thinking of how to do that, I thought it was really the right time to put it all in the wider perspective of risk-benefit and to remind the Committee that one of the driving forces behind these efforts in CBER and specifically in OVRR that started almost two years ago is the new development in the HIV vaccine development field.
		And I think we all are very aware of the fact that the HIV epidemic is still continuing unabated and with the 16,000 new infections a day, that there is quite an increased disillusion with the ability of antiviral therapies to either curtail the epidemic or to cure infected individuals.
		And I think there's increased hope and belief that the appropriate vaccines that will be tailored to countries around the world will eventually lead to the scope of these epidemics.
		And we're very excited.  I think we're having a very important period during this development stage because many investigators both in academia and in companies have taken up the challenge of developing new vaccine approaches to HIV.
		And what I tried to list here is some of the new viral vectors and some other approaches that have been developed and are in various stages of development, and most of them still in preclinical, but some are already in the clinic.
		And there was a whole lot of viral vectors that are currently under development starting with pox viruses, such as canarypox, MVA and the NYVAC.  You've already talked about the Adenovirus 5, and they both can be used as both replication competent and nonreplicating vectors.  Adeno-associated virus, the Venezuelan equine encephalitis vectors, Seliki Forest virus, and Herpes virus.  This is not an inclusive list, but are all types of nonreplicating vectors that are under development.
		And activated HIV vaccines are definitely under consideration and new ways to inactive the virus to increase the level of safety are under development in multiple labs, and we shouldn't forget recombinant plasmid DNA vaccines, as well as purified protein, peptides and lipopeptides under development.  
		There are several bacterial vectors that are under development, and in combination with all of them are novel adjuvant, cytokine, and co-stimulatory proteins.  Many of the new vaccine approach will include dual or with triple modalities, which have been termed prime boost approach with one type of vaccine that may be used for the priming, and then a second modality will be used for boosting, and the hope is that the appropriate arm of the immune response will be generated to, indeed, protect infection, and if not the initial infection, at least reduce the initial viral load and slow the progression of the disease.
		So new HIV vaccine may require novel substrate, and you heard a lot about the need for complementing cell lines for nonreplicating viral vector vaccines, and both the 293 and the PER.C6 are good examples of them because they are providing in France the E1A, 1B genes that are missing in the vectors.
		They may also be used for optimal production of recombinant live, attenuated viruses, and definitely new novel cells are required for production of the activated HIV vaccines.
		So what could be the advantages of designer cell substrate as we move forward into these new classes of cell substrate?  As you heard during the day, designer cell substrate as we define them are derived from either primary cells or from well characterized diploid cell lines.  
		The cellular localization is achieved with nontransformed genes that can be either viral or cellular derived, and therefore, the essence of the immortal line genes and/or their product can be closely monitored during vaccine production and final product characterization.
		I would like to then focus your attention specifically on Adeno. 5 E1 transformed designer cells, and again summarize the advantages that you've heard during this day.
		So Adenovirus 5 in cells is non-oncogenic in humans.  For the more adenovirus or Adeno. 5 E1A/B transformed cells are not tumorigenic in uni-competent (phonetic) animal models.  The reason is that the Adeno. 5 E1 expressing cells are killed quite efficiently by cytotoxic T cells and by natural killer cells.
		In addition, as you heard from Dr. Cook, Adeno. 5 E1 expressing cells are highly sensitive to mediators of apoptosis, such as TNF alpha.
		We also know that Adeno. 5 from swollen (phonetic) cells are only weakly tumorigenic and immunodeficient athymic nude mice, as you heard from Dr. Andre Lewis, with a TPD-50 of around 6.5 times ten to the sixth cells required to see tumors in about 50 percent of athymic nude mice.
		We also heard that Adeno. 5 transformed HEK cells, 293, have already been used in the production of adenovirus based vector for gene therapy, and there's quite a large number of Phase 1 trials, and there was a considerably amount of safety data.
		However, you also heard that when we move into this new class of cell substrate, the designer cell substrate, there are still potential safety concerns, and I just want to reiterate them here.
		Incomplete medical history of the original tissue  and incomplete documentation of the tissue culture ingredients used in the propagation of the cell substrate may be of concern, as well as the long history of immortalized cell may result in exposure to adventitious agents and potentially to TSE/BSE agents due to undocumented bovine ingredients in the culture medium.
		So now we'd like to summarize briefly OVRR approach and reiterate what you've heard early on.  Do we really believe that extensive safety testing should be conducted on the new master cell banks independently of the vaccine vector, and the rationale is that the same MCB can be used for production of multiple product.
		If an adventitious agent is detected in the master cell bank, it is important to document its removal during product processing and purification and its absence in the final product.
		As you heard from Dr. Krause, some viral vaccine vectors may interfere or reduce the sensitivity of certain safety assays.  We would also like to recommend that sponsors should be encouraged to place the results of the new master cell banks in the public domain in order to increase public confidence in the safety of the new cell substrate.
		And I'd just like to briefly summarize the testing that we have discussed during the day with regards to master cell bank tumorigenic and oncogenicity studies.  It is useful to actually test the intact cells and to determine their TPD-50, and in order to do that, one needs to use several cell doses and observe nude mice for five to six months because different cells have different kinetics of developing tumors.
		With regards to the high molecular weight that's extracted from the cells, it's important to conduct oncogenicity studies to establish the inability of oncogenic sequences, viral or cellular derived to cross-tumors in animal models.
		With regard to adventitious agent testing, as you heard from Dr. Krause, in addition to the standard assays, it's important to try to incorporate new state-of-the-art assays for detection of agents that can infect human cells as needed and as become available.
		We think it will be important to test cell lysates in order to detect occult oncogenic viruses and the way to approach probably to inoculate the animal species, for example, the newborn hamster and the rats that was shown in the nice table that Dr. Krause showed you, and to observe them for about five to six months.
		With regard to  the potential risk of TSE and BSE, it may be important for all new cell substrates, including designer cell substrates to sequence the PrP gene of the MCB to exclude the presence of the familial variant that was described by Dr. Priola, and to test for the presence of protease resistant PrP protein by sensitive Western blot.
		As far as residual DNA is concerned, a concerted effort should be made to reduce the amount of cell substrate derived DNA in the final product to less than ten nanograms per human dose, and as you heard from Dr. Keith Peden earlier, that may be translated into probability of less than one in ten million or 100 million, probability of transmitting an oncogenic sequence per ten million or more human doses.
		For vaccine administration via the oral route, as was also mentioned earlier, higher level of residual cellular DNA may be allowed, especially if studies demonstrated known tumorigenic potential.
		So I would like to end by actually presenting the Committee with several points to discuss.  Please discuss the adequacy of OVRR approach to the evaluation of designer cell substrates for use in the manufacturing of viral vaccine.  You can make specific reference to tumorigenicity and oncogenicity studies, residual cell substrate DNA, potential contamination with adventitious agents, including occult oncogenic viruses and TSE/BSE agent, and feel free to discuss any additional safety concern that you may have.
		Thanks.
		ACTING CHAIRMAN DAUM:  Dr. Golding, thank you very much.
		With your permission I wonder if we could leave this last --
		DR. GOLDING:  I have one for an overhead.  It may be easier to --
		ACTING CHAIRMAN DAUM:  That would be great.  Whatever is audiovisual's pleasure, and we'll use that as soon as we come back from our break and leave it up for the whole Committee discussion.
		Questions for Dr. Golding's presentation only, please?  Ms. Fisher.
		MS. FISHER:  All of these tests that you want to have performed and these assays, is the manufacturer going to be doing this?  Is FDA going to then be retesting?  I mean, how is it going to be insured once something is come up with, you know, that's actually going to be followed?
		what kind of oversight will there be on the testing?
		DR. GOLDING:  Well, this is actually part of the normal development through the procedures.  When they manufacture and develop a new cell substrate or a new vaccine that is made in a new cell substrate, they will usually ask for a meeting with the agency in the form of a pre-IND.
		During this time any new safety issues regarding the cell substrate or the vectors are discussed, and we start to get dialogue regarding the type of additional testing that are needed.  It's quite understood that this type of test has to be done and presented.  The result has to be presented in the IND application, and we are looking very carefully at the results of this test, and if we find that they are inadequate, we have to make a place to stop the beginning of Phase 1 trials.
		But we are looking at each product and at the target population that it's designed for.  We look how much tests were already done and how much tests we still need to be done, and based on very extensive internal discussion and discussion with the sponsors, a decision is then made whether to stop the initial clinical trials before everything is done or whether to allow progress or at least initiation of small Phase 1 trial and try to then -- and require the company of the sponsor to complete additional safety studies before going to much larger scale studies.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		I think we'll move on then to the open public hearing portion of the meeting.  Is there anyone in the audience that would like to address the Committee at this time?
		(No response.)
		ACTING CHAIRMAN DAUM:  In the absence of recognizing a rush to the microphone, I would like to declare us in recess for 15 minutes.  It's 3:25.  We will reassemble at 3:40.
		Thank you.
		(Whereupon, the foregoing matter went off the record at 3:27 a.m. and went back on the record at 3:44 a.m.)
