UNITED STATES OF AMERICA

+ + + + +

DEPARTMENT OF HEALTH AND HUMAN SERVICES

PUBLIC HEALTH SERVICE

FOOD AND DRUG ADMINISTRATION

+ + + + +

CENTER FOR BIOLOGICS EVALUATION & RESEARCH

+ + + + +

VACCINES AND RELATED BIOLOGICAL PRODUCTS
ADVISORY COMMITTEE MEETING

+ + + + +

THURSDAY
NOVEMBER 19, 1998

		The meeting took place in the Versailles Rooms I and II, Holiday Inn, Bethesda, Maryland, at 8:00 a.m., Patricia L. Ferrieri, M.D., Chair, presiding.

PRESENT:

PATRICIA L. FERRIERI, M.D.	Chair
NANCY CHERRY	Executive Secretary

ADAORA A. ADIMORA, M.D.	Member
REBECCA E. COLE	Member
ROBERT S. DAUM, M.D.	Member
KATHRYN M. EDWARDS, M.D.	Member
DIANNE M. FINKELSTEIN, Ph.D.	Member
HARRY B. GREENBERG, M.D.	Member
CAROLINE B. HALL, M.D.	Member
ALICE S. HUANG, Ph.D.	Member
KWANG SIK KIM, M.D.	Member
STEVE KOHL, M.D.	Member
GREGORY A. POLAND, M.D.	Member
DIXIE E. SNIDER, Jr., M.D., MPH	Member

	INVITED PARTICIPANTS:

		DON BLAIR, Ph.D.
		ROBERT BREIMAN, M.D.
		THEODORE EICKHOFF, M.D.
		THOMAS FOLKS, M.D.
		STEPHEN HUGHES, Ph.D.
		MARGARET JOHNSTON, Ph.D.
		NEAL NATHANSON, M.D.
		MICHAEL OXMAN, M.D.
		GEOFFREY SCHILD, Ph.D.
		WILLIAM WOLD, Ph.D.
		SIDNEY WOLFE, M.D.

	SPEAKERS:

		DR. LESLIE BALL
		DR. NORMAL BAYLOR
		DR. KATHLEEN CLOUSE
		DR. KAREN ELKINS
		DR. LYDIA FALK
		DR. CARL FRASCH
		DR. ANTONIA GEBER
		DR. ARIFA KHAN
		DR. PHILIP KRAUSE
		DR. ROLAND LEVENDOWSKI
		DR. ANDY LEWIS
		DR. DOUGLAS PRATT
		DR. BECKY SHEETS

	ALSO PRESENT:
		
		CAROLYN HARDEGREE, M.D.

A G E N D A

Page

Call to order, Announcements and	4
	Administrative Matters, Introductions

Tribute to Dr. Mary Lou Clements-Mann	9

Open Public Hearing

	Update on Approved Products	12
	Dr. Norman Baylor

	Update on reverse transcriptase	13
	activity in chicken cell derived vaccines
	Dr. Arifa Khan

	The evolving use of animal cells as	29
	vaccine substrates
	Dr. Andy Lewis

	Evolving cell substrate issues as they	63
	pertain to investigational vaccines
	Dr. Becky Sheets

	Experience with therapeutics derived	92
	from mammalian cells
	Dr. Kathleen Clouse

