DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

XENOTRANSPLANTATION SUBCOMMITTEE

Thursday, June 3, 1999

8:30 a.m.

Holiday Inn
Bethesda, Maryland

PARTICIPANTS

Hugh Auchincloss, Jr., M.D., Chairperson
Gail Dapolito,, Executive Secretary

MEMBERS

Prem S. Paul, D.V.M., Ph.D.
John M. Coffin, Ph.D.
Claudia A. Mickelson, Ph.D.
Jonathan S. Allan , D.V.M.
Martin S. Hirsch, M.D.
Richard Kaslow, M.D., M.P.H.
David Onions, BVSC, Ph.D., MRCVS, FRSE
Harold Y. Vanderpool, Ph.D., Th.M.
Daniel R. Salomon, M.D.
Leroy Walters, Ph.D.
Nicholas W. Lerche, D.V.M.
David Sachs, M.D.
Ms. Abbey S. Meyers

CONSULTANTS

E. Steve Woodle, M.D.
Mr. Antonio Benedi
William G. Lawrence, J.D.

GUESTS

John Conte, M.D.
Robert E. Michler, M.D.
Ralf R. Toenjes, Ph.D.
Marian Michaels, M.D., M.P.H.

CDC

Louisa E. Chapman, M.D.

NIH

Mary Groesch, Ph.D.

FDA
Louis Marzella, M.D., Ph.D.
Karen D. Weiss, M.D.
Carolyn Wilson, Ph.D.
Eda Bloom, Ph.D.
Philip D. Noguchi, M.D.
Jay P. Siegel, M.D.

C O N T E N T S

Welcome and Introductions, 	4

Conflict of Interest, Gail Dapolito,	7

Introduction, Jay P. Siegel, 	10

Topic I:

Porcine Endogenous Retrovirus Update,
   Carolyn Wilson, Ph.D.	12

Xenotransplantation: A Scientific Basis
   for Risk Assessment, John Coffin, Ph.D.	28

Guest Presentations:

In Vivo Expression of Porcine Endogenous
   Retrovirus in Pigs Analysis of Non‑Human
   Primates Transplanted with Porcine Organs
   for Evidence of Cross‑Species Transmission
   of PERV, Gillain Langford, Ph.D., Imutran, Ltd	51

HepatAssist Liver Support System Containing Porcine
  Hepatocytes Case Study: Bioreactor PERV Analysis and Risk
   Assessment, Zorina Pitkin, M.D., Circe Biomedical	61

Surveillance of PERV Infection in Exposed Persons,
   Walid Heneine, Ph.D. CDC	70

Porcine Endogenous Retrovirus Testing in Patients
   with Pig Fetal Neural Cell Transplants,
   Jonathan Dinsmore, Ph.D., Diacrin, Inc.	80

Development of Assays for Monitoring Baboons and
   Humans for the Transmission of Porcine Endogenous
   Retrovirus, John S. Logan, Ph.D., Nextran, Inc.	84

Retrospective Patient Study ‑ Testing Strategy and
   Methods, Khazal Paradis, MDCM, FRCP(C), Imutran, Ltd.	97

Questions from the Committee 	105

Guest Presentation:

Nipah Virus Outbreak, Malaysia
   Dr. Khabbaz, CDC	174

Committee Discussion 	193

II FDA Xenotransplantation Policy Development
   FDA Perspective
   Eda Bloom, , Ph.D., FDA	221

C O N T E N T S
PAGE

Guest Presentation:  Immunoisolation Technology
   Taylor Wang, Vanderbilt University	227