		ACTING CHAIRMAN DAUM:  One of the nice things about having our information presented to us in discrete bits this morning is that we had an opportunity to discuss a lot of issues related to those that the FDA wishes to hear our advice about.
		And so now comes a time rather than perhaps needing to start from square one to be sort of well immersed and understanding what some of the issues are and concerns.
		There are no actual votes today, but there are issues for discussion, and they're nicely summarized on this slide, which I asked Dr. Golding to put up for us again, and I'd like to just focus a little bit on how we proceed here.
		Please discuss the adequacy of OVRR's approach to the evaluation of designer cell substrates for use in manufacturing of viral vaccines.
		Now, this morning's presentations were divided into three discrete kinds of concerns, one, the tumorigenicity and the oncogenicity studies; secondly, the residual cell substrate DNA concerns; and, third, of course, the adventitious issue concerns.
		So I would like to have or invite Committee members and consultants to begin this discussion by lumping those three things and talking about whichever of those issues you would like to bring up and discuss.
		We have most of our, perhaps even all of our speakers from this morning available as resources, and I think the issues have a lot of commonality to them.
		If we don't get good discussion on all of the issues, then I might take the Chairman's prerogative of refocusing the discussion and doing them sort of one by one, but let's sort of see what we get.
		For consultants that are new to this process, I really would like to hear at some point during the discussion from everyone at the table, and we will help people remember to shut off their cell phones and beepers, and also to participate in the discussion at some point.
		So with that, Dr. Decker cannot wait for a comment.  So I'll begin with him first.
		DR. DECKER:  Well, Bob, as you know, I've got to meet a prior commitment.  I have to slide out early.  So I'll say my piece now and then be gone.
		I'll try to take --
		ACTING CHAIRMAN DAUM:  We're grateful.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  I mean for the first part.
		(Laughter.)
		DR. DECKER:  You know I have your home number.
		(Laughter.)
		DR. DECKER:  The first question or the first issue laid in front of us was the reminder of the nearly half century old bias against using immortalized or neoplastic cell lines for production of vaccines or biologicals, and one implicit if not explicit question was whether the time has come to overturn that prejudice and to exploit these based on the new developments and the marked dramatically improved capacity for understanding and auditing them, and I think the answer to clearly is yes.
		If we're going to move forward, it's time to take advantage of these things, but then, of course, it has to be done with I would love as a writer to say absolute, but as a scientist I know that's ridiculous; so as near to absolute assurance of safety as we can humanly attain at our present level of scientific development.
		With respect to the three issues raise, I think the one that's of probably the broadest general concern is the adventitious agent one, but I think that's one that is by no means particular to the designer cell substrates, and I think that we would approach them or we would want to see FDA approach them with the same level of caution and thoughtfulness as they do with respect to any biological in the developmental substrates coming forward.
		And so I in my own mind, I pretty much set that one aside with respect to the specific issue, which is the designer cell, and for them we've got more particularly the residual DNA and the oncogenicity questions, which are legitimate, but it seems to me that we have more than adequate tools and techniques to address them, and it seems to me that the OVRR's approach to this is perfectly adequate.
		So I'm quite comfortable with the way these things are progressing now.
		ACTING CHAIRMAN DAUM:  I take it these comments are from your perspective as an industry representative here, and some people have raise issues this morning about how the agency and industry are interacting about these issues, and so are you comfortable with that from a point of view of -- I mean some of these bars are pretty high here.  It sounds like people are going to be asked to jump through who are making designer cell vaccines.
		And from an industry perspective, does that sound like something you think people who are making them will comply with, can comply with, must comply with?
		DR. DECKER:  Well, that's an interesting question, and let me back up on that because although I'm here as the industry rep., and I try to be conscious of that all the time, obviously you get the whole person, and I've been in industry for six months and in academia for 20 years.  So I guess you get that balance of it.
		Before I came to this meeting, as before every meeting, I E-mailed my colleagues at all of the major vaccine companies, reminded them that the meeting was coming, and asked them to instruct me on anything they thought was industry relevant.
		I received zero replies.  So, therefore, I come uninstructed.
		(Laughter.)
		DR. DECKER:  And largely uninformed until I heard this morning's meeting, other than with my background  in vaccinology.
		And because of that, you're not getting the response of the head of our production or the head of our research labs.  You're getting the head of our Medical Affairs Department responding, and as such, I see nothing here that doesn't strike me as within the bounds of reason.
		That doesn't mean at some later meeting I won't have some information or instructions that I can share with you concerning a technical problem that's arisen that it's hard for industry to respond to, but right now you're getting my response mostly as a vaccinologist, and it seems reasonable.
		ACTING CHAIRMAN DAUM:  Okay.  Thank you for that.
		But anybody else in the Committee can now feel free to chime in or our consultants on any one of these points that you wish.  Yes.
		DR. WOLFE:  These are general comments on the points that at on the board.  About a year and a half ago at one of these meetings I commended CBER generally, this group particularly on the remarkably good research they had done, and there was at that point some question about adequate funding to keep doing what they're doing, and it sounds like that's at least somewhat better.  It's never as good as it should be.
		But given this whole -- as most, if not all of you know, this was originally part of NIH, still physically there, was not even a regulatory agency until 1971 or two, whatever the year was.  It can be looked on as a research.
		The first question is adequacy of OVRR approach.  From a research perspective, I think it's excellent.  Some cutting edge studies are being done, things that Phil talked about, nonspecifically amplify nucleic acid when you're sort of hunting for adventitious agents that haven't been identified before.
		But to reflect the questions that came up several times this morning, what's the match between that and the first product or the first products that are coming through the hoop?
		And this morning it was is it just a recommendation; is it would it be nice to do or what?  And I think that although we will and I will certainly direct these questions tomorrow at the sponsor when there's a presentation, it would seem to me that there are certain at least state-of-the-art diagnostic, if you want to call them, techniques for either tumorigenicity or adventitious agents or for residual cell substrate DNA which are far enough along that they should be just automatically requested of the company as part of their package.
		There are other ones that are in development, and even though we don't have the final word on how sensitive and specific they are and so forth, there should be some consideration given to having the company at least do a certain amount of those.
		And I think that's really the dilemma.  It isn't so much, particularly after what we've heard today, as to whether OVRR has done a good job.  I think we would all agree they've done a tremendous job, but how does this translate into the regulatory aspect of the agency?
		I think that's a difficult question, and I certainly would like us to move in terms of our recommendations in the direction of as safe as possible, never, you know, 100 percent safe, but as safe as possible, and that means sort of pushing the companies to take advantage and be required for their specific products to do all that has already been done.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Wolfe.
		Dr. Aguilar-Cordova.
		DR. AGUILAR-CORDOVA:  I would echo that.  Some tremendous research was presented here, but addressing the issues directly posed there about tumorigenicity, oncogenicity studies, and designer cells, I would caution that we might get a false sense of security based on this designer classification of these cells.  The only thing they have that's really designer is that they are designed to be able to propagate a virus.  They weren't  transformed or immortalized on purpose by that gene.
		And in fact, from the data that we saw today, it seems like it requires multiple events to occur to become a tumorigenic agent.  E1A and E1B was presented to be a very poor transforming, you know, tumorigenic agent.  So therefore, this cell might probably have multiple event.
		And I think it would be dangerous to have the false sense of security that if we cannot detect E1A and E1B as a contaminant, that that would be any better than not detecting anything else from an unknown transforming event, and thus, I think that designer cell classification should be looked at carefully.
		On the other hand, we were also shown that it is a very unlikely event that this transforming or oncogenic activity would come through from the minute amounts of contaminating DNA that are currently allowed on a per dose basis, and given that very unlikely event that I believe was ten to the minus eight, ten to the minus ninth range, then the additional events that might have occurred in those cells really probably don't change the probability of or the risk of that contaminating DNA since one in ten to the ninth is probably no different  than five in ten to the ninth.
		But lastly I would then follow Dr. Decker's, that the adventitious agent portion of this discussion would probably become the most critical of all of those since that was apparently logs of difference in risk.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		We have Dr. Kohl, then Dr. Hughes.
		DR. KOHL:  I'd like to go back to something Dr. Decker said, and if we could achieve a consensus on that, it would make it easier for me and maybe for the Committee.
		I'm having trouble with at least three issues that we're being asked to discuss, and Mike suggested, and I think I concur, that the adventitial agent issue regarding designer cells, other than the fact that they're around longer and maybe have a slightly or some undefined increased risk of being infected with something, but other than that, I don't think we have been presented with anything suggesting that the adventitial agent issue in designer cells is any different than the adventitial issue in any new cell substrate.
		So the question is:  should we be discussing that pertinent to this or can we leave that issue alone and let CBER go through their routine adventitial issue discussion, which they have to do for every cell line, and move to the two things that seem to be pertinent to designer cells in particular?
		ACTING CHAIRMAN DAUM:  I would respond by saying that I hear a consensus building for the concept that the adventitious agent issue is not unique to these designer -- I hate that term -- designer cell lines.  Nevertheless, we have been asked to comment on whether the new cell lines we've heard about today in a generic kind of way -- how to address these issues, and more importantly, is the approach that's being taken adequate to the concept?