	Studies on safety of cell lines	112
	Dr. Philip Krause

	Items for the Committee to discuss	141
	Dr. Andy Lewis

P-R-O-C-E-E-D-I-N-G-S
(8:10 a.m.)
		CHAIRPERSON FERRIERI:  I'd like to call the meeting to order.  
		If you could all take your seats.  Those of you who are sitting up here at the table, if you could find your seats, please.  And this is an open session, and so we don't have to validate anyone at this time.
		I'd like to start by introducing myself, Patricia Ferrieri, from the University of Minnesota Medical School, Departments of Lab Medicine and Pathology and Pediatrics.  And I'm the Chair of the Committee, and I'd like to have introductions of everyone at the table.
		If you could state your names and your institution, we'll start at my far right and work around.
		Dr. Greenberg.
		DR. GREENBERG:  Harry Greenberg, Stanford University in the Palo Alto VA Hospital.
		DR. EDWARDS:  Kathy Edwards, Vanderbilt University, Nashville, Tennessee.
		DR. SNIDER:  Dixie Snider, Centers for Disease Control and Prevention.
		DR. POLAND:  Greg Poland, Mayo Clinic, Rochester.
		DR. KIM:  Kwang Sik Kim, Children's Hospital, Los Angeles.
		DR. HALL:  Caroline Hall, University of Rochester, New York.
		DR. KOHL:  Steve Kohl, University of California, San Francisco.
		MS. COLE:  Rebecca Cole, Consumer Representative from Chapel Hill, North Carolina.
		DR. DAUM:  I'm Robert Daum from the University of Chicago.
		MS. CHERRY:  Nancy Cherry, FDA.
		DR. BREIMAN:  Rob Breiman, National Vaccine Program Office.
		DR. NATHANSON:  I'm Neal Nathanson from the Office of AIDS Research.
		DR. EICKHOFF:  Ted Eickhoff, University of Colorado.
		DR. OXMAN:  Mike Oxman, the University of California, San Diego, and the VA Medical Center in San Diego.
		DR. FOLKS:  Tom Folks, Center For Disease Control and Prevention.
		DR. WOLFE:  Sid Wolfe, Public Citizens Health Research Group.
		DR. WOLD:  Bill Wold, St. Louis University.
		DR. BLAIR:  Don Blair, NCI, Frederick.
		DR. JOHNSTON:  Peggy Johnston, NIAID.
		DR. HUGHES:  Steve Hughes, ABL BRP at the Frederick Cancer Research and Development Center.
		CHAIRPERSON FERRIERI:  Thank you very much.
		Some of the members are missing, and we'll introduce them as they join us.  
		I'd like now to turn the meeting over to Ms. Cherry for various administrative matters.
		MS. CHERRY:  Well, I, of course, have the conflict of interest statement, or the meeting statement, to read into the record.
		This announcement is read into the record at this meeting of the Vaccines and Related Biological Products Advisory Committee on November 19 and 20, 1998.  For this meeting, FDA has invited a number of consultants and guests to participate in the meeting.
		The Agency has determined that the services of the guests are essential to the committee discussions.  No temporary voting privileges have been extended at this meeting.  
		Dr. Broome, although listed on your roster of participants, was unable to be with us today.
		Screenings were conducted to prevent any appearance, real or apparent, of conflict of interest in the meeting discussions here at this meeting.  		Based on the agenda made available and on relevant data reported by participating members and consultants, it has been determined that all financial interests and firms regulated by CBER that could be affected by the Committee's discussions have been considered.
		In accordance with 18 USC 208, some members and consultants required and have been granted general matters waivers.  These waivers permit Drs. Edwards, Greenberg, Hall, Huang, Hughes, Kilbourne, Kohl, Murphy, Poland and Wright to participate fully in all Committee discussions.
		Dr. Daum has been granted a waiver permitting him to participate fully in the Committee discussions on cell substrate issues.
		The following reported interests are being made public to allow meeting participants to evaluate objectively any presentation and/or comments made by guests seated at our table:
		For the session on cell substrate issues, Dr. Donald Blair is employed by NCI; Dr. William Wold reported grants with NCI and collaboration with Immunex; and Dr. Michael Oxman reported collaboration with Merck and the VA Cooperative Studies Program, research grants from Merck Consulting supported by Merck and SmithKline Beecham, and possible future collaboration with Dr. Ann Arvin.
		For the session on live influenza virus vaccines, Dr. Robert Chanock is employed by NIAID in the Laboratory of Infectious Diseases.
		Copies of all waivers and appearance documents addressed in this announcement are available by written request under the Freedom of Information Act.  
		In the event that the discussions involve specific products or pharms not on the agenda for which FDA's participants have a financial interest, the participants are aware of the need to exclude themselves from such involvement, and their exclusion will be noted for the record.
		With respect to all other meeting participants, we ask, in the interest of fairness, that you address any current or previous financial involvement with any firm whose products you wish to comment on.
		That's it.
		CHAIRPERSON FERRIERI:  Thank you, Nancy.
		I would like to pay tribute to Dr. Mary Lou Clements-Mann, who was a member of our Committee.  And this is on behalf of the other Committee members, as well as FDA.  
		Mary Lou and her husband, Jonathan Mann, died on September 2nd on Swiss Air flight 111 when it crashed off the coast of Nova Scotia.  She was on her way to an AIDS meeting at WHO in Switzerland.  Mary Lou made many contributions to our Committee. 
		We enjoyed particularly her incisive judgement, her analyses, her thoughtfulness, and the twinkle in her eye when she spoke.  
		She was Professor at Johns Hopkins University in the Department of International Health in the School of Hygiene and Public Health and was known for her vast efforts in the field of vaccines, had contributed greatly to studies of diarrhea and respiratory pathogens, and was, in more recent years, actively involved in AIDS vaccine development and implementation.
		I would like to dedicate a poem to her by the late Polish poet Wislawa Szymborska, the 1996 Nobel Laureate for Literature.  And this poem is from a volume View With a Grain of Sand.  It's titled "Nothing's a Gift."
		"Nothing's a gift, it's all on loan.  
		I'm drowning in debts up to my ears.  
		I'll have to pay for myself 
		with myself, 
		give up my life for my life.
		Here's how it's arranged:
		The heart can be repossessed, 
		the liver, too,
		and each single finger and toe.
		Too late to tear up the terms,
		my debts will be repaid,
		and I'll be fleeced,
		Or, more precisely, flayed.
		I move about the planet
		In a crush of other debtors.
		Some are saddled with the burden
		of paying off their wings.
		Others must, willy-nilly,
		account for every leaf.
		Every tissue in us lies 
		on the debit side, 
		Not a tentacle or tendril 
		is for keeps.
		The inventory, infinitely detailed,
		implies we'll be left 
		not just empty-handed
		but handless too.	
		I can't remember 
		where, when, and why
		I let someone open 
		this account in my name.
		We call the protest against this
		the soul.
		And it's the only item 
		not included on the list."
		I'd like everyone to stand for a moment of silent prayer or kind thoughts for the memory of Mary Lou and Jonathan.
(Whereupon, a moment of silence was observed.)
		CHAIRPERSON FERRIERI:  Thank you.  Please be seated.
		We'll move now to the open public hearing.  Ms. Cherry will take over for this.
		MS. CHERRY:  At this time, we have opportunity for anyone who wishes to make a statement.  There is a microphone in the center here.
		No one had contacted me in advance, and I see no one coming forward, so I will declare the open public hearing closed at this time.
		CHAIRPERSON FERRIERI:  Thank you, Nancy.
		We'll move then to Session I, an open session on approved products, and we'll have an update from Dr. Norman Baylor from FDA.
		Dr. Baylor, will you step forward?
		DR. BAYLOR:  Good morning.  
		I wanted to briefly go over a couple of approvals that we had since our last meeting.  The first one is an acellular pertussis vaccine absorbed, and the trade name for this product is Certiva.  It was approved on July 29th of this year.
		And it's a vaccine for the first four doses in the series for DTaP.  It's for the immunization of infants and children six weeks of age to seven years of age, prior to the seventh birthday.  		This vaccine is manufactured by North American Vaccine, Inc. in Beltsville, Maryland.  And the D&T components of this vaccine are manufactured by Statens Serum Institute in Copenhagen, Denmark.  
		The other product license application that we approved since the last meeting is for the rotavirus vaccine.  This is a live, oral, tetravalent vaccine.  The trade name of this vaccine is RotaShield, and it was approved at the end of August of this year.
		And it's for the immunization of infants two, four and six months of age.  The first dose can be given as early as six weeks of age, but the initiation after the six months of age is currently not recommended in the package insert.
		This vaccine is manufactured by Wyeth Lederle Laboratories, Inc. of Marietta, Pennsylvania.
		We also had -- I don't have a slide for it, but we had one major approval of a supplement for the reissuance of a license for BCG vaccine manufactured by Connaught Laboratories, Ltd. in Toronto, Canada.
		And that's all I have for you.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Baylor. 
		We'll now move into our longer session for the morning -- it's also open -- on cell lines for viral vaccines.  And we'll begin with the update on reverse transcriptase activity in chicken cell derived vaccines presented by Dr. Arifa Khan from FDA.
		DR. KHAN:  Good morning.
		Today I would like to present an update on the reverse transcriptase activity that's present in chicken cell derived vaccines.  
		I would like to present some of the studies related to this topic that were presented to the WHO earlier this year in April, as well as some additional studies done in my lab related to -- that addresses some theoretical concerns that remained with regards to the presence of the RT activity in the vaccines.
		In the first slide I would just like to give a brief background related to the chicken RT activity and with regard -- and I've also indicated publications up to date with regards to what has been done on this issue.
		Initially Böni, et al., in 1996, published that low level reverse transcriptase activity was detected in all chicken cell derived vaccines using a highly sensitive PCR-based reverse transcriptase assay called PERT, which can detect one to ten virions.
		This initial finding was reported to the WHO, and then additional studies were done by several laboratories in Europe, as well as the U.S., including the NIBSC, the CDC, as well as labs in the FDA to confirm this initial finding, as well as to expand on this and to evaluate the use of the PERT assay and other PBRT-related assays for analysis of vaccines.
		And these studies have been done and published by Robertson, et al. in 1997 and by Dr. Peden's lab by Maudru, et al. in 1998.  
		And after confirmation that there was a reverse transcriptase activity present in all chicken cell derived vaccines, the important question, of course, was whether this RT activity was associated with a retroviral particle; and, more importantly, whether this retrovirus particle could infect and replicate in human cells, and therefore be of public health concern.
		Studies done by other groups are summarized here.  The RT activity was found to be associated with retroviral particles of two distinct avian endogenous retroviral families designated as EAV and ALV.
		In addition, studies done in Dr. Robertson's lab at the NIBSC and in my lab, in the FDA, demonstrated that the RT activity originated from the chicken cell substrate and that no replication competent retrovirus was found using a variety of different human cell lines including human PBMCs.
		And interestingly, I've indicated in the last bullet that, about 20 years ago, similar RT activity was reported using the traditional assays available at that time which was produced from CF cultures and that was particle associated and replication defective for chicken cells that were tested at that time.
		These studies, as well as other information, were reviewed by the WHO early this year, and they, as well as the FDA, continued use -- recommended continued use of the vaccines made in chicken cells, and this report is published in July of '98.
		Okay, because there were retroviral particles associated with the RT activity, there was still a theoretical possibility that the virus could enter the cell and infect the cell and the sequences integrate into the human DNA.
		The concern was because, for retroviruses, there are examples in which retroviral-induced oncogenesis can occur by insertional mutagenesis.  And I've listed some examples here in which case some retroviruses can activate protooncogenes and, in more rare cases, inactivation of tumor suppressor gene can occur.
		So to address this theoretical concern, my lab initiated studies, in collaboration with Dr. Shahabidene in my lab, to look at the infection and integration of EAV-related sequences using human PBMCs.  
		The next two slides indicates the strategy that we used.  There were two sources of material that we analyzed.  
		We analyzed the chicken embryo fibroblast supernatant, which we found to contain high levels of the RT activity; as well as we used a control production lot of the measles vaccine that was equivalent to the vaccine that we indicate as MVE, or measles vaccine equivalent.
		In terms of the CF supernatant, what we're looking at are the native retroviral particles that will be produced from the cell substrate, as well as contaminating DNA that's produced from the primary culture due to cell lyses.
		And this cellular DNA also contains endogenous retroviral sequences, which are a normal component of the host DNA.  
		In terms of the MVE, we would predict that there would be native retroviral particles in there, as well as endogenous retroviral DNA from the cellular source.  In addition, there is a possibility that there could also be potential pseudotypes present due to formation of pseudotypes with the measles vaccine virus and the retroviral sequences.
		Okay, we used two approaches to analyze the integration question.  In one case, we treated the CF supernatant and the MVE samples with DNAse to remove any of the endogenous retroviral DNA sequences.  And then the treated inocula was -- then PBMCs were exposed to the treated inocula for 24 hours, and cellular -- and total cell DNA was analyzed by direct PCR for EAV sequences.
		In another strategy, which would be more specific for analysis of integration, was ALU-PCR.  And in this case, we used the untreated inocula CF supernatant or the MVE and exposed PBMCs for 24 hours, and then DNA was prepared and analyzed by ALU-PCR.
		I'm going to go more into the details of the ALU-PCR when I describe the results.
		In the next two slides I'm going to describe our results with the first strategy of direct PCR analysis of the DNAse treated samples.
		Okay, in this slide, which is seeing our analysis of uninfected cells -- do I have a pointer?  Okay, in the first lane of each three panels are the uninfected PBMC control DNA with each set of primers.  The primers that we used was EAV, and we also used CRE, which is a highly repetitive chicken element, as a control for the DNAse treatment.
		And we used amphotropic MuLV to demonstrate that a known retrovirus that can infect PBMCs was not affected with the DNAse treatment.  
		In this panel, as you can see, without the DNAse treatment, the EAV primers can detect -- produce a very intense activity, a very intense PCR band.  However, after DNAse treatment, we don't detect a band, indicating that this activity -- that this amplification was due to the presence of the DNA and not due to the presence of infections retrovirus.
		In this case, using the CRE primers, we can also demonstrate that the contaminating cellular chicken sequences are removed with the DNAse treatment and these bands are also seen in the PBMC background of the inoculated DNA.
		And, as you can see in the ampho, there's no effect of infection with a known retrovirus with and without DNAse treatment.
		The next slide shows similar analysis using the MVE sample.  And, as you can see again in the -- there is high amount of EAV detectable sequences present in the untreated sample.  Whereas, upon DNAse treatment of the MVE prior to inoculation of the PBMCs, then this activity is removed.
		And the results of the CRE indicate that the DNAse treatment worked and that you also remove this contaminating DNA.  
		Because the sample also had measles vaccine present in it, we also analyzed the RNA from the same experiment to demonstrate that there was no affect of DNAse treatment on the ability of the measles vaccine virus to infect the cells.
		You can see that here with and without DNAse treatment.  And also the ampho control shows no effect of the DNAse treatment.
		So these results indicate that the CF supernatant and the MVE do not contain sequences that can be detected after DNAse treatment upon inoculation of PBMCs.
		Then, to further confirm these results, we also utilized the ALU-PCR strategy.  And first I just want to go over the strategy briefly.  And again, this is a very complicated strategy, and I've indicated one of the references that we've used here.  
		In addition, there's another reference by Minami, et al. in Genomics in 1995.  And basically, the conditions that we've used are described in those two papers, and we'd be happy to discuss further if anyone wishes to.
		But the reason we used ALU-PCR is because there are about one million copies per haploid genome of ALU elements in the human DNA.  The ALU elements are about 300 bases, and their average occurrence in the DNA is about every three to six KBs.
		So therefore, if an exogenous sequence integrated into the human genome, there would be a chance that there would be -- that they would integrate upstream or in the vicinity of an ALU element.
		Therefore, this strategy was utilized to analyze the integration of EAV sequences in the adjacent -- in regions that have ALU elements.
		As you can see, to do a complete analysis for one retroviral element, you need to use a combination of four sets of primers indicated as A, B, C, D; and the reason being because, for the retrovirus, you need to analyze integration for both of the LTRs.
		This is the five prime and the three prime LTR, and you need to find out whether this LTR is integrated upstream or downstream of an ALU repeat element, as well as you want to be able to detect the integration in either orientation of the ALU element.
		So therefore, a combination of using these four primer sets will analyze for integration of a retroviral element located upstream or downstream of an ALU element in either orientation of the ALU element.
		Now, for simplicity -- for the purpose of simplicity, I'm only going to present limited analysis of our ALU-PCR, but we have done the analysis using all the four sets of primers for each of the samples that I'm going to show you.
		And also, I should mention that the strategy was optimized based upon the published papers so that the conditions were -- so that the sensitivity was increased and there was -- the conditions were such that there was diminished amplification of ALU-ALU detection, which can also occur.
		And also we used conditions of long PCR so that we could include any insertions that occurred up to six KBs or more from the ALU element.
		Okay, these are the results using the control amphotropic MuLV retrovirus.  Up here is a diagram showing that two sets of primers were used that would analyze integration of the three prime LTR or the five prime LTR with respect to the ALU.
		And the results indicate here that, in PBMCs infected with the MuLV shown in lane two, we could see unique bands that were indicative of integration, as compared to the uninfected PBMC control in lane one.
		Now, one would expect to see more than one band because the integration occurs randomly and the distance with respect to the ALU would be different.  		And to confirm the origin of these bands, we did sequence this band here and we found, by sequencing, that there was the amphotropic LTR with adjacent spiking sequences and then the ALU element.
		Then we applied this strategy to analysis of EAV sequences in the measles vaccine equivalent sample, and I'm only going to show you the results from one of the four sets.  But using all the four sets, we have no evidence for integration as compared to the control PBMCs.
		This is an ethydium bromide stained gel showing that, because of the nature of the primers, you can get a smear.  However, we see no -- we saw no bands that distinguished the inoculated sample from the uninoculated sample.
		To further confirm that there were no low level integrants present in this fuzziness, we hybridized with a specific probe and, again, there was no detectable sequences that were specific to the probe.  And this is a positive control for the probe.
		And so these results indicate that -- along with the other results that we have, that there is no evidence for integration using the ALU-PCR technique.  These are just summarized in the next slide.
		So basically, I should also mention that, in terms of the sensitivity of the two assays that I've shown, in terms of the direct PCR assay, our sensitivity currently -- we can detect ten copies of EAV sequences.
		In terms of the ALU-PCR, we can detect 100 copies.  But we are currently evaluating the limits of detection for each of these assays using relevant standards.  We have created a standard for the ALU-PCR that contains retroviral LTR and ALU elements that we have a relevant control for that PCR.
		So, in conclusion, the data thus far indicates that there is no evidence of integration of EAV sequences in human DNA using the CF supernatant or the MVE, thus indicating the inability of native EAV particles to infect human cells.
		And our ongoing studies are directed towards doing similar analysis of ALV-related sequences that are present in the vaccine prep, as well as to do extensive characterization, molecular characterization, of the endogenous ALV and EAV sequences in the MVE because we need to understand how representative are our primers with regards to detection of other endogenous retroviral sequences that are present.
		Basically, there's very limited sequence data available on the EAV sequences, so we need to generate additional data to demonstrate that the primers are representative to that family of sequences.
		Thank you.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Khan.
		Because we're ahead of schedule a bit, we do have time for the panel to ask a few questions.   Is there any member?
		Yes, please.
		For the recording person, it would be helpful if you announced your name first and then she would be able to record that.
		DR. FOLKS:  Yeah, I'm Tom Folks, Centers For Disease Control and Prevention.
		Couple of questions.  You went to a lot of trouble and rigors to get rid of the contaminating DNA from the prep.  Did you just look for unintegrated sequences after the infection step of the PBLs?
		DR. KHAN:  Okay, basically we're looking ‑- in the DNAse treated protocol, we're looking for both unintegrated and integrated because that's total cell DNA.  So we would pick up either.
		DR. FOLKS:  Okay, but you used ALU.
		DR. KHAN:  Okay, the ALU is for the --
		DR. FOLKS:  For integrated.
		DR. KHAN:  -- integrated, yes.  That is specific for that.
		DR. FOLKS:  Okay, but you didn't show any information where you just looked for unintegrated DNA.
		DR. KHAN:  Okay, the ALU-PCR is directed for integrated DNA.  The DNAse treatment and direct PCR analysis would detect unintegrated and integrated.  So they're two different procedures.  Maybe I'm not clear about the question, or I don't think I've answered it.
		DR. FOLKS:  Well, I mean, you satisfy, I think, by getting rid of the contaminating DNA, you show that.  But then do you go and show -- I mean, you've taken a lot of effort now to go to the next step to look for integrated, and I didn't see information that indicated that you still could have some slip through and integrated sequences would be present.
		DR. KHAN:  Yeah, I guess -- again, I think the -- I guess our major concern was for integrated sequences.  And I don't know, you know --
		DR. FOLKS:  But the ALU treatment is -- study is -- I mean, it works well with MuLV.
		DR. KHAN:  Yeah.
		DR. FOLKS:  But in something that might be much lower at an infectious nature, especially in a pseudotyping scenario, you might have much less integrated material.
		DR. KHAN:  Right, that's why we need to determine the sensitivity, the detection limits, of the ALU-PCR. 
		Again, I should mention that we did select the ALU-PCR strategy to look for specific integration because, in terms of strategies for looking at specific integration, this strategy is more sensitive than others -- for example, inverse PCR.
		So this is more sensitive in terms of that type of strategy.  
		But just to clarify, in terms of the ALU integration, the sample was not DNAse treated.  We're looking at the whole -- we're looking at the native untreated sample.  But I agree, I mean, that strategy was not meant to pick up integrated; it picks up integrated, whereas the other one would pick up both.
		CHAIRPERSON FERRIERI:  We have another question from the table.
		DR. HUANG:  From Alice Huang. 
		You mentioned that your PBMC cultures were exposed to the various materials for 24 hours and then you tested them.  Did you test for any longer time?  Did you go for 48 or 72?
		DR. KHAN:  Yes, actually in my initial studies, which I did not present here, we did do 48, and we also did not find any integration by ALU-PCR.  		Then I went back to choose 24 in case -- you know, we're really looking for effects of nonproductive infection.  And I was concerned that maybe, you know, if there was some effect -- some deleterious effect to the cell or something due to an initial integration event, some theoretical effect, that maybe we would be missing it, so therefore we went back to the 24 hours.
		But longer than that is difficult because the measles vaccine virus itself lyses the culture in about three to four days.  And I should mention that the dose that we are analyzing is 40 human doses equivalent, so it's a high dose with regards to the infection.
		CHAIRPERSON FERRIERI:  Any other questions?  Anyone from the audience wish to comment or ask a question?
		Thank you, Dr. Khan, we'll move on with the program then.  
		Dr. Andrew Lewis will begin the series then on the evolving use of animal cells as vaccine substrates.  
		I'm sure we'll take up the slack then.  I'll just repeat the rules of our activities here.  To speak, you can raise your hand and hopefully I'll see you, and then announce your name, please.  		
		Dr. Lewis.
		DR. LEWIS:  Good morning.
		I'd like to begin the discussion this morning that will occupy us for the rest of the morning by introducing myself to the Committee.  I'm Andrew Lewis, the head of the DNA Virus Laboratory in the Division of Viral Products.