Open Public Hearing	243

FDA Perspective	247

Open Public Hearing	254

Committee Discussion	260

P R O C E E D I N G S
Welcome
MS. DAPOLITO:  I would to welcome the subcommittee participants and guests, and all here, to this meeting of the FDA Xenotransplantation Subcommittee. I am Gail Dapolito, and the Executive Secretary of the subcommittee, and a designated federal official for today's proceedings.
I would like to begin by stating today's meeting will be conducted as a subcommittee meeting of the Biological Response Modifiers Advisory Committee. As is evident, this is a subcommittee on xenotransplantation. This was listed in the Federal Register Notice.
There are two notable differences between a subcommittee meeting and a full advisory committee meeting. One is that there are only two standing advisory committee members present at the table ‑‑ one right now. They will be introduced to you in a moment. The other difference is the that report that will be furnished by the subcommittee following today's discussions will be approved by the parent committee, the Biological Response Modifiers Advisory Committee. Other than that, today's discussion will proceed as would a full advisory committee meeting.
At this time, I would like to introduce today's participants seated at the table. I will begin on my left: Dr. Prem Paul, Iowa State University; Dr. John Coffin, who will be joining us soon, Tufts University School of Medicine; Dr. John Conte, the Johns Hopkins Hospital; Mr. Antonio Benedi, Transplant Recipient International; Mr. William Lawrence, United Network for Organ Sharing. Mr. Benedi and Mr. Lawrence are participating today as patient representatives of the subcommittee. Dr. Robert Michler, Ohio State University Medical Center. Next we have Dr. Claudia Mickelson, Massachusetts Institute of Technology. Next to Dr. Mickelson is Dr. Ralf Toenjes, Paul Erlich Institute; Dr. Jonathan Allan, Southwest Foundation for Biomedical Research; Dr. Marian Michaels, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh. Going around the table, Dr. Martin Hirsch, Harvard Medical School, Massachusetts Medical Hospital; Dr. Richard Kaslow, University of Alabama at Birmingham; Dr. David Onions, University of Glasgow; Dr. Harold Vanderpool, University of Texas Medical Branch; Dr. Daniel Salomon, the Scripps Research Institute.  Dr. Salomon is a current member of the Biological Response Modifiers Advisory Committee.
Next to Dr. Salomon when he arrives; he is running a little late; Dr. Hugh Auchincloss, Harvard Medical School, Massachusetts General Hospital. Dr. Auchincloss is the chair of this subcommittee and a current member of the Biological Response Modifiers Advisory Committee and in his absence, until he arrives, Dr. Salomon will be participating as the chair of the subcommittee. Dr. Leroy Walters, Georgetown University, Kennedy Institute of Ethics; Dr. Nicholas Lerche, University of California; Ms. Abbey Meyers, National Organization for Rare Disorders. Ms. Meyers is a recent past member of the Biological Response Modifiers Advisory Committee. She is participating today as the consumer representative of the subcommittee.
Proceeding around the table, Dr. David Sachs, who also will be joining us later, Harvard Medical School, Massachusetts General Hospital; and Dr. Steve Woodle, and he will be coming in later this morning too, University of Chicago. Dr. Woodle also is a past member of the parent committee. Dr. Mary Groesch is representing the National Institutes of Health. Dr. Louisa Chapman is representing the Centers for Disease Control and Prevention.
The FDA is represented today by Dr. Louis Marzella; Dr. Karen Weiss is not here; Dr. Carolyn Wilson; Dr. Eda Bloom; Dr. Philip Noguchi; and Dr. Jay Siegel.
There are two participants listed on your roster who are unable to make it this morning, Dr. Ronald Desrosier and Dr. Manikkam Suthanthiran.
As a courtesy to the participants and to your neighbors in the audience, we ask that cellular phones be turned off in the conference room. Please go outside in the lobby if you would like to use your cellular phone. And, we ask that pagers be set on the silent mode.
A couple of final announcements ‑‑ the questions for the committee discussion today may be found in the stapled materials where the agenda is on the front page. In addition, or the speakers here today there is a speaker ready‑room across the hall if anyone needs to use that.
As you have noticed, on this floor of the hotel we are having a little bit of an air‑conditioning problem. They are trying their best to keep this room cool.  So, if you will bear with us, we hope we can keep it as comfortable as possible.
I would like to make just one final request, the FDA isn't known for having a whole lot of money so, unfortunately, we can only provide coffee and refreshments for members. I would ask you to reserve the side table for members.
Conflict of Interest
Thank you. Dr. Salomon, with your permission, I will read the meeting statement. The following announcement is made part of the public record to preclude even the appearance of a conflict of interest at this meeting. Pursuant to the authority granted under the Committee Charter, the Director, Center for Biologics Evaluation and Research, has appointed the following participants as temporary voting members: Dr. Hugh Auchincloss, Jonathan Allan, John Coffin, Martin Hirsch, Richard Kaslow, Nicholas Lerche, Abbey Meyers, Claudia Mickelson, Prem Paul, David Sachs, Daniel Salomon, Harold Vanderpool, Leroy Walters and Steve Woodle. In addition, Mr. Antonio Benedi and Mr. William Lawrence have been appointed as temporary voting members.
Based on the agenda made available, it has been determined that the agenda addresses general matters. All financial interests in firms regulated by the Center for Biologics Evaluation and Research which have been reported by the subcommittee participants and consultants as of this date have been considered. In accordance with 18 USC 208, the following participants have been granted a general matters waiver which permits them to participate in the committee discussions: Dr. Martin Hirsch, Dr. David Onions, Prem Paul, David Sachs and Daniel Salomon. Also, the waiver approved for Dr. Hugh Auchincloss on July 10, 1995 is applicable for this meeting.
The following guest participants have been invited to participate in today's discussion by the Chair of the Xenotransplantation Subcommittee: Dr. Robert Michler, Dr. Marian Michaels, Dr. John Conte and Dr. Ralf Toenjes.
With regards to FDA's invited guests, the agency has determined that the services of these guests are essential. The following interests are being made public to allow meeting participants to objectively evaluate any presentation and/or comments made by the participants. Dr. Louisa Chapman is employed by CDC.  She and other CDC employees are involved in collaborative research monitoring for evidence of porcine endogenous retrovirus infection, and performing confirmatory testing in exposed persons or firms that could be affected. No financial support is received.
Dr. John Conte is Director of Johns Hopkins Heart and Lung Transplant Program. Colleagues in this program are involved in trials supported by a firm that could be affected by the discussions. Some of Dr. Conte's patients could be enrolled in these trials.
Dr. David Cooper reported that he receives consulting fees from Biotransplant, and is a science advisor for Biotransplant.
In the event that the discussions involve other products of firms not already 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 public record. A copy of the waivers is available by written request under the Freedom of Information Act.
With respect to all other meeting participants, we ask in the interest of fairness that they address any current or previous financial involvement with any firm whose products they wish to comment upon.
I will turn it over to you, Dr. Salomon.
DR. SALOMON:  Well, I would like to welcome everybody today, and the meeting begins with Dr. Jay Siegel making some introductory remarks for the FDA.
Introduction
DR. SIEGEL:  Thank you, and I would like to welcome the members of the committee as well as the members of the audience. The FDA and its sister agencies in the Public Health Service, NIH and CDC, have been committed through the process of dealing with xenotransplantation to seeking the best in scientific input, as well as to having an open process with public discussion of critical issues, and we look forward to this meeting as helping to fulfill both of those goals.
This is a particularly important and also challenging area of biological product development. At this particular meeting we have what I consider to be a rather varied and interesting agenda that, as often is the case with xenotransplantation, spans the broad range of scientific and medical disciplines that come into play in conducting and carrying out this research.
We will have three topics, the first being an update on issues of porcine endogenous retrovirus, a topic we have been discussing with this committee and in other public fora for a little over two years now, is that right?  And, an update on additional findings and recent policy developments and seeking further input in that area.
We will be discussing PHS and FDA policy guidelines regarding xenotransplantation, something we have also had in development and discussion for I guess going on for five years now. Specifically, we are going to be touching on an important aspect of that, which has perhaps not been well discussed till now, which is the impact of some of the varied approaches to xenotransplantation and varied characteristics of xenotransplantation protocols on how they may impact appropriate public health and policy measures to ensure that they are conducted in a safe manner.
Tomorrow we will have what promises to be a very informative and intriguing discussion on issues in clinical development of solid organ xenotransplantation.
So, again, I am really excited by this agenda; I am looking forward to the advice we will receive, and I welcome you and all and thank you all in advance for your participation.
DR. SALOMON:  Thank you, Dr. Siegel. Well, that officially then introduces us to the first topic, which is an update on porcine endogenous retrovirus. I have been reminded that before opening this part, because this is a public hearing and in the nature of a free and open exchange with the public, it is appropriate at this point that if anyone in the audience would like to make any comments, any introductory comments or questions to the group that they may step forward at this point. There were no formal previous requests but, again, that doesn't prevent anyone from stepping up at this point.
I guess this is the point in the wedding where you go, "if there are no objections to this union," ‑‑
[Laughter]
‑‑ of course, the mike is not going away; the public is more than invited or encouraged to step up at any key point and contribute to this discussion. That would be great.
Then, we will open the first topic, porcine endogenous retrovirus update, and the introduction and a perspective will be given by Dr. Carolyn Wilson of the Office of Therapeutics Research and Review for CBER.
Topic I: Porcine Endogenous Retrovirus Update
[Slide]
DR. WILSON:  Again, I am going to also welcome the members of the committee and the audience. As already introduced, this first session of this two‑day meeting is meant to update the committee on pertinent scientific and medical data on the in vitro and in vivo biology of porcine endogenous retrovirus.
[Slide]
Although there have been a number of scientific publications of data in this area, we also, at the FDA, have received data from sponsors and that data has come in, in response to a letter that was issued in October of 1997. At that time, all sponsors of porcine xenografts were placed on clinical hold pending, first, the development of assays for detection of infectious retrovirus in porcine xenografts. They were asked to submit that data for review by the FDA; secondly, to develop assays to detect evidence of infection by this retrovirus in recipients of porcine xenografts. Again, this data was asked to be submitted for review. In addition, they were asked to develop contingency plans in the event of a positive result in the testing of patients and to update the informed consent documents in order to acknowledge potential risks presented by the porcine endogenous retrovirus.
It is the data that the FDA has received in response to the first two points of this letter that will be the focus of today's presentations and discussion. In particular, when this committee last met in December of 1997, t here was a lot of interest expressed by committee members to hear what the results of monitoring of patients were going to be. So, you will see that on the agenda there are a number of presentations by sponsors of their results from looking at patients, and there will be an emphasis in the discussion section on this topic as well.
[Slide]
Since Dr. Coffin isn't here yet, I will proceed with my talk and then he will talk after me. So, the purpose of my talk is to provide an overview on the data that FDA has received from the sponsors in response to that letter. In addition, I will try to also incorporate data that has been published in the scientific literature and, in the last few minutes of my talk, discuss some data that we have generated here at CBER.
Of the initial ten INDs that were placed on clinical hold in October of '97, six have been able to adequately address the issue posed in that letter. The data that they have presented to the FDA on their infectivity assays and their assays to monitor patients is what the focus of my talk will be today.
[Slide]
This is a summary of the data we have received from the sponsors, looking at their particular porcine xenografts. On the left are the various tissues that have been examined, and we asked them to look primarily for infectious virus, since this is an endogenous virus, to look for sequences that would have been positive.
The hepatocytes have actually been tested by more than one sponsor. This is just a compilation of the data by several different sponsors. One sponsor in particular, though, in addition to doing a culture assay also looked directly from reverse transcriptase activity. This is an enzyme that is uniquely expressed in a cell that is infected with a retrovirus.
In a number of the lots that they examined, the majority were negative for RT activity. A small minority did have what I would term borderline positive results. In this particular assay the cut‑off for a positive result is two‑fold above background, and in these few lots that were positive it was greater than two‑fold but less than three‑fold above positive.
In culture assays, though, all the hepatocytes have been negative. This is culture with human 293 cells and in some cases also with a porcine cell line called ST, and all the results from these assays have been negative.
One sponsor has also done an additional analysis. In their particular product they load hepatocytes into a device that allows for ex vivo perfusion and, since this device provides some level of a barrier they wanted to determine whether or not that would be a barrier to transmission of PERV if it were there. That analysis is negative, and the sponsor will be presenting the details of that study in a presentation later this morning.
In a culture assay with neuronal cells, again, there was no isolation of infectious virus, and one sponsor used peripheral blood mononuclear cells because their product is actually a whole liver that is used for ex vivo perfusion. This was a surrogate for that. In this case, they activate the cells with phytohemagglutinin and interleukin‑2 and, again, the results were negative.
[Slide]
Now, in the published literature there are several other tissues that have been examined by culture assay, and some are in contrast to the results on the previous slide. PBMCs that were activated with phytohemagglutinin and phorbolmyristic acid were positive for expression of virus and actually did express virus that was infectious to a human cell line.
In addition, a group reported in The Lancet last year that primary cultures of porcine aortic endothelial cells also expressed a virus that is directly infectious for a human cell line.
In the case of islet cells and kidney cells, there was one report, looking just for reverse transcriptase activity directly in those primary cultures, and it was negative in the case of islet cells and positive in kidney cells. In that report they didn't correlate those findings with infectivity studies.