		So while I agree with your point, I think the agency still needs our opinion.  So I'd like to continue to leave it on the table as an issue, although I think you're right personally that it's no different than any new cell substrate.
		I have Dr. Hughes, Ms. Fisher, then Dr. Katz.
		DR. HUGHES:  I'd like to respond a little bit to the point that was raised a few moments ago about whether or not designer cell substrates are, in fact different than transformed cell lines derived by other means.
		And I think the first thing that's worth discussing is that the cell line that's transformed in vitro by whatever means is not necessarily equivalent to a tumor cell, and one of the things that's particularly important if one is talking about the collaboration of a number of genetic changes necessary to create a frank tumor cell for the kinds of things we're discussing here, the development of cells that are permanent in culture, it may not be necessary to have as many change, and that I think automatically distinguishes the kinds of things we're discussing here in terms of creating designer cells certainly from cell lines derived from tumors from animals or humans, and I think that's an important distinction.
		And the other thing, I think, that matters in that regard is that particularly if in the creation of the cell in vitro, the designer cell, if you like, if one does set the system up in such a fashion that the controlling gene -- and this is the point I tried to make this morning -- can be shut on or off, one can ask then by shutting it off whether the cell returns to what one might call a normal phenotype or not, and there are some relatively simple assays which are probably beyond the scope of the discussions we're having today to ask whether or not those cells are reasonably normal in terms of their behavior.
		And I think you can get at by doing things of that sort whether or not there are additional changes that are associated with the establishment of those cell lines, and I think that's one of the reasons that that type of experimentation is worth doing.
		And finally, to just comment very briefly on what's on the list and to respond to some of the other points that were made earlier, I think in a sense the question is not even so much whether we should proceed in this direction, but more of a question of how we should proceed.  In fact, not whether or not we should move in this direction, but how, and what are the tests that are necessary to provide the responsible margins of safety that we hope always, whether we're involved in creation of receiving of vaccines, that we think we deserve.
		And I think it's a question -- the real question is to think very carefully and to try as best we can to determine the best ways to be safe, and I think for some of these issues and for some of the things that we need to test for that the answers are reasonably straightforward.
		Not all of the work is necessarily done yet, but I think we know more or less how to do it.  I think some of the other questions are more difficult, and probably in a sense one of our tasks ought to be to try and think carefully about not whether to do it, but how to do it.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		I have Ms. Fisher, then Drs. Katz, Griffin, Goldberg, and Coffin.
		MS. FISHER:  Well, it appears that the risk assessment of residual DNA infectivity and tumorigenicity and how much should be allowed is almost entirely dependent upon the assumption that the population to be injected with this DNA is immunologically competent or what is being termed as, quote, normal.
		But hundreds of millions of humans have genetic predisposition to cancer and autoimmunity or are suffering from active cancer and autoimmune disorders, and it doesn't appear to me that there's been enough consideration given to biodiversity that narrows the definition of what is normal, and that this has an impact on the validity of the animal studies conducted so far and affects the premise that what counts most is how much DNA rather than who is being exposed to it.
		And I think that before we walk down this road, a lot more has to be known about the differences between people and their response to this DNA.
		ACTING CHAIRMAN DAUM:  I want to make sure I understand you clearly.  Are you speaking about expanding the scope of the animal research?
		MS. FISHER:  Yes, because to truly give informed consent, you're going to have to know more.  Individuals are going to have to know if they have a higher risk because of their, you know, predisposition potentially.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Katz.
		DR. KATZ:  I was going to debate with Michael Decker, and I'm sorry he left.  I would turn ‑-
		ACTING CHAIRMAN DAUM:  As I am.
		(Laughter.)
		DR. KATZ:  I would turn his neutral position into an even more positive one.  I think there's less concern with these cells than with primary derived cells.  The problems we had with SV40, with avian leukosis virus.  We didn't use Sudimangabese (phonetic) or chimpanzees.  So we didn't run into HIV, but all of the potential adventitious agents have been from cells from a natural source, from other primates or other non-human sources.
		Whereas I think these are much better defined and much better tested.  I would give them a more positive approach as what' being done and what's potentially available is much more reassuring than were we to use another primary cell line.
		ACTING CHAIRMAN DAUM:  Thank you.
		I have Drs. Griffin, Goldberg, Coffin, Blair, and Diaz.
		Dr. Griffin, please.
		DR. GRIFFIN:  Well, I think that with respect to oncogenicity, first of all, we know at least the 293 cells can cause tumors in mice.  So by definition the cells themselves can cause tumors at least in some animal models.  So, therefore, the issues become whether there's something else besides which are really the issue that Dr. Hughes has just made, whether there's something else besides the adenovirus transforming genes that are in the cell lines that we should know about, and the only way we're probably going to be able to figure that out is if we can knock out in some way the function of those genes and see if those cells still can cause tumors in mice.
		So something else that we should be worrying about is there, but then the issue become  really then the DNA issues because you aren't going to be injecting cells as a part of what you give as a part of a vaccine.  Presumably you can be sure of that.
		But then the quantities of DNA and having very good data on how many nanograms or the real quantitation in mice of different varieties that have both increased susceptibility to tumors because of oncogene mutations and also increased susceptibility because they're young or immunodeficient in one respect or another, including immunodeficiency in NK cells, not just T cells.
		So skid mice or something that perhaps is even more susceptible than a nude mouse.  So I think that it's really moving toward just a clear understanding on characterization of these cells and this quantitation issue, which is what it's all going to come down to at the end in looking at the final product basically.
		And the other point is that, which really hasn't been brought up because it's in many ways the easiest to deal with, is a real risk obviously is recombination in these cell lines, and that the product that you can actually get viable virus out, and the engineering that's going on right now will probably deal with that issue.  So that makes it very unlikely, but that is in the other tumor models at least where even though adenovirus is probably not that oncogenic in humans, but in other situations where you really have the increased risk is if you get recombination.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Goldberg.
		DR. GOLDBERG:  First of all, I'd like to commend the agency for the excellent presentations.  It's really clear and basically are allowing me to ask questions, which I couldn't do from just the reading.
		I think the idea of having a framework for the testing of adventitious agents is absolutely right on.  It's very important, but I think even now it's time to start looking at the assumptions and looking at the results that you've observed under various modeling assumptions, whether it's in vitro or in the early animal work.
		I mean, it's very hard to say "never," and statistically you never can.  there's always a finite probability of something occurring, and therefore, I think it's very important that you start to think in terms of the different underlying truth that could produce data, such as the data that are observed.
		You're also using batteries of tests as I see it, and you can come up with rules for combining the batteries that would give you more assurance and reduce the probabilities that you're missing something, and I think that some of that needs to be done.
		It can be done crudely in terms of worst case analysis.  Everything is under the worst possible scenarios that you could think of.  What would your numbers look like, and then you accumulate them, but there are more sophisticated ways to do that, but that's a way to start.
		You can also design sequences of the testing that may be able to make it more efficiency for manufacturers and for you to be able to work your way through based on some of these results, and those things all need to be worked out.  We can't do it here, but they are things that I think need to be thought about.
		The other issue is the carcinogenicity studies.  I think that that needs to be done, but the system obviously would need to be stressed to the limit because these are very low -- the likelihood of any of this occurring is very low.
		ACTING CHAIRMAN DAUM:  You're breaking up, Dr. Goldberg.  Can you speak right into the microphone for us?
		DR. GOLDBERG:  Okay.  I think you have to stress the CARCI studies, I mean, and do them in the way that you do standard carcinogenicity for any drug, which can be much longer than five or six months.  You need to push the thresholding doses that you're using to see whether or not anything does occur at some dose, and then you work your way back to what might be a safe threshold.
		ACTING CHAIRMAN DAUM:  I think that the idea of trying to mathematically model risks of worse case scenarios is one that it does really need to be developed in a more systematic way than we've heard today, and then I think once those models are developed, it's a separate issue of whether we -- and by "we" I mean the most global "we" I've ever said in my life -- we, the public and the people sitting at this table and the FDA and the manufacturers and mostly the people who are going to receive these vaccines then need to decide whether or not they accept those risks, theoretical or not.
		But I think modeling them with all of the information we do have, incomplete as it may be, is a wonderful concept that I'd like to hear more about.
		DR. GOLDBERG:  I just have one more comment.
		ACTING CHAIRMAN DAUM:  Okay.
		DR. GOLDBERG:  Which is I think that then you evaluate the risks we're going to have to make some distinctions about a preventive vaccine that's given to children versus a therapeutic vaccine for  serious illness, and there will be some gradations of acceptable risk which will also play into the evaluation.
		ACTING CHAIRMAN DAUM:  I have Drs. Coffin, Blair, Dias, Faggett, and Kohl, and now Myers and van der Eb.
		Dr. Coffin, please.
		DR. COFFIN:  Okay.  First I wanted to comment to Ms. Fisher's comment on the experiments that are being done regarding the oncogenicity of oncogene DNA in experimental animals.  These are -- and as to how they would play into the issue of variation of immune confidence in the public at large.