		Before joining CBER in 1995, I spent some years in the National Institute of Allergy and Infectious Diseases studying viral carcinogenesis using adenoviruses, SV40, ANU-SV40 hybrids, as well as a variety of transformed cell lines as experimental models.
		Since joining CBER, I've had the privilege to work with the organization in the capacity as a head of a group concerned with cell substrate issues.  		During the past three years, this group has focused on issues associated with xenotransplantation, the presence of reverse transcriptase activity in chicken embryo fibroblasts that Dr. Khan has just reviewed for you, with SV40 as a possible human pathogen, and with issues associated with the possible use of tumor cells as substrates for viral vaccines.
		It's this issue that will -- that's going to occupy our attention for the rest of the morning.  		Over the past year or so, CBER has received inquiries and requests about the use of cells derived from human or animal tumors as substrates for viral vaccines to be used prophylacticly in the general population.  It's my task, in the next few minutes, to introduce the Committee to the challenge CBER is facing in addressing such requests.
		In addressing this challenge, CBER is being asked to move beyond the precedent that was established back in 1954 against the use of cell lines established from tumors, especially human tumors, as cell substrates for viral vaccines.
		A number of factors are motivating the interest in moving beyond the 1954 precedent.  I've listed what we consider to be the most important of these factors on the next slide.
		These factors include the development of whole virus or traditional vaccines to human immunodeficiency virus; bioengineering approaches to viral attenuation in vaccine development; the rapid development of vaccines to emerging viruses such as the H5N1 influenza virus that appeared in Hong Kong last winter; progress in understanding carcinogenesis and in detecting adventitious agents; and finally, experience with highly purified biologicals that have been derived from various types of neoplastic cells.
		Based on these motivation factors and the fact that CBER must respond to inquiries and requests regarding the feasibility of using tumor cells to develop and produce vaccines for general use, for this morning's presentations we'd like to initiate a dialogue with the Committee that we hope will prove useful in dealing with future issues related to the use of neoplastic cells as vaccine substrates.
		For my presentation this morning, I'd like to point out that I'm going to use the term "neoplastic cells" quite broadly to refer to all types of immortalized cells, including continuous cell lines that are either non-tumorigenic or tumorigenic, as well as those cell lines that are established from mammalian tumors.
		As we go forward with the business of addressing the use of neoplastic cells as vaccine substrates, I'd also like to point out that it will be necessary to schedule discussions about specific issues as future meetings with the Committee in closed session.
		As the presentations today proceed, we'd like the Committee to keep in mind the issues that we'll be asking you to discuss at the end of the talks.  These issues are presented in the next slide.
		Following talks this morning, we'd like the Committee to comment on the approach that we'll review for you in the talks this morning that are being considered in CBER to evaluate the use of neoplastic cells that are proposed for use in the manufacturing of viral vaccines.
		And we'd also like for you to consider any additional items related to today's presentations or to issues related to the use of vaccines developed or produced in neoplastic cells that the Committee finds appropriate.
		We thought it would be appropriate to begin our discussions of a change in precedence in the use of cell substrates by first defining for everyone why it's necessary to use animal cells to produce viral vaccines, and then reviewing the types of cells that are currently being used to produce these products.
		The next slide shows why it's necessary to use animal cells to produce viral vaccines.  Viruses in general, as shown in this slide, can be thought of as infectious nucleic acids, either RNA or DNA, which can reproduce themselves only within the context of living cells.
		Based on these characteristics, viruses can only be isolated and propagated in viable cells.  Thus, many of the aspects of a study of viruses, including the development of vaccines to protect humans from viral infections, can only be accomplished using those living cells that are both susceptible to viral infection, as well as viral replication.
		A critical parameter in the use of living cells for vaccine production is that the cells must not only be able to grow the infecting virus, but they must be able to grow it to sufficient concentrations to make vaccine production feasible.
		In the next two slides I've listed the types of animal cells that are available to produce viral vaccines.  Intact animals that are used to produce vaccines include embryonated chicken eggs to make influenza vaccines, and calves that were used in the past to make smallpox vaccines.
		The use of intact animals obviously bypasses the need to use tissue culture for vaccine production.  Primary cells represent unpassaged tissue culture cells that are established from embryonated eggs or from animal organs.
		These include cells established from chicken embryos used to produce measles and mumps vaccines, and cells from the kidneys of African Green Monkeys that are used to produce polio vaccines.
		Diploid cell strains represent cell cultures established from normal human lung tissues.  Due to their lack of immortality, these strains should be passage for a limited number of times in cell culture.  They are non-neoplastic and they are not tumorigenic.
		Examples of these cells include the WI38 cell strain, the MRC5 cell strain, and the FRhL cell strain.  A variety of vaccines are made in these types of cells which include polio, rubella, hepatitis A, rabies and the rotavirus vaccine.
		Continuous cell lines also represent cell lines to establish some tissue culture from the normal organs from nonhuman primates or rodents.  
		However, continuous cell lines differ from human diploid cell strains, and that, during passage in tissue culture, they become immortal; and therefore they share characteristics with neoplastic cells.
		In addition, sometime during prolonged tissue culture passage, immortal cell lines can become tumorigenic.  That is, the cells from high passage levels will produce tumors when they're injected into susceptible animals.
		The VERO cell line, established from the normal kidney of an African Green Monkey, is an example of an immortal, non-tumorigenic, continuous cell line.  However, VERO cells can become tumorigenic if passage level is greater than 146.
		Following this, VERO cells will produce progressively growing tumors when they're injected into immunodeficient nude mice.  
		Some polio vaccines are made in VERO cells.  The Chinese hamster ovary cell, or CHO cell, and baby hamster kidney cell line 21 represent continuous cell lines that are not only immortal, but tumorigenic as well.
		Thus far, such cells have been used as substrates only to produce highly purified biologicals.  
		Tumor cell lines represent cell lines established by growing cells from humans or animal tumors in tissue culture.  The cells in these lines are immortal and are usually tumorigenic.  The only human tumor cell line currently in use to produce a biological is a Namalwa cell line.
		This cell was established from a B cell lymphoma from a patient with Epstein Barr virus-induced Burkitt's lymphoma.  It's used to produce an interferon product that's licensed in Europe.  
		Other continuous cells lines that offer potential as vaccine substrates are normal cells that are transformed in tissue culture into immortal and possibly tumorigenic cells.  The 293 line of adenovirus transformed human kidney cells in the COS-1 line of SV40 transformed African Green Monkey kidney cells are representatives of these types of cells.
		There are currently no licensed products that are made in virus transformed cells.  
		The use of embryonated eggs in cells grown in tissue cultures to produce vaccines has evolved slowly over the past 60 years.  
		With the discussions today in their historical perspective, in the next two slides I review for you some of the important events in the development of cell substrates for vaccine production that have occurred during the past 60 years.
		In 1933, Drs. Woodruff and Goodpasture grew the fowlpox virus on the chorioallantoic membrane of embryonated chicken eggs.  Subsequent work found that the embryonated chicken eggs were susceptible to a wide variety of viruses.
		And as I've shown you on the previous slide, influenza, yellow fever, smallpox vaccines, as well as the vaccine for horses against encephalomyelitis virus, were all developed in the embryonated chicken egg before 1950.
		The innovations of Woodruff and Goodpasture were followed 16 years later with a demonstration by Enders, Weller and Robbins in 1949 that polio viruses would grow and produce virus-specific changes in human kidney cells growing in tissue culture.
		This study introduced the first reliable method to grow viruses in test tubes, set the stage for the tissue culture era of virology, and provided a major breakthrough in the growth of viruses for vaccine production; a breakthrough that I don't need to remind you that we're still exploiting today.
		In 1953, the first lots of inactivated polio vaccine were produced in rhesus monkey kidney cells.  
		And in 1954, in response to the need to develop adenovirus vaccines to protect military recruits in boot camp against acute respiratory disease, the Armed Forces' Epidemiology Board met and determined that only normal cells were acceptable as substrates for viral vaccines, thus establishing the precedent that we're addressing today.
		In 1961, Hayflick and Morehouse described the long term culture of human diploid cell fibroblasts, showed that such cells were susceptible to polio virus infection, and initiated the discussions that human diploid cells might be more suitable for vaccine production than primary cells from animal organs.
		In 1967, the World Health Organization Committee on Cell Culture accepted human diploid cells as substrates for vaccine production.  In this same year, Van Wezel grew cell lines on microcarriers, a technique that greatly expands the surface area available for cell growth in any culture container.
		In 1977, interferon was produced in concentrations sufficient to be clinically useful using cultures of lymphoblastoid cells obtained from human B cell lymphomas.  And in 1978, the possibility of using continuous cell lines as substrates for biologicals was introduced at a meeting at Lake Placid, New York.
		In 1981, Montagnon, Fanget and Nicolas produced the polio vaccines using viral cells grown in microcarrier cultures.  In 1987, the World Health Organization accepted the use of continuous cell lines for the production of highly purified biologicals and biotechnology products, including the polio vaccine.
		And now, in 1998, we're confronted with new technological advances and the need to pursue the development of all types of HIV vaccines.  This makes it necessary to consider how to approach the use of continuous cell lines and tumor cell lines as possible substrates of viral vaccines.
		And in the late 1970s and early 1980s, when confronted not only with the ability to produce therapeutically useful amounts of interferon for Epstein-Barr virus-induced human lymphoblastoid cells, but the potential to use hybridoma technology to produce therapeutically useful quantities of monoclonal antibodies and the ability to develop a wide variety of products using recombinant DNA technology, regulatory agencies were challenged to weigh the need for these new products with the possible risks that were associated with the use of continuous cell lines derived from normal tissues and cell lines derived from animal tumors to produce these products.
		The question was, how to proceed?  A successful approach evolved over a period of about ten years, and patients are now benefitting from a variety of products made possible by a combination of these scientific and regulatory innovations.
		The innovations in regulatory review that permitted the clinical use of highly purified biologicals produced in continuous cell lines was based on the considerations that I've listed in the next slide.
		This approach was based on the following points.  First, those concerns that were specifically associated with the use of continuous cell lines were identified.  
		These concerns included the possible presence of adventitious agents and the possibility of transferring abnormal biological characteristics to recipients by way of occult viruses, residual cellular DNA, or residual cellular proteins.
		Methods were needed -- or they were needed and developed to address each of these concerns theoretically and experimentally.  These methods were then applied to develop estimations of the risk posed by the concerns.  
		And finally, these concerns and methods were addressed and developed in scientific forums.
		In considering how to approach the use of continuous cell lines and cells derived from tumors as substrates for traditional vaccines, we've reviewed the approach that was used in managing the regulatory issues associated with the introduction of continuous cell lines as substrates for highly purified biologicals.
		Following my talk, Dr. Sheets will present a more comprehensive outline of this approach, and then Dr. Clouse from the Office of Therapeutics will review the experience with this approach in evaluating biotechnology products.
		The success with the introduction of the use of continuous cell lines provided an example of how to approach the new regulatory challenge of evaluating the use of neoplastic cells as vaccine substrates.
		Using this example, we are considering the approach I've outlined in the next three slides.  The approach consists basically of five components.  We'll begin with the process similar to that initiated in 1987 to evaluate continuous cell lines.
		To begin this approach, it's first necessary to identify the specific issues that need to be addressed.  I'm going to outline these issues that are related to the use of neoplastic cells in the slides that follow this one.
		Once the issues are identified, the next step is to develop theoretical and experimental methods to evaluate each issue.  Dr. Krause is going to outline some of these methods in his talk later this morning.
		To make the development of methods to evaluate each issue useful for regulatory purposes, it's necessary to develop criteria to establish the possible level of risk associated with each issue. In addition to developing a plan, it's necessary to discuss and develop not only the concepts, the issues, but all aspects of any approach in public scientific meetings and workshops.
		The specific issues that are associated with the development of tumor cells as vaccine substrates we've tried to present in the next two slides.  
		In this table, I've listed the general issues in the left-hand column, the nature of the concerns that are generated by these specific issues in the middle column, and references to data that generated the concern in the column on the right.
		In developing a table, we reviewed, as best we could in the time available, the literature dealing with possible risk related to neoplastic cells as vaccine substrates.  The table was then constructed by listing all of those issues that would appear to have raised concerns.
		Such a comprehensive list is based on the idea that it was essential to begin our approach from as broad a base as possible, as any issue judged to be irrelevant could be considered and dismissed.  This is the complex slide.  
		And time doesn't permit, and it's not our purpose today, to present a detailed review of each issue in the scientific documentation of why it's included in the table.  Many of these issues will be the subject of detailed discussions at future meetings.  
		Today I'm only going to mention the general issues and the specific concerns that are related to them, and very briefly mention why it's necessary to define some of these concerns.
		As you can see, the first general issue includes tumor cell contamination with the possible induction of tumor allografts.  At first glance, this concern may seem trivial, as it's quite easy to document the removal of viable cells from vaccines.
		What is noted in Southam & Gross, human tumor cells have, on very rare occasions, been grafted into allogeneic humans.  So of course it's going to be important to demonstrate and document that any vaccine produced in a neoplastic cell contains no such neoplastic cells.
		Adventitious agent contamination with the possible transfer of known or unknown infectious agents is a concern for all biologicals and probably doesn't need any further discussion.
		The formation of recombinants between vaccine virions and adventitious viral agents present in cell substrates occurred in adenovirus vaccines that were prepared in rhesus monkey kidney cells in the 1950s.
		Before the discovery of SV40 in 1960, humans were immunized with adenovirus vaccines that contained adenovirus SV40 hybrid viruses.  The rate of recombination between adenoviruses and SV40 in monkey cells is not known; nor do we know anything about the ratio between the hybrid and non-hybrid adenovirions that were present in any of the vaccine preparations that were used at that time.
		As recombination among a variety of viruses and cells co-infected in tissue culture, it is not uncommon.   This is an issue that certainly will need further consideration.
		The possible activation and transfer of occult or unusual agents as a consequence of vaccine production in neoplastic cells is a complex topic.  Studies have shown that prolonged passage in tissue culture can induce the expression of endogenous viruses.
		And other studies have shown that viruses, especially retroviruses, can parasitize the genome of large DNA viruses such as herpes viruses.  The efficiency with which such events might occur in neoplastic cells and in vaccines is unknown.
		An unusual viroid-like agent has been associated with transmittal of a lymphoma in Syrian hamsters.  Epidemics of infectious lymphoma have occurred in several different hamster colonies.  Whether such agents are present in humans is unknown.
		Continuing down the list, we have contamination with cellular DNA and the possible transfer with this DNA of oncogenic or infectious genetic information in the form of activated oncogenes, integrated viral oncogenes, or viral genomes, as well as proviruses.
		Dr. Krause will have more today about evaluating this concern in his talk later this morning.
		Now, based on recent developments in the fields of transmissible spongiform encephalopathies, when considering the presence of residual cellular materials, the possible transfer of residual cellular proteins containing prions must be considered.
		In addition to these issues, the possibility that vaccine virions will package cellular nucleic acids or rescue integrated viral genomes or pseudotype endogenous retroviruses with an efficiency that could lead to the transfer of biologically relevant quantities of oncogenic genetic information needs to be considered as well.
		The possibility that the instability of the genome of the neoplastic cells growing continuously for long periods of time in tissue culture might enhance any of these events must also be weighed.
		Now, in assessing how to address the variety and complexity of the issues that need to be considered in managing proposals to use neoplastic cells as vaccine substrates, the most important consideration, of course, is how to develop methods to measure risks and use those measurements to evaluate product safety.
		Our approach to the safety issue is to look at the possibility of assessing the level of risk posed by each concern quantitatively, establishing the chance of occurrence or the probability of a worst case scenario for each, and using such data to weigh the relative risk of any product produced in neoplastic cells.
		The approach we are considering is presented in the next slide.  
		To simplify the discussion, we're designating this a defined risks approach.  What we're proposing is that a defined risk approach could accomplish the goal of providing a quantitative estimate of the level of risk posed by each concern, as well as a means of quantifying the overall, or cumulative, level of risk posed by the product itself.
		Establishing both concern associated risk levels and cumulative risk levels will then provide methods to better judge the suitability of any product for the public health situation it's designed to alleviate.
		And again, in his talk later this morning, Dr. Krause is going to address this aspect of our approach in more detail.  
		Now the manner in which we plan -- or we would hope to implement such an approach to evaluating regulatory issues associated with proposals to use neoplastic cells as substrates for vaccines is presented in the last slide.
		To implement this plan, we would hope to review the problem and develop a working document that outlines the issues and concerns and how they may be addressed.  We would hope to hold workshops on the issues and possibly have the document be the subject of one or more discussions at those workshops or of a workshop independently devised to consider the document itself.
		The document would then be revised based on the outcomes of these discussions, and it would be presented to the Committee, this Committee, for review and comment.  Following those discussions, we would hope that CBER could develop some more formal policy on the use of tumor cell lines as vaccine substrates.
		This is the end of what I have to say.  I'd be happy to try to address any questions you might have before I turn the podium over to Dr. Sheets.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Lewis.
		I guess I would caution the panel that, because it is an open session, that there may be some questions Dr. Lewis will be unable to answer if they're too specific and deal with proprietary issues.
		Dr. Greenberg.
		DR. GREENBERG:  Andy, thank you for a very organized look at this.
		When you're defining risk, it seems to me that perhaps you want to define it compared to a standard.  And wouldn't your standard be your diploid cells and risk would be the increased risk above and beyond what we have already determined to be acceptable?
		DR. LEWIS:  Dr. Greenberg, the definition of what is an acceptable and an unacceptable risk, I think, is an issue that will create considerable discussion.  At this point in time, I think that we're not at a stage where we can answer that question with any confidence.
		And I think that risk is going to be a shifting target.  It's going to depend on the nature of the product, the nature of the problem the product is designed to alleviate, and the complexities of the individual concern that that risk is attempting to measure.
		I think the concept at this point in time is that we would hope to be able to use experimental data to define what has been -- what the risk might be and how it has been altered as a result of the manufacturing process, or at least addressed, and then be able to use that to estimate what the risk factor might be.
		Beyond that, I think it's very hard to get a handle on it without further consideration.  So I don't really think I can answer your question satisfactorily is what I'm trying to say.
		CHAIRPERSON FERRIERI:  Dr. Adimora.
		DR. ADIMORA:  People have a variety of concerns about vaccines, people being the general public.  To my knowledge, the cell substrates in which the vaccines are grown has not been one of their concerns, one of their major concerns, to date.
		But I was -- but it conceivably could be in the future, particularly if continuous -- if other continuous cell lines are used or tumorigenic cell lines are used more frequently for vaccines.  
		And I was wondering what you thought about that, about the potential for the public's concern, and if there were going to be any ways to address any potential concerns?
		DR. LEWIS:  I think that, in contrast to what you say, there has been tremendous concern over the history of cell substrates both in the public and in the scientific community about the introduction of new -- of the use of any substrate and the introduction of new substrates.
		There was a tremendous concern associated with the polio vaccine that was developed in rhesus monkey kidney cells associated with SV40, the SV40 infection.  Two years ago we were one of the sponsors of a meeting that were dealing with the follow up to those concerns.
		Those concerns continue today.  The introduction of the diploid cell strain was accompanied by a ten year -- a more than ten year debate on the safety of using human cells as a substrate for vaccines.
		So there's -- anytime a new substrate has been proposed, it's a major alteration in our thinking and requires -- and has generated considerable concern.  Now, sometimes this concern has reached the public through the media; sometimes it has not.
		DR. ADIMORA:  Well, see, that's what I was talking about.  I'm aware that there's been tremendous concern on the part of the scientific community, but maybe I've missed -- I guess I must have missed it, but I hadn't been aware of tremendous amounts of concern in the general media on the part of the general public, and there conceivably could be much more than there already has been.  
		That's what I was referring to.  I'm aware of the scientific community.
		DR. LEWIS:  Well, I think there has been in the past.  Now, what happens in the future, we'll have to see.  
		CHAIRPERSON FERRIERI:  Other questions?
		Dr. Snider.
		DR. SNIDER:  Dixie Snider.
		Andy, thanks a lot for that very nice presentation.
		It seems to me that there may be some assumptions that are being made in taking this approach, and it would be useful to make them explicit.  Because if we're talking about a new cell substrate, the issue comes up is how do you decide on whether to use a new substrate rather than an old one.
		And there, it's not only just the ability of your virus, let's say, to grow in that substrate, but how efficiently it does that, and so you get involved in some cost.  
		And I'm just wondering how the model takes those things into account, because most of the things you talked about really focus on we have already made a decision to assess a new substrate, and it seems to me it skips over a couple of questions.
		DR. LEWIS:  I don't think that I meant to give the impression that any of this is cast in stone.  What we're trying to do is develop an approach for purposes of discussion that can be used as a target to shoot at.
		I don't think there have been any concrete decisions to implement anything that I've said at this point in time.
		DR. SNIDER:  Okay, well, I mean, I guess my suggestion then would be that somehow in the model, as it moves along, some criteria be established on when it would be appropriate to even begin to talk about a new cell substrate as opposed to an older one.
		What would be reasons to move to a new substrate as opposed to one that's already been extensively used for which we have, presumably, fairly extensive safety data?