[Slide]
Now to move on to data regarding recipients who have been exposed to porcine cells, either ex vivo or in vivo, all sponsors to date have, at a minimum, performed analysis of peripheral blood mononuclear cells looking for pig retrovirus sequences by DNA PCR.
In addition, one sponsor has also supplemented that information with use of a Western blot that analyzes development of antibodies that would be specific against a retrovirus. The remaining sponsors are in various phases of development of serologic methods.
A third approach for looking for PERV transmission in vivo, which actually was suggested by committee members at the last meeting, was to look for plasma viremia. At this point, no sponsors have developed or incorporated that method.
[Slide]
What I was going to show on the next slide are the results first from DNA PCR analysis. This is a summary table of all the negative results that we received from sponsors by DNA PCR. On the next slide I go into a little bit more detail on the few positive results we have obtained.
Because at the time of issuance of the letter some patients in certain clinical trials had already been treated, you will see that the data is sometimes referred to as retrospective and those patients may be several months to several years post treatment, and I call active those patients that were treated subsequent to the issuance of the letter and, so, are monitored at the time of treatment forward.
So, in the first example the product are hepatocytes which are within a barrier. Retrospective analysis of 29 patients that have been treated has all been negative, and these patients were examined anywhere from two months to five years post treatment with hepatocytes.
In the 13 patients that have been treated subsequently, 12 of those have been negative. Actually, this number is now greater. Since I made this slide last week the sponsor updated. There are additional 9 patients who have been treated that are also negative at the early time points.
For a product that uses whole liver for ex vivo perfusion, 2/4 patients who have been treated had negative results. In a retrospective analysis of 24 patients treated with fetal neural tissue all of the results have been negative, and this analysis was done on patients who had been treated anywhere from 2 months to 2 years post treatment.
On the right, you can see I just list the relative sensitivity of the DNA PCR assay in each case.
[Slide]
This is the analysis of the positive results. In the single positive in the clinical trial using hepatocytes with a barrier, this patient had a positive result that was obtained at a sample taken immediately post treatment. Subsequent samples, taken at 1, 2 and 3 months post treatment were all negative. Additional analysis of the positive sample from mitochondrial DNA using porcine specific sequences also yielded a positive result. So, the interpretation of this kind of finding is that you are most likely detecting porcine cells rather than truly an infected human cell.
In the second case of the two patients that were treated with whole liver perfusion, in the first there was a time point at six months which was initially negative. A subsequent sample at 12 months was positive, and then subsequent to that the 17‑month sequel has been negative.
In the second patient there was a positive result at two weeks and one at two months time points post treatment, and a negative at 11, 17 and 20 weeks.
In both of these particular cases there was insufficient DNA to go back to test either as a repeat for the pig retrovirus sequences or to do additional analysis for mitochondrial DNA. So, it can't be ruled out that these results may represent microchimerism. The other possibility is that at the time points when the positive results were found these samples were collected at a relatively short period of time, a similar time period from both of these patients and the samples were actually analyzed in parallel. So, at that point the sponsor was concerned that it could be a false positive due to introduction of a contaminant into the sample, and has changed the protocol and, as you can see, all subsequent samples have tested negative. So, the other possible interpretation of these results is that it is a PCR contaminant. Of course, the third possibility is that it represents a true positive.
[Slide]
This is from the published literature. There were two reports last year in The Lancet where, again, retrospective analysis was done. This was performed by the CDC in a cohort of ten patients treated with pancreatic islet‑like clusters, in Sweden. These patients were anywhere from four to seven months post‑transplant. The CDC used a variety of methods to analyze these patients. They did DNA PCR of peripheral blood cells. They looked for serum viremia by both RT‑PCR and by PCR‑based RT assay, which is a highly sensitive way to detect the viral enzyme reverse transcriptase, and by Western blot to look for antibodies.  In all cases, all samples were negative. Walid Heneine will be going through this data in more detail, as well as additional data that CDC has generated from retrospective studies.
In the second report, from Robin Weiss' laboratory, two patients who had been exposed to porcine kidneys for short‑term ex vivo perfusion were also analyzed. Again, these are retrospective samples taken anywhere from six hours to three years post treatment, and by both DNA PCR of PBMCs and to look for neutralizing antibodies the results were negative.
[Slide]
So to summarize, the results that we have obtained from sponsors ‑‑ this is just another way of looking at it by exposure of the recipient, in the case where patients were exposed through short‑term ex vivo perfusion and there was a barrier between the cells and the patient's fluids, in the retrospective analysis 29 patients were negative. In short‑term follow‑up patients, actually as I said, this number of 12 has been updated and is now 21 total patients who are now negative, and there was the one positive result which is most likely due to microchimerism.
In the second type of exposure, which again involves short‑term in vivo exposure but in this case is through whole liver so there is no barrier ‑‑ there is a mistake on this slide, there are actually 2 negative patients and 2 positive patients.
In the third type of exposure which involves in vivo direct implantation of cells, we have long‑term survival of the cells and there is no barrier between the porcine and human cells, 34 patients examined retrospectively have all been negative.
[Slide]
Now, I want to again reiterate that this is a summary of data we have received based on DNA PCR analysis, and I just want the committee and the members of the audience to be aware that there are some inherent problems with DNA PCR analysis of PBMC. For example, this type of analysis when you have a low‑level infection, something that might look like microchimerism could, in fact, be a low‑level infection.
Secondly, the inherent rate of false‑positive results in PCR is another problem that needs to be overcome. Third, there is data suggesting that human PBMC may not be a natural target for infection. So, this may not be a reasonable place to look.
[Slide]
I wanted to just quickly go through some data that we at CBER have generated, where we have tried to infect human PBMC in vitro and we were unsuccessful. So, we actually looked at a variety of hematopoietic cell lines to determine whether or not there was some lineage that we were missing in our in vitro culture conditions that was more susceptible than others. As you can see, the cell lines that are highlighted in blue were the ones that did become productively infected, and the results from that data is shown in the bottom half of the slide.
[Slide]
But we were able to infect cell lines representing T cell, B cell and myeloid lineages. We then used these cell lines that became positive as a producer virus, with the idea that perhaps these may be better adapted to growing in hematopoietic cells, and tried again to infect PHA‑blasts of human PBMCs and, after an 8‑week culture period, they remained negative by RT‑PCR. As a positive control we were able to infect 293 cells and we know the cells lines were productively infected. So, this is just a note of caution that PBMC, at least in vitro, don't appear to be susceptible to infection.
[Slide]
In the last few minutes of my talk I wanted to quickly go through some data that we have generated at CBER, looking at porcine plasma‑derived Factor VIII ‑‑ plasma‑derived product. This, actually, is something that we have done in conjunction with CDC, and it was brought to our attention by CDC when they contacted us about a year ago to say that they were looking at lots of this particular product, called hyate C, and found that there was PERV RNA present when they looked by RT‑PCR.
This particular product is used in hemophiliac patients who develop inhibitors to the human Factor VIII. So, it is used in a small minority of hemophiliac patients and it is really a treatment of last resort for these patients. So, at that time we didn't want to prematurely take this product off the market. We certainly notified the Hemophiliac Society that there was this data but we felt that it was really imperative to do additional analysis. So, we did confirmatory testing of CDC lots and did additional testing to look at whether or not these results correlate with infectious virus present in this product.
So, of five lots that CBER received from CDC, we did confirmatory testing and we, like the CDC, also found that they were positive for pig retrovirus RNA. We also used a PCR‑based reverse transcriptase activity assay. Keith Peden and Tom Maudru, from the Office of Vaccines, did these assays for us and they also found that they were positive.
[Slide]
So, to determine whether or not the product also contains infectious virus we took six lots of hyate C, and we started with lyophilized vials that had been stored at minus 70. So, I wanted to emphasize that these had never gone through a round of freeze/thaw which may limit your ability to detect infectious virus.
We directly re‑suspended them according to the manufacturer's instructions and then inoculated them into three different cell substrates, human 293 cells, feline PF‑4 and porcine ST cells. These are three cell substrates that have been shown to be susceptible to infection by PERV. As a positive control we included dilutions of PERV to look at the sensitivity of these three cell lines. The human 293 and the feline cell lines could detect out to 1:1000 dilution, although ST at 1:100 became positive. We also did direct inoculation of hyate C with PERV to control for a potential inhibition of detection of virus if it were present. Then we took these same six lots and directly analyzed them for viral RNA and for reverse transcriptase activity.
[Slide]
These results are shown here. These are the results of the PCR‑based RT assay performed by Tom Maudru and Keith Peden. On the Y axis are the units of activity in pico units porcine endogenous retrovirus microliter. The blue bar on the right represents tissue culture fluid from one of our cell lines that is productively infected. So, it is a positive control. All the lots tested, which I have just randomly labeled A through F are positive. They all have detectable levels of RT activity in this assay. As you can see, they are all 4‑5 logs lower than our tissue culture fluid.
[Slide]
This was our positive control where we spiked hyate C into the lots. The set of bars labeled "none" are cells that were directly inoculated with PERV without hyate C and A through F again are lots of hyate C that were inoculated with PERV.
As you can see, we were able to recover virus in all of those with the exception of lot D, where we were able to get positive results but there was some significant inhibition in that particular lot.
[Slide]
We carried the cultures of the hyate C without PERV in them for a total of 8‑9 weeks. We analyzed these cultures every 2 weeks for viral RNA expression by RT‑PCR and in all cases they were negative. At the end of the culture period, cellular DNA was analyzed by DNA PCR and, again, these were negative.
[Slide]
So, we concluded from these studies that although there is viral RNA and viral reverse transcriptase present in final product, there is no evidence for infectious virus in the final product and that, most likely, the positive results for RNA and RT activity represents inactivated virus that is not removed during manufacturing.
[Slide]
So, I just wanted to finish with a quick summary of what I have told you this morning. First, analysis of various porcine tissues, both by sponsors and what has been published in the literature, shows that expression of infectious virus is tissue specific. Not all porcine tissues express infectious virus, and that most likely also the differentiated or activated state of the cells may impact whether or not infectious virus is present. These results aren't surprising. It is quite similar to what is seen, for example, in the murine endogenous retrovirus system.
Looking at transmission of PERV in recipients, to date most of the data that we have seen from sponsors has relied on DNA PCR analysis of PBMCs. Most of this data has been negative. The very few positives that we have seen are most likely due to either microchimerism or problems of a false positive or contamination in the PCR reaction.
I showed you data suggesting that human PBMCs, at least in vitro, are not susceptible to PERV infection. We don't know whether or not that translates to the in vivo situation, but it certainly highlights a need for developing other methods to analyze patient samples.
Finally, in the porcine Factor VIII story, although we were able to detect viral RNA enzyme in this product, there was no evidence for infectious virus in this particular product.
I am going to stop there. I think we are waiting to have questions at the end of all the speakers. Correct?  And, I saw John Coffin come in during my talk, and I wanted to welcome Dr. Coffin. He is a member of this subcommittee and he was a co‑organizer of this year's Banbury Conference on Xenotransplantation. He has kindly agreed to provide a summary to you today of the findings of that meeting in order to bring to bear any emerging scientific data. Dr. Coffin?
Xenotransplantation: A Scientific Basis for Risk Assessment
DR. COFFIN:  Thank you very much, and I apologize for my late arrival. I am not quite yet used to the 270 traffic.
[Slide]
Last month, Robin Weiss and I organized the meeting at the Banbury Center, on Long Island, to discuss what you see here ‑‑ Xenotransplantation: A Scientific Basis for Risk Assessment. This meeting was organized partly as part of the Hastings Center, which is a bioethical think tank in New York. The meeting, although small, ranged over a number of different areas, including applications and challenges of xenotransplantation; a lot of talk on ethical issues; discussion of regulatory and policy issues; and, finally, discussion of infections, particularly viral, disease risk associated or potentially associated with xenotransplantation technology.
My discussion today will be limited only to the last of these points because that is certainly all that I feel qualified to discuss for sure. I want to make several other caveats. This was a very small focus workshop. Many people with interest in this field could, of necessity, not be present. So, this is not by any means a comprehensive report but an update simply of what was discussed at this meeting. A lot of what was discussed is in the nature of review, and a lot of what I will present is in that nature. A portion of it will overlap what you have just heard from Carolyn, who was at the meeting, and a fair amount of the data she presented here was presented there. Finally, quite a number of the people whom I am going to quote, to my dismay, are in the audience or on the board and, so, if I misquote you, you can come up to me quietly afterwards and pat me on the shoulder, or if I over‑quote you or anything else I will take some measure of responsibility for doing that.
[Slide]