		These experiments are being deliberately designed to minimize the immune competence of the animals that are being injected.  They're going into either newborns, weanlings, or into nude mice and, if necessary, other animals of this type will be being brought in.  So they're being set up to be to a first approximation the worst case scenario in terms of what the immune competence of any potential human recipient would be, at least as close as we can come in straightforward animal models and animal models that are reasonable to deal with and don't have a very high background tumor incidence, for example.
		Regarding the issues on the board, the tumorigenicity and oncogenicity studies of the cell substrate have to be done, but I have a lot of trouble seeing how you use the information you get from them because if you have a cell line that does not give tumors in animals, but yet it's a cell line, do you come to the conclusion that that cell line is necessarily safer and you can relax the stringency with what you have to do other testing because of that?
		And on the other hand, I'm not convinced that we can come to the conclusion that a vaccine prepared from a cell line which is much more tumorigenic necessarily carries with it, just because of that fact, carries with it a greater potential to contain DNA which will cause bad oncogenic consequences in the recipients of the vaccines.
		So we have to know this information, but I just don't see how it's used or what its relevance will actually be to what the final decisions and the final way we think about a product are.
		One thing we haven't considered very much with these that is special to the designer cell substrates in the system that's been particularly under discussion and to others regarding retrovirus helper lines and so on is the issue of recombination.
		There has been a lot of bad experience with that in retrovirology.  I'm not sure that the adenovirus people have gotten anywhere near the levels of the sort of bad things that have been discovered with people generating helper cell line after helper cell line that couldn't possibly give replication competent recombinants and finding that sure enough they do, and it wasn't until things really have to be subdivided in a much more firm way in retrovirus systems than they are in these systems to actually reasonably insure that there are very low levels of recombinants.
		this will, of course, need to be  discussed in much more specific terms, but I think it's an issue that we have to keep in mind.
		And another issue that is specific for vaccines, viral vaccines and live viral vaccines, is the possibility that the virus itself can bring in -- can over and above the DNA fragments that are present, can actually encapsidate or recombine in cellular DNA and enhance the introduction of that DNA into cells.
		If these were propova virus vaccines, propova virus is very well known to encapsidate genome size fragments of cell DNA and bring them in.  Adenovirus, I don't know where they tend to do that as much, but I suspect there's some of that, and I think that's an issue that may have to be looked at separately that hasn't really arisen in our discussions.
		As far as the issue of DNA contamination in these products and the possible oncogenes, I don't see how we can treat these cell lines any differently than we would any other cell line as far as the standards that have to be applied, which we don't completely know what they are yet, I think, because we don't really have -- I don't think we have the data yet that we need to really judge the risk of this.
		I think the risk is extremely low, but I don't think we have the data to really put any quantitation on it, but I don't see why there should be a difference between DNA, between viruses grown on any kind of cell line, one to another, as to the way this is treated.
		ACTING CHAIRMAN DAUM:  Thank you very much, Dr. Coffin.  You raised a lot of new points that we haven't really addressed in detail yet.
		Dr. Blair, then Dr. Diaz, Dr. Faggett, Kohl, Myers, van der Eb, Minor, and Aguilar-Cordova.
		Dr. Blair, please.
		DR. BLAIR:  I think to follow up, I think I'm probably a little more comfortable that they don't form tumors than John.  Maybe I would agree that we may not know what that means, and certainly you can't be less careful with the material that comes from a cell like that, but I think the closer the cell is to a normal what we seem comfortable with, which is a normal diploid cell, the closer it is to that, the more comfortable I would feel, and I think the more comfortable the public will probably feel as a whole.
		In terms of the biodiversity issue, I think there is an attempt in some of these experiments to test tumorigenicity of things that we believe to be tumorigenic to try and establish some sort of baseline that we are trying to do this in a variety of different backgrounds, as many as to stress the system in such a way as to get a positive response from which we can then determine some sort of sensitivity and hopefully then some level of confidence in negative responses.
		But I guess it is very difficult to use the rodent or anything else as a real model for all of the varieties of human genetic background that these vaccines will go in, and ultimately we have, I guess, to be as safe as we can be in the model systems and develop as good a model system and as testable a system as we can to test the risk.
		But there will probably always be a, you know, some risk of some situation, and people will have to be aware of that as these things go out.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Let's move on to Dr. Diaz, please.
		DR. DIAZ:  Well, firstly, I'd like to comment that I think that the approach to the evaluation of these newer cell substrates is very thoughtful and certainly based on what data is available currently.
		And with that in mind, we always worry about the unknown, and I think it's very important that the FDA makes recommendations on what types of testing ought to be done to at least give us as good a feel for the safety of a particular product as is capable at that point in time.
		And yet I also feel that it merits perhaps even making some perhaps even more specific recommendations about what types of tests ought to be done at what time point and how perhaps often they might need to be repeated or at what time frame they might be repeated in dealing with some of these cell lines.
		Because of the length of time involved in some of the tests, I would hate to get to a point in time where we're down the road and recommendations were somewhat open to interpretation, and then we come down the road and suddenly the question arises, and we're at a point in time where we have to deal with less information about that product than we could potentially have had.
		So I recognize the issues about requiring versus recommending and yet perhaps putting some time or at least some recommendations based on certain intervals would be meritorious.
		Switching gears a little bit, the usage of some of these cells and particularly the usage of vaccine constructs in viruses like adenovirus or perhaps there may be other viruses to which this might even be more applicable, but viruses that are somewhat ubiquitous in the general population.  
		I think despite the fact that issues about recombination can probably be hopefully dealt with and ruled out.  I think the question is still going to come up at some point in time with these products when they're used in humans, and suddenly there's an infection perhaps in an immunocompromised host or viral products, viral genes or viral gene products found in tumors in humans, and the question will come up in a vaccinated person:  is this related to vaccine or is it related to a wild type virus occurring.
		And what I haven't heard is any discussion, and perhaps it's not possible from a molecular standpoint, but any discussion about having some kind of marker or the need to have some kind of marker in these constructs that would be able to when that situation does arise to answer that question, whether those products or that infection is a result of recombination with the vaccine virus and/or whether it's wild occurring.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Faggett.
		DR. FAGGETT:  I  agree with my colleagues that there's been a high quality of the presentations today, and I really appreciate it, and I especially appreciate the historical perspective from my colleague Dr. Sam Katz, a survivor of potentially an SV40 infection.
		Stay well, Sam.
		But I think this has really been an open and honest discussion of very technically challenging concepts.  There's been a lot of good science present, and to include admission of limitations of that science.
		I think we as a panel are now better able to make evidence based decisions with a better understanding of the evolving nature of this science.  I think OVRR has been very adequate in its approach to the evaluation of designer cell substrates, to include all of the topics listed.
		I agree with Dr. Hughes that this is an opportunity to really bring on line the state of our testing to minimize risk contamination by adventitious and other agents yet unknown.
		I agree with Ms. Fisher that we do need to keep in mind that as we take the animal study results and try to apply them to a diverse target population, that there are a lot of other ethical considerations as well as scientific that we have to consider.
		But I think as a primary care provider I'm very comfortable that we're in the process of really looking at all available data, and in so doing, we'll be a lot more comfortable to recommend to our patients that, indeed, no stone was left unturned by us.
		And I'm really impressed that safety has remained a top priority in this discussion.  So often we have the science separated from the safety, and it's an afterthought.  In this instance I'm seeing safety as a primary concern, and I think with that approach we'll be better able to really anticipate any questions in the future.
		I think the results of our recommendations will be better accepted because of this in depth examination, and I truly thank you for the presentations today.
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Kohl, I think you're up.
		DR. KOHL:  We've heard a little bit about refining the risk assessment, I think, and trying to get finite kinds of numbers, and it's a little bit deja vu.
		I was looking at the September '99 panel discussion, which several of the members here were on, and I was struck by Dr. Sedivy's statement.  Hopefully I'm not killing his name.  "It is worth trying to do calculations so long as you do not believe the final numbers."
		(Laughter.)
		DR. KOHL:  And I guess I wanted to know from the people who were there and maybe anybody who's wiser than I am.  Have we moved further from that statement or are we still pretty much there?
		It's apparently just an exponentially multiplication system, and as we get further along in multiplication, the numbers get fuzzier and fuzzier.
		ACTING CHAIRMAN DAUM:  I would like to actually begin the -- I think you raise an interesting point, and I'd like to begin and ask Dr. Hughes we'll stay focused on this subject for a moment, but I think the numbers are one more approach to understanding what kinds of situations in terms of safety we're dealing with here.
		I don't think the numbers are going to solve the problem or provide security that makes us forget about all the other things we've heard about and talked about today, but I think if you're talking about ten to the minus 39th versus ten to the fourth, I think that gives you a certain difference in how you think about what we're talking about here.
		So I pulled those two numbers out of a hat as a potential illustration, but I think what you quoted as being said a couple of years ago was right on the money.  As long as we don't take them too seriously and believe that we've defined something that we really have no idea what we're talking about,b ut it still is useful to get us in the range and give us a little more piece of the puzzle, a little more frame.
		Dr. Hughes, you probably have something much more erudite to say.