		DR. LEWIS:  I think we have to deal with the requests that are made to the organization for a regulatory consideration.  And so I think that our response to that -- what we have to do is respond to those requests.
		And we're trying to develop some sort of systematic approach to think about how to respond to those requests.  
		The substrate that's used by the manufacturer will, of course, depend on those two things that you said.  First of all, it has to -- the virus has to replicate and has to be of sufficient quantity to make the product useful.
		And the safety issues would then have to be considered based on the nature of that material.  We would hope that they would select materials that would be more amenable or pose less of a risk, but that may not always be the case.
		But we can't -- we have no way of advising, I think, against what they do at this point in time.  
		Dr. Hardegree or Dr. Eagan, would you all like to comment on that aspect of the selection process?
		DR. HARDEGREE:  I think some of this discussion may best follow some of the points that Dr. Sheets may want to make.  But I think it's fair to say that we are facing these issues now.  
		I mean, we would -- Andy has posed the development of some papers and some applications through workshops, but we are being faced with new cell substrate usage everyday with different products.  		And we are obviously in -- not at the point of considering licensure of those products, but some small Phase I studies may be underway with some of the cell substrates that are being talked about, and are certainly being talked about globally.
		These are not just issues related to the U.S.  The WHO is also facing this issue and wanting to continue this discussion through workshops as well.  And I think that this is the purpose of getting this out today, is to try to get some of the points that you believe that we need to address on the table.
		CHAIRPERSON FERRIERI:  Thank you.
		DR. SNIDER:  I don't want to belabor this too much, but it does seem to me that, although FDA can't tell the manufacturers what to do, and shouldn't, that some discussion and perhaps some guidance might evolve from the meetings you're talking about with regard to some of the trade offs.
		Because, after all, if we're talking about a cell line that has an extensive safety record and only a slightly higher yield, let's say, on the new substrate and so forth, then one might be inclined to go -- to encourage the use of the more traditional substrate, is the only point I was getting at on that.
		The other point I wanted to make is I think, in thinking broadly about this, this is not to downplay any of the things that you're talking about here, but I think inevitably these issues -- some issues are going to emerge after the fact. 
		Some new scientific data is going to become available or some claim is going to be made.  And so the post marketing surveillance aspect of this I bring up again as something that we really have to pay attention to and try to ensure that we have adequate resources for.
		CHAIRPERSON FERRIERI:  Much of this will be quite appropriate as we go into the big discussion after the presentation.  So we have time for two other questions.  Two people already had up -- had their hands up.  Dr. Kohl and then Dr. Daum.
		Steve.
		DR. KOHL:  Steve Kohl.
		Thank you again, Dr. Lewis, for your presentation.
		It strikes me that many of the issues you've raised in your two issue tables don't necessarily strictly apply to tumorigenic lines.  I mean, these are concerns that, if anything, are maybe even heightened in whole animal use, in primary cell lines.
		And I think it's very important that this not be seen as a giant leap into a great unknown area, but as our improved understanding of risks that we've actually been dealing with for years and years sometimes without knowing it.
		DR. LEWIS:  I'd say absolutely.
		CHAIRPERSON FERRIERI:  Dr. Daum.
		DR. DAUM:  I think that we've clearly been focusing -- well, we have been focusing almost exclusively on this issue of risk.  And I think that the questions that people are looking for feedback on are both scientific and, ultimately, as someone raised over here, social issues as well.
		But I think there's more than risk to deal with here because there's also an issue of benefit.  And I think that there's a variety of very real concerns that you pointed out so nicely in your presentation, and then comes width between the lines on many of your slides a number of theoretical concerns.  
		There's probably lots of things about these processes and these new ideas about how to make products and vaccines that we don't know much about.  And I would submit that it's impossible to know, a priori, where all those potential problems are.
		And the corollary of that, or the inference from that, is it's impossible to really accurately gauge what the risk is.  And I'd like to at least introduce the idea that, while it's tempting to say let's not take any risk and come to the idea of let's not use neoplastic cells for anything, I think we also need to keep an eye on the potential benefit.
		And the thinking about risk and benefit will certainly evolve as ideas -- we allow the scientific and social communities to go forward with thinking about this.  
		To just conclude with one example that I think was really helpful, at least for me to think about, is when there were 30 or 40 or 50,000 children in a year being paralyzed each year by polio, I think people would have been willing to take some risks, if you will, with development of a vaccine quickly and get it on the market.
		And, in a sense, that's what happened.  The trade off is that there's a few cases of vaccine-associated paralytic polio.  Would someone 40 years ago have been willing to trade the 50,000 for six or eight?  Of course they would.
		And then the thinking evolves to another point where the 50,000 are gone, and now we say it's socially unacceptable to have these six or eight.  We have a new problem that we want to deal with together.  So I think the thinking about what is risk and what is benefit is going to evolve with each idea that you introduce.
		And it's important, I think, that we look at the benefit as well as considering the risk at the same time.
		CHAIRPERSON FERRIERI:  One last question.
		Dr. Wolfe, please.
		DR. WOLFE:  When I was asked to come to this meeting, I was told that it has arisen because of some pending applications, the details of which there's no need to discuss this morning.  But I'd just like -- at the risk of continuing the belaboring that Dr. Snider mentioned, I agree with the point he's making.
		A very thoughtful process has just been laid out involving public discussion, a draft of some proposals, revising it and everything.  And what I'm concerned about is that the thoroughness and careful consideration of this process may get affected by the need to respond to these pending applications.
		As you all know, in parts of the FDA there are these prescription drug user fee set deadlines.  Tomorrow is a deadline for some product that's been submitted for treating diabetes, for example, and they have to respond.
		And my own knowledge is much more in the area of prescription drugs than biologics, and the question I have really is, is the thoughtful process you're talking about, which I think needs to be gone though -- in addition to the point that Dr. Snider made is, what is on the benefit side, the point you just raised -- exactly what is the benefit to be derived from leaping off of this 40-some year old, 50-some year old dictum about we shouldn't be using human tumor cell lines?
		Is this process going to be artificially speeded up and made less thorough because of these pending applications or not?  And this is a question really for the regulators, and maybe it's going to be discussed later.  But I'm very worried about that because I think that -- with or without these specific applications having been filed, it's a matter worth discussing and worth discussing very carefully.
		And it may take a long time both to answer the question is there clear evidence that we are willing to take some measured risk because of the clear evidence of the benefit, and not just an economic benefit that you can produce these things faster and cheaper, which is a benefit, to be sure, but other kinds of benefits.
		I'm just worried about the whole time frame of this because it is propelled now by these pending applications.
		DR. LEWIS:  I think the only way that I can respond to that, based on what I reviewed, is that every time these discussions are introduced about changing substrates, there is a substantial debate.  		The introduction of the diploid cell, that took ten years.  It took ten years to get continuous cell lines together.  And I think it's very unlikely that anything precipitous would happen here under any circumstance.  
		The regulatory process certainly has its deadlines, and they have to be addressed, but safety is the issue here.  And the regulatory process, at least from my perspective, has to be convinced that safety is being met, and I think that would be the overriding concern.
		CHAIRPERSON FERRIERI:  I'd like to see --
		DR. LEWIS:  So I think it could be precipitous, but it's unlikely.
		CHAIRPERSON FERRIERI:  Dr. Hardegree, did you want to say something?
		DR. HARDEGREE:  I think it's important that we're talking about time lines that differ from ‑- when we have to deal with IND processes, we have to think about the time lines that we need and whether or not materials can go forward, whether they go on hold because of safety issues, whether we have an adequate information base for that.
		For the license application, we have user fee deadlines as well that are dealing with the review process.  And I have to emphasize that it is an obligation to review and not approve.  So that we have to be certain that the products that we are looking at are safe and effective based on the material that has been submitted.
		And if we need to do additional cycles of review after we do our initial reviews within the time lines, then that's what we have to do.
		CHAIRPERSON FERRIERI:  I think we can amplify some of these issues when we go into the longer discussion period.
		Thank you, Dr. Lewis.  
		We'll move on to the presentation by Dr. Becky Sheets from FDA on evolving cell substrate issues as they pertain to investigational vaccines.  And she's going to use a high tech presentation here, I see.
		DR. SHEETS:  Good morning.
		I think that a lot of the questions that were brought up just now in the discussion I'm going to be addressing in my talk, so hopefully you'll find answers.  If not, I'm sure we can have further discussion.
		Good morning.  I'm Rebecca Sheets, and I work in the Viral Vaccines Branch of the Applications Division in the Office of Vaccines at CBER.  
		Today I will be presenting to you the safety and regulatory issues concerning cell substrates for the production of viral vaccines, and providing you with an example of how CBER has begun to address these issues for novel substrates proposed by manufacturers.
		CHAIRPERSON FERRIERI:  Excuse me, Dr. Sheets.  We need to have the slides moved up some.  
		Is there someone here who can adjust the machinery?
		DR. SHEETS:  I'm not sure -- we've already put it on --
		CHAIRPERSON FERRIERI:  That's as high as it can go?
		DR. SHEETS:  -- the Tower of Babel.
		CHAIRPERSON FERRIERI:  Can the audience see the slides?
		PARTICIPANT:  Sixty percent.
		CHAIRPERSON FERRIERI:  Well, simplistically, if you just add something underneath it, it will raise the slides.
		DR. SHEETS:  Is that a little bit better?
		CHAIRPERSON FERRIERI:  Not really; not for everyone in the room.  It has to be higher.  Let's move that back a little and it's --  it's getting better.  You're working in the right direction. 
		This is the best use of Holiday Inn crockery that I've ever seen.
		(Laughter.)
		How does the audience feel about this now?  Great.
		PARTICIPANT:  It looks relatively safe.
		DR. SHEETS:  That's important.  I don't want to lose this important Government property here.
		Okay, I'll continue.  Next slide, please.  No, back one.  Page up.  Thank you.
		The authority that CBER has for regulation of investigational vaccines is provided according to Title XXI of the Code of Federal Regulations in Part 312.  This regulation grants CBER the authority to ensure product safety before investigations can be conducted in humans.
		CBER does not have the authority to dictate to sponsors what product they should manufacture or in which cell substrate they should manufacture it.  And CBER must provide guidance to sponsors on how to demonstrate safety.
		It is also important to understand that CBER licenses products for intended uses or indications.  CBER does not license cell substrates per se; nor, for that matter, adjuvants per se.  
		These are components, or raw materials, used in the manufacture of products.  It is the final product that has been demonstrated to meet an intended use which is licensed by FDA, and each product is reviewed on a case by case basis.
		Next slide.
		Guidance for industry is currently available for characterization of cell lines used to produce biological products.  In addition to the 1993 so-called "Points To Consider" document published by CBER, some viral vaccines, specifically those not made in primary cells, are covered by guidance promulgated in 1997 by the International Conference on Harmonization, or ICH, in the Q5D document.
		Neither of these documents specifically address the use of transformed or tumor derived cell lines.  Rather, they address the use of diploid cell strains and continuous cell lines.  I'll describe the differences between these substrates shortly.
		Next slide.
		But first, what is meant by characterization of cell lines?  Each manufacturer must characterize the cell substrates banked and used in production in their own facilities.  This includes a description of the history of the isolation and banking of the cell substrate.
		The isolation may have been performed prior to the manufacturer's receipt of the substrate.  Also, they should provide the description of how they prepared cell banks.  They should describe the growth characteristics of the cells, and they should test the cells to determine the karyology and tumorigenicity.  		Importantly, they must assess their cell banks for freedom from adventitious agents.  I'll describe this further.
		Next slide.
		Tests to be performed to characterize cell banks include the determination of the karyology of the cells.  Furthermore, tumorigenicity is assessed by injecting cells into immunosuppressed rodents to monitor tumor formation.
		Colony formation in soft agar can also be assessed.  These tests apply to cells which are not of rodent origin, as all continuous cell lines of such origin are tumorigenic. 
		Next slide.
		Adventitious agent tests include those for bacterial and fungal sterility; cultivatable and non-cultivatable mycoplasma; and for insect cells, spiroplasma.  And when appropriate, mycobacteria can be detected in guinea pigs or culture. 
		Finally, viruses can be detected by in vitro and in vivo tests, specifically viruses such as those which cause acute or lytic infections, and those which cause latent infections like retroviruses and other oncogenic viruses.
		Some of these adventitious agents are readily amenable to detection, such as bacterial and fungal sterility.  Some are more difficult to detect, but well established methods for detection are available, such as mycoplasma or acute viruses.
		And some are problematic to detect by currently validated detection assays.
		Next slide.
		Tests for adventitious viruses include those performed in vitro.  Monolayer cultures of at least three cell types are used, including cells of the same species and tissue type as the cell substrate being tested, human diploid cells and monkey kidney cells.
		These cultures are assessed for hemadsorption and hemagglutination at the end of the culture period.  Also, if the growth medium or other components to which the substrate is exposed are animal derived, then tests should performed on the raw materials according to Title IX of the CFR, Part 113.
		Furthermore, bovine derived products should be certified to have been obtained from herds free from bovine spongiform encephalopathy.  
		Next slide.
		Tests for adventitious agents also include those performed in vivo in adult and suckling mice; in embryonated hens eggs; and, when appropriate, in guinea pigs, rabbits and/or monkeys.
		Next slide.
		Additional tests may be required.  For rodent substrates, antibody production tests in mice, rats or hamsters (referred to as MAP, RAP or HAP tests) are performed.  Also, an infectivity test to detect lymphocytic choriomeningitis virus, or LCM, should be performed.
		For human substrates, it may be appropriate to screen for Epstein Barr virus, cytomegalovirus, or hepatitis B or C viruses by in vitro techniques such as PCR.  The appropriateness of these tests is supposed to be considered on the basis of the tissue source and donor medical history.
		Next slide.
		Also, when appropriate, tests for oncogenic or latent viruses should be considered to detect papilloma viruses, adenoviruses and herpes viruses.  
		Finally, and importantly, tests for retroviruses must be performed.  These tests include transmission electron microscopy, assays to detect reverse transcriptase activity, and infectivity assays.
		Next slide, please.
		When CBER reviews an IND for a viral vaccine, we make safety evaluations based on several considerations.  We consider the intended use.  Is the product intended for prophylaxis or therapy?  What is the target population? 
		For example, is the sponsor intending to use the product in healthy infants?  What is the route of administration of the product?  Is it to be given parenterally or mucosally (for example, intranasally or orally)?
		How many doses will be given cumulatively?  And what is the severity of the disease being targeted?  Is the medical need currently unmet?  
		And finally, we consider safety on the basis of whether the substrate is intended for production of the final product, or was used in the history of product development (for example, in the isolation or passage of viral seeds).
		Next slide, please.
		We also consider safety of the product on the basis of the extent of purification, or viral clearance, during production.  Many viral vaccines are simply culture supernatant harvested from the production cells and sterile filtered.
		Some products, however, undergo significant purification procedures.  
		Furthermore, we consider the extent of inactivation of the product.  Live viral vaccines undergo no inactivation because the vaccine virus itself would be inactivated and thus rendered ineffective.
		In contrast, inactivated vaccines undergo significant inactivation procedures which can be validated to destroy infectivity of both the vaccine virus and many potential adventitious viruses.
		Next slide.
		CBER takes into account the whole history of potential exposures of the final product to animal derived substances when considering product safety.  Safety is considered on the basis of the substrate used for production.  
		What is the source species, and what contaminants could have come from that species?  And what exposures would have occurred during production?  For example, could human operators expose the cell substrate to human viruses, and is the substrate susceptible for replication of those viruses?
		We consider the raw materials used in production such as any antisera used for selection or purification, and medium components used in cell cultures such as serum or trypsin.
		Are these raw materials animal derived, and what are the source species?  What contaminants could have come from those species?  And we consider the passage history of the viral seeds, including the cell substrates in which they were isolated and passaged, and the raw materials to which they were exposed during isolation.
		The viral seeds may be isolated in cells different from those used in production, and they may have been passaged through several different species cells for the purpose of attenuation.
		We consider all of these exposures in product safety considerations.
		Next slide, please.
		The types of cell substrates, which are the focus of today's discussion, include continuous cell lines, which are cells which have survived extended culture passages and do not senesce.  Thus, they're considered to be immortalized.
		Such cell lines can be banked, unlike primary cells, and thus can be characterized.  Proposals have also been made to use transformed cell lines such as 293 cells which are derived from a human embryonic kidney, but then transfected with adenovirus Type 5 sequences which transform them to become tumorigenic.
		Tumor derived cells have also been proposed either for production or for isolation of viral seeds.  Examples include HuT78 cells derived from a human T cell lymphoma, HeLa cells derived from a human cervical carcinoma, and C127I cells derived from a mouse mammary tumor.
		What are the characteristics of continuous cells?  
		Next slide.
		They're generally constituted of a heterogeneous or nonclonal mixture of cells which have a selective survival potential.  They have accumulated mutations during extended culture which have given them this selective survival capability.
		Consequently, they're generally aneuploid.  In other words, they no longer maintain a diploid karyology.  They are immortalized in that they apparently grow forever without senescing.
		And importantly, for this discussion, they can be tumorigenic or they can remain non-tumorigenic.  This characteristic can depend on the passage level and the number, location and types of mutations.
		For example, VERO cells are generally not tumorigenic at the passage levels used for vaccine production, although they can develop this capability at higher passage levels.  Additionally, because these lines are constituted of heterogeneous mixtures, different banks of the same substrate can have different characteristics.
		For example, at the same approximate passage levels, some banks may be non-tumorigenic, whereas other banks may be capable of forming tumors in animals.  Thus, it is critical that cell banks be assessed on a case by case basis by each manufacturer.
		Next slide.
		Transformed cell lines are those which have been treated by viral infection or transfection of oncogenes or viral genes, or by chemical methods which result in alteration of their genotype and phenotype.
		Transformed cell lines can be tumorigenic even though they're not necessarily derived from a tumor.  293 cells were derived from normal tissue, but were transformed with sequences from an oncogenic virus.
		Importantly, transformed cells are generally transformed by a known event such as infection with an oncogenic virus or treatment with chemicals that result in specific kinds of mutations.  The mutations in these cells can potentially be characterized.
		Next slide, please.
		In contrast, tumor derived cells are directly isolated from tumors of human or animal origin.  Generally, the events causing the tumor to form are unknown or incompletely known.  Tumor derived cells are generally aneuploid, or non-diploid.
		These too can be heterogeneous.  For example, clones can be isolated from tumor derived cell lines which have lost the tumorigenic phenotype.  So simply because a cell line is tumor derived does not mean that it is tumorigenic.
		These characteristics do not equate.  Again, tumorigenicity must be assessed on a case by case basis.
		Next slide, please.
		The cell substrates proposed to be used to produce investigational vaccines will be shown in the next few slides.  Investigational vaccines are proposed to be made in the same cell substrates as currently licensed products, as well as novel substrates or novel uses of currently used substrates.
		The Committee has received a table listing the substrates used to manufacture vaccines currently licensed in the U.S., to which you may wish to refer.  		Please keep in mind that the examples I will give may be either from active investigational new drug applications, or INDs, or they may be from products of which we are aware that are in the pre-IND or preclinical stage of development.
		In other words, they have not gone into humans yet.
		I've not listed the disease indications nor manufacturers for these examples because that is proprietary information which cannot be discussed in an open forum such as this.  
		However, I will attempt to identify the uses and will indicate the type of vaccine, be it recombinant, live, attenuated or inactivated; and the proposed use, whether it's therapeutic or prophylactic.
		Next slide, please.
		Simple substrates or unicellular organisms such as E-coli, salmonella typhae or BCG are proposed for manufacture of recombinant vaccines either as substrates for prophylactic and therapeutic vaccine candidates or as live vectors for prophylactic vaccine candidates.
		Insect cell lines are also proposed for use to manufacture recombinant vaccine candidates for prophylaxis.  Primary cells are proposed for manufacture of live attenuated vaccine candidates, live vectors, or recombinant subunit vaccine candidates for prophylaxis and therapy.
		Diploid cells are proposed for manufacture of live attenuated prophylactic vaccines, often for infant use.  
		I realize that I didn't say -- when I'm referring to therapeutic indications, these are therapeutic vaccines.
		Next slide.
		A number of continuous cells are proposed for use.  CHO cells and BHK-21 cells are used for approved therapeutic biologicals either approved in the U.S. or abroad, and are proposed for manufacture of recombinant subunit or recombinant live vectored vaccines for prophylaxis and therapy.
		Madin-Darby canine kidney cells are proposed to manufacture inactivated vaccines for prophylaxis. 
		Next slide.
		Importantly, among the proposed continuous cell substrates for investigational vaccine candidates are VERO cells.  Although there is only one U.S. licensed vaccine made in VERO cells, and it is an inactivated vaccine, sponsors perceive VERO cells as a preferred substrate.
		They have proposed use of VERO for live attenuated vaccines, both conventional and recombinant, for prophylaxis often in healthy infants.  VERO cells have also been proposed for the manufacture of live vectors for prophylaxis and therapy and inactivated recombinant vaccine candidates for prophylaxis.
		The use of VERO cells for live viral vaccines has a large regulatory impact in terms of the number of potential products affected.  This is a novel use of a substrate that's used for currently approved vaccine.
		Next slide.
		Other proposed substrates for investigational vaccine candidates include virus transformed cell lines to produce recombinant live vectors for prophylaxis and tumor derived cell lines for inactivated, live, attenuated and highly purified recombinant subunits for prophylaxis and therapy.
		Next slide.
		Finally, tumor derived lines are proposed for isolation of recombinant viral seeds for live attenuated prophylactic vaccine candidates.  
		This example is important because HeLa cells, which are believed to have been -- to have contaminated in overgrown, early cultures of Hep2 cells, are known to contain human papilloma virus Type 16 sequences, the probable source of tumor formation.