So, the infectious disease risk ‑‑ as I said, we focused really pretty much only on viral diseases but we can break it into two different types, first, exogenous viruses and by that I mean agents which are not transmitted genetically or transmitted horizontally from one animal to another or vertically during birth but which do not form part of the genome and, secondly, and as you have already heard discussed from Carolyn and the reason for the greatest concern at this point, endogenous retroviruses, particularly the porcine endogenous retroviruses or PERVs, also called PoEV and PoERV. This is my particular preference but I am not going to impose that.
Several different areas were discussed ‑‑ biology and distribution, testing for infection, and Carolyn just gave you an overview of that, as well as risk assessment of these viruses. So, these are the areas that I plan to share with you in the next few minutes.
[Slide]
First, there were several examples of exogenous virus infections that might be of some interest to think about that were presented at the meeting. I will say here that I am not going to go talk by talk; I will jump around from one speaker to another but I will put attributions as we go along. Parrish discussed the rather striking situation of the origin of the canine parvovirus, which was originally a cat only virus, and apparently a few mutations, somewhere in the vicinity of 1976, or at least first detected in 1976, occurred, and within a span of a very few years this virus had spread worldwide, even into rather remote wild animal populations. There has been a series of subsequent revolutionary events that have virtually replaced this virus. So, the potential for sort of mutant viruses with extended host range, given the probability of viruses like this to be deposited in feces and carried onto airplanes and then spread around the world, which is the likely mode by which this particular virus has been spread, is certainly very well documented in this case.
The second kind of example, and this is also an example of a virus which with a relatively small change in its genome, a series of small changes, has changed its host range so that it can spread to completely different animals and then, in fact, can subsequently change and spread back again into the original cat population.
In the case of arenaviruses, which is a family of viruses found worldwide, particularly in rodent populations, many of which are benign ‑‑ this was discussed by Mike Buchmeier ‑‑ despite very high titers, some of which, for example Lassa, are high virulent in humans but don't in general spread to a great extent from one human to another. Also viruses in that group are hemorrhagic fever and hanta viruses of the New World.
Hanta viruses is a particularly interesting case. They are prevalent in many parts of the United States, particularly in the north east and, in fact, in Baltimore a study showed that there is a 5 percent total incidence of this virus in people, and this tends to be particularly in households with infected mice. So, there is a lot of ongoing introduction of this particular virus into the human population but never taking hold.
[Slide]
A case that wasn't discussed but I thought I would throw on here because it is of particular interest I think, and actually I am sort of sorry we didn't have somebody presenting it at the meeting, is J ALV, which is a recent avian leukosis virus that only has shown up really in the last few years. Avian leukosis virus is widespread in commercial bird populations causing a certain amount of mortality but under relative control. However, there are endogenous proviruses in chickens related to these, some of which have apparently been in the bird population for quite a long time in the genome, and are quite old and are thought to be pretty much completely defective but sometime, probably not too long ago, there was a recombinant combination of that between this exogenous virus and one of these old ones which provided a new envelope gene, therefore, a new host range. This virus probably spreads both vertically and by vaccination where one takes the same equipment and does thousands or hundreds of thousands of chickens. This virus has spread throughout particularly the broiler industry and has caused hundreds of millions of dollars worth of damage in the last few years. So, there may be an object lesson here that might be worth keeping in mind.
David Onions talked about large numbers of porcine viruses, 70 or 80 I believe that are known, and discussed the ones that for xenotransplant should be of specific, special concern, for example, viruses which cause known zoonotic infections and can go from pigs to humans; viruses that, although not known to cause such infections, can replicate human cells; viruses that can undergo abortive infection and, therefore, are potentially oncogenic; or viruses that are known to change host range or potentially able to do so; and also viruses that cannot be easily eliminated by hysterotomy; and barrier breeding such as parvoviruses and circoviruses and CMV, and these are all of particular concern.
[Slide]
I am going to talk about measures that they are discussing for derivation of clean animals. It seems impractical to maintain animals, like mice for example, under completely germ‑free conditions but deriving a breeding stock under conditions where there is the least possibility for the passage of infectious agents and maintenance under conditions that are as clean as absolutely possible, including health screening of staff so that infection isn't brought in by the people working with the animals, including subtleties such as recent vaccinations may well have the risk of bringing in viruses that could infect the animals and subsequently, of course, be passed on; and then close monitoring.
A point that can be taken about many of the viruses that are of concern is that we do not seem to have yet adequate tests for this purpose, good quantitative either serology or PCR or other tests. So, there is a considerable amount of development work required to ensure that this exogenous virus problem is as well contained as one thinks it should be.
[Slide]
A couple of anecdotes ‑‑ well, not anecdotes but small number of examples from some recent studies involving baboon transplants where Marian Michaels looked for baboon cytomegalovirus in several transplants, one of which was transiently positive but subsequently disappeared, and Jonathan Allan talked about a couple of studies looking at baboon foamy virus where he could find some DNA, no seropositivity and the DNA was probably due to microchimerism although, as was pointed out in the previous talk, it is a very difficult issue to deal with, very hard to sort out in the case of where one is looking for DNA to distinguish virus in infected cells from virus that has newly infected recipient cells.
[Slide]
There was a fair amount of concern expressed in the meeting that perhaps disproportionate attention is being paid to endogenous virus issues and one should look more closely at the exogenous infection issue.
It was also noted that there is considerable challenge in detecting and controlling these viruses; that new viruses are appearing on the scene. There has been a new porcine virus that is also fatal for humans. It appeared in Malaysia a few months ago, and this continues to happen and constant vigilance is required for this.
But all in all, I think the feeling ‑‑ at least the feeling I came away with was that with proper controls risk of infection of exogenous passage viruses are likely to be less than the very severe risk of infection of the currently used allotransplant technology and, therefore, at least from the standpoint of exogenous viruses the benefit, given proper controls, of xenotransplantation is likely to be quite high.
[Slide]
Then, of course, there was considerable discussion of endogenous viruses, particularly the porcine endogenous viruses. I will just give a little bit of background before I go into this. Endogenous viruses are retroviruses that have found their way in a DNA form into the germ line. They are found in all vertebrate species and humans carry thousands of these. We all probably have pretty much the same complement of them since, as far as we know, all the viruses in humans were introduced some number of millions of years ago and I think we are pretty much genetically homogeneous in that respect. That is not true of many other species, including pigs, where the viruses were introduced into the germ line relatively recently in evolutionary terms and, in some cases, some of these proviruses ‑‑ there are in the group we are talking about perhaps 30 or so per animal, depending on the strain. Some of these can clearly yield infectious virus. You just heard Carolyn talking about some particular cases of that.
In cats, just as an example discussed by Steve O'Brien, there is the virus RD114. RD114 is an interesting story for those of us who were around back in the early '70s. It was found as a virus that was produced by human tumor lines that had been originally generated by passage through kittens. It was believed, and a press conference was held ‑‑ of course, this was the early days of the Special Virus Cancer Program ‑‑ this was believed originally to be human retrovirus and was very rapidly shown not to be, but to be a xenotropic virus of cats, that is, a virus that could not infect cat cells efficiently but could infection human cells and was readily transferred into these cells and infected these cells in the xenotransplantation, if you like, of a human tumor in kittens.
A very similar phenomenon was seen in mice where I, as chair of the IVC over the years, have had a number of people walk into my office with human tumor lines that were pouring out of really lovely C‑type retroviruses and usually the first question I asked these people was were these tumors ever passed through mice. Human tumors passed through mice have a very high probability of having been infected by an endogenous mouse virus and then subsequently expressing that virus. In fact, there are some commonly used cell lines around that have that property, some of which may be being used for genetic engineering purposes.
This group of viruses is present only in the genus to which domestic cats belong but no other groups, but it is related to endogenous virus in baboons which implies that there was some cross‑species transmission event, perhaps from baboons to cats but there may have been some intermediates, and so on, some two million years ago.
So, this gives you a feeling for how these viruses come in and can be passed on in the germ line only to reappear at rather awkward moments later on when you present the opportunity in this particular coculture for having viruses of humans and cats together. I should also point out that there has never been any evidence of infection in humans with this virus by handling of cats or, so far as I know, in veterinary surgical procedures. So, it doesn't mean this virus, once it got in, could be spread from person to person but you certainly create an opportunity here for infection that may not otherwise occur in natural circumstances.
[Slide]
Porcine endogenous virus is a murine leukemia related endogenous virus. I believe this group is now called gamma retroviruses, found in multiple copies in all pigs examined, and related sequences can be found in all suidae, in all pig‑related species. Infectious virus is released by most porcine cell lines, except for the ST line which was mentioned before, and this, as Carolyn was discussing, is therefore a useful cell line for testing infectivity of these viruses and it can be released, as you have already heard, as activated lymphocytes.
Three principal subgroups have been identified by sequence testing of the envelope gene which includes both sequencing host range and interference testing. These are called A, B and C. A and B can detect human cell lines reasonably well. C, which actually I believe is not really identified as a virus but only as an envelope gene, can affect almost only pig cells, with the exception of one human cell line. So, these two viruses, in the rather confusing terminology that was originated with mouse retroviruses, could be referred to as polytrophic viruses. It should be pointed out that just because they have the same host range doesn't mean they use the same receptor for infection. Interferon studies indicate that these two viruses actually use different receptors.
Multiple copies of these proviruses are found in all pigs tested. C is found in high copy  number in mini pigs and either less or not at all in other strains, and there is considerable polymorphism from one individual to another.
[Slide]
This is my memory of an unrooted phylogenetic tree relating these envelope genes. So, it gives you the feeling actually that as you sequence you can detect more and more of these, like C1, B1 and A1, which are not necessarily known to belong to infectious proviruses but which relate to the known infectious endogenous sequences, the As, Bs and Cs, and as one sequences this thing will certainly bush out more and more as we go along, and there may well be more subgroups hiding in there. A and B are the main ones that are seen as being produced by the cell lines that I mentioned.
[Slide]
As Carolyn pointed out and as I think she discussed earlier, virus isolated by the induction of lymphocytes can adapt in some way to human cells, either by getting more infectious or by segregating out less infectious virus from the population.
The most efficient isolation actually starts ‑‑ at least some cell types require starting with cocultivation with pig cells and then passage through human cells, raising the possibility that in many cases there may be some additional either pseudotyping by, let's say bringing in additional envelope genes, or actually combination events where the pig cells between pig viruses may actually be giving rise to the most infectious virus types for humans. These viruses are not only present in pigs as endogenous sequences, David Onions showed that you can detect low‑level viremia in at least some pigs.
As I said, there are a large number of proviruses, at least 30 or so per animal, many of which are different from one animal to another, that is to say, they are integrated at different sites. So, they are different coviruses probably with some different biological properties. Many of those, probably most of those based on our experience with other endogenous retroviruses, are likely to be defective in one way or another.
At least six proviruses are common to all pigs and, therefore, could not be bred out if one were to start on an ambitious breeding program to remove proviruses at different chromosomal sites. Whether these represent infective ones we don't know yet.
In addition to the C type or gamma retroviruses, if you like, if one starts looking, as with all other species that we know of, at least all other mammalian species and virtually perhaps all other animal species, if you go looking you can find other types of endogenous proviruses. For example, PCR probes that were general for reverse transcriptase genes and some viruses turned up that resembled groups of endogenous viruses that are found in large numbers in humans and other types of species, D type or also related to mammary tumor viruses. Whether these represent infectious elements as they would in some monkey species or non‑infectious viruses which would be the case in humans, remains to be determined but we have probably only scratched the surface in terms of endogenous provirus‑like elements in pigs or many other species.
[Slide]
There was a certain amount of discussion of the testing for these in animals and in human recipients. Potential tests include serology. For example Western blot for the p30 gag kapsa protein has been developed; use of RT‑PCR plasma, PCR in lymphocytes and amplified RT assays, a very sensitive assay for RT‑containing virions.
Carolyn discussed the issue of mitochimerism and the difficulty of detecting that, and that still remains an issue and there was actually a fair amount of discussion on the problems of separating out whether you are detecting an infected human cell or an infected pig cell.
Most testing of patient samples to date is negative. I think we are going to hear a lot more about that in this meeting, including some patients who have received extracorporeal perfusion or allocell transplants. There are a number of monkeys that had heart or kidney transplants, and a large series of patients and others exposed to pig products, although there are some hints that perhaps there may be one Western blot positive individual. Maybe we will hear more about that.
[Slide]