		DR. HUGHES:  I very much doubt that, but I'll be happy to give you my prejudices.  I was one of the people, and I'm sure you know if you've looked at the text, who expressed some skepticism of some of the calculations, not in the sense that I don't think they have some use, but as was mentioned a little while ago, because there are uncertainties in each of the numbers that goes into the calculation, you tend to expand the uncertainty issue as you multiply out.
		And I think that was exactly what motivated us to try and get together with the FDA to try and derive in the animal models which have their limitations, and I think we would be the first to admit that, but to at least for the animal models get some numbers that have much less uncertainty.
		And I suppose I would apologize in some sense in the name of the federal government that it hasn't been a faster process, but as I think everyone who does science understands, the first thing you have to get is some money, and that part of the problem is solved.
		And I think if -- and I'm sorry to say that I think it will probably be another year, but I think in another year we will at least be able to give in rodent models some clear preliminary data, and I don't think that's going to solve everything, but I think it will give us more confidence that we know what we're trying to deal with, and I think that will make us feel even more comfortable or less comfortable with what we're doing.
		But I will always feel more comfortable if I have data that I believe has a firm numerical basis, and I think that's the goal of the experimentation that's being done with the NCI and the FDA.
		ACTING CHAIRMAN DAUM:  Thank you.
		I think we've spoken to Dr. Kohl's point a little bit, which is well taken, and Dr. Myers is next, then Dr. van der Eb, Minor, Aguilar-Cordova, and Kim, and there's actually a couple more.  That will get you.
		DR. MYERS:  I really like the risk modeling approach because even if the numbers are soft, at least put them into some sort of perspective.  As you say, ten to the four is a lot different than ten to the 39.
		It seems to me the two things that are different about designer cells, a lot of people have commented that the cell substrate or the rules for cell substrate, whether we're talking about a new diploid cell line or others are pretty much the same, and one is Dr. Daum's point about what is different about the designer cell is what we put into it, and if we could ablate that function and see that the cells revert to what we expect, I think we learn a great deal about the stability of the cell substrate.
		But I'd like to say something about the adventitious agents before we completely leave those.  I agree with Sam that diploid cells and now continuous cell lines would be, I would think, much safer from an adventitious perspective.
		We don't want to forget that these cells are somewhat different in that they are derived from fetal neural cells, and so as we're developing the assays to look specifically for adventitious agents, we maybe should be targeting them specifically for vertically transmitted agents.
		And so, Phil, as you were giving your model of the spiked VZV assay that you did, I was think about if, in fact, you were dealing with trigeminal ganglia and looking for VZV, the sensitivity of the assay and the difficulty of trying to find a latent virus in that type of setting.
		So while I think the likelihood of adventitious agents -- the risk would be much reduced as we get more experience with these types of cells.  I do think as different cell substrates come along, we should think about their origin and target our seeking assays towards in this case vertically transmitted and neurotropic agents.
		ACTING CHAIRMAN DAUM:  Thank you, Marty.
		Dr. van der Eb.
		DR. VAN DER EB:  I would like to add a comment on the opportuniticity (phonetic) issue.  Two, nine, three cells are oncogenic in nude mice.  They are weakly oncogenic, and so are the PER.C6 cells.  Both cell types of weakly oncogenic in immunodeficient nude mice.
		The BRK cells, baby rat kidney cells, formed by Adenovirus 5 in our hands are just as weakly oncogenic as the human cells, and in fact, if I remember correctly, but I am not absolutely sure if I am correct, also the BRK cells -- so this is the baby rat kidney cells -- transformed by oncogenic Adeno. 12 are also not that much more oncogenic than the Adeno. 5 cells.
		The fact that Adeno. 12 transformed cells are oncogenic in immunocompetent animals and are, therefore, called oncogenic viruses is due at least in part to the fact that E1A of Adeno. 12 can switch off the Class 1 C antigens, the transplantation antigens in the transformed cell, but basically they do not seem to be very much more oncogenic in nude mice.
		I remember many years ago there has been an experiment where in SV40 transformed human diploid skin fibroblasts, the SV40 large T antigen gene was switched off, and that was due to a temperature sensitive mutation in the large T antigen, and after several packages these cells, when you switch them to the nonpermissive temperature so that the SV40 large T is no longer functional, the cells return to a more or less normal phenotype and, in effect, stop the fighting, so indicating that in SV40 at least transformed cells after a number of passages, but not hundreds of passages, but maybe 20, not much more has occurred.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		Dr. Minor, please.
		DR. MINOR:  Firstly, I think that the studies on the tumorigenicity/oncogenicity of the DNA are very, very welcome, I think.  I think the existing data are a bit anecdotal, and I'm quite impressed that they seem to agree with each other as Keith Peden demonstrated, and I think it would be nice to have some real data on these things that you can actually put your hand on your heart and believe.
		(Laughter.)
		DR. MINOR:  With respect to tumorigenicity, in general, it seems to me that while you have designer cell lines where you put in a particular gene and that results in transformation, nonetheless, you don't really know the full story.
		I mean, I think there's a difference between the retinal transformed cells and the kidney transformed cells, for example, and it's not clear to me why one goes relatively easily or straightforwardly and the other one doesn't.
		So it does seem to me that you don't really know the fully story about why the PER.C6 is the way it is and why the HEK cells are not perhaps the way it is.
		And then I think going on with what John Coffin just said, I think the question of degree of tumorigenicity is something that really needs to be carefully thought through.  I'm not sure how tumorigenic a cell line would have to be before you decided it wasn't acceptable or if it could ever be unacceptable depending on how tumorigenic it is.
		And that's why I think the DNA issue is really a very important one to get to grips with.  There are clearly DNA delivery systems like cells, for example, which you wouldn't want to have stuffed into your vaccinees.  Oncogenic viruses you wouldn't want.  Viral nucleic acid I think you wouldn't want particularly.
		I'm not sure what happens when you get down to oncogenes and other bits of DNA as well.  It seems to me there's a bit of a data gap there in terms of do you really worry about them or not.
		So I think I very much welcome the DNA studies which are being done by CBER.  I think they're all to the good.
		One thing I wouldn't like to slip through the net actually is the adventitious agent question.  It seems to me that considering the viral testing, I don't think there is anything special about these cells in terms of what you would actually do to look at them.
		The TSE, however, I was a bit horrified by this.  My country, as you may be aware, has become increasingly obsessed with BSE over the last 20 years, and the approach which has always been taken, adequate or not, is really to look at the nature of the materials which are going into your culture, system, and so on, this being regarded as the best place to put your resources and the most effective way, if you like, of resolving any issues that arise.
		And the impression I had from what was said here is that it's now considered at least relatively straightforward perhaps to get a cell in culture infected with a TSE agent or to get a cell in culture spontaneously producing the TSE agent, and for me this is a basic shift in the paradigm.
		I think it's going to be a very difficult thing to actually do that, which doesn't mean that it shouldn't be done, of course, right?  But I would like to not let that one just go by on the nod because I think that's actually a matter of some concern, I think, and it would be a need to balance, if you like, the good against the possible consequences of doing that.
		For example, if you wanted to go and test calf serum in cows, it would take you seven years and ten million pounds, which is a lot of dollars, as well.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		Next is Dr. Aguilar-Cordova, please.
		DR. AGUILAR-CORDOVA:  So I just have three quick responses or comments to what's been said.
		ACTING CHAIRMAN DAUM:  Please.
		DR. AGUILAR-CORDOVA:  One is with regard to what Ms. Fisher has spoke to, and that is the target population, and obviously I think that there needs to be some consideration of that, and I would hope that at the early stages of this one would not use very sensitive or very skid population, for example, or leap frog mainly (phonetic) patients or such, and I'm more concerned with that.
		However, there are still two different concerns there.  The product and the contaminants, and even within the product one must be a tad bit concerned, but we must keep in mind that the potential for RCA or replication competent adenovirus in the product, that may come in through the product versus what may be in the environment and at what levels might be tolerated or acceptable since we're all exposed to some RCA most of the time.
		The second was with respect to the testing that was proposed by Dr. Krause and the technology that's being used, and it's fabulous actually.  I really like that use of random primers probably to just detect little pieces of DNA in the supernatant.
		Now, and another thing that may be considered, and I don't know if your group is doing anything, but just to throw it out there, it would be new technology like micro chip analysis, and in fact, that might even be something that would be considered in the TSE or BSE type of environment as to whether the presence of such infectious agents may lead to different genomic expression profiles that might be detectable that way.
		And lastly, I hate to beat a dead horse into the ground, but I guess often they're the easiest ones to beat.
		(Laughter.)
		DR. AGUILAR-CORDOVA:  And this has to do again with the designer cell state, and I was looking at the glass here in front of me, and it sort of prompted the analogy.
		I think when we're talking about tumorigenic cell lines, defined as cells that can form tumors in nude mice, as Dr. Hughes was mentioning, it's perhaps a series of events that were captured in vitro, and that they may not reflect what events have happened in vivo, but they still lead to tumor formation in the nude mouse.
		And if one has a glassful of candies like this and one more candy puts it over the edge, just because you know what that one candy that put it over the edge is doesn't preclude the fact that there's still a whole bunch of other candies in there, and if you take that one candy out, it won't go over the edge anymore.  So it won't be tumorigenic anymore, but all of the other candies are still there, and the only difference in the designer cells, again, is that you know that one candy.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Kim, it's your turn.