		However, these cells are not proximal to the product.  They are proposed for isolation of the viral seed, which will then be propagated in a continuous cell line for production.
		Next slide.
		Why would a sponsor propose to grow viruses in novel substrates such as continuous, transformed or tumor derived cell lines?  Such cell lines have a growth advantage almost by definition.  They survive better in culture than primary or diploid cells.
		They can be more readily adapted for growth in large scale bioreactors or fermenters, or in serum-free medium.  This facilitates commercial scale production.  And use of serum-free medium helps eliminate a potential source of adventitious agents.
		In fact, in some cases, including the example I will soon give, it may be the only feasible way to achieve commercial scale production.  And cell lines can be engineered to produce novel products that might be infeasible otherwise.
		Also, one should consider that it is possible for such substrates to be characterized, unlike primary cells.  Thus, it is feasible that a safer product might result.
		Next slide.
		Additionally, often viruses can replicate to significant higher titers in such substrates resulting in higher yield process.  This allows commercial scale production and may result in a cheaper product.
		In some examples, viruses cannot replicate at all in other substrates.  Continuous, transformed or tumor derived cell lines may be the only sensitive substrate for particular viruses.  In part, this may be because the substrate provides necessary genes to support viral replication.
		An example of this is the adenovirus vectors being studied for gene therapy.  Such adenovirus recombinants are defective for replication, so they cannot be grown in WI38 cells as are the licensed adenovirus vaccines.
		But they do replicate in 293 cells which provide the missing adenovirus genes.  
		Next slide.
		So why are we concerned about cell substrates?  Well, because they are a source of contamination of the products manufactured in them.  They can be the source of adventitious agents, the source of tumorigenic potential, and the source of residual cellular DNA which can have both infectivity or tumorigenic potential.
		Dr. Krause will talk later about this a little more.
		Next slide.
		Adventitious agents are a concern for any cell substrate.  Earlier I described the types of agents to be screened and the tests for these.  However, some substrates present novel concerns as they may contain express viruses such as exogenous oncogenic viruses or recombinant viral elements.
		Or, the cellular DNA from the substrate may be the contaminant.  This DNA may contain proviruses or viral genomes which could then transmit infectivity.
		Next slide.
		Oncogenicity, or potential to cause tumors in recipients, is a major concern for considering use of those cells that have tumorigenic potential.  
		But what are the potential mechanisms for transmitting tumorigenic potential from cell substrates to final products, the viral vaccine?
		As you heard earlier from Dr. Lewis, these include the cells themselves, cellular proteins including oncoproteins and growth factors, adventitious oncogenic viruses, and cellular DNA.  I'll go into each of these in more detail.
		Next slide.
		Cells which have been assessed to be tumorigenic in animals could quite potentially be oncogenic in humans.  However, even the least purified viral vaccines are generally filtered to remove potential bacterial contamination and cellular debris from production.
		This filtration would also remove intact substrate cells.  Cellular proteins might be concerning, but they have no way to replicate, so they would not persist in the recipient and would only be able to exert briefly any possible effect before being degraded in the recipient.
		Next slide.
		More concerning is the potential for exposure to adventitious oncogenic viruses.  The primary reasons for this concern are that screening methods for these viruses are difficult or relatively insensitive, and that there may exist currently unknown or occult agents that have never before been detected despite use of current technology.
		And finally, concern has centered on cellular DNA which could be inserted into the cells of the recipients and transmit the tumorigenic potential.  Cellular DNA could contain activated oncogenes, or it could activate oncogenes or inactivate tumor suppressor genes in the recipient by insertional mutagenesis.
		Cellular DNA could also contain the proviruses or viral genomes of oncogenic viruses.  The methods for assessing tumorigenicity of cellular DNA are problematic.  Dr. Krause will discuss this further later.
		Next slide.
		Now that I've listed a host of potential problems, you might ask why anyone would consider use of these substrates.  Therefore, I'll give you a specific example to shed light on the rationale for possible use of neoplastic substrates.
		HuT78 or similar human tumor derived or virus transformed T cell lines have been considered for development of traditional approach HIV vaccines; in other words, live, attenuated or inactivated HIV.
		Consequently, CBER has begun to develop policy to guide sponsors in the safe development of such candidate vaccines.  To these ends, a workshop was held in 1996 at the National Cooperative Vaccine Development Groups for AIDS Annual Meeting in Bethesda, Maryland.
		This workshop helped identify issues of concern.  The proceedings were published in AIDS Research and Human Retroviruses.  
		In addition, as you've heard from Dr. Lewis, CBER is proposing to hold a future workshop to discuss use of neoplastic substrates in a more global manner.  
		Next slide.
		Production of live, attenuated or inactivated HIV vaccine candidates will require the use of cells in which HIV can replicate.  
		Human peripheral blood mononuclear cells pose problems for commercial scale production and would require lot by lot screening of donors and cells for adventitious agents.  Lot to lot consistency may also be an issue.
		Human tumor derived or virus transformed T cells support the replication of HIV, and I'll discuss this more on a moment.  Alternative substrates, which might seem preferable, have not, to date, been developed.
		Even if such an alternative substrate were engineered, it is unclear that commercial yield of HIV would be obtainable or whether the receptors would remain express long enough for HIV to infect and replicate in such cells.
		Next slide.
		Human T cell lymphoma derived or virus transformed T cells lines have been proposed by various potential sponsors.  Tumorigenicity remains a concern, including the following issues:  residual cellular DNA and the imperfect methods available for measuring tumorigenicity.
		Inactivants in the case of inactivated vaccine candidates might actually decrease or eliminate tumorigenicity.
		But there remains an unknown significance for vaccines if the starting material (the cell substrate), but not the final product (the vaccine), has tumorigenic potential.  Assays for tumorigenicity of cells, DNA and the final product are problematic, as will be discussed by Dr. Krause.
		Furthermore, purification levels achievable -- next slide, I'm sorry -- for a multi-component virion are unlikely to be as high as those for single recombinant protein.  Adventitious agents are, of course, a concern for every cell substrate.
		But in the case of HuT78 cells, there is a concern because these would be of human origin; because tumor viruses such as HTLV1 may have caused the tumor, it may be difficult to detect residual sequences by current screening methods; and because non-vaccine retroviruses may be present which could pseudotype with the vaccine virions or recombine with the vaccine provirus to form new viruses of known pathogenicity.
		Next slide.
		Now I said I would give the specific example for the purpose of demonstrating why one would consider using neoplastic cells for vaccine production, and thus far I've only talked about the issues of concern for risk, but there is a large potential for benefit.  
		As you well know, on a global basis, greater than 30 million people have already been infected with HIV.  The epidemic is continuing to grow globally with little sign of abatement anywhere.  Approximately 16,000 new infections occur daily.
		And very few, if anyone, survives once infected with HIV, and there remains no demonstrated cure.  
		The therapies available that prolong life are unaffordable for the majority of infected individuals because greater than 90% of them live in developing countries.  Thus, a safe and efficacious vaccine to prevent new infections would have a huge, global, public health benefit.
		Next slide.
		But why try live, attenuated or inactivated HIV vaccines?  Well, because there are only a few recombinant vaccine candidates reaching the Phase III of clinical development.  The Baltimore Committee and others have expressed the need to simulate development of more approaches to increase the potential candidate pipeline.
		Most efficacious viral vaccines are live, attenuated or inactivated viruses.  These approaches have been proven for other viral diseases, and they provide a multi-component immunogen which presents the immune system with multiple targets.
		This may be of critical importance in the case such as HIV for which there remain no known correlates of disease protection.
		Next slide.
		CBER weighs risk and benefit in the evaluation of vaccines.  We have begun to develop policies to assess risk from tumorigenic or tumor derived cell substrates in response to manufacturers' requests for guidance.
		This has included issue identification, proposals for research to assess risk sources, development and promulgation of test methods, and establishment of criteria for acceptable levels of risk which is dependent upon the intended use.
		Next slide.
		CBER has considered how to measure risk.  Ideally, a substrate should be free from viral contamination; but if the substrate is contaminated, then viral clearance or inactivation during production processes should be validated.
		Tumorigenic potential could be estimated by measuring the quantity, integrity and state of residual cellular DNA.  The WHO has recently revised their guideline of an upper limit of 100 picagrams of cellular DNA from continuous cell lines, which was based on scientific data available at that time, to ten nanograms.
		Furthermore, determining better methods to measure tumorigenicity of DNA would help in risk estimation.  Because the cells themselves are the most obvious source of tumorigenicity, as is demonstrated in animals, validation of cell clearance provides a measure of safety.
		Next slide.
		To summarize, currently CBER has no specific regulation prohibiting the use of a particular cell substrate for the production of viral vaccines.  Our regulatory authority is granted to ensure product safety.
		CBER also has a public health mission "to facilitate the development of new and improved, safe and efficacious vaccines."  Thus, CBER must work with manufacturers to provide guidance on how to meet the regulations; in other words, to demonstrate product safety.
		This is a guiding principle behind the FDA Modernization Act approved by Congress last year.  
		Next slide.
		Traditional approach vaccines -- in other words, live, attenuated and inactivated viral preparations -- remain the most proven approaches to development of efficacious vaccines.  These types of vaccines must be prepared in cell substrates.
		Next slide.
		There still exists significant public health needs which encourage manufacturers to consider novel and potentially more problematic product approaches because, for one thing, the easy things have been done already; and as old disease are controlled, like smallpox, polio and measles, other existing diseases gain in importance to be conquered.
		And as HIV and other emergent diseases have taught us, new diseases do arise.  Many of these emergent diseases, like HIV, are not simple to conquer.  
		This concludes my presentation.  I was going to introduce the next speaker at this point; but I understand that we're going to have a break now, so I'll finish here.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Sheets.
		I think we'll keep the questions for a later period.  And we will go into our scheduled break.  We will resume promptly at 10:25.
(Whereupon, the foregoing matter went off the record at 10:07 a.m. and went back on the record at 10:25 a.m.)
		CHAIRPERSON FERRIERI:  Could we all assemble now, and the Committee members and other invited guests please come to the table?  We've had a nice, long break now of almost 25 minutes.
		We'll move into the rest of the session, if you could please take your seats.
		We're going to keep to the schedule, and questions then can be held for the -- after the two presentations.  I think that will work best, and everyone can judge what the priorities are.
		For those of you who are celebrating today's National Smoke Out, I want to encourage you to stay with it.  I know what it's like, and certainly congratulate any of you who have decided to do that today.
		If we keep the breaks too long, then you might crave cigarettes more, so we'll try to restrain ourselves.
		The next talk is experience with therapeutics derived from mammalian cells, and Dr. Kathleen Clouse from the Office of Therapeutics, FDA, will speak.
		I'm sorry if I didn't pronounce your name correctly, Dr. Clouse.
		DR. CLOUSE:  I'm Dr. Kathleen Clouse from the Division of Cytokine Biology in the Office of Therapeutics Research and Review.  
		Within the past 20 years, the biotechnology industry has developed a wide range of products for many subacute, acute and often life threatening indications.  
		Over 40 safe and effective products have been licensed by the Center For Biologics Evaluation and Research, and many of these have been derived from neoplastic cell substrates.  The success of these biologics has been achieved, in part, due to efforts taken to assure product safety.  
		I have been invited to speak to you today about our experiences and practices in the Office of Therapeutics Research and Review in dealing with safety issues concerning recombinant therapeutic products that are derived from mammalian cell lines.
		Now, aspects of my talk are also applicable to issues concerning recombinant subunit vaccines, but differ significantly from issues that are pertinent to live, attenuated or inactivated vaccines.
		As shown here, there are major differences between licensed CBER vaccine and therapeutic biologic products.  First of all, the intended use.  For vaccines, it's for prophylaxis, obviously; whereas, for therapeutics, it's been used for treatment of often very acutely ill people.
		So the patient population in general for vaccines is healthy, versus ill for therapeutics.   
		The dosage schedule and route of administration can differ.  For whole vaccines, you would dose, in many cases, based on infectious units.  For biologic therapeutics, you would dose based on units of biological activity in some cases and, in other cases, based on mass.  
		And the dosage can get as high as milligram quantities.
		The schedule also differs.  In vaccines, you give them for a very short period of time with long term effects; whereas, with therapeutics, although initially they were used for short term treatments in acutely ill patients, we're now starting to treat more chronically ill patients on a long term basis.
		The routes of administration differ for both vaccines and therapeutics.  Now non-recombinant vaccines cannot achieve the same level of product purity that is achievable with recombinant therapeutics or subunit vaccines, and that is because you're dealing, in general, with one single purified protein for your recombinant therapeutics.
		Now the licensed recombinant products within the Office of Therapeutics have been expressed in the following substrates:  nine products have been licensed that are expressed in E-coli, two from yeast; but the need for more complex protein processing has resulted in the use of mammalian cell substrates.
		And what we find is there are actually 18 products that have been licensed from mammalian cell lines, but these are pretty much limited to two cell lines at this point:  CHO cells, Chinese hamster ovary cells; and the SP20 murine myeloma cell line.
		Now, 11 of the 18 products have been expressed in CHO cells and seven in the SP20, or subclone, cells.
		Now, this reflects a common practice in industry that, once something has been licensed successfully using a particular cell substrate, the inclination is to continue to use it because more and more knowledge has been accumulated regarding the safe use of these cell lines.
		However, there are situations that arise when certain products are not amenable to expression in the existing cell lines.  And this leads to scientists going to different cell substrates, and then needing to provide more documentation to the agency regarding the safe use of these substrates.
		Now the licensed therapeutics that have been derived from mammalian cells to date include tissue plant plasminogen activator, or Alteplase; erythropoietin; DNAse, which is marketed as Dornase Alpha; interferon beta 1A, or Avenex; and, more recently, a fusion protein that consists of the Fc portion of human IgG1 and the (p75) TNF receptor which is marketed as Enbro; and we have 13 monoclonal antibodies.  
		Of the monoclonal antibodies, about seven have been expressed in the SP20, but at least six have been expressed in CHO cell lines.
		Now the documents available through CBER concerning cell substrate use include the ICH guidance document on cell substrates and the "Points To Consider" document that were referenced by Dr. Sheets.
		But, in addition, there's a "Points To Consider" document that came out in 1997 regarding the manufacture and testing of monoclonal antibody products.  And most importantly, from my talk, is an ICH guidance document on viral safety evaluation of biotechnology products that are derived from cell lines of human or animal origin.
		This guidance document pertains to recombinant biologic therapeutics and also is applicable to recombinant subunit vaccines, but does not apply to whole vaccines or gene therapies.
		Now, the current approach to working with cell lines to produce biological therapeutic products focuses on production, identification and characterization of the cell substrate; but also on validation of the manufacturing process for removal or inactivation of adventitious agents, and also testing of the bulk drug substance in the final finished drug product to assure safety for the patient.
		Characterization of the cell lines intended for use in the manufacture of biologics includes documenting the history and general characteristics of the cell line, establishing a cell banking system consisting of a master cell bank and working cell bank, and implementation of quality control testing.
		Now, the same regulations apply to therapeutics as to vaccines, and these were discussed in detail by Dr. Sheets, so I will not go into them again.
		But, in addition to characterization of the host cells with regard to the cell substrate issues, their source (phenotype and genotype), detailed information must be provided to the agency regarding the expression vector system for recombinant therapeutics.
		The source and restriction map of the gene construct has to be provided.  The source, origins of replication, the promotors, enhancers and any antibiotic resistance genes present in the vector have to be identified.
		The final gene construct, the cloning process for generating it, has to be provided, the information.  And the cloning and actual establishment of the cell line also must be provided.
		Now, once the cell line has been cloned and the master cell bank and working cell bank has been generated, they are tested as described by Dr. Sheets.  But, in addition to testing of the master cell bank and working cell bank, for therapeutics there's extensive production cell testing.
		Now, the production cells are derived from -- directly from the working cell bank and, in general, extensive testing is done throughout the manufacturing process.  
		But at least once the production cells are tested at the peak of their in vitro life span, and extensive testing is done with regard to purity for all of the adventitious agents described by Dr. Sheets, and also for cell substrate stability.
		And we're looking for consistent production of the product throughout the life span of the cells.  We're also looking for retention of the production capacity during long term cryopreservation of these cells.
		And also, at the peak of their production, we're very interested in the genetic construct stability.  
		So part of the extensive testing that is done is to sequence the genetic construct that's present in the master cell bank and comparing it to that that's present in the production cells at the peak of their in vitro life span.
		A quality control of the cell substrates that are used for production includes cell culture media where you monitor additives derived from animal sources, any antibiotics, growth factors and so on; and again, emphasizing management of the cell cultures, looking for product consistency and constant adventitious agent testing; and also specific testing on the unprocessed bulk drug and the processed bulk drug and the final drug product.
		The companies will set lot release specifications for the final bulk drug substance and drug product, and they will also set specifications for parameters to monitor in vitro production and management of the cell cultures.
		The quality control testing for biologics again includes testing for bacteria and fungi, mycoplasma, viruses, both adventitious -- and one that's omitted from there -- and also endogenous retroviruses.
		Tumorigenicity testing is actually not required for the therapeutic products licensed to date because it's not required for continuous cell lines derived from rodents since they've already been proven to be tumorigenic.
		However, I would like to emphasize addition practices that are employed for -- with regard to virus monitoring and to reduce -- that are geared at reducing the risk of virus contamination in biologic products.
		And this is the concept of viral validation.  Now, the risk of viral contamination is a feature that's common for biologic products that are derived from cell lines.  And as mentioned before, the contamination can come endogenously from cell substrates that express virus particles, and you can have contamination from adventitious virus.
		And the concern, obviously, is that these infectious agents could be transmitted to patients and could be infectious for the patient.
		So there are three complimentary approaches that therapeutics uses for the control of potential viral contamination.  First of all is to select and test the cell lines and raw materials for the absence of viruses.
		And again, this was dealt with in detail by Dr. Sheets.  Secondly, to assess the capacity of the production process to actually clear infectious viruses should a contaminant be introduced that you do not pick up on.
		And also to test the product at appropriate production steps again for the absence of adventitious virus. 
		Now, no single approach will necessarily establish the safety of a product.  First of all, there's an inherent limitation to quantitative viral assays that are used in that their ability to detect low viral levels pretty much depends on the size of the sample.
		So the confidence that virus is absent from a product may not really result from direct virus testing.   However, you can achieve a certain level of confidence by showing that the purification process itself can remove or inactivate viruses.
		So the type and extent of viral tests and viral clearance studies in general will depend on the extent of the cell bank characterization and qualification that was done, the nature of any viruses that were detected in this characterization, the culture methods that are used for product production and all of the media components that are used, the results of viral testing after cell culture, and again, the ability of the process to clear the viruses.
		Now, validation of viral elimination requires the following:  
		First, you have to select appropriate viruses for the testing.  The manufacturer has to prepare a scaled down manufacturing system.  They have to do an analysis of step-wise virus elimination.
		They have to determine whether virus elimination is due to physical removal of viruses or inactivation of viruses.  If inactivation of virus is one of the steps, then kinetics of inactivation have to be determined.
		And then, at the end, the manufacturer has to estimate the overall combined effect to generate a final figure for the overall viral clearance for the system.  In the next few steps I'll discuss each of these individually.
		As far as the viruses used for viral clearance studies, first of all, they should resemble viruses that could contaminate the product.  And they also should represent a wide range of physicochemical properties so that you can adequately assess the ability of the system to remove any potential viral contaminant.
		They should include relevant viruses.  And by this, these are viruses that are either identical to or are the same species of viruses that can contaminate the cell substrate and have been identified as contaminants.
		For specific model viruses -- and these are viruses that are used when relevant viruses are unavailable.  And relevant viruses may be unavailable if they either cannot be grown to a high titer or if they're too infectious to be used for viral validation studies.
		And generally, the specific model viruses are closely related to the known and suspected viruses.  They're generally in the same genus and family.  In addition, nonspecific model viruses should be used.
		And these viruses are used for the purposes of characterizing the capacity of the manufacturing system to inactivate or remove viruses in general; in other words, to characterize the robustness of the manufacturing processes.
		And the nonspecific model viruses should possess different properties.  The properties should be varying DNA and RNA genomes.  You should have representative viruses that are enveloped and non-enveloped.  They should have low to high physicochemical resistances.
		For example, resistance to acid pH is one parameter.  They should range in size.  Very often they range from 20 nanometers to 200 nanometers.  And all of these should be viruses that can be grown to high titers to give an adequate indication of the level of virus clearance that can be achieved.
		Now, once the viruses have been selected ‑- and generally you select three or four model viruses for each viral validation study that's done ‑- a scaled down manufacturing system should be set up outside the normal manufacturing facility.
		And this is deliberately to prevent the introduction of any virus into a manufacturing facility that is operating under good manufacturing practices.  This also enables performance of the viral testing by qualified staff with virologic expertise.
		Generally what happens is there's a collaboration between the manufacturer and an outside company that does viral testing.  The manufacturer actually provides the scaled down model system and employees that actually operate this.