Finally, as I think everybody should be aware and is one of the reasons we are having meetings is that the real risks for this infection are unknown. In order for a PERV transmission to actually result in an event of concern a number of steps have to occur ‑‑ spreading infection of the recipient from an initial production of virus infecting a few cells of an individual, spreading infection has to occur. The event has to lead, at least in some individuals, to pathogenesis. If it didn't, it perhaps wouldn't be of great interest although it would certainly be of some concern. Finally, transmission from one individual to another ‑‑ if the virus only gave rise to the sort of pathogenesis one usually sees with this type of retrovirus where a fraction of individual recipients had some sort of malignancy at some very long time after the event, this might well be an acceptable risk in cases where the initial event was life‑threatening and there was no choice. The risk or the concern of what happens if a transmissible virus is created is uncovered or created by this step is obviously much more serious. The Stoye scale was brought up where each of these events is considered to be progressing from likely to unlikely to very unlikely to very, very unlikely, with some sort of undefinable scale attachment as you go along.
I think there was a general feeling at the meeting, and perhaps that is something that will be discussed again, that it would be very valuable to develop some kind of animal model for PERV infection so that, should there be some problems ‑‑ at least as far as was discussed at that meeting, there wasn't a feeling that the progression of the development of transplant technology should be held up waiting for this model, but that this should be a parallel track and that it would be important, should there be evidence for infection and so on, to have some sort of model to be working from even if it wasn't considered to be the optimal model.
I think with that I will close and thank very much Carolyn Wilson and Jonathan Stoye for helping put this presentation together. Thank you very much.
DR. SALOMON:  Thank you very much, John and Carolyn. I am actually doing very well on time, and I ask for the chairman's prerogative to ask some questions at this point which are not traditionally on the agenda. We do very much want to stay to time so that gives us about five or so minutes that we could open up for some questions.
DR. VANDERPOOL:  I have one question that is more common sensical and it certainly may be important for the public members who are here, and I would appreciate it, John, if you or Carolyn could comment on what you mean by infectivity. It is really on your second to the end slide. You distinguish between infectivity, pathogenesis and transmission. But I think for commoners who are non‑specialists infectivity sounds really serious. It sounds like you have a disease and you can pass it on. Could you, for the sake of the public and other persons present, make some of those distinctions a little finer for us?
DR. COFFIN:  I should preface by saying that we and other animals get infected with many agents that we actually never recognize the infection of. So, infectivity and disease induction are two very different things, and that is particularly true in retroviruses where there are many retroviruses, including the relatives of HIV in monkeys actually which can infect individuals and never give rise to significant disease.
So, it is a very important distinction, and there are two levels of infectivity that are important in this particular case. One is at the local site where there are cells of the donor, the pig for example, immediately adjacent to human cells. Whether some virus gets transmitted to those cells immediately adjacent to the first step, and so first infection. Then, the second step is whether those cells can pass virus on to the rest of the body.
So, there are two very different issues here, the issue of whether you can locally infect a few cells and whether then that goes on to create a spreading of infection that can actually spread through the whole body, very large numbers of cells in the body and create much, much greater opportunity for any kind of disease effect, if that is going to happen.
The infection of a few cells I think is probably, given the models we have to work with, considered to be not an unlikely event. Widespread infection is probably considered at this point to be much more likely but, again, we don't know what the scale is. Infection of a few local cells is much harder to distinguish than widespread infection would be, leading to viremia, virus in the blood, infection of cells at different system locations.
DR. HIRSCH:  I have a question for Carolyn. I think you importantly suggested that human PBMCs might not be infectable with PERVs and, yet, a lot of requests are being made to test human PBMCs. My question is have you done investigations of subsets, like CD4 cells, CD8 cells, monocyte macrophages within PBMCs because, as you know, in other systems like HIV sometimes the CD4 cells become more infectable when you take the CD8 cells out.
DR. WILSON:  No. That is a good point, we haven't tried those manipulations. Instead, we tried going back to just cell lines and lineages to first get an idea if what certain lineages might be more susceptible, but that is a point well taken.
DR. ONIONS:  Carolyn Wilson very elegantly showed the difficulty of infecting human peripheral blood mononuclear cells. The caution I have is that we know that certainly in animal models we find widespread action of peripheral blood mononuclear cells that are very difficult to infect in vitro, just as human cells are with PERVs but the root of infection has to be through a stem cell. So we should be cautious in dismissing using PBMCs as a target just on the basis that they are quite difficult to detect in vitro.
DR. SALOMON:  Yes, Dr. Allan?
DR. ALLAN:  I have a question for Carolyn, and this is probably something that we can discuss during the meeting, which is the question of contamination with PCR because when you get a positive result, obviously, the issue is whether it is contamination or not and if we get positive at one time point and negative at another time point do you dismiss it as contamination?  How do you address the issue of contamination?  Do you do multiple samples?  Because if you put it into the realm of contamination then you are sort of dismissing ‑‑ you may be dismissing all PCR results and why do anything?  I mean, it is really difficult.
DR. WILSON:  Well, that is why I didn't eliminate the possibility that it wasn't a true positive result. I don't think the data at this point ‑‑ because there weren't enough samples to go back and do repeat testing, it can't be eliminated as a possibility.
DR. ALLAN:  Because you could have instances, especially in microchimerism, where you get a positive at one time point and you get a negative at another time point.
DR. WILSON:  Yes.
DR. SALOMON:  Dr. Michaels and then Dr. Mickelson.
DR. MICHAELS:  Another question for Carolyn, I don't know if you have this data; it might be something that we will need to bring up with some of the sponsors later but, again, bringing up some of the limitations which you have shown with the PBMCs, have any of the sponsors looked at other tissue samples of, for example, some of the patients who had the hepatocyte barriers if they then later underwent an allotransplantation for the liver is the native liver available that could be tested, or has it been tested?
DR. WILSON:  Nobody has done those types of experiments.
DR. SALOMON:  Dr. Mickelson?
DR. MICKELSON:  Just a general question maybe for Carolyn and other experts on the panel, a lot of the assays seem to depend on reverse transcriptase PCR which, in the context of the patient, means if you can pass it you are already looking at a situation where infection has been established, which is unfortunate. Is there some other way or some other assay that could be used to give you a hint that something might be happening before there is established infection in the patient?  Can you look for the appearance of antibodies in patients against PERVs or something?  It seems that if you have an analysis that only gives you a positive once something has happened, maybe if you had an earlier step assay that could be done ‑‑ I would like some discussion of that.
DR. SALOMON:  Yes, specifically to that I would say for the record it was a recommendation that was, I believe, accepted by the FDA from this board the last time we met that we should put an emphasis on developing serological assays parallel to PCR technology based assays.
DR. MICKELSON:  I just didn't see that in some of the information that was here, but just the earlier you can move on into the event the better it is for patients.
DR. SALOMON:  There was mention in Dr. Coffin's slides of a Western blot study. John, do you want to comment on that?
DR. COFFIN:  Well, I will ask David to comment on it. He is the one who has been developing it.
DR. ONIONS:  Well, I can comment on the serology in two senses. One is that we have been using recombinant p30 and we are now putting a recombinant ending to screen patients for antibody. We have looked at well over 200; the 300 on John's slide includes controls and validation. We know that the assay doesn't pick up antibody to HIV and HTLV so that we are confident of its specificity. We do have patients that do have antibody. We have confirmed that there is antibody binding by using mass spectrometry techniques. This is actually probably bound to p30. So we are confident there is antibody to p30. What we don't know is whether that epitope could be due to cross‑reactivity. So, we can't dismiss the possibility that this is quite spurious. If you screen people for HTLV capsid antibodies, you will find families that appear to have antibodies to that and, yet, there is no other evidence that they are HTLV infected. So, we need some caution there.
But just in terms of using serology, I think it is an important adjunct in testing but my caveat is that, again, this is not HIV. In the gamma retroviruses normally antibody is associated with a recovery phase rather than with concurrent active infection. Usually it is a secondary event. So, I am not sure it is useful in a diagnostic sense in the human that it is a very early indication rather, in my view, it is an indicator of exposure and often of recovery. So, it is a rather different indicator.
DR. MICKELSON:  Then, what would you suggest as an indication for early stage?
DR. ONIONS:  Well, we know that the earliest phase that we see in animal models and human models where you take amphytropic virus and put it into primates, or you look at cats, that the first thing that you see is a transient infection where you see virus in the plasma at a low level. You then get infection, usually in the bone marrow, and then you get a higher titer plasma viremia and also virus in cells. So, using, as John hinted, developing techniques to look for virus in the plasma is an important adjunct in testing, and one that I would encourage.
DR. SALOMON:  I am afraid, just to stay on time, we are going to have to go forward. This brings us to the second phase of this morning in which we have invited some guests for presentations, and the first presentation is from Dr. Gillian Langford, from Imutran, entitled, In Vivo Expression of Porcine Endogenous Retrovirus in Pigs Analysis of Non‑Human Primates Transplanted with Porcine Organs for Evidence of Cross‑Species Transmission of PERV.
Guest Presentations
In Vivo Expression of Porcine Endogenous Retrovirus in Pigs
Analysis of Non‑Human Primates Transplanted with Porcine
Organs for Evidence of Cross‑Species Transmission of PERV
DR. LANGFORD:  Thanks.
[Slide]
I want to talk about two studies that we have done at Imutran and in collaboration with GTI which is a company based here, in Washington, primarily to look at expression of endogenous retroviruses in the pigs that we are planning to, or hope to use in clinical transplantation, and a study that we have done where we have analyzed non‑human primates that have been transplanted with organs from pigs to look to see if those primates have been infected by the virus.
[Slide]
The studies that we have done to look for expression of the virus have really been performed to supplement the data from the in vitro studies. We have analyzed expression in out pigs using three different techniques, the Northern blot analysis, transmission electron microscopy and an assay that looks for an enzyme that is associated with the virus.
[Slide]
We have done Northern blot analysis on families and non‑related pigs, from about ten different pigs within our herd of animals, and what we have done is both Northern blot and RT PCR analysis and we have taken a variety of tissues from our pigs to see whether we could see viral mRNA expression.
What we have found is that when we look in a variety of tissues ‑‑ the heart, lung, liver, kidney and spleen ‑‑ we see viral expression.
We have also established an in situ hybridization technique which we have used to look in neural cells from our pigs, and we have found that by in situ we can see viral expression of our mRNA expression as well.
Interestingly, what we have found is that the highest level of expression, and this is from tissue to tissue within our pigs, is within the lung, and this is consistent between all the pigs that we have looked at. We see high levels of expression in the lung and much lower levels of expression in the kidney and heart tissues.
[Slide]
As well as looking for mRNA expression, we wanted to look to see if we could see viruses actually being released from our pig tissues, and we did this by transmission electron microscopy. What we did, we took a series of tissues from our pigs and analyzed them by TEM. In addition, from some of the primates that have been transplanted with the pig organs we took a variety of tissues from those primates and also analyzed those by TEM.
The results that we found when we looked in the tissues from our pigs were that there was no evidence that we could see any viral‑like particles when we looked in the heart, kidney and bone marrow. In addition, from our primates when we looked in the spleen and lymph nodes, again, we couldn't see any evidence of virions being produced.
[Slide]
However, when we took serum from our pigs we actually saw evidence of viral‑like particles by TEM analysis, although when we did the immunogold analysis these TEM particles didn't bind antibodies against them, the recombinant gag, or antibodies directed against the whole virus.
However, because we had seen these TEM results of these viral‑like particles in porcine serum we went ahead and analyzed porcine serum using the product‑enhanced reverse transcriptase. This is a two‑stage assay. When exogenous RNA is added to a test sample, if the test sample contains reverse transcriptase the RNA is converted to cDNA, and then that cDNA is amplified. So, if you get a positive signal in your PERT assay, this indicates the sample contained reverse transcriptase.
[Slide]
As I said, what we did was to take a whole series of serum samples from our transgenic pigs. We fractionated those serum samples through a sucrose gradient to ensure that we were really looking at viral particles, and then we analyzed those samples using the PERT assay. What we found is that we had positive results, indicating that we may have a low‑level viremia in our pigs.
[Slide]
So, what we wanted to do was really to assess whether that PERT activity was associated with infectious virions. So, we did this using infectivity studies in which we cultured human cell lines which were known to be permissive for PERV. In this example we used human 293 cells in the presence of porcine serum for up to ten passages. After this, we harvested the human cells and analyzed them by PCR to see if there was any evidence of infection.
[Slide]
What we found is that when we looked at these human 293 cells there was no evidence of infection. So, that led us to conclude that although we can see PERV activity in our pig serum, this isn't necessarily associated with infectious virions.
[Slide]
As I said at the beginning, not only did we look for viral expression in our pigs, we have also analyzed samples from both baboon and cynomolgus monkeys that have been transplanted with transgenic pig organs to look for evidence of infection in these primates. At the moment there is some debate about the validity of using primates to look for cross‑species transmission of the virus, primarily because there is very little in vitro data that shows that primates are permissive or the cell lines are susceptible to infection by PERV.