		DR. KIM:  Well, I guess looking to the question about whether approaches are adequate, I think that this question certainly is a moving target.  As we heard today, there are many new assays, and animal models are being developed to look at the older issues related to oncogenicity and particularly adventitious agent contamination issues.
		So I think, again, I'm sure that these issues will come back on another basis as we gain more data and experiences.  So certainly I think we can address what we have today, and based on that I think I'd like to support the idea of, you know, having some sort of mathematical model, which we talked about earlier, because, again, there are data being generated in our models and questions have been raised whether that is relevant to humans or not, and again, there are some biologic and theoretical concerns with information coming out from in vitro, as well as animal models.
		I think the best we can do is to try to come up with some sort of worst case scenario, and again, as many people have said, that shared that information with the public and that have the public aware of all of these exercises have gone, you know, before the material has been presented to them.  So they certainly will be up to date on these issues.
		And then lastly, I would also like to see some utilization of current advances made in the genomics, and I think it will be fun to look at some of these issues that were raised and so on and see what are, you know -- if something is entirely benign, then I would hope to see that there are no changes, but if something is coming up, then up and down, you know, that might, you know, imply some potential of some biologic concerns.
		ACTING CHAIRMAN DAUM:  Thank you, Kwang Sik.
		I put my name down on the list, and it comes up now.  So I would like to ask a question.  Dr. Peden, I think it was -- is he still here?  There he is.  Good.
		Dr. Peden -- my eyesight is bad, too -- Dr. Peden, I think you raised the issue of SV40 follow-up or someone.  Dr. Krause.  Excuse me.  I was curious as to -- I was taking note of your comments about SV40 now having been found in several patients with human cancer, and I was aware that that was true and wondered how much of a surveillance that finding has prompted.
		In other words, is there an ongoing screening of human tumors for SV40 among recipients of vaccines?
		We've heard the issue several times today about the need for not short-term observation with vaccines that may have oncogenic potential.  Here's an example of something that might need a very long-term follow-up, and what's being done about that?
		DR. KRAUSE:  Well, Dr. Lewis can probably add to my comments on this because he's been working in this field longer than I, but there have been -- the fact that people were exposed to SV40 was noted fairly early on.  So it was possible to follow people over the long term and do different kinds of studies to determine whether different kinds of tumors appeared to be more likely in people who were exposed to one of the vaccines versus not, and also compare people from different regions where different vaccines which had different likelihoods of being contaminated were administered.
		And so the epidemiological data exists, but such as they are, these are not studies that, I guess, are designed to pick up very low risks, and a confusing aspect of the SV40 in tumor issue is that some of the SV40 has also been detected in tumors of people who are too young to have received these contaminated vaccines.
		So I think that's complicated.  There have been very many published studies.  I don't know.  Is it over 50 at this point?  How many published studies have found SV40 in one tumor or another?
		DR. LEWIS:  It's over 50, less than 60.
		(Laughter.)
		DR. KRAUSE:  Okay.  Of course, these studies, in general, have used PCR, which is a method which is certainly subject to contamination, but there's a very large number of laboratories, many of them independent laboratories that have found this.
		There also are a few negative studies as well, and so it's difficult to know what to make of it, except one thing is clear, that it's a potential problem.
		I think Robin Weiss wrote a very nice review -- I think it was in Nature -- of the book The River, which basically said regardless of whether or not any of this stuff is right, the fact is if it isn't right, it's just because we were very lucky, and so I think that that may well apply here as well.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		I have Dr. Stephens, Coffin, Priola, Ms. Fisher.  So we'll go next to Dr. Stephens.
		DR. STEPHENS:  It's getting late, and most of my comments have already been made.  I do have one issue, again, regarding a number, and that's the ten nanograms issue that repeatedly comes up.
		Dr. Faggett this morning raised the question in terms of the WHO standard change from .1 to ten, the '88 to '90 recommendation, and I just wanted to hear some further discussion.  I think Dr. Lewis had his hand up at one point and could comment.
		It seems to me that less is better, and for all of the issues that we've already discussed today, and again, while did we change that standard from .1 to ten nanograms?
		ACTING CHAIRMAN DAUM:  Dr. Lewis, would you comment?
		DR. LEWIS:  Yes.  I think one of the driving forces behind the change in that recommendation was the fact that concepts of carcinogenesis evolved from the late '80s through the middle '90s.  Generally it was felt that one oncogene was one transformed cell based on the virus models, but I think the work that was done on carcinoma of the colon by Burt Vogelstein and carcinoma of the breast perhaps by several other people whose names I can't recall right at this moment, but the concept evolved during this period of time over about eight years from one gene being involved in neoplastic development to multiple genes being involved in neoplastic development.
		Now, when the data that was used to compute the ten picogram limit was based on a one-hit model; when you change that to a two-hit model, you basically increased or reduced the risk factor by the square of that risk factor, and the risk factors that I recall were around, I believe, one -- based on the one-hit model, about one in ten to the 11th or ten to the 12th, and so you double that, and now you're up around one in ten to the 20th.
		And I think based on that, I think that change in concept was one of the driving forces behind the increase in the level of DNA that was proposed.
		DR. STEPHENS:  Can I pursue this just a minute?
		ACTING CHAIRMAN DAUM:  Yes.
		DR. STEPHENS:  Because we're dealing in this particular instance, I think, with a one-hit model, EA-1G, in essence, which is being used to transform cells.  Other events likely occur as we've heard today after that one event.  So why not limit the issue to a one-event model rather than going to a more liberal standard, if you will.
		ACTING CHAIRMAN DAUM:  Anyone at FDA like to comment on Dr. Stephens' point?
		DR. LEWIS:  Well, I think in terms of the adenovirus E1A, that gene, while it transforms cells, when you have it in its maximal capacity to infect humans and to spread in the population as a virion, the viruses are not oncogenic.  So in an artificial system you do get transformation, but when you put it in a situation where it spreads through the population, and in fact, most of us sitting around this table are probably carrying Adenovirus 2 or Adenovirus 5 in our peripheral blood monocytes.
		So there's no -- and people who have searched very diligently for the presence of adenovirus DNA in various kinds of human tumors, and there's no evidence that it's there. 
		So I think for these reasons, we're not as concerned about the adenovirus E1A gene, especially if it's isolated, as we are about some other things.
		Jim might have something to say about that.
		ACTING CHAIRMAN DAUM:  Does Dr. Cook or Dr. Golding have their hands up?  If it's about this point, we'll go now. If it's not, we'll go in line.  This point?
		DR. COOK:  Yeah, I'd have to go now because if I wanted until the end of the line, I'd totally forget what I was going to say.
		ACTING CHAIRMAN DAUM:  It's happening to all of us, sir.
		(Laughter.)
		DR. COOK:  So I would like to respond to this specific point, and that is it isn't fair to think about E1A as a one-hit model.  The only way that E1A can do anything in terms of a measurable immortalizing event is what we all use in the laboratory, which is to put E1A into 3T3 cells or something like that, and you can get colony formation by using E1A with a neomarker or something like that alone.
		If you try to do an E1A alone primary cell transformation, and Dr. van der Eb can comment about this, it's virtually impossible.  The only person I know who's -- I mean, you can find a rare, a very rare cell line, but it's going to be several orders of magnitude, many orders of magnitude less efficient than E1A plus X, and X can either be a previously altered cell line that's been immortalized so that now you can get a colony formation or it's a complementing oncogene like E1B or ras or polyoma middle T, but thinking of E1A as a single hit is probably inappropriate.
		DR. STEPHENS:  Yeah, I guess part of my concern had to do with the polyoma virus data where low levels, nanogram or picogram amounts, can be an issue in terms of infectivity, not necessarily -- but the combination is, I assume, what I was concerned about.
		DR. BLAIR:  Right, but I think you have to envision polyoma or any of the polyoma viruses like SV40 or polyoma as the equivalent of E1A plus E1B, considering what they can do. SV40T or polyoma T can do a couple of things, and E1A has to have E1B to do those two things.
		So E1A is probably only half as good.  It's probably only part of a transforming gene.  Dr. van der Eb really is the world's expert on this, but that's my take.
		ACTING CHAIRMAN DAUM:  Dr. Golding, did you want to speak to this issue?
		DR. GOLDING:  Yeah, I think that it's important to remember that part of our approach is not to look -- I think we would recommend that the Committee does not look at one aspect in isolation.  So I think to get sort of hooked up on the ten nanogram versus .1 nanogram is really not -- it's only one thing that we recommend.  That, I think, was derived from some of the worst case scenarios that Dr. Peden described.  If you are to translate it into what the probability of transmitting an oncogenic gene, such as activated res, you're talking about something in ten to a million, in 100 million human doses.
		But in addition, because we're dealing with designer cell substance, because we know what was put into them to immortalize them, you have the additional safety of knowing that that particular gene is not in your final product.
		So you'll have a way of following the product through the purification, as well as looking at the final product to make sure that it's not there.  So to your best ability you're saying whatever we put in these cells to start with is not in the product.  It's not in the vaccines that go into people.