		The viral testing group provides the high titer virus and actually does the virus isolation and testing as it goes through the manufacturing process.
		When they generate a scaled down manufacturing system, they have to validate to show that they -- this adequately represents the production procedures.  It has to be scaled down proportionately, and all of the production parameters have to be duplicated, including such things as buffer flow rates and so on.
		And finally, if it's impossible to identically or accurately scale down any aspect of the manufacturing process, the manufacturer has to determine the impact of the production deviations on the viral clearance studies and how they may reflect the outcome of the studies.
		So for analysis of step-wise virus elimination, first it's desirable to assess the contribution of each major production step.  And sufficient virus should be present in the material at each step.
		Generally we try to select a virus -- or the company selects a virus that can be grown to at least 108 particles per ml.  The virus should be added to in process material at each step.  
		Generally what's done is the manufacturer provides virus -- or the manufacturer provides drug substance from at least three lots of material at various steps in the production.
		And from this, when the material has been run through the purification process, quantitative infectivity assays should be done.  And these should be shown to have adequate sensitivity and reproduceability.
		Now, it is important -- because virus elimination can consist of physical removal versus inactivation, both of these serve as a mechanism for virus reduction.  It's important that, if there is virus elimination at any step, the mechanism of the loss of viral infectivity be determined.
		If inactivation of virus does occur, then samples need to be taken at different times and an inactivation curve should be constructed so that the kinetics of virus inactivation can be determined.  
		And the purpose of this is to make sure that all of the parameters are in place to make sure that the product is exposed to the appropriate conditions so that any virus that's present would be inactivated.
		Now, in order to determine the overall viral clearance, statistical analysis has to be used on the data, and the results should be statistically valid to support the conclusions that are drawn.  And the statistics that should be used are described in Appendix 3 of the ICH viral validation document.
		So, in order to estimate the combined effects of the viral inactivation and clearance, they need to quantitatively estimate the overall level of virus reduction that's achieved.  And it's important that they show that there is an excess capacity for viral clearance within the system.
		So this means that they have to compare the amount of virus that's eliminated to the amount of virus that's present, let's say, in the unprocessed bulk drug substance.  
		For murine cell lines, since there are frequently endogenous retrovirus particles, this amounts to processing portions of the bulk drug substance and using transmission electron microscopy to determine the viral burden.
		Now, this only applies to endogenous viruses.  If there are adventitious viruses present, you don't proceed and process the bulk drug substance into final drug product.
		From this, they need to calculate the estimated number of virus particles per dose, and that's described in Appendix 5 of the ICH document.  And they also need to calculate the level of virus reduction, and these calculations are described in Appendix 4.
		So you can see the utility of the document.
		But there is one note that I would like to make, and that is that a number of factors in the design and execution of viral clearance study can actually lead to an incorrect estimate of the ability of the process to remove virus.
		And these are also discussed in detail in the ICH document, and you can use this to see what you need to avoid.  
		Now, the process of viral validation can actually be used to determine -- or used for risk assessment, but that's not the intent of the document.  The intent was actually to enable the manufacturer to provide confidence that any virus that entered the product, in all likelihood, would be cleared and would not appear in the final product that is released.
		So, in addition to doing extensive viral testing and validation of viral removal, we also have to validate removal of the cell substrate.  First of all, they have to document removal of all the media components.
		If fetal bovine serum is used, for example, in general the companies will monitor bovine IgG and bovine serum albumin levels to make sure that, in the final product, these are virtually undetectable or at extremely low, non-immunogenic levels.
		The same is true for any growth factors or antibiotics or inducing agents that are present in the media.  You also have to look for removal of cell derived proteins.  	
		And this is done, in general, by establishing a host cell protein assay where a manufacturer takes non-transfected cells, prepares a mock antigen, and immunizes animals, generates an antibody, and sets up an ELISA that's capable of detecting host cell proteins.
		And for this, they will set lot release specifications and monitor each lot of product that's produced.  But in addition, they have to control for host cell proteins that may not be picked up in this assay, but may co-purify with the recombinant product.
		And to do that, what's frequently done is they will run an SDS polyacrylamide gel and use silver stain and identify all of the bands that appear in the silver stained gel.  They look for primary product band, they look for breakdown products, and they look for aggregates of the product.
		If there are other major bands that cannot be attributed to the product that is desired, they have gone to great lengths to identify this, even so much as eluting the protein and sequencing it to identify it.
		If it is a frequent contaminant that co-purifies, then specifications have to be set for release to make sure that this protein is present in low levels or, alternatively, an added step in the manufacturing process to remove this particular protein.
		And finally, they have to monitor for removal of host cell DNA.  We previously had set limits of less than ten picagrams, but in most of the guidance documents the requirement has been removed because it does vary somewhat depending on the nature of the product that is being manufactured.
		So finally, what we can conclude is that, in the 12 years using licensed biologics and in the years preceding that, during their development as investigational new drugs, no cell line derived biological therapeutic product has been implicated in the transmission of viruses.
		Also, there is no known adverse events that have occurred that can be attributed to cell substrate exposure; however, this is very difficult to determine.  
		Also, what I wanted to emphasize and what we hope to gain from this advisory committee meeting is that the complexity of cell substrate issues is increasing in biologic therapeutics, and the issues are also paralleling the complexity of the vaccine cell substrate issues as we enter into gene therapies and the proposed use of therapeutic vaccines to regulate immune responses.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Clouse.
		We're going to move on to the next speaker.  And then, following Dr. Krause's talk, we will have a little time to ask questions of the three preceding speakers.  
		This talk is studies on safety of cell lines.
		And if Drs. Sheets and Clouse could be available close to a microphone so that, when we get to the questioning period, that you would be readily available.
		MS. CHERRY:  We can -- if you can find a seat at the table when we get to the questions.
		CHAIRPERSON FERRIERI:  There are also a lot of seats here in the front row, and there are two tall microphones.
		DR. KRAUSE:  The goal of this talk is to discuss our initial approach to thinking about how to apply laboratory data to the assessment of cell substrate risk.
		And before I get started, I'd like to give credit to Anamaria Serig-Honigman, who is a post doctoral fellow in my laboratory who performed the experiments I'm going to show you from my lab; as well as to Keith Peden in the Laboratory of Retrovirus Research whose help in formulating this talk was indispensable.
		We would like to develop a quantitative approach that enables us to use laboratory data to assess risks associated with cell substrates.  The change in precedent against the use of neoplastic cells as vaccine substrates needs to be based on objective scientific data that can be used to evaluate levels of risk.
		As Dr. Lewis pointed out, this could be implemented by quantitatively assessing the level of risk posed by each issue, establishing the probability of a worst case scenario for each issue, using data to evaluate the risks individually and cumulatively, and using the data then to assess the relative risk of the product.
		In considering the laboratory evaluation of cell substrate safety, the first question is, what materials need to be tested, and by whom?  Both manufacturers and CBER will have to perform tests on novel cell substrates and products.
		Some of these tests may need to be performed on each lot of product.  Additional laboratory studies to answer more general questions will also need to be done.  Because these studies will provide general information, they should be performed by public institutions.
		It is very important that information relevant to safety issues be viewed as objectively obtained and readily available to all.  Any product produced in these novel cell substrates will need to be tested extensively for the presence of adventitious agents.
		While the focus of our discussion today is neoplastic cells, it should be noted that equal or greater concerns related to adventitious agents may be raised regarding novel primary cell substrates.  
		Testing for adventitious agents in novel cell substrates includes the tissue culture and animal tests that Dr. Sheets described.  In many cases, it may also be necessary to develop new, specific tests for other agents.  
		For example, over the last years, CBER developed tests for SIV, HIV and SV40 which were applied to polio vaccines grown in primary monkey cells in order to help address concerns that this vaccine might be contaminated with those viruses.
		This is an example of a PCR test that we developed in my laboratory to detect SV40 in vaccines.  As you can see from the right-hand panel, this test is capable of detecting between one and ten copies of SV40 DNA in a vaccine sample.
		The left-hand panel shows four samples, marked one through four, in which SV40 was not detected.  The lanes marked V represent negative controls which were performed in every other sample.  		We tested 60 lots of OPV using this method, all of which were negative for SV40.  Phil Minor, at the NIBSC in Britain, has also performed extensive testing of polio vaccines using PCR-based methods and also found the polio vaccines used in the UK to be free of SV40.
		An obvious approach to ensuring the freedom of viral vaccines from adventitious agents is based on the validation of viral clearance, as described by Dr. Clouse.  This assumes that measures that could clear adventitious viruses are taken.  
		While these methods work for highly purified products, they are very difficult to implement for live virus vaccines for which attempts at purification may affect the viability of the product.
		One area in which further research might improve our ability to detect adventitious agents in cell substrates is the development and implementation of nonspecific assays to detect unknown viruses.  In implementing such assays, it's important to understand the sensitivity with which they rule out the presence of potential adventitious viruses.
		One example of a nonspecific assay is the polymerase chain reaction based reverse transcriptase assay, or PERT, which can detect all retroviruses, including those that have not yet been discovered.  This assay may also be combined with tissue culture methods.
		Currently performed tissue culture assays are, in general, nonspecific.  It is possible that broadened tissue culture assays may provide better sensitivity to detect adventitious agents in novel cell substrates, or that improved animal models -- for example, incorporating the use of immunocompromised animals -- might improve the sensitivity of such testing in a nonspecific way.
		We are also interested in considering nonspecific molecular assays for detection of adventitious agents in cell substrates.  Examples of nonspecific molecular tests that could detect unknown viruses are shown on this slide.
		One approach is to use degenerate primers specific for various viral species.  For example, in each of the past few years, new herpes viruses have been discovered using degenerate PCR primers derived from herpes virus polymerase sequences.
		Another approach is to use PCR-based subtraction methods such as those that have been used to find viral sequences in diseased human tissues to examine vaccine or cell substrate samples for the presence of nucleic acids that could represent adventitious viruses.
		A third approach is to purify nuclease resistent nucleic acids from a vaccine or cell substrate sample and use generic primers to non-specifically amplify those nucleic acids that are resistant to nuclease digestion.
		The protein capsids of many viruses protect their nucleic acids from such digestion, so that this method might be able to identify contaminating encapsidated nucleic acids.  
		For any of these methods, it would be important to understand the sensitivity to detect known viruses before their ability to detect unknown viruses can be evaluated.
		According to the recently revised WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals, the amount of cellular DNA in biological products should be limited to ten nanograms per dose, an increase from 100 picagrams per dose.
		This limit is meant to apply to continuous cell lines and not to products given orally or products derived from microbial, diploid or primary cell culture systems.  The ten nanogram figure was derived by considering data in theoretical calculations regarding the tumorigenicity of injected DNA.
		However, it should be noted that, for live viral vaccines in other less highly purified products, it may not be possible to limit the total amount of DNA to ten nanograms.  While CBER is attentive to WHO guidelines, CBER evaluates products on a case by case basis in determining appropriate limits for cell substrate DNA.
		Thus, the question is raised, what data would be required to provide assurances regarding safe quantities of residual cellular DNA for vaccines produced in these novel cell substrates?
		Potential tumorigenicity of DNA from cell substrates is one factor that has limited the quantity of DNA in biological products.  Although cellular DNA from neoplastic cells has never been shown to be tumorigenic in animal models, injected intact tumor cells may be tumorigenic in animal models and in humans.
		Of course, some DNA from these cells would persist in vaccine products.  
		As you can see, the ability of different types of cells to form tumors in animal models varies substantially, ranging from tens of cells for endometrial carcinoma to millions of cells for 293 cells required to induce a tumor in half of -- or in nude mice.
		The TPD50 represents the dose of cells required to induce a tumor in half of the injected animals.  
		One question that CBER will need to answer is whether the relative tumorigenicity of the cells that are used should affect CBER's assessment of the risk associated with the DNA that comes from those cells.
		Another question is whether the history of the cell line, or the mechanism of transformation, if it is known, should influence CBER's assessment of this relative risk.
		This slide lists several additional approaches that might be used to better understand the potential tumorigenicity of residual DNA from neoplastic cells.  
		These include the identification and validation of improved animal models for tumorigenicity testing -- for example, highly immunosuppressed animals or transgenic mice that are deficient in p53 or the constitutively expressed oncogenes such as RAS.
		At this point, it is not clear whether such models would detect oncogenic DNA with greater sensitivity than other animal models.  Further investigation of tissue culture transformation assays, like NIH 3T3 cells, either alone or in combination with animal models in which transformed cells are injected into animals to look for tumors, might be another useful approach.
		One could also consider assessing the tumorigenicity or infectivity of cell substrate extracts which could detect occult, known tumor viruses.  These methods could be developed in a quantitative fashion for optimal use in risk assessment.
		Another issue associated with cell substrate DNA that has not been discussed as thoroughly as the tumorigenicity risk is the potential for this DNA to be infectious.  Viral genomic DNA is infectious when it is injected into animals.
		Moreover, tumor cells and primary cells may harbor latent viruses and thus contain viral genomes.  And DNA used from cells used to produce retroviral vaccines are a special case because they may contain retrovirus genomes for that reason as a byproduct of vaccine production.
		Several factors may influence the assessment of the tumorigenicity and infectivity risk associated with residual DNA.  
		These include the total quantity of DNA in the vaccine; the number of doses to be given; the size of the DNA, where larger DNAs might not get into cells as well and smaller DNAs might be small enough to interrupt or not to encode intact genes; sequence-related properties of the DNA (for example, whether it encodes a virus or not); the number of copies of potentially infectious or tumorigenic sequences per cell; and the state of the DNA.
		The state of the DNA includes such factors as whether it is associated with chromatin, whether it is integrated into a cellular genome, whether it is linearized or circular, etc.
		For the considerations raised on this slide, although it is likely that they all have an effect on the infectivity or tumorigenicity of residual DNA in a vaccine, these studies have not, in general, yet been performed in a quantitative fashion that would permit us to apply a quantitative risk assessment model.
		This slide summarizes data taken from the scientific literature regarding the infectivity and tumorigenicity of viral DNA for several different viruses when injected into several different animal models.
		And there's one mistake on this slide.  For polyoma virus, when the TPD50 was calculated, hamsters were used and that's not written on this slide -- baby hamsters.
		Using the approach outlined in the manuscript that is in your background package, on the right-hand side of the table I have calculated the theoretical risk associated with a product that contains one microgram of cellular DNA that contains a single viral genome per cell.
		This calculation accounts for the dilution of the viral genome in the cellular genome and assumes that viral genomic DNA is as infectious or tumorigenic when incorporated in cell substrate DNA as it is when it is linearized and injected directly.
		This risk estimation also assumes the total risk is directly proportional to the amount of DNA injected.  The estimated risk of an infection associated with this theoretical product thus would range from as high as one in 8,000 for polyoma virus DNA to one in tens of millions for other DNAs.
		An improved understanding of the relative infectivity of different types of DNAs might assist in developing tests to ensure that dangerous quantities of infectious DNAs are not in biological products produced in novel cell substrates.
		One other comparison may be useful.  For example, for polyoma virus DNA, it appears that this DNA is quite a bit more infectious that it is tumorigenic.  
		This suggests that much of our effort in understanding the risk associated with DNA and biological products should be focused on understanding and minimizing the risk of infectivity.
		It should be pointed out that direct assessment of the tumorigenicity and infectivity of residual DNA associated with a particular product may be impractical.  This is because, in order to achieve meaningful safety margins, very large quantities of DNA would need to be purified and tested in multiple animal models for infectivity and tumorigenicity.
		In the example on this slide, if a dose of a product contained one microgram of residual cellular DNA, assessment of a million doses in a single type of assay would require testing of one gram of DNA.  Moreover, this approach may require use of more animals than is practical.
		That is why I believe it is important to take a more general approach and understand the potential underlying infectivity and tumorigenicity of the different types of DNA.
		A third concern regarding the manufacture of viral vaccines in novel cell substrates is the possibility that the vaccine virus might package components of the substrate cells.  This includes the potential for packaging cellular DNA, recombining with endogenous sequences, or pseudotyping endogenous viral sequences.
		A full assessment of this risk may require additional experimentation designed to understand the rates at which these sorts of events occur in the absence of selective pressures.  
		One approach is to quantitatively assess recombination or packaging rates using -- by examining the packaging of reporter genes, such as antibiotic resistent genes that are expressed within a sample cell substrate.
		Another approach is to directly quantify cellular DNA that may be packaged within viral particles.
		The latter approach is illustrated by this experiment from my laboratory in which we examined the ability of herpes simplex virus to package the cellular DNA of HeLa cells, which are a cervical carcinoma line which contain about 50 copies of the human papilloma virus Type 16 genome per cell.
		We performed polymerase chain reaction of nuclease resistent viral DNA representing the contents of viral capsids to detect HPV sequences.  When HSV was grown in HeLa cells, we detected HPV sequences associated with the HSV capsids.
		Our negative control was HSV grown in VERO cells, which do not contain HPV DNA, that was then added to uninfected HeLa cells.  The negative control demonstrates that the HPV DNA we detected was not simply a contaminant during the process of purifying the viral capsids from the HeLa cells.
		Further experiments to define the quantity of cellular DNA that is packaged per virion could be performed using this system.  Similar quantitative approaches could assess other types of viral packaging of cellular components at potential recombination rates between the vaccine viruses and endogenous sequences.
		In summary, I've tried to outline laboratory approaches that could be used to better assess safety issues associated with novel cell substrates, including neoplastic cells.  
		These include extensive testing for relevant potential adventitious agents, development and implementation of improved nonspecific methods to detect new adventitious agents with non-sensitivity, the use of quantitative approaches to assessing DNA infectivity and tumorigenicity, and the assessment of viral -- rates of viral packaging or recombination with pseudotyping of cellular sequences.
		In addition, quantitative risk assessments could and should be performed along similar lines for other issues as required.
		Thank you.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Krause.  If you could stay at the microphone for a minute or so.
		We'll take questions from the panel that are related to his presentation for information.
		Yes, Dr. Greenberg.
		DR. GREENBERG:  Thanks for that very nice presentation.
		You've outlined a program that requires a lot of laboratory experimentation to go forward.  At previous meetings of this Committee, we've been informed about lack of funds to do experimental work, and I just wonder where all of this work to define how to define risk is going to happen?
		DR. KRAUSE:  That's an excellent question.  As you know, the amount of research resources that CBER has to perform studies like this is greatly limited and has become increasingly limited from year to year.
		I think that a lot of the reason that I am presenting these kinds of studies to your group is to get a sense from this Advisory Committee of the relative importance of doing these kinds of studies in coming to decisions regarding whether or not to use novel kinds of cell substrates so that then appropriate cases can be made to have these kinds of studies done in one place or another.
		DR. GREENBERG:  Can I just ask a follow up?  Are manufacturers themselves investing in trying to do this type of work as to form the basis of moving forward?
		DR. KRAUSE:  To my knowledge, this general kind of work is not being done by manufacturers.  On one of my earlier slides, I pointed out that I think it's important for these kinds of studies to be done in public institutions where nobody will suspect the objectivity of the research and where one can be certain that all of the work is in the public domain.
		So that if one manufacturer does studies, the question then becomes what's the motivation for that manufacturer to provide that information to assist another manufacturer in getting a competing product approved.
		So the answer is, to my knowledge, these kinds of more general studies are not being done by manufacturers.
		CHAIRPERSON FERRIERI:  Yes.
		DR. HALL:  Caroline Hall.
		This is fascinating talks and excellent, and I'm amazed at the amount of effort and work that has gone into this.  
		Is it all right to go ahead?
		CHAIRPERSON FERRIERI:  Yes, we're beckoning to one of the other members of the Committee.
		Sorry, Caroline.
		DR. HALL:  No, that's all right.  
		But I'm also concerned that, in this -- sort of as I listen to this, this mire of conundrums of how you're ever going to put all these potential risk factors in to have a priority of risk or set up these standards -- that, in doing this, are we using other standards that are, in actuality, assumptions?
		And this is what I mean by this, is that we have assumed that such things as the route of inoculation -- that there are certain risks factors; that systemic may be more dangerous, say, than orally, etc.; that more -- a greater inoculation dose or repetitive dose may be more dangerous when, in actuality, could it be an individual circumstances that a greater antigenic boost is actually safer.
		Or that the inactivation procedure is good, it gets rid of the adventitious agent or inactivates it, but that the process may actually be worse than the adventitious agent, etc.
		So that what I'm asking is, in some of these sort of -- what I think are basic standards that we utilize, are they being also put in potential standards and considered in the individual cases?
		DR. KRAUSE:  I agree with you completely that they should be, and that, in many cases, of course, you can't address those kinds of questions directly.  On the other hand, many of those kinds of research questions can be addressed in animal models.