We have actually generated some in vitro data that shows that both rhesus and chimpanzee cell lines are actually susceptible to infection by PERV, and we think that supports the model that we have used.
[Slide]
So, basically, the model that we have employed has involved taking samples from baboons or cynomolgus monkeys that have been transplanted or orthotopically with transgenic hearts and kidneys. All the primates that we analyzed samples from were actually immunosuppressed, and the survival times of the primates that we took tissues from were between 9‑99 days.
[Slide]
As well as taking terminal necropsy samples from our primates and analyzing those by PCR and Western blot analysis, we are also collecting serum samples from our primates so that we can analyze them by RT‑PCR to see if, when we transplant the porcine organ, it stimulates the release of additional virions into the primate serum.
[Slide]
This slide describes the testing strategy that we have used to analyze the tissues that we have collected from our primates by PCR. We start the analysis by doing a PCR based on the pol gene of the virus. If the result from this PCR analysis is negative we can conclude that the sample hasn't been infected. However, if we get a positive result we don't know if that result was due to microchimerism or natural infection of the primate tissue that we are looking at.
So, to distinguish between microchimerism and infection we have developed a second PCR assay based on centromeric repeat sequences that are present in all pig cells. We know that there are approximately 2.5 thousand copies of this repeat centromeric sequence so we know that we have developed a highly sensitive assay to detect pig cells in our primates.
If when we do this PCR assay we don't detect any centromeric sequences, then we can conclude, because we have a positive PCR result for the virus, that the primate has been infected. However, if we get a positive result for the centromeric sequence, then we are in the position where we have to resolve between microchimerism and infection.
[Slide]
We have tried to resolve between these two possibilities of microchimerism and infection by developing quantitative PCR assays. What these assays involve is determining the number of copies of PERV sequences in the sample and the number of copies of centromeric sequences.  When we have those two values, we then determine the ratio of PERV to centromeric sequences.
What we know from analyzing a variety of our pigs is that we know the ratio of PERV to centromeric in our donor pigs. So, when we look in our primate sample, we can then compare that ratio to the ratio that we find in the pig cell. These are just examples. If you have microchimerism, this ratio of PERV to centromeric sequences would be the same as you would find in a pig cell. Whereas, if you had an infection this ratio would be altered. You would expect to see a higher number of PERV sequences relative to the number of centromeric sequences. So, if this ratio is altered we can conclude that the tissue that we are looking at has actually been infected.
However, there is a caveat to this testing approach that we have used. If you have a very low‑level infection in the presence of microchimerism the value of PERV sequences that you see would only increase a fraction, and statistically you wouldn't be able to distinguish between this ratio and the ratio that you would see for microchimerism. So, it is possible that if you have microchimerism and a low‑level infection you really wouldn't be able to discriminate between those two possibilities.
[Slide]
This slide shows some of the work that we have done to validate the PCR assays that we have used, and this work is really all being done at GTI. This graph shows that when we use between 10 to 106 copies of plasmid containing the viral sequences that there is a linear relationship between the number of copies of the virus and the threshold signal at which you would consider the sample to be positive.
[Slide]
We know that by adding spiked or by adding plasmid containing the virus to a background of human cells that we have been able to determine sensitivity of our assay, and we know that if we spike in one copy of the virus to 500,000 human cells we can actually detect that virus. However, because of Poisson distribution we actually claim that the limit of detection of our assay is ten copies of virus per 500,000 cells.
The centromeric assay that we have detected has a slightly different sensitivity than the PERV PCR assay that we have detected. Here we can detect 10 copies of the centromeric sequence porcine endogenous retrovirus 500,000 cells.
[Slide]
As well as analyzing the primate samples by PCR, we have also taken the terminal serum samples and analyzed them by Western blot analysis to see if there have been any antibody responses. The Western blot assay that we have used involves using either recombinant gag protein or whole PERV virus isolated from infected human cells. As a positive control we have antibodies that have been taken from rabbits that have been immunized with either recombinant p30 gag protein or the whole virus.
[Slide]
So what are the results that we have seen from the analysis of our primate samples?  What we have done is taken a whole series of tissues from our primates. Predominantly we have looked in spleen and lymph node samples, and we chose those samples initially based on the premise that if we expected to see microchimerism in allotransplantation, they would be the tissues that you may expect to see it in. But in addition to looking at spleen and lymph node samples, we have also looked in heart, lung, liver, kidney and testes samples from our primates.
What we found is that when we look at the ratios of PERV to centromeric sequences in all the tissues that we have analyzed, and that is a total of 100 tissues so far, we don't detect any evidence of infection in those tissues. However, what we did find, and what we were most surprised to find is that in the majority of the tissues that we analyzed there was evidence of pig cells. We found both PERV and centromeric sequences.
What we don't know at the moment is whether these cells are really indicative of true microchimerism or they just released from the pig cells during the rejection process.
[Slide]
As well as the PCR results, we have Western blot results from 40 of the primates that have been transplanted with transgenic organs, and in all these Western blots we haven't detected any evidence of anti‑PERV antibodies.
[Slide]
So, in conclusion, although we have seen PERV activity in the serum from our transgenic pigs, the in vitro coculture experiments we have done, where we have passaged human cells in the presence of porcine serum, suggest that we don't have any infectious virions present in our pigs.
In addition, when we have analyzed primates by both PCR and Western blot analysis we haven't had any evidence of infection in these primates, and that is, we believe, now supported by our in vitro coculture results.
[Slide]
Finally, I would just really like to acknowledge the people who were involved in these studies. Thank you.
DR. SALOMON:  Thank you very much for staying on time. I think it is obvious to everyone that Dr. Auchincloss has joined me, to my right, and with that I will yield the chair to him.
DR. AUCHINCLOSS:  Dan, thanks very much for filling in, and I apologize to the FDA and my committee members for being late. I believe we are moving on now to Zorina Pitkin, Circe Biomedical.
HepatAssist Liver Support System Containing Porcine
Hepatocytes
Case Study: Bioreactor PERV Analysis and Risk Assessment
DR. PITKIN:  Good morning, everyone.
[Slide]
This presentation concerns in vitro PERV analysis and risk assessment of the HepatAssist liver support system containing porcine hepatocytes. There are three parts in this presentation. First, we will introduce the HepatAssist system and its specifics. Then, we will have a brief overview of in vitro and in vivo PERV studies. Finally, we will present a case study on in vitro PERV analysis of the HepatAssist system.
[Slide]
To start with the introduction of this system, the system has been designed to support patients with acute liver failure until the liver regeneration or organ transplantation. As an update, we have conducted a Phase I/II clinical study to assess safety and tolerability of the system in patients with acute liver failure indication. It was completed in 1997. We are in the process of conducting a Phase II/III multicenter, randomized clinical trial that was initiated in 1998.
[Slide]
On the right‑hand side you see the picture of the HepatAssist system, and on the left‑hand side you see the schematic of the machine. During HepatAssist system therapy a patient's plasma, collected through an apheresis machine, is circulating through the bioreactor containing porcine hepatocytes. In the bioreactor the hepatocytes are segregated behind a hollow fiber membrane and perform many of the metabolic functions of a healthy liver. This is the cross‑section of an individual fiber, which is magnified, and the plasma is circulating through the lumen of the fiber. The cells are placed outside the membrane, and the membrane is not permeable to the cells. So, the case study on the PERV assessment will be focusing on this bioreactor.
[Slide]
With regard to the cells placed in the bioreactor, the cells are cryopreserved after isolation which allows for extensive quality control testing prior to use in the clinical setting.
[Slide]
This is the schematic of the quality control testing of cryopreserved hepatocytes, with the time scale which shows the time it takes to release cryopreserved cells for the clinical duration. In the blue boxes you see the critical steps of cell processing from herd qualification to cryopreservation, and the cells are released for clinical use only when all in‑process and final release criteria are met. In‑process and final release testing includes bacteriology, viral testing, microbiology and functionality testing.
[Slide]
It is a closed system and this is a picture of one aliquot of cryopreserved hepatocytes.
[Slide]
With regard to the treatment that patients are receiving with the HepatAssist system, there are 5 billion viable cells, hepatocytes that are introduced in each treatment and the hepatocytes in this system are segregated, as I said, behind the hollow fiber. Each bioreactor is 100 percent integrity tested, and the pores of the membrane are 0.15 micron size. Thus, the hepatocytes are not in direct contact with human cells during treatment.
[Slide]
The duration of the treatment is 6 hours administered daily to a patient, again, until either liver regeneration or organ transplantation. Based on current experience and the first study, on average there are 3‑4 treatments per patient.
[Slide]
Now that I have introduced you to the system, I will just present a brief overview of in vitro studies studies ‑‑ PERV assessment; the strategies for risk analysis concern two parts, in vivo and in vitro testing. With regard to in vivo testing of patients, we have conducted three prospective testing on 29 patients' PBMCs. The testing was done by DNA PCR and all patients were found to be negative for PERV. The exposure time was 3 months to 5 years post‑treatment.
With regard to the prospective testing, the testing is ongoing and it is a critical component of our protocol that all patients are to be tested at various time points. Patients' PBMCs are being tested by DNA PCR, as well patient serum is being tested by Western blot analysis.
[Slide]
With regard to in vitro testing, the hepatocytes were tested by cocultivation with 293 cells and there was no evidence of infectivity in human 293 cells cocultivated with human hepatocytes for longer than 35 days. Finally, we conducted a PERV assessment of the HepatAssist system.
[Slide]
The goal of this in vitro PERV assessment of the HepatAssist system was to test the system by the reactor that is used clinically for PERV infectivity and, second, to investigate whether the membrane in the bioreactor provides a barrier to PERV transmission.
As far as the study design, in order to simulate clinical conditions under which the system or the therapy is being delivered to the patients, we seeded the same number of porcine hepatocytes behind the hollow fiber membrane, and the hepatocyte bioreactor was perfused with culture medium. However, the duration of the experiment was 24 hours, which is 4 times longer than the clinical administration of the therapy. That was done to simulate the increased exposure to the cells.
[Slide]
As a positive control, we placed PK‑15 cells behind the hollow fiber membrane, and the design of that positive experiment was exactly the same. Five million PK‑15 cells were used and the bioreactor was perfused with the same culture medium with 10 percent of serum, and the perfusion time was 24 hours.
[Slide]
This is a schematic of the experimental setup where the culture medium was used instead of plasma as in the clinical setting. It was perfused through an oxygenator and hollow fiber cartridge, and it was collected every two hours and then pooled together. The only difference with the clinical administration was that we did not use charcoal column as we believed that it could potentially interfere with the virus.
[Slide]
So, the medium was collected every two hours and then pooled together, and concentrated by a factor of 1000 by ultracentrifugation, where the cell supernatant from the cell compartment was not concentrated.
[Slide]
As far as the infectivity test, the circulating medium collected and cell supernatant were incubated with human 293 cells for 35 days. As a control they used human 293 cells, and the positive control was human 293 cells infected with PERV.
[Slide]
In the infectivity analysis, at the end of the cocultivation period human 293 cells were analyzed for PERV infectivity by DNA PCR; with negative control, DNA from MRC‑5 cells; and positive, genomic DNA from porcine PBMCs.
[Slide]
Now I would like to present the results of this study, first on the hepatocytes experiment. The media from the HepatAssist bioreactor containing porcine hepatocytes showed no evidence of PERV infectivity in human 293 cells inoculated with either cell supernatant or circulating medium which was 100 times concentrated.
[Slide]
As to the PK‑15 cells, on the contrary, the media from the bioreactor containing these cells showed evidence, as expected, of PERV infectivity in human 293 cells inoculated with both cell supernatant or 1000‑fold concentrate of the circulating medium.
[Slide]
However, in the PK‑15 experiment the circulating medium showed at least a 5‑log reduction ‑‑ and I want to emphasize it is a qualitative assessment in infectivity in human 293 cells when compared to PK‑15 cell supernatant in the bioreactor.
[Slide]
I would like to show you the picture of DAN PCR analysis for PERV infectivity, and want to point your attention to 4 particular lanes, lane 8, 8, 10 and 11. Lanes 10 and 11 represent PK‑15 supernatant unconcentrated, incubated with 293 cells, and that represents a very strong signal. Whereby, lanes 8 and 9 represent concentrated 1000 times circulating medium incubated with 293 cells. Lane 8 ‑‑ there is no signal that could be seen on this autoradiograph, and lane 9 shows a very faint signal. So this is our qualitative assessment of reduction in infectivity.
[Slide]
In conclusion, the experiments with the HepatoAssist bioreactor containing hepatocytes showed no evidence of PERV infectivity in the human 293 cell line. Second, the HepatAssist bioreactor membrane reduced PERV infectivity by a factor of at least 5 logs using PK‑15 cells.
[Slide]
Thirdly, the membrane based bioartificial organs may help, it is our belief, to reduce the risk of zenozoonosis such as PERV transmission. Further, membrane based bioartificial organs segregate xenogeneic cells, thus preventing direct contact with patient tissues. Finally, cryopreservation of animal cells allows for conclusive quality control testing in this setting.
[Slide]
Now I would like to acknowledge my colleagues at Circe Biomedical and people we have collaborated with in the past, Primedica Corporation and Q‑One Biotech. Thank you very much for your attention.
DR. AUCHINCLOSS:  Thank you. We will move on now to a presentation fm the CDC by Walid Heneine, and I probably massacred the way you pronounce your last name.