		And in addition, we would like to see a reduction on total amount of DNA to a dose that's really reduced the risk of any unknown oncogenic sequence to very, very low probability.
		ACTING CHAIRMAN DAUM:  You know, I think the committee is not hooked up on this one issue because I think we recognize that you can't just say as long as it's below ten we don't care what's in it, but I think the issue that Dr. Stephens is trying to explore is what prompted a change, and a change was made, and it's intriguing to us because the issues that prompted it may help us in our deliberations.
		So I think that's why we're sort of exploring it here.
		Dr. Krause, do you want to speak to this issue?
		DR. KRAUSE:  Yeah, just two very brief points.  One of them is in addition to the data that Dr. Lewis mentioned, there also is a lot of data that wasn't available about the amount of DNA that's present in blood, and so if you consider the amount of blood in the form of transfusions that people are exposed to without adverse effect, I think that also had an influence on the WHO and changing their limit.
		The other caution that I would add is not to over interpret the polyoma virus infectivity data, and the reason is because mice are very susceptible to infection with polyoma virus such that it does not take very much polyoma to infect a mouse.
		And if there were a virus that infectious for humans, it would be very, very unlikely that such a virus would not yet have been discovered.  So the kind of as yet unknown agents that one would be worried about would be very unlikely to parallel that situation.
		So I think the polyoma virus example is very useful as a worst case type scenario, but I don't think that you can take that number and apply it directly to the human situation.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. van der Eb, did you want to speak to this issue or has everything been said?
		DR. VAN DER EB:  I think so.  I just would like to add that E1A, indeed, it can practically not transform cells, and the reason is that E1A is a very strong inducer of apoptosis in cells.  So you need E1B in order to neutralize that effect.
		And as to the ten nanograms of DNA, this is ten nanograms of turtle chromosomal DNA, of course.  It would be a very different issue if the ten nanograms were only activated ras oncogene or something like that.
		ACTING CHAIRMAN DAUM:  Thank you.
		I'm going to return to my general list now, and did you want to speak to this very issue?
		DR. KETNER:  Yes.
		ACTING CHAIRMAN DAUM:  I'm sorry.  You go first.
		DR. KETNER:  It hasn't been mentioned I don't think that as far as DNA is concerned and the transfer of oncogene size matters, and so it's of interest, I think, to know what the nature of the DNA in these perhaps is likely to be.  If it's small pieces of DNA, a couple hundred base pairs, then it's much less likely to pose a possibility of transferred an activated oncogene or E1A or even ras E1A plus E1B.
		So if the preparation of the product involves, for example, DNA of the lysates, the hazard is very much reduced.
		ACTING CHAIRMAN DAUM:  Okay.  I have Dr. Coffin, Dr. Priola, Ms. Fisher, and then I have a question, and that's all the people I've recognized so far.
		Dr. Coffin.
		DR. COFFIN:  As it turns out, I also wanted to speak to the issue on the floor.
		ACTING CHAIRMAN DAUM:  A double header here.
		DR. COFFIN:  The question I had in a sense relates to that.  It goes in a slightly different direction.
		First, my memory of the change in the standard was that there are also some practical issues involved, such as the ability of the technology at the time to measure small amounts, I think, if I remember correctly played -- and you can correct me on this.  There may have been some other issues as well as what was, in fact, in practice achievable in any given real life prep.
		In my memory of the discussions around this, of which I was present at one or two of them, not everything, was that there are also some issues like that in addition to the theoretical issues that were important.  This goes actually to the point also about the size of the DNA, is if it wouldn't for the future be worth considering a more directed standard since we have the ability to do PCR assays and, you know, quantitative PCR assays now that are within the range of anybody that's making vaccines, whether it wouldn't be worth down the road considering developing an assay that's based on numbers of copies of some standard sequence that's present in all cell substrates that might be present as measured by some standard assay, a sort of true copy number standard which would then factor in the fact that most of the DNA is probably degraded and very, very unlikely, you know, a few hundred base pair pieces, and extremely unlikely to be possible to be reassembled into an intact gene in the course of this.
		That's just sort of an aside.  
		It was pointed out earlier, and this is something that was remarked on, I think, during Dr. Peden's talk about sort of the remarkable concordance of the numbers of copies of DNA that came out of all of these different studies. 
		I was struck by that, too, but then on a little reflection, I realized that that's actually an artifact of the numbers because almost everybody uses somewhere around a couple of micrograms of DNA in these studies, and all of the samples on the studies that were reported gave positive results, and since in all of the molecules the genomes were about the same size, so all of them -- considering that lot together, I think an expected result that once you go through the same multiplication with numbers that are about the same to start with, you end up with the same result at the end, whereas, in fact, the underlying biological differences could have been extremely large while all of the numbers that are on those charts or all of the final conclusions that are on those charts should have had less than or equal to in front of the numbers.
		Maybe in some cases it was really ten to the 13th molecules, but less than or equal to ten to the 13th molecules could be ten to the fifth molecules or ten to the fourth molecules.
		So I think the concordance that appears there is actually a little bit of slight of hand, inadvertent slight of hand on the part of the analysis that was done.
		ACTING CHAIRMAN DAUM:  Thank you, Dr. Coffin.
		Dr. Priola.
		DR. PRIOLA:  Yeah, I'd like to break entirely with this train of discussion and return very briefly to the comment Dr. Minor made about contamination with adventitious agents, and particularly with the TSE, and that is that it has become -- it's apparently much easier to infect cells with TSE agents than we originally thought, and I know that he's aware of this.
		Experimentally we've learned a bit better how to do that.  It's still quite unpredictable and quite difficult to do, and under the circumstances that are being discussed here with these PER.C6 cells and exposure to fetal bovine products that might be potentially contaminated with BSE, we're talking about logs of difference in terms of exposure to infectivity.
		So when we do these things experimentally, we expose these cells to enough infection, infectivity to kill ten million mice, and if you look at the very few studies that have been done looking at TSE infectivity in blood, there's enough in there to kill a handful of mice.
		So we're talking about very, very different situations.  So under the conditions being described here, I would certainly say that it's unbelievably difficult to passage TSE infectivity in that regard, even though we are getting better at it.
		I wish it were as easy as it might be, and I'd also like -- yeah, but I'd also like to very briefly address Dr. Aguilar-Cordova's comment about using microray analysis to perhaps pick up other markers of TSE infection that might be useful in a situation like this, and it's an excellent idea.
		And I know that recently there have been reports, for example, that there is an erythroid differentiation marker whose expression level has changed in TSE infected animals, and those are the sorts of novel approaches that would be extremely useful in these situations, but may take several years to develop.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		Ms. Fisher.
		MS. FISHER:  Well, I would like to return to the SV40 issue.  SV40 has been cultured out of brain, bone, and lung cancers of children born to parents presumably who were exposed to polio vaccines contaminated with SV40, and there is a suggestion by some researchers that there was horizontal transmission perhaps involving exposure to SV40 DNA, and my question is:  are the animal studies looking at successive generations of mice exposed to residual DNA for tumors?
		ACTING CHAIRMAN DAUM:  Thank you.
		Dr. Krause or someone from FDA want to take that one on?
		DR. PEDEN:  No, I don't think we know that, and we certainly hadn't factored it in, but maybe we could consider it at some stage.
		The trouble with the mouse experiments, of course, is you normally are going to sacrifice the mouse, but we could in the future design experiments to address that.
		And while I'm on the microphone, I just wanted to say in defense of those numbers there were a limited number of studies, John, that did go down in the dose response.  Now, admittedly there weren't that many, but there were some.
		So we do in some cases have a lower limit, albeit in very small studies.  So it may not be quite such a slight of hand as you imply.
		ACTING CHAIRMAN DAUM:  Okay.  We're getting to the point where I would sort of ask people to really as they contemplate comment, whether it's been said before already or whether it's something new, and also direct your attention to the last line of the slide, which is to discuss any additional safety concerns besides the ones that are raised.
		Ms. Fisher raised the one of passage, placental passage across to subsequent generations.  Does anyone have others?
		We're trying to sort of sum up.  So we're looking for new points for the top issue and comments about additional safety concerns, and then we can come to closure on this, I hope, fairly soon.
		Dr. Coffin, please, and then Dr. Moulton.
		DR. COFFIN:  Just to make sure it gets on the list.  I said this before, but I just want to put it on the list.  One is the recombination issue, which I think really was additional to that, and the other is packaging of host cell DNA into otherwise empty adenovirus virions or virions of any virus vector that's being used.
		The second case is particularly worrisome in a sense because, as compared to DNA that's floating around free, because you're giving that DNA a very efficient means not only of getting into the cell, but getting into the nucleus of the cell, and so I think that's something that ought to be looked at, at least looked at experimentally to see what the issues really are.
		ACTING CHAIRMAN DAUM:  Thank you.
		I can reassure you that every word is being recorded, and from what I've seen of my E-mail since I've chaired this Committee, read very carefully.
		Dr. Moulton and then Dr. Aguilar-Cordova.