		And I guess, again, that's one of the reasons that we're here today is to get an assessment from you, as the Advisory Committee, about what kinds of information one really should have in order to proceed, from my perspective, not only with tumor cells or with neoplastic cells, but also with other novel cell substrates.
		But, in any event, since neoplastic cells are the focus of the discussion today, to make that big step.
		DR. HALL:  The assumption is they're worse.
		CHAIRPERSON FERRIERI:  We won't preempt that part of the morning program that Dr. Lewis will be presenting to us, so we will confine the questions and comments then to information.
		Dr. Snider.
		DR. SNIDER:  Thank you.
		I add my comments of congratulations and appreciation for the quality of the presentations today.
		With regard to tumorigenicity, one thing that is not clear to me about what CBER proposes to do is whether investigating the mechanism would be a part of what CBER would propose to do or would this be done elsewhere?
		It seemed to me, when the issue was discussed, that it does make a -- it may, at least, in a lot of cases, be important to know the mechanism in order to perform the appropriate kinds of tests.
		So what is CBER's plans in that regard?  What would you like to do?
		DR. KRAUSE:  My own bias is that, at least at first blush, attempting to assess the relative risk of one cell versus another, that if you understand at least something about the mechanism by which a cell line became immortal for one cell and you understand nothing about it for another cell, there's some solace to be taken in that knowledge in that there might be a lower relative risk for the cell lines for which you have that understanding.
		That may not always be true and, in many cases, that kind of understanding is illusory.  I think that, you know, understanding at a basic level the mechanisms by which cells become tumorigenic or oncogenic is probably beyond the scope of the kinds of things that CBER or perhaps even the NIH can hope to understand in a very short period of time.
		But I think that CBER will need to call in all appropriate outside expertise on those kinds of issues and will need to take that kind of expertise -- those experiments into account.  And it may well be that, after doing that, experiments that should be done at CBER will suggest themselves.
		CHAIRPERSON FERRIERI:  Dr. Lewis, would you like to add to that response?  No.
		Another point, Dr. Snider?
		DR. SNIDER:  Well, I just wanted to follow up and say that I was thinking that, you know, this would be a role for NIH and for academia.  And therefore, if that's the case, it seems to me CBER might consider having another meeting to try to foster the kind of research that is going to be necessary to discover these mechanisms.
		DR. KRAUSE:  I'm hoping, and I think Dr. Lewis is hoping as well, that that would be one of the outcomes of the public discussion of these issues.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Krause.
		Dr. Kohl had a question for Dr. Sheets.
		Do you still -- would you like to raise that, Dr. Sheets?
		DR. KOHL:  Thank you.
		Dr. Sheets, we talked at the break.  We kind of have breezed by the issue of protein problems and, in particular, the question of prions.  And I wondered if you could address that.
		DR. SHEETS:  Yes, this is Dr. Sheets.
		Is this on?  Can you hear me?  Okay.
		I think that we are certainly not ignoring the issue.  This would be an issue possibly for all kinds of cell substrates because the normal gene is present.  However, it is possible, in some of these more novel substrates, that mutations could have occurred.
		It's problematic to assess this risk.  There aren't really good assays that are commercially available or validated for assessing products.  But it's certainly not an issue that we're ignoring.
		I didn't want to go into a lot of detail about it in my presentation because it is such a problematic issue.  So Dr. Lewis did include briefly that this is something we're cognizant of.
		CHAIRPERSON FERRIERI:  Any other questions for Dr. Sheets?
		Questions for Dr. Clouse, the presentation on the therapeutics, experience with therapeutics?
		Yes, Dr. Oxman.
		DR. OXMAN:  I just have a comment and a question for Phil Krause following his very excellent presentation.
		I've been impressed that we're getting very good at looking for agents that we can -- that we know about, but I'm obviously concerned about the agents that we don't know about.  And you commented on the use of degenerate primers as one approach to using PCR for finding things we don't know the name of or the gene structure of yet.
		I wonder if you could expand on that a little bit.
		DR. KRAUSE:  Well, obviously the use of degenerate primers is only going to be useful for identifying unknown agents that we know relatives of.  		But if you take a family of viruses and can identify conserved sequences within that family, and often that turns out to be within the DNA polymerase chain, which seemed to be among the best conserved sequences in DNA viruses at least -- and, of course, RNA viruses don't have DNA polymerases.
		But I think that those kinds of primer pairs that can detect members of a class of viruses and thereby extend our ability to detect new members that are somewhat related to, but different from, all viruses could be extended.
		The example that I gave in the talk was that of herpes viruses where each of the last few years, when I've gone to the International Herpes Virus Workshop, a new herpes virus has been discovered in some animal using precisely this strategy of using these -- of first suspecting its existence, and finding an appropriate sample.
		And second, then using degenerate primers that have been selected based on careful sequence analysis of all of the known sequences of herpes viruses, and then identifying them, and then sequencing the span between the primers and find that, in fact, it's different from any known herpes virus.
		And then once one has a probe, you can go on and do more work.
		DR. OXMAN:  That's a very fine and powerful approach to members of families we know about.  And I just think this is one area that we need to expand on a little bit, and that is the agents that we don't know about yet.
		DR. KRAUSE:  Right.  The two other types of experiments that I described that I think have some chance of identifying agents that we don't know about, one of them relates to completely non-specifically amplifying nuclease resistent nucleic acids.
		Most viruses that -- have protein capsids that will protect the viral nucleic acid from nuclease, RNAse or DNAse digestion.  
		And the PCR methods that are now available enable you to non-specifically, using random primers or selected random primers or other similar such methods, non-specifically amplify DNA that's in a tube.
		And we all know that, of course, also from the O.J. Simpson case.
		And so the idea of taking a cell substrate or a sample in which one suspects that there's an adventitious virus and then first completely nucleasing it, getting rid of all the free DNA and RNA, then perhaps ultracentrafuging it to purify encapsidated nucleic acids, and then releasing those nucleic acids and non-specifically amplifying them may provide the way of identifying those kinds of things.
		The other method, which is somewhat more complicated, that I alluded to is attempting to use subtractive techniques to find new viruses.  
		And again, since I'm a herpes virologist, my greatest experience in this comes from the herpes virus field, but the Karposi's sarcoma herpes virus was discovered using a very powerful PCR-based subtractive technique called representational difference analysis.
		And so the key there is finding a sample that's worth subtracting from another in order to see what's left over.  And one approach might be to -- if one is attempting to detect non-cytopathogenic viruses, to inoculate a sample onto tissue culture and then subtract early time points from later time points to see whether any nucleic acids have amplified over time.
		Or, alternatively, if one goes back to the nuclease resistent nucleic acid concept and subtracts away the vaccine strain or something, then we might be able to find something else that's left over.
		So these are general concepts that I think might be used.  But I agree with you completely that more research in this area is potentially very helpful.
		CHAIRPERSON FERRIERI:  Thank you, Phil.
		We have a question from Dr. Brieman.
		DR. BREIMAN:  And I think this question is for Dr. Sheets, but I'm not totally sure.
		In that wonderfully colorful piece that you gave us from Maurice Hillemann, which was from 1978 and is an argument in favor of production of biologics in cancer cells, he seems to make the point there that the primary purpose for moving ahead with these sorts of cell lines is really for developing subunit vaccines.
		And, in fact, he makes the comment that "there is, to my knowledge, no present important live virus vaccine need for which primary cells or diploid cell strains will not provide an acceptable answer."
		The impression that I'm getting from the earlier discussion is that one of the driving forces for this is the ability to produce HIV vaccines and make them affordable, you know, for use in developing countries and so forth.
		So is this statement no longer applicable; that one would have to move to these new sorts of cell lines to develop, say, live vaccines?  
		I guess the other issue is that he goes on in this piece to talk about dragon killing and kind of -- it colorfully again talks about the -- you know, what sorts of things one could then do if you were producing a subunit vaccine, which includes all sorts of inactivation measures, which again wouldn't apply to, you know, a live virus vaccine.
		I'm just wondering what's changed since this?
		DR. SHEETS:  Well, certainly Dr. Hillemann is quite a colorful character.  
		I think that it is true that early uses of some of these types of novel substrates were for recombinant purposes.  Certainly that's why the therapeutics field is much further ahead than we are in considering the risk benefits for such things.
		However, there are becoming more and more increasing uses which I can't tell you all of the specific indications, but certainly people are considering using -- I believe it's in the literature -- the Madin-Darby canine kidney cells, which is a continuous cell line, for influenza.
		Certainly influenza is grown in eggs.  But if we want to be able to break free of the restrictions of -- for influenza vaccination production -- vaccine production, which is very complicated timing-wise -- you have to get the hens produced.
		Then the hens have to be producing the eggs.  Then you have to have each batch of eggs come in so that you can produce a lot of vaccine.  So all the timing is very critical.  A lot of that would be alleviated with use of continuous cell line.
		So that's one purpose.  The HIV example that I gave you, if you want to grow live HIV for further inactivation or for a live attenuated vaccine, it's most likely going to be viable to do so in a tumor derived cell line.
		Although it can be grown in primary cells, it's unlikely that commercial scale yield would be able to be achieved, and lot by lot testing for adventitious agents would be a huge burden.
		So I think there are multiple examples.  And we are certainly being approached all the time.  We have several applications for things, and also sponsors who approach us before they submit an application for advice.
		So yes, this is changing.  More and more novel approaches are being tried.
		CHAIRPERSON FERRIERI:  Thank you, Becky.
		Dr. Greenberg.
		This is our last question, and then I'm going to turn it back to Andy Lewis to present the focused issues for discussion.
		DR. GREENBERG:  This is basically a comment and a suggestion based on what Mike Oxman mentioned.
		I also am worried about the agents that can't be -- that aren't known.  And I would simply say that the Defense Department and DARPA is investing really very large amounts of monies for other reasons in finding agents that aren't known, and that there might be some partnering that FDA could use for some of this very new technology to, in this case, look for agents in these biologics, especially using micro arrays and things like that for looking for signature transcriptional responses in cells.
		So maybe it would be good for somebody to talk to DARPA.
		CHAIRPERSON FERRIERI:  Thank you, Harry.
		We will flip that into one of the two key issues.  That's a very important item.  We can amplify on that.
		Andy.
		The rest of the hour now will be devoted to these two focused items.
		DR. LEWIS:  We thank you for your attention this morning.
		In the presentations that we've made before the Committee, we've tried to outline the manner in which living tissues needed for vaccine manufacture have been selected over the past 60 years.
		Through the development of the polio vaccine, the selection process was serendipitous and opportunistic.  
		Following the discovery of SV40 in rhesus monkey cells that were being used for vaccine production in the 1950s and the development of the human diploid cell strains in 1961, the selection process shifted into a more deliberative phase that we're acknowledging today.
		As a result of this process, only six types of cell cultures from the eukaryotic species have been selected as substrates suitable for vaccine manufacture.
		Due to technological advances and new disease causing microbes, once again it's necessary to approach the possibility of expanding types of tissues that can be used for vaccine production.
		And once again, the question is, how to proceed?  
		In a first attempt to address this question this morning, we've shared with the Committee our initial thoughts on how we might go forward.  At this stage of the process, we think it's premature to ask the Committee to address questions regarding the use of specific neoplastic cell lines as vaccine substrates.
		We thought it better to ask the Committee to comment generally on the issues regarding the use of neoplastic cells as vaccine substrates, and, within the context of these deliberations, to address the two items that I presented in my third slide this morning and that I'm going to present again on the next slide.
		So in this regard, we ask the Committee to please comment on CBER's concept and approach to evaluate neoplastic cell lines that are proposed for use in vaccine manufacturing, and then consider any additional items related to today's presentations or to issues related to the use of vaccines manufactured in neoplastic cells that the Committee finds appropriate.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Lewis.
		Well, let's open it up and address the first question, and that is the general concept of the use of neoplastic cell lines.
		Who would like to lead off?  Any volunteers?
		Great, Dr. Kim.
		DR. KIM:  Yes, this is Kwang Sik Kim.
		My concern is that I don't think we understand clearly the basis of tumorigenicity.  And without having a complete information and knowledge on these issues, it will be very difficult to speculate what is going to happen.
		Good example is this morning we heard that RT activity has been detected in chicken derived cell lines.  And, you know, I think that a similar kind of findings will be obtained from other cell lines, including tumor derived cell lines, and I don't know what to do with that information.
		CHAIRPERSON FERRIERI:  Well, that occupied several of our Committee meetings prior to your joining us, Kwang Sik, so it threw many people into a turmoil.
		Any amplification on that point, those who might share this concern at the table?
		Yes, please, Dr. Folks.
		DR. FOLKS:  Tom Folks.
		That's occupied a lot of our time at CDC as well.  And just to make a comment, and maybe it's moving more to point two, but clearly I think that when you introduce or you find new agents associated with anything, or you iatrogenically attempt to change something -- and I'll use xenotransplantation as an example where we're heavily invested in that as well in looking for the transmission of something new or unknown, how do you go about knowing where you're stepping?
		And about the only thing -- or one of the things you can do is surveillance, clinical surveillance.  And I haven't heard a lot about that.  And again, I think our purpose of this meeting is to talk about substrates.  
		But clearly, as we move beyond substrates and we start to think about application of the substrates into humans, we want to be sure that there is some type of random -- limited random surveillance, clinical surveillance, that follows anything like this.
		And we've been surveying the reverse transcriptase activity, associated type activity, that children have been receiving over a number of years with the measles, mumps, rubella vaccine that you're alluding to.
		And clearly, there are no sequences, no avian leukosis virus, or EAV type sequences that we can find in children post this type of vaccination.  Likewise, we're developing other surveillance tools like Western Blot in serology to survey children, and again we find no real conversion of children to these chicken viruses.
		So, you know, the concern is real.  And whenever you find something new -- and assuredly, as Dr. Krause alluded to with representational difference analysis, this is a powerful, powerful tool to find new things that are going to appear in all types of substrates in vaccines that we're currently using.
		We have to be cautious as to what they really mean, and surveillance may be one of the only ways we can do it.
		CHAIRPERSON FERRIERI:  Let me refocus the first question though, Dr. Folks.  Would you care to comment on CBER's current approach to evaluating these neoplastic cell lines?  As an opener, is it reasonable?  Is it, at the moment, based on the technologies you're familiar with, sufficiently comprehensive?
		DR. FOLKS:  Highly reasonable.
		CHAIRPERSON FERRIERI:  Highly reasonable?  I hope some of you will forgive me for calling upon you, but I think of you as our experts for the Committee.
		Dr. Wold, would you care to address this point of what your opinion is of CBER's current approach to the evaluation of these cell lines?
		DR. WOLD:  Wold or Wolfe?  Bill Wold.
		The question has been raised -- if I could make a comment first -- as to why bother to consider cancer cell lines and other cell lines when there are lines that have been worked -- that have worked well in the past.
		There are a number of reasons.  First, many viruses don't grow well and cannot be genetically manipulated in the WI38s and cells of that nature.  Secondly, the new technology available has created many new opportunities to develop genetically engineered vaccines and recombinant vaccines.
		And those kinds of genetic manipulations have to be done in cells that are easily studied in the laboratory.
		And thirdly, I think if you considered using other types of cell lines, you would engage in an entirely new community of researchers interested in vaccine development and biological development.
		For example, academia, many of whom don't want to become involved in this kind of research because they don't have the resources, and it's just too much trouble to get involved.
		So I think if it were easier to do the kinds of experiments that we can do now and we'll be able to do in the future, it would be better for everybody.
		To comment specifically on the point, I think the experiments outlined are very reasonable.  They're really taking the worst case scenario for, you know, contamination of DNA, protein when there isn't a great deal of evidence that there's a lot to be concerned about with any of these particular concerns.
		But, nevertheless, the kinds of experiments that are proposed should be done, and I think they're reasonable.
		CHAIRPERSON FERRIERI:  Dr. Blair, do you have an opinion?  Would you share your opinion with us, please?  I'm sure you must have an opinion.
		DR. BLAIR:  No, I think the -- there clearly, in some cases, may be advantages to some of the neoplastic cells in the sense that one can clone them and characterize them to a much greater extent than you can the continuous cell lines or primary cells.
		And I think that you -- that the approaches that were described to test for those things we know about and to test for -- you know, for the limits of our detectability of some of those things are the kinds of -- are the approaches that you have to do.
		I'm not sure how to test for those things we don't know about and whether we already know examples of everything that's out there such that, if it were in one of our established lines, we would have noticed it by now.
		I don't know, but I think the approach to establish the minimal limits and the limits of how much of any potentially hazardous or potentially hazardous material might be present in the output of some of these established lines should -- is certainly a worthwhile approach.
		I don't know whether there's the equivalent of an RFP within the FDA to get some of this work done on the outside, but that's certainly the kind of an approach that might get it done.
		CHAIRPERSON FERRIERI:  Thank you.
		Dr. Wolfe.
		DR. WOLFE:  Assuming that the point that Dr. Snider raised, which is, in every instance that there's been an application, now or in the future, clearly the only way to go is with human or animal tumor cell lines, which may or may not be the case.
		It may be that, in some cases, that's the way to go.  And other places, for economic or reasons that aren't really that health related, the company has chosen to go that way.  
		But assuming that, at least in some of these instances, we would all agree that the way to go is to use human or animal tumor cell lines, it seems that the very careful approach both at the research level for developing the methodology, and then, when and if these are considered for human trials, the research to check out from the Government's standpoint, is very well thought out, but it is extremely resource intensive.
		And so I don't think we can answer the first question without assurance because we're being asked -- or the people on the Committee permanently -- I'm just here intermittently, I guess -- are being asked is this okay without guarantees that there will be the resources.
		It seems that it would be better to have the resources used in house -- it may be necessary to augment that with RFPs or maybe convincing pieces of the DOD or NIH, with its now $13 billion dollar budget, to be interested in it.
		But I think we have to get some assurance that there will be the resources.  Otherwise, nice idea, but no implementation could be really dangerous because it would -- going back to Dr. Krause's point, these things are too important to be left to anything other than public and publicly accountable institutions.
		And if the budgets aren't there for that, they will be left to private institutions, and I would not have the trust in those institutions to do the very thoughtful kind of processes being described there.
		CHAIRPERSON FERRIERI:  Oh, I agree with you completely and think that we need considerable public advocacy to see that the resources are available.  These are very vital.
		I'm targeting Dr. Hughes.  I wonder if you'd like -- with your position as Head of the Molecular Carcinogenesis Laboratory, if you would like to add your perspective to question one.
		DR. HUGHES:  I'm pleased certainly by what I've heard this morning.  I think the approach in general is thoughtful.
		CHAIRPERSON FERRIERI:  Is your microphone working, Dr. Hughes?
		DR. HUGHES:  I don't know how to comment on that.
		(Laughter.)
		CHAIRPERSON FERRIERI:  We'll try to fix it.
		DR. HUGHES:  I think the first thing though that needs to be kept very firmly in mind if one is considering neoplastic -- using neoplastic cells is not the risk in the absolute sense, but, in effect, the comparative risk relative to normal cells.
		And the reason for pointing that out is that, someone who fiddles around with retroviruses in one form or another, I think it's only fair to point out -- and I think the experience with the avian system that we heard something about this morning is illustrative -- that you are not going to be able to avoid, even with normal cells, some potential risk.
		I think if you're contemplating using neoplastic cells, and I think that's a reasonable thing to contemplate, one needs to think very carefully, wherever possible, about the route by which those cells became transformed.
		And I think it's important to think differently about virally transformed cells and cells that are transformed by some relatively well understood bits of DNA damage, loss of suppressor genes or activation of some endogenous oncogene like NIC.
		And I think, as was outlined, and I think nicely, one needs to think about the relative risks in any derived agent-reagent, whether it comes from normal cells or transformed cells, at the protein DNA and viral level.
		And certainly my prejudice, which I think reflects what was discussed this morning, is that the risk of protein is relatively modest.  The risk of DNA is less well understood, but probably also relatively modest if one excludes virally transformed cells.
		And I don't think anyone is really seriously contemplating using those.
		And the real problem is viral.  But I would point out, and I think this echoes a bit what Don Blair said, it may, in some ways, be easier to assess the contamination and the risk of viral contamination with a well characterized permanent cell line than it is with normal cells.
		And I certainly think that one needs to be cautious in detecting "unknown agents."  But that, I point out, is at least as big a problem, if not a bigger problem, if one is getting, you know, eggs or tissues from here, there and everywhere.
		It's an unavoidable issue.  And I think the final point, which I don't believe was discussed, is that maybe some thought should be given, since there was discussion of HIV, to differentiating agents or reagents that are intended for use in immunocompromised individuals and those that are not.
		CHAIRPERSON FERRIERI:  Thank you.
		Back to Dr. Wolfe and then Dr. Johnston.
		DR. WOLFE:  Yeah, a quick comment, which was I think that your statement that you don't think anyone is seriously considering using virally transformed cells is not correct.  
		I think that one of the -- from the discussion, as I have read it this morning, that that is one of the things that's being considered.
		And, for the same reason as you, I would be concerned about that.
		DR. HUGHES:  Well, I think it's very important to distinguish when one uses the term virally transformed.  I would not call 293 virally transformed.
		DR. WOLFE:  You wouldn't?
		DR. HUGHES:  That is a virus that is transformed by a modest component from adenovirus.
		DR. WOLFE:  Okay, so you --
		DR. HUGHES:  And I think that there's really no reason to expect that if you -- that you could resurrect an infectious agent out of a 293 cell since the 293 cell does not contain the vast majority of adenoviral sequences.
		And I think that one could easily argue that the only way that you're going to recover something like an infectious adenovirus is to add an infectious adenovirus, and I think that's a different sort of situation which one should guard carefully against.
		DR. WOLFE:  I thought you were talking about the --
		CHAIRPERSON FERRIERI:  Thank you, Dr. Hughes.