Surveillance of PERV Infection in Exposed Persons
DR. HENEINE:  Thank you for inviting me.
[Slide]
I would like to start first by going over some diagnostic strategies that we are using at the CDC to look for PERV infection in exposed persons or animal models, and then move quickly to the applications of these assays and what data we have obtained by using those assays.
One major point in the discussions today is the different strategies we could use, and what we have been doing, developing and evaluating at the CDC lab is both molecular and serologic approaches for the diagnosis. On this slide we see a listing of the assays we have to detect PERV in the peripheral blood lymphocytes using primers and probes derived from preserved PERV sequences available to us to date.
We have also developed a PCR assay to detect pig‑specific mitochondrial DNA to evaluate whether there is any evidence of microchimerism in the sample. What is not mentioned in the slide here is the assays we have using the PERV primers and probes to look for PERV RNA as a marker of productive infection or viremia, and we have applied those to the analysis of serum or plasma.
In addition, we recognize the importance of looking for antibodies against PERV as an adjunct approach for diagnosis, and we have also developed a Western blot assay which I will mention to you again later.
I have added here that in the case of a positive result by PCR and serology we should not stop there. We should also try to see if we can isolate virus from infected individuals.
[Slide]
The issue of microchimerism, as you have heard this morning, is very important for adequate diagnosis of PERV infection, and what we have developed is a simple method. The way we have adopted it is to use dilution of peripheral blood mononuclear cells and not DNA extracts of PBMCs. Rather, before extraction of DNA use dilution of these peripheral blood mononuclear cells and then go back and test those dilutions for the presence of PERV sequences and for the presence of pig mitochondrial sequences, and then look at the titer of the positive signal that you will see.
Here, I have highlighted two scenarios that you may see. In the case of a chimerism the titer of PERV sequences will be equivalent to that of the pig mitochondrial DNA because it will reflect the presence of any pig cell in the dilution.
In the case of an infection you will see a higher titer of PERV sequence compared to the pig mitochondrial DNA sequences. This scenario, for example, reflects low level microchimerism in the presence of PERV‑infected human cells. Again, I reiterate here that the key point is not dilution of the DNA but dilution of the cells, and we usually do a two‑fold dilution so that you can very quickly determine what state you have.
[Slide]
Let me go back a little bit further to the serologic assay we have used and that we reported last month in Transplantation, with validation. The Western blot assay we use is a whole‑cell lysate assay of 293 human cells infected with PERV. We use as a diagnostic marker the p30 activity of the gag protein. For seroreactive samples we usually run another Western blot that has a control antigen, which is the uninfected 293 cells, to make sure that any reactivity we see is due to the viral protein and not to any non‑specific reactivity associated with cellular 293 proteins.
This is an example of how we applied this assay on the evaluation of antibodies to PERV in pig islet‑cell recipients which we have previously reported, as mentioned this morning.
[Slide]
I can go back and show you some of these assays that we have applied. For example, here, looking for PERV in humans that have received pig liver perfusions, we have looked at four patients. All four have been consistently negative by PCR. We have done the serology. The first three have also been seronegative. This is the time post‑exposure. In patient "D", the fourth patient, we have seen a very weak seroreactivity at two months after the exposure but then afterwards we were not able to detect any reactivity at seven months, and we have now, two months later, another time point here. There was no molecular evidence of PERV in this patient, which led us to interpret this transient seroreactivity as not evidence of infection but maybe as evidence of exposure to viral protein.
[Slide]
This is the pig islet‑cell recipients, the ten patients we have looked at. These are some characteristics that I would like to go over very quickly with you. This is the number of islet‑cell clusters that they have received, and this is the evidence of prolonged survival seen in some of them, which was the detection of C‑peptide.
We have also looked in a different way for evidence of xenograft survival by looking at the mitochondrial pig DNA in the plasma, and we have seen a strong correlation between detection of C‑peptide and presence of pig mitochondrial DNA.
Nevertheless, despite the evidence of prolonged xenograft survival, none of the ten patients had evidence of PERV infection by the molecular and serologic tests that we have done.
[Slide]
So, so far from this limited surveillance, we can conclude that the data are reassuring. However, we would like to highlight that generalization of these results to other types of exposures is limited, and we need to evaluate PERV transmission by type of xenograft. However, our studies illustrate the minimum standard for laboratory surveillance for PERV.
[Slide]
I would like to move to another study we have done that was mentioned by Carolyn this morning, which is looking at the evidence we found of PERV in porcine Factor VIII products. This study, actually, began when we looked for evidence of reverse transcriptase activity in the plasma of pigs. This was part of the Swedish collaboration we have had for pig islet recipients. When we looked at the plasma of the recipients for any evidence of RT activity that could be a marker of the presence of any retrovirus, we did not find any RT activity, however, when we looked at the controls, the pigs that we have tested, we detected very easily RT activity. These are duplicate results from two pigs. This is HIV‑1 infected plasma. There were really equivalent signals.
[Slide]
So, that indicated to us that there is some level of viremia, as you have heard this morning, in pig plasma and, therefore, led us to analyze products of pigs that are made from pig plasma. We analyzed the porcine Factor VIII that is used now in licensed product for use for hemophiliacs.
[Slide]
Again, this is more specific information on the delectability of PERV RNA in pig plasma, and 18/20 that we have looked at were positive. Of these 18, 16 also had detectable RT activity by the Amp‑RT assay. This is another PCR‑based assay. Again, the correlation between detection of both markers was positive, and that indicated to us that we need to look a little bit closer into this product.
[Slide]
So, we tested 13 lots of Hyate C ‑‑ this is the name of the product ‑‑ for both PERV RNA by RT‑PCT and also we tested for RT activity.
[Slide]
We were able to detect the PERV RNA in all the lots tested, 13/13 for this sequence; again, 9/9 for the pol; and 5/5 for the env.
[Slide]
I will show you an example here. You see very strong signals here only when you reverse transcribe. When you don't reverse transcribe you don't see anything indicating that it is RNA in origin.
[Slide]
This is an example from the pol RT‑PCR product.
[Slide]
We also wanted to see whether these sequences are associated with particles. As you have heard this morning, you can do that by looking at reverse transcriptase activity, which is a particle associated enzyme. We were able to detect RT activity in many lots. However, some of these lots had evidence of inhibition of the assay so we had to play around with the testing procedure by pelleting and dilution of the product to be able to dilute out the inhibitor.
[Slide]
However, we were able to demonstrate the presence of RT activity in 10/13 lots tested.
[Slide]
As Carolyn said, we were also interested in seeing whether there is any infectious PERV and we carried out experiments by inoculating some of these lots into susceptible cells, 293, HT1080, and these cultures are still ongoing and, so far, we do not have any evidence of infectious PERV detected that could be isolated in these systems.
[Slide]
The key question here is do we see any evidence of transmission to hemophiliacs that have used this product?  We collaborated with the Hematologic Diseases Branch at CDC that had previously collected a lot of samples from these recipients, and we tested a total of 111 samples and 88 were from the Factor VIII recipients and 23 were from unexposed hemophiliacs. We did blinded testing for antibodies to PERV.
[Slide]
None of the 88 samples were found to be positive; all were negative, as well as the 23 other controls. This is an example of one of these experiments about the seronegative findings we have.
[Slide]
We could try to speculate on what could explain those results, and keep in mind here the scenario we have or the experiences we have in that population with the HIV‑contaminated human Factor VIII, how HIV was transmitted to that population while, in this case, we have had no evidence of transmission so far.
[Slide]
So, you could look at the explanations for the lack of PERV transmission as related to the product itself, like possible presence of the infectious PERV ‑‑ possible absence of the infectious PERV in the product. We don't have evidence for that from the infectivity studies done by us and the FDA. This could be due to a little or no infectious PERV in the pig plasma originally. You have heard some data also from Gillian about the lack of any detectable infectious virus in pig plasma.
However, we still don't know very much about the negative findings in these assays, and what confidence we need to put into negative results using our knowledge today and the information in this culture system. So, we need to take this with caution and say that the negative finding may not confirm really absence of infectious virus.
We could also think that maybe there was some infectious virus originally but that it has been lost or reduced during the manufacturing. The manufacture of that product includes cryoprecipitation, polyethylene glycol precipitation and freeze drying which all can reduce infectivity of retroviruses. However, this manufacture does not include any inactivation by heat or by detergent and this may explain how we were able to detect RT activity in these samples.
Also, there may be factors that are host related and that maybe PERV, which is present in this product, could have been infectious but then these PERVs were quickly inactivated by the complement system that we have that can be triggered by our naturally occurring anti‑gal antibodies. So this could protect the hemophiliac recipients from infection.
[Slide]
I would like to stop here and recognize our collaborators at CDC. This is the team that is involved with all this work, and some collaborators also from other studies outside the CDC. Thank you very much.
DR. AUCHINCLOSS:  Thank you very much. I am sure it is true for the other members, but my questions are building up for the end of the presentations but right now we are going to take a break for about ten minutes and we will start promptly at 10:45 to resume this session.
[Brief recess]
DR. AUCHINCLOSS:  We will resume with a presentation from Diacrin by Jonathan Dinsmore.
Porcine Endogenous Retrovirus Testing in Patients with
Pig Fetal Neural Cell Transplants
DR. DINSMORE:  Thanks a lot.
[Slide]
My task today is just to present the patient testing data that we have generated to date. I wanted to spend a few minutes going over the application that we are involved in using pig cells, and we are using fetal pig cells and these are for treatments of contractable neurologic diseases. The cells are actually implanted stereotactically directly into the brain.  Animal studies and patient data indicate that we may achieve and maintain long‑term survival of these cells. These are stable grafts that maintained within the patients.
[Slide]
The patient data that I am going to summarize for you applies to three separate applications that are in clinical trials currently. One is for the treatment of Parkinson's disease. This is a Phase I safety study. It involves 12 patients. The transplant, as I said, involved fetal pig neuronal cells. These patients received transplants to one side of the brain. A total of 12 million cells were implanted. There are two modes of immunosuppression utilized in this study. Six patients received systemic immunosuppression with cyclosporine and six patients received cells that were treated with a F(ab) fragment, a monoclonal antibody, and this is a proprietary methodology that Diacrin is developing.
[Slide]
The other trial that I will be presenting today is for a trial in Huntington's disease. This is a fatal disease for which there is no currently available treatment. Again, it is a Phase I safety study and 12 patients were involved. Actually, there is an error on this slide.  The dose of cells was 24 million cells. Again, it was a unilateral transplant to one side of the brain. Again, two groups of patients, six receiving cyclosporin and six receiving antibody‑treated cells.
[Slide]
A third trial that I will present patient data for involves the treatment of focal epilepsy. So, this is patients who have intractable focal epilepsy that are refractory to available drug treatments. Again, it is a Phase I safety study. There are three patients enrolled currently in this study of a planned six. Again, fetal pig neural cells with stereotactic delivery directly to the brain, four million cells. In this case, all the patients received antibody‑treated cells.
[Slide]
A little bit about the patient samples that were tested. These were tested at Primedica Labs in Washington. PBMCs were the test sample utilized. For the Parkinson's and Huntington's disease patients these trials were initiated prior to the institution of testing for PERV so we utilized our archival samples which were frozen at minus 70. For the epilepsy patients the trial was initiated after the initiation of PERV testing and, therefore, those patients were tested on a real‑time basis as these samples were collected. For all of these, additional blood samples from the patients are reserved for additional testing, for serological testing or for testing with assays that may be developed as we go forward.
[Slide]
The assay procedure is simple. You have seen it before with the other presentations. DNA is extracted from the test sample. PCR is performed. We utilize the primers which recognize the protease region of the PERV. These were primers which were developed and published in 1997 in Nature Medicine. After the PCR, there is separation of the PCR products from the gel. It is then transferred to a membrane and then probed with a fluorescein tagged probe. After the PCR there is a third oligonucleotide which is used as a probe on a Southern blot and the detection is via a light detection exposure to x‑ray film.
[Slide]
These are the primers that we utilized. These are the PCR primers and this is the probe that is used on the Southern blot.
[Slide]
These assays were validated first before initiating testing on patient samples; to confirm that they were negative, these primers on human cells, human PBMCs and human cell lines with integrated human retroviruses. These same samples were concurrently run with positive controls for human DNA. They were all positive with human beta‑globin primers. All of these samples were samples containing pig cells, either pig PBMCs from the pigs we used for isolating cells or from pig cell lines.
[Slide]
That is just summarized here on a table with the pig cells being positive for the PERV primers, the human lines all being negative but positive for the beta‑globin sequences, and human samples that have various amounts of pig DNA spiked are all positive for both probes.
[Slide]
This is just an example of what you see on these Southern blots after running these assays. So, human PBMCs are negative. Human PBMCs with various amounts of added pig DNA are positive. Pig PBMCs are positive, with various amounts of pig PBMC loaded. Human cell line and cells lines with integrated human retrovirus are all negative. Run on the same gel are the positive pig controls. So, with the validation of the assay we went forward with patient testing.
[Slide]