		DR. MOULTON:  Okay.  Just a few points.  One is I don't think I've heard anyone commend the risk analysis that was done for TSE/BSE, and actually I was pretty well impressed by that.  It was basically a combination of prevalence and dilutional calculations, and you know, if I had a one in 1,000 risk of getting HIV next year, I wouldn't hesitate to line up for a vaccine based on this product.
		So that didn't bother me.  I guess also because I'm not from the U.K.
		But on the other hand, I think I did hear a lot of people saying that we do need more data for the oncogenicity question because those calculations are basically a linear extrapolation from a dozen unfortunate chickens, and we do need some more information there.  
		And the other thing is that a calculated risk is not the same thing as a managed risk, and it would be good to make sure that we have a good plan down the line for how to manage these risks because at the end of the day, we're not going to know what they are very closely, and there are things we can do to manage them, and these include longer term follow-up of patients who are in Phase 1 and Phase 2 trials, as well as enhanced surveillance methods and efforts at the point of introducing vaccines into the population.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		Dr. Aguilar-Cordova.
		DR. AGUILAR-CORDOVA:  Yes.  An issue that hasn't been brought up and trying to think of novel --
		ACTING CHAIRMAN DAUM:  Thank you.
		DR. AGUILAR-CORDOVA:  -- this is something that we've looked at in the gene therapy and discussed at RAC quite often, and that is the shedding of vectors, and of course, we're looking at it in a small population, but if one thinks about a larger population, there will be many of those that would receive the vaccine that may have active adenoviral infection or get active adenoviral infections through that period and then co-package or carry the new vaccine and shed that to the environment in general.
		That may be a positive or it may be a negative.  I'm just putting it out there as a risk factor that is certainly possible.
		With regard to packaging of cellular genes, that is not a very efficient issue with adenoviruses.
		ACTING CHAIRMAN DAUM:  How efficient do you need to be though?  That's the question.
		Thank you very kindly.
		Dr. Cook and then Dr. Priola, Dr. Kohl, Dr. Faggett.
		DR. COOK:  I just want to respond to Ms. Fisher's point about SV40 being cultured from a variety of different types of tumors.  It's probably good to set the record straight and say that wasn't the case.  In fact, SV40 in most of these studies has been detected only by fairly high PCR cycling, and so it's sequenced detection, not viral infection that's being detected in these tumors.
		ACTING CHAIRMAN DAUM:  Thank you for clarifying, Dr. Cook.
		Dr. Priola, please.
		DR. PRIOLA:  Yeah, I want to bring back up one issue that struck me this morning from Dr. Hughes' talk, and that is how, of course, we all know that passage of tissue culture cells over time can change the properties of the cell, and that the discussion, if I've been following it correctly anyway, seems to be concentrated on approval or trying to set up tests that could be done to eventually get these cells approved for production of vaccines.
		But if that approval occurs, is it worthwhile to consider occasional follow-up testing, not perhaps as rigorous as the initial testing, but occasional follow-up testing to insure that the properties of the cells that had been characterized in the beginning are maintained?
		ACTING CHAIRMAN DAUM:  Does someone want to address that?  Dr. Golding?
		DR. GOLDING:  Yeah, I think one of the procedures that are in place is to require the sponsor to establish master cell bank at the very early stages of passages, and therefore, all of the characterizations that are done on that master cell bank should remain in place, and only this master cell bank is used for future production of new lots rather than the concept of just continue to passage the cell.
		If a new master cell bank has to be established, then, of course, all of the tests have to be done again.
		DR. PRIOLA:  Well, okay.  Then so just as an ignorant question on my part because I don't know much about this, when cells are pulled out for vaccine production, how long would a cell line such as these PER.C6 cells be maintained once it's pulled from the master bank to produce the vaccine?
		So I'm assuming that it would be for a very limited amount of time.
		DR. GOLDING:  That's correct.  You have a master cell bank.  Then you have working cell banks which are used for expansion for production of a given lot, and a lot of the testings are done on them as well, and that's the end, and then the next lot has to start again from another --
		DR. PRIOLA:  Thank you.
		ACTING CHAIRMAN DAUM:  Thank you very much.
		Dr. Faggett.  Did you not speak yet?  I'm sorry.  
		DR. FAGGETT:  Dr. Kohl is first.
		ACTING CHAIRMAN DAUM:  I apologize.  I crossed his name off by accident.
		DR. KOHL:  No respect.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  On the contrary.
		DR. KOHL:  Thanks, Bob.
		One of the early things we heard about the PER.C6 cells being described was their construction to avoid recombination, and I've been around long enough to have some skepticism that that's another one of those 100 percent things.
		We haven't talked about that much.  What do the experts feel about how, you know, fail safe these cells are in terms of not being able to recombine?
		ACTING CHAIRMAN DAUM:  Dr. van der Eb?
		DR. VAN DER EB:  You can never exclude formation of RCA in any cell type.  There is always the chance that non-homologous recombination takes place.  So you can never exclude that.  It is only far less likely.  That is all I think we can say.
		ACTING CHAIRMAN DAUM:  That makes it a simple piece of this puzzle, doesn't it?
		Dr. Faggett, please.
		DR. FAGGETT:  Thanks, Bob.
		Back to Dr. Hughes' point about the assays that are going to be available.  They mentioned that there's a lot of experimental assays being looked at.  What's being done to really accelerate FDA approval of those kind of assays? 
		So that I think that's a critical part of assuring safety issues.  That's my additional concern.
		ACTING CHAIRMAN DAUM:  Anyone want to comment from the agency on that?
		DR. KRAUSE:  Yeah, I'm not sure that specific assays that address scientific points require -- maybe I'm saying the wrong thing -- but I don't think they require FDA approval.  FDA approves products that are used in diagnostics of people, but if there is an assay which will have value in assessing a cell bank or a cell substrate or a vaccine, that assay can be done without some kind of an independent approval process of that assay.
		DR. FAGGETT:  Well, I think my real question is what is being done to really accelerate availability of those assays.  I think I did misspeak.  I'm speaking specifically experimentally.
		DR. KRAUSE:  You know, I think there are several approaches.  One of them, of course, is to try and do research at FDA which improves people's familiarity with these kinds of assays so that they understand them and how they behave.
		Another one is when products come in for discussion, to discuss the possibility of doing these kinds of assays with the manufacturers and the sponsors, and if it appears as though the assays are going to be of some value, to either use some data generated at FDA to encourage them to do it or perhaps to make those kinds of encouragements even without internal data.
		But if an assay looks as though it's promising and has a real chance to answer a question that is important in a regulatory sense in a reasonable amount of time, then we're not precluded from asking for that to be used.
		ACTING CHAIRMAN DAUM:  Thank you very kindly.
		I think we may be sort of partied out.
		(Laughter.)
		ACTING CHAIRMAN DAUM:  I would like to make a couple of comments perhaps before we close.  One of them is that I think that judged by the Committee's comments and consultants' comments this afternoon, that we're generally very impressed with the effort that the agency has made to deal with the issues that are on the slide.
		There's lots of unknown questions, and there's lots of unknown issues, but one doesn't get the feeling that there's lots of unaddressed issues, and I think the agency is to be commended for taking an aggressive and thorough stance toward beginning to look into this very difficult issue.
		I think one of the things that we did talk about today several times in addition to lots of fine tuning of efforts underway is the idea of more mathematical modeling and risk assessment, if those are the correct terms to use, Drs. Goldberg and Moulton, to get some sense of what kinds of risk we're talking about as quantified, although as Dr. Kohl correctly points out, we don't want to take them too seriously or put over emphasis on that one approach.
		I think one of the background issues that was hinted at several times was that the diseases that this research is intended to culminate in and prevent are absolutely devastating and are killing many millions of people all over the world, and that's why at first glance one might throw up their hands and say, "Well, look.  We just don't want to take any risk and we're going to just stop this until we know everything."
		But then the disease commands us, I think, to continue to develop this, and I'm drawn to the example of polio in sort of closing, which people in the room know a lot more about than I do, particularly Dr. Katz.
		But when first vaccines were licensed against polio, there were 50,000  children a year being paralyzed in the United States.  And an admittedly imperfect vaccine was put out that itself caused polio, very rarely, and produced a lot level residuum of disease in this country.
		At that time I think that even if this Committee were today addressing that very issue, that that tradeoff would be worth it.  That 50,000 children not being paralyzed every year would be worth an imperfect vaccine that we didn't know everything about.
		Naturally once the disease was virtually eradicated and there were no cases occurring from wild type virus anymore, then we had a different situation where we had to readdress that very issue.
		But here we have a disease that is epidemic in many parts of the world, and we have vaccines that show some promise in helping to deal with that enormous disease burden, and I'm very impressed with the effort gathered in this room today to begin to quantify and address and conduct research into making those issues as minimal as they possibly can be.
		Are there any other comments from Committee or consultants?  
		I think the Committee has been wonderfully interactive today and up front with comments.  Our consultants have been fantastic in terms of getting issues raised and discussed.
		Speak now.
		(No response.)
		ACTING CHAIRMAN DAUM:  We're adjourned.
		Thank you very much.
		(Whereupon, at 5:17 p.m., the meeting in the above entitled matter was adjourned.)