		Dr. Sheets, do you want to add to this discussion?  And then we'll go back to Dr. Johnston.
		DR. SHEETS:  I did want to clarify.  Certainly there are people considering 293 cells for the production of adenovirus recombinants.  
		The other thing is there are people considering use of HeLa cells, which we're -- we are presuming, because this is cervical cancer and there is human papilloma virus Type 16 in there, that that was the -- one of the transformation events.
		So you shouldn't discount that these aren't being considered.  
		Now, I can't comment on what the products are or whether -- what the risk-benefit ratio would be, how FDA is viewing such applications or potential applications, but it isn't a truth to think that no one out there is considering them.  They certainly are.
		CHAIRPERSON FERRIERI:  Dr. Hughes.
		DR. HUGHES:  It is -- you may know more about this than I.  When you speak of HPV being present, are those -- how well characterized are the genomic elements in HeLa?  Are they intact?  Do we have whole viral information?  Do we know?
		CHAIRPERSON FERRIERI:  Dr. Krause.
		DR. KRAUSE:  You know, the trouble with studying HPV is the lack of good tissue culture models in figuring that kind of stuff out, but there are about 50 copies of the HPV genome in a HeLa cell.  		And, to the extent that has been looked, it appears as though all the components are there.  Whether they're completely normal HPV Type 16 or there's something wrong with them, I'm not sure has been looked at.
		But they appear normal based on the limited studies that have been done.
		CHAIRPERSON FERRIERI:  Dr. Johnston, thanks for your patience.
		DR. JOHNSTON:  Yes, if I may, I'd like to have a question answered and then provide a comment.  And this is a question to Andy Lewis.
		Andy, you presented earlier a very well thought out plan for developing a policy here at CBER, and I'm wondering if there's been given any thought to the time line that it will take to accomplish that plan?
		DR. LEWIS:  There's been some consideration of that.  I think that the understanding that I have right now is that there may be resources available to begin with an international workshop on cell substrates sometime between now and next October, probably late in the summer or September.
		In terms of developing a working document within the organization, I think we have outlined for you what the major components of that document might be.  It's not in hand at this point in time, and there's really no serious discussion as to a time frame as when that could be available.
		But, realistically, I think we could probably have something within six months or so, if that were the way we would go.  Beyond that, I don't think we have any perception as to a time line.
		DR. JOHNSTON:  Okay, thank you.
		My view is that neoplastic cell lines definitely need to be considered aggressively, particularly for situations where there are no alternatives.  And I think that the methods, the plan, the approach that has been outlined today is highly reasonable.  
		The resource issues are probably not insignificant; however, I think in general, when one is talking about prevention, we tend to undervalue those technologies, and perhaps this is one area where that undervaluing needs to change and that resources need to be made available to accomplish these tests in an expeditious manner.
		Now, whether or not one can rely on NIH, I think, is questionable.   There is a new vaccine study section, but the degree to which it will look favorably upon very applied applications is yet to be determined because there's just no track record there.
		So I would caution FDA to first look within before looking without.
		Now, my concern is actually in contrast to that raised earlier by Dr. Wolfe, and it gets back to a comment made by Dr. Daum earlier, and that is one of risk-benefit ratio.  Risk will never be nothing.  We all know that.  
		And my concern is not so much that FDA will be precipitous, but that it may be too slow.  And I guess one thing I'm uncertain about is where in the equation the risk-benefit discussion will take place.
		Will this be at a product by product level?  And what will happen in cases -- and I'll use the case of HIV vaccine since it was raised -- where the risk-benefit ratio itself will probably be dominated by issues other than cell substrate?
		And that is the safety of the product itself, as well as the huge potential benefit that it may have.  
		So that is one example where there may not be alternatives to other cell substrates, where there could be substantial risk issues involved in the product itself, where there could be an enormous benefit.
		And I guess my concern is I would hate to see things not progress because cell substrate issues have not yet been addressed and a policy has not yet been -- you know, something that would be widely applicable to everything has been formalized.
		CHAIRPERSON FERRIERI:  Well, that's very well articulated.  Before we take a question from Dr. Nathanson, I wonder if Dr. Hardegree or a member from the Agency would like to respond to Dr. Johnston's point?
		DR. HARDEGREE:  Well, I think that you've heard before that we probably have to consider benefit-risk on a product by product category.  And so that, as people are bringing us specific problems and issues, each of those are being considered.
		And I think you also heard Andy say this morning that, as we have a specific product that we need to bring to this Committee, that we can do that in a way that we may need to address.  
		But it's time to get the discussion going about what the issues are, and so that everybody can be thinking about these together as opposed to a commercial sponsor alone thinking about it with FDA.
		CHAIRPERSON FERRIERI:  Thank you, Carolyn.
		Dr. Nathanson, I was going to call upon you, but you've volunteered.
		DR. NATHANSON:  I just want to emphasize what several people have said, Peggy Johnston and also Sid Wolfe, about -- number one, I would hope -- the world desperately needs an AIDS vaccine.  
		I would certainly hope that you would put some teeth into the response to this, number one, by including some statements about a time line and about resources.  And I certainly don't think it should be left to RFPs and grant applications.  
		This is something that is an applied problem, and there should be money that is specifically put there and a very directed program to accomplish this.  And I think that could be a very useful recommendation, so I just wanted to emphasize that.
		CHAIRPERSON FERRIERI:  Well, thank you.  I think this is just what Dr. Lewis and other members need to hear from us, and the recordings here are taking this all in.
		Yes, Dr. Edwards.
		I need to tell you, Kathy, when you called me the other day, my secretary wrote it down as Dr. Edwards Vanderbilt.  And I said I don't know any Dr. Edward Vanderbilt, but then I figured it out.
		Kathy.
		DR. EDWARDS:  Thank you.
		Well, first of all, I'm very impressed with the FDA presentation.  And it's been certainly lucid and has clearly shown the quality of the science that exists at the FDA in spite of tremendous financial constraints.
		And I think that that really is a tribute to the productivity and the intellect and hard work of people.  And certainly that funding does have to be enhanced in this area because clearly this is a mission of CBER and it's very, very important.
		Every day I labor in the trenches of trying to convince parents that vaccines are safe, and obviously that is becoming more and more difficult to do.  
		So that I think it is really imperative that, from this meeting, there is a public sentiment and there is real concerted effort to get the funds to the FDA to continue this important work to assure that vaccines are safe.
		And so that I really think it's a very important practical issue that must be done.
		CHAIRPERSON FERRIERI:  Thanks, Kathy.
		Dr. Snider and then Dr. Wolfe.  
		No?  Okay.
		Dixie.
		DR. SNIDER:  Several comments.  I guess, in summarizing my advice to the FDA, I would say that I think that the emphasis that they placed -- where they placed the emphasis today is understandable, but there are things on the front end and on the back end that need to be looked at just as carefully, need to be discussed publicly.
		And, for example, the whole business about justifying using certain cell lines, I personally am in favor of it, but the point is that there needs to be some discussion around the alternatives, as I said, for traditional substrates as opposed to novel substrates and the whole risk-benefit thing we've been talking about, you know, compared to what alternatives.
		And it seems to me that really needs to take place early on, especially from a manufacturer's point of view, before there is too much investment in a particular approach.  And so I think that needs to be given more consideration.
		And then, to just echo again what I said earlier and what Tom has said, I think on the back end -- I mean, no matter what we do in terms of monitoring production and monitoring safety during the early phases of development, there are going to -- new issues are going to come up or old issues are going to hang on.
		And so there is the issue of monitoring folks during the randomized trials and during post marketing surveillance.  And it raises all the issues again of what mechanisms are you going to use to monitor people who have been recipients of these vaccines.
		And I think the public's going to want that kind of an assurance.  And it's not too early to begin to think about some paradigm for that, as well as a paradigm for, you know, checking out whether certain proteins or viruses or cells or DNA slips through in a manufacturing process.
		CHAIRPERSON FERRIERI:  Thanks, Dixie.
		DR. SNIDER:  And finally, the tumorigenicity thing I want to bring up again because -- on the record, that it's not just NIAID, it's NCI.  But somehow, I think it's important to understand mechanisms and mobilize the research community around conducting some studies to find out mechanisms.
		I think it's going to be a challenge.  And maybe NVPO can help, maybe others can help, but it's important to engage on this issue.
		CHAIRPERSON FERRIERI:  Thanks.  That overlaps very much into the second question, and I'm grateful for your doing that.  A couple other points.  First Dr. Huang, and then Caroline Hall, and then Dr. Daum.
		DR. HUANG:  When I look at CBER's approach to evaluating neoplastic cell lines, I basically see two major worries.  One is tumorigenicity and the other one is adventitious agents.  
		I'm quite comfortable with the first approach and the worry about tumorigenicity.  I think that, in fact, some of the tests may be overkill.  This is in particular when we begin to think about more recent knowledge of how cancer cells are actually formed.
		It's a progression, multi-step progression.  
		And I believe that one of the papers that we got earlier to read really did mention this.  And so, on that particular line, if we see oncogenes or sequences of oncogenes or partial oncogenes around, I'm not terribly worried about that aspect.
		However, with adventitious agents, I would just like to add my bit to the worry about those agents that might cause slow viral disease or neurological disease that we don't know a whole lot about.
		And more specifically, I think that, of the cell lines that were used to test for adventitious agents, I would suggest that one of the cell lines that be used and which is used regularly in diagnostic laboratories is the 293 cell line which will pick up many humantrophic agents that other cells may not.
		So that's a very specific sort of suggestion.
		CHAIRPERSON FERRIERI:  Thank you, Alice.
		Dr. Hall.
		DR. HALL:  I just want wondered if Dr. Lewis could explain or clarify a little more about the document that will be available in about six months.
		You said the time line.  What this will contain or how it will be utilized.  Is this something that would be given to a potential manufacturer and then the burden is upon them to get these tests, to do these tests?  And it brings up the questions of standardization, etc.
		DR. LEWIS:  The perception that I would have of the document -- it might be ready in six months -- would be something that we'd be discussing internally and that we would be having other people look at in any type of forum that's necessary.
		And if we had and we organized a meeting, say, sometime in August or September, if that document were ready, certainly it could be a part of a session or part of a day or whatever for discussion at that meeting.
		I think to -- my perception would be that it would be premature to have this as a guidance document at this point in time at this stage of its development to be submitted to industry.  That would only be ready after these discussions, and perhaps even after more discussions.  
		Because once we get into this process, it may be somewhat open ended.  And how long it might take to come up with a guidance document I think might be unpredictable.  Certainly I don't think we would stop having to deal with these problems as they come up, and we'd just have to manage as best we can.
		But again, if the question is to when there may be a guidance document ready, I think it would not be ready in six months.  That would be my perception.  Dr. Eagan or Dr. Hardegree may have other things to say about that.
		CHAIRPERSON FERRIERI:  Thank you, Andy.
		Dr. Daum.  And appreciate, Bob, if you might segue into the second question.
		DR. DAUM:  Yeah, I think somewhere between the first and the second, but certainly there will be some second question comments.
		In sitting here sort of thinking about all of this, it seems to me that there's sort of three orbs or circles that are -- need to spin all at once to try and get arms around these issues.  And one is the -- what is the need to pursue this new technology?
		And we haven't heard as much about that as I would like partly for -- mainly proprietary reasons.  But we heard some of them in theoretical context.  
		But I think the need is a crucial thing because I think there's an education process here that's got to go on to sell the scientific community and the public beyond about the need for new technologies.
		Comes with the need for new technologies is careful monitoring strategies, sort of sphere number two.  And I've been very impressed this morning at the sincerity, at the thoroughness and at the scientific integrity of plans to monitor these new technologies as they go forward with everything that's known about what to monitor.
		But I think that there's also another sphere sort of orbiting around here which we're all trying to come to grips with somehow, is that comes with any new technology risks that we don't know much about at this point.
		And I think that for people to accept these risks and believe that they're important to take is going to again come around to the education process about what is the need and what is driving pursuit of these new ideas.
		And so I would like to add something that I haven't heard much so far, is that there needs to be some kind of education process of the scientific community and perhaps of the public beyond about the need for new technologies and new ideas and what's being done to monitor them carefully.
		So, to me these three things are sort of spinning around at once.  And the vast majority of what we've heard and are able to comment on is what is CBER's approach to new technologies with existing ideas and looking for existing transmissions, for instance, of infectious agents.
		And there's a couple of areas that they're, by virtue of the beast, unable to comment on.  And I think education and acceptance by us, that the risks may be worth it and let's go forward together with our eyes wide open has to be the approach.
		CHAIRPERSON FERRIERI:  Thanks, Bob.
		Dr. Oxman and then Dr. Eickhoff.
		DR. OXMAN:  Just to expand a little bit on a point that Dixie Snider mentioned a couple of minutes ago, most of what we've seen so far is an approach to dealing with a vaccine produced in a potentially neoplastic substrate and then trying to minimize the risk.	
		One of the best ways of minimizing the risk is to start off by choosing the better of two or three or ten substrates and how to do that.  And I think more effort needs to be put into that.  
		I realize it's difficult, because of the position of the FDA, to dictate what substrates people should use.  I've been around long enough to see that many substrates are chosen quite at random and serendipitously.
		And once an investment is made, often by accident, people are reluctant to change the substrates.  There are -- neoplastic cell substrates cover a very broad territory.  
		And the risks involved in manipulating an already well characterized transformed cell, for instance, by inserting a gene that would express CD4 on its surface to make it suitable as a substrate for HIV, this would be a much lower risk and an easier thing to deal with in terms of guaranteeing safety than an uncharacterized or a much less characterized neoplastic cell line.
		So I think emphasis on the choice of substrates prospectively would be very useful.
		CHAIRPERSON FERRIERI:  Thank you.
		Dr. Eickhoff.
		DR. EICKHOFF:  Thanks.
		I'd like to thank the presenters this morning, as others have already done, for really a very elegant series of discussions of the current state of using neoplastic cell lines for vaccine production.
		I think it is a -- as others have said also, it's a very reasonable approach.  And I think also it's a very necessary approach because one gets the impression, at least I did, that this whole approach is really a bit of a freight train that is pounding down on CBER and we better start learning how to deal with it now rather than waiting for some product license application that uses this technology and then suddenly there will be a real problem on our hands.
		The process that is proposed -- as I said, it's a very reasonable one.  I think it should -- obviously the science is going to be the determinative discussion.  But it should also be a very public process, I think, and I would really like to emphasize that.
		Not only because public advocacy is probably going to be necessary to provide the necessary resources, which are uncertain at best, but also because -- and here I'd like to take off on what Dr. Daum said because I think this is terribly important.
		There's going to have to be an enormous amount of public education.  The public today is a very different public than it was 30 years ago when polio vaccine came on the market and the response was, "Does it work?  Here, give it to me."
		There were no concerns about safety for obvious reasons, and today there are.  The public, for a whole variety of reasons which we don't need to go into now, has become much more distrustful of the vaccine enterprise, if you will.
		At least many segments of the public are. And when they hear about vaccines produced in neoplastic cells, that will set some people's teeth on edge.  And so I think the public education process is going to have to be enormously important.
		CHAIRPERSON FERRIERI:  Thank you, Ted.
		I was volunteered as a youngster for the early Salk trials, so I feel I'm very sensitive to these issues.  I undoubtedly have a little SV40 around and I don't worry about it at night, but I do care a lot about what we're doing now and in the future certainly.
		Other comments?
		Yes, please.
		DR. SCHILD:  I feel very privileged to have been able to come to today's debate.  I think the FDA has done a superb job in presenting, with great clarity, the complexities and urgency of these issues.  And I think their proposals are warmly received.
		One thing is key, and that is the need for more regulatory research.  And I think it's absolutely right that Government funded bodies have the major role in this field of research.  Industry would like to do more, but I think getting the new products out is a major priority.
		These issues are of global significance.  And there is -- I'm sure there is good spirit of global cooperation.  I think the various regulator authorities in the world will work together on issues and share out the challenge.
		One other thing I'd like to mention is that all biological products of the type we've been discussing today are really very much the function -- their characteristics are very much a function of the cells in which they were produced.
		The measles vaccine made in chicken cells doesn't work in the same way exactly as measles vaccine made in human diploid cells.  Influenza virus grown in one substrate is different from that grown in another.
		And when we're considering the first part of this, evaluating in neoplastic cells, we have to take into account also the characteristics of the products produced in those cells and how cells affect those as part of that consideration.
		Thank you.
		CHAIRPERSON FERRIERI:  Thank you, Dr. Schild.  I'm so pleased that you could join us on the panel today. 
		Let's keep up this crescendo here.
		Dr. Greenberg.
		DR. GREENBERG:  Well, at the risk of being repetitive, I am in agreement with much that is said here.  But I think we have to remember that we, here on the Committee, have listened to, in my span on the Committee, about a year and a half of decreasing funding for any form of research, and we're hearing a very important need.
		And everything I have learned up to this point would say that the resources are not presently available to carry out this need.  And so, if there's any single message that I would like to give from this Committee -- to the Government from the Committee, is that those resources have to be available.
		Otherwise it's sort of silly to be asking us this question.
		CHAIRPERSON FERRIERI:  Well, I agree completely.  And I like to think that comments like yours and others will be picked up by the national media.  We're frequently bugged so much about the things we discuss here.  We can't control what the translation is.
		So, if you're out there in the audience, I hope you're hearing this, that you're quoting members who have said it.  This is the third or fourth time.  We are all very worried about the financial structure of CBER, other elements of FDA as well.
		And this is an example of the vitality, the critical need to back this up.  Otherwise we shouldn't be in the business, in my opinion, if we can't pursue this in depth. 
		And a great deal of money is wasted in Government for things that I am politically sensitive to not enumerating for you all today, but I think that the nation's health is the subject here that we're gripping with.
		Yes, Dr. Adimora.
		DR. ADIMORA:  I wanted to, very briefly, revisit the issue of the risks and benefits in various target populations, and just revisit the issue of what would be the arena in which these issues would be decided.
		Specifically, I think what I mean is that there are risks and benefits of vaccines that are manufactured in these continuous cell lines, and the extent to which these risks and benefits change -- the extent to which these risks and benefits change certainly varies with the anticipated target population.
		For example, in HIV, if one thinks about the critical need for an HIV vaccine, there are clearly very different risks and benefits associated when you're thinking about a child growing up, say, in some part of Central Africa who might have, for example, a 50% chance of ultimately dying of HIV at some point in his or her life compared to the same risk of a child growing up in suburban America.  
		On the other hand, risks and benefits can certainly vary among inhabitants of even the same country -- obviously within different subpopulations of the same country.
		I'm normally very conservative by nature, but one concern that I have is that some of the -- I'm sort of uncertain how to say this, but I'm concerned that intense focus on -- and I'm not saying that this is not -- that this is unwarranted, but I think that I worry about the extent to which intense focus on very small risks in some populations may inhibit the development of vaccines in populations in which such vaccines could do immediate good to huge numbers of -- to people who are at incredibly high risk.
		And I'm not sure how to approach this issue or even who should approach it, but I just thought that it was a reasonable issue to throw out concerning your questions for number two.
		CHAIRPERSON FERRIERI:  I think that if Mary Lou had been with us, she would have been able to relate to that point and be a champion of what you have just said.
		Other points? 
		Dr. Lewis, have you heard enough today that will help you all?  We'll start the collection tin in a moment.
		(Laughter.)
		But I would like to be able to sum up very briefly.  I want to congratulate the panel, the permanent members of the Committee, and the wonderful guests we had today to make this very dynamic beyond my hopes and expectations, I might say.
		And so I hope that FDA has benefitted from it.  I think the critical things that we have heard today that relate to the current approach of evaluation include the reasonableness of the approach, how critical it is to take these approaches; and that, although some things may appear to be in excess, most of the approaches can be defended.
		The need for the financial resources is evidence.  We don't have confirmation or affirmation that those resources will be available, and we need to recruit public advocacy in the strongest way and their interest in education of the public to be on the same band wagon so that we're all together as this moves forward.
		We all have an investment in this.
		The other items that were brought up include a need to better understand the mechanisms of what we are dealing with and what is being studied, not just a simple pragmatic approach, and if the resources could accommodate pursuing that.
		And we can't be at all dependent that other elements of the Federal Government such as NIH will be able to pursue that, so we're going to have to be very creative in thinking of how we're going to understand this and support basic understanding of these lines.
		Alternatives have been suggested as well.  Although the focus has been neoplastic cell lines, that we consider other novel, creative substrates as we move forward.  Monitoring strategies of these new technologies have also been emphasized, and trying to move forward in a very prospective way in examining alternative choices.
		And the choice of substrate need not be confined to the historical past.  And although those who come to FDA already have chosen their cell substrate, it doesn't mean that the word can't get out from now on that don't be confined and fettered by the past.
		We look forward and throw back to FDA the need to see a document that you would prepare internally, and would encourage its review at a special session, for example, of the workshop that is being proposed by early fall.
		I think that sums everything up, but I think that the inclusion of the public in this process is very vital.  And I encourage whatever mechanisms we might have of public relations, other ways of disseminating information that can be understood by everyone for people who do not do molecular biology by day and night.
		So, I'd like us to break for lunch.  And those of us who are included in the closed session at 1:30, we will start promptly.
		Thank you all very much.
		(Whereupon, the proceedings were adjourned at 12:26 p.m.)