This summarizes the results for the Parkinson's disease patients as well as control samples which were taken from Diacrin employees. The time sampled is listed here. They are all negative for PERV sequences; all positive with the pig DNA spiked samples.
[Slide]
There were similar results for the Huntington's samples, again, taken at various times post‑transplant.
[Slide]
Then, for the epilepsy patients, as I said, there is real time testing. So, baseline, three weeks, two months, three months and six months. More samples than this were actually collected but these are the samples that were tested, and they have all been negative. There is one missing here because a blood sample could not be drawn from the patient on that day.
[Slide]
This is just a representation of what one sees on the Southern blot analyses. There are duplicate samples here of patient PBMCs that are all negative; those same samples spiked with various amounts of pig DNA are all positive.
In addition to these patient tests that I have shown you, we have an ongoing Phase II clinical trial for Parkinson's disease. It is a blinded trial, therefore, I am not presenting the data on those patients here. PBMCs from those patients are being tested in real time and, to date, all of those samples have been negative as well.
One final note, although I am not going to present it here, we have done the cocultivation assay that you have heard about today with fetal pig cells, and those have been negative for the presence of any infectious virus being activated from the donor cells themselves. That is it.
DR. AUCHINCLOSS:  Thank you very much. Our next presentation is by John Logan, from Nextran.
Development of Assays for Monitoring Baboons and
Humans for the Transmission of Porcine Endogenous Retrovirus
DR. LOGAN:  Thank you.
[Slide]
I would like to talk today about the development of assays for monitoring both baboons and humans for the possible transmission of porcine endogenous retrovirus sequences, and I would really like to split my talk into actually three parts. In the first part I would like to talk a little bit about experiments done in vitro in cells and culture and coculture assays looking for potential transmission. I would also like to talk about looking in human patients who have been exposed to porcine livers and extracorporeal circuit. Then I would like to spend the last part of the talk talking a little bit about baboons that have been transplanted with various pig tissues.
[Slide]
To talk first of all about the issue of coculture, we performed a series of coculture experiments really to look at the possibility that transmission of PERV sequences from both hepatocytes and also stimulated lymphocytes from our transgenic pigs using human 293 cells as the indicator cell line. Our positive control in all these samples was PK‑15 cells, either cocultured with 293 cells or virus produced from PK‑15 pigs infected directly into 293 cells. The lymphocytes themselves were stimulated with PHA and IL2.
[Slide]
This is just a summary of that data. We actually analyzed the data in a number of different ways. We looked at both hepatocytes that were non‑irradiated and irradiated hepatocytes, and stimulated lymphocytes, as well as PK‑15 cells. We looked for the production of virus after 35 days of coculture in the 293 cells and we assays for the production of infectious virus using standard RT assays, as well as RT‑PCR assays of the supernatant at the endpoint of 35 days. All of these assays of hepatocytes, irradiated hepatocytes or lymphocytes from the transgenic pigs were all negative.
When we looked by DNA PCR at the end of the 35‑day period, the results in the case of the lymphocytes were clear. There were no PERV sequences; no porcine genomic sequences. In the case of the non‑irradiated hepatocytes in one set of experiments it was negative. In the other set of experiments it was positive for PERV sequences. The results were positive for pig genomic DNA. In the case of the irradiated hepatocytes, one set of experiments was negative.  One was very weakly positive with PERV. It was variably positive for porcine genomic sequences.
Our conclusion essentially from this slide is that we certainly could see no evidence of the presence of infectious virus isolated from either hepatocytes or lymphocytes from pigs.
[Slide]
We then went on and we looked in various assays for both patient and baboon samples. We looked at two sets of assays. The first set of assays was the assays you have heard about in detail this morning, which are really PCR‑based assays looking at various probes for the PERV genome, either protease regions or envelope regions. We also developed an antibody‑based test to try and look for the presence of antibodies, either in patients or various human populations or in baboons after transplantation of pig tissues, to see if that could be another methodology to look for potential evidence of exposure to virus.
[Slide]
If you look first at patients that underwent extracorporeal liver perfusion, in general the patients were all negative using PCR‑based analysis of peripheral blood mononuclear cells.
[Slide]
There was one exception to that. That one exception we saw in two patients that were analyzed at the same processing time point but at different points, and those patients turned out positive in the PERV assay.
We then had a problem with getting enough DNA to see if they were positive for porcine genomic sequences and we couldn't equivocally say whether they were positive or negative. At subsequent time points for both of those patients were seen to be negative, and we felt the most reasonable explanation at that time was a contamination issue although, as Carolyn said this morning, we certainly could not exclude the possibility of a transient low‑level infection.
[Slide]
We have modified our PCR technique in order to make sure we reproducibly get enough DNA for analysis. We actually store DNA now as well as lymphocytes. And, we have modified our DNA prep method for lymphocytes to get DNA more efficiently amplified.
[Slide]
Let me change gears a little bit and talk about the development of an antibody‑based test. Clearly, the PCR‑based technology is very useful in terms of level of sensitivity but can suffer from problems of contamination, and we wanted a second methodology that we could utilize to look at potential infection sites throughout the body, as well as a method which would give us complementary data in the case of a PERV positive. For that reason, we tried to establish some serological‑based assays.
In the case of PERV this is a real challenge because, as you well know, there are no animal model systems which we can elucidate what potential levels of antibodies or time periods that we would obtain. So, we attempted to do this by trying to assemble various components of the assay in order to try and validate that we were capable of detecting anti‑probe antibodies.
[Slide]
To do that we required an antigen source. In the first instance we used recombinant gag protein in a number of different systems, bacterial and mammalian. We have produced anti‑gag antibodies against the antigen produced in those systems. Then we tried to development an assay format using these antibodies as positive controls which would actually detect the presence of antibodies.
[Slide]
Let me turn now to the production of antigen. Just illustrated here are two systems that we utilized in E. coli. One of them was a fusion system with a gag coding sequence used to the protein A region. We could produce fusion protein containing gag. The second system was used as GST fusion, again with a recombinant gag sequence, and again, we could produce antigen and purified anti‑antigen. Those antigens then were used for two different immunization strategies, one in mice for production of monoclonal antibodies and the other in rabbits for production of polyclonal antibodies. In addition to that, we also expressed the gag sequence in CHO cells as a separate indicator system for the development of antibodies against protein.
[Slide]
As we look at this system, what we have done here is we have taken two cell lines, one of Chinese hamster ovary cells, the second of Chinese hamster ovary cells which actually express the gag antigen. In this case the gag antigen has a myc tag on it to verify that we could detect protein expressed in these cells.
In this case, this is mouse immunization. Obviously, the mice before exposure to the immunizing antigen has no detectable antibody either in CHO gag or CHO control cells. With immunization we see a specific increase in antibody binding to the CHO gag cells but not to the CHO control cells. In the case of the myc, that is just a control to show that we can detect this antigen produced in the cell.
[Slide]
We went on to look more closely at these antibodies. In this case we used five different test subjects. One was PBS media. one was a monoclonal antibody isolated against gag. One was rabbit pre‑immune serum; and one was rabbit post‑immune serum. With an ELISA type format, we then used five different antigens. We used Chinese hamster ovary cells; CHO cells with the gag protein; basic E. coli cells and E. coli cells that contained the GST gag fusion, as well as purified GST gag fusion protein.
We looked first at monoclonal antibody lanes. We looked first in CHO cells. Obviously, monoclonal antibody doesn't bind CHO cells. The CHO cells expressed in gag cells by significant binding. It doesn't bind to E. coli extracts but it does bind to E. coli extracts containing the gag fusion protein as well as the purified gag antigen.
In the case of the rabbit pre‑immune serum, we see no binding to any of the substrates. In rabbit post‑immune serum, no binding to CHO cells; significant binding to CHO gag cells. However, in the case of rabbit post‑immune serum, this is immunized with protein A gag fusion which has E. coli protein so we do see production of E. coli antibodies but there is some stimulation on the GST gag E. coli cell.
[Slide]
This is just a summary of series of monoclonal antibodies that we have isolated against the gag protein, the polyclonal antibodies that we have utilized. All of them bind in an ELISA type format. Some of them bind quite well on the Western blot format, and some of them actually identify infected 293 cells in fluorescent‑based cell assays. So we felt we now had a good resource of antibodies in order to start the process of trying to develop an antibody‑based test.
[Slide]
We expressed three different PERV gag fusion proteins. We confirmed integrity of the gag reading frame by sequencing peptides. We then immunized mice and rabbits with this fusion protein.
[Slide]
We tried to development an assay. We decided initially to utilize PERV‑infected human 293 cells as a source. Clearly, the PERV‑infected 293 cells produced more of the gag antigen, produced envelope and, therefore, in theory we could see production of many different potential antibodies produced.
[Slide]
This is just a very quick look at some data to show that we can, in fact, detect specific binding to PERV‑infected human 293 cells, and these are 293 cells obtained from Robin Weiss' group. We looked at rabbit pre‑immune sera. There is no binding to either uninfected 293 cells or to 293 cells which have been infected with virus and PK‑15 cells. With rabbit post‑immune sera, no binding to 293 cells, but significant binding to infected 293 cells. And, the same with monoclonal antibodies, no binding to 293, good binding to 293 infected cells. So, we thought that these cells would be good in an ELISA type format initially just to look for the production of antibodies against PERV.
[Slide]
We decided to do that initially looking at patient samples, and this is actually looking at patients' post‑liver perfusion. We are actually looking at post‑allografts. These patients are on immunosuppression. We looked initially at the anti‑porcine response that these patients have against pig cells in general and not just against PERV. What we see is that in the first few day post‑perfusion a rapid induction of anti‑gal antibodies with IgG and IgM. We actually failed to detect any significant level of binding to other pig antigens. So, the antibody response seemed dominated, at least in this case, by alpha‑gal antibodies. So, these patients saw porcine tissue, and that porcine tissue stimulated an immune response.
[Slide]
We looked now in an ELISA type format to see if these patients over time had expressed antibodies which could now recognize PERV‑infected cells. We looked first with PK‑15 virus‑infected 293 cells. This is rabbit serum pre‑immune and post‑immune, and we see a significant binding to 293 cells. If we now look at these patient samples over time, both IgG and IgM, we see no difference in binding between uninfected and infected 293 cells.
If we look in a Western blot type format where we now take extracts from 293 cells, either infected or uninfected, and we also do an additional test where we take uninfected 293 cells and add pertussis toxin ‑‑ we add pertussis toxin to show that these patients can, in fact, have antibodies that we can detect.
We looked first in the case of the monoclonal antibody to look at infected 293 cells versus uninfected 293 cells. We see binding to gag‑related peptides in infected cells but not uninfected cells.
What is consistent in this patient and also in the other patient is that we no specific binding or no antibodies with specifically recognized bands present in only 293 infected cells as opposed to 293 uninfected cells. We do see good binding to pertussis toxin.
[Slide]
We also went on and apart from analyzing these patients, we also looked at the human population in general in a small part, and we looked at two given populations. We took a random human serum population, and also a population which should have greater exposure to pig cells. In this case, we looked at swine practitioners. These are veterinarians who specifically work with swine on a day‑to‑day basis and have reported during the last year to have lots of blood contact with pigs.
If you look in an ELISA type format again, these are the control antibodies. We see good binding only to infected, not uninfected 293 cells. But in case of either the random human population or the swine practitioner serum we see really no evidence of any difference in binding between infected and uninfected 293 cells.
[Slide]
We have also done that same experiment using a Western blot format. I show you a few examples of that here from the random population as well as the swine practitioner population and, again, there is no difference in binding between infected or uninfected 293 cells.
[Slide]
We then also went on to look at pig to baboon xenografts ‑‑ and I told you the xenografts have actually been done some time ago ‑‑ to look at DNA PCR‑based techniques as well as antibody‑based techniques to see if we could detect presence of PERV sequences. Realizing that there is some controversy over whether PERV could infect baboon cells or not, and I think we have heard today results both ways in this particular assay, at least with regard to one primate species, we felt, however, it was still worthwhile to look at these samples rather than to wait to have conclusive evidence either way.
[Slide]
We looked first of all, and this is just one example of many that we have done ‑‑ we decided to adopt a slightly different regime in which we looked at xenografts that in this case had only been in for a few days, and then looked at both the antibody response of the baboon against the xenograft after a heterotopic heart transplant or the response against specifically PERV antigens in 293‑infected cells. After removal of the xenograft, the baboon in this case was maintained on immunosuppression for a further 90 days. So, we put the xenograft in with immunosuppression, removed it and continued the immunosuppression.
What we see is that there is actually a lack of fairly strong anti‑pig response in these baboons against pig tissue. It is dominated again by alpha‑gal response. I only show the alpha‑gal response in this case.
However, if we look at the ELISA technique to pick up potential anti‑PERV antibodies, the controls show good binding again. However, in the case of various time points, looking at 293 infected and uninfected cells, we see no difference in binding. So we see in the baboons again an anti‑pig response showing they can respond to pig antigen but no anti‑PERV response.
[Slide]
We have also looked by DNA PCR. In this case we show lymphocytes. This is just an indication of the sensitivity. It essentially goes down 1 cell in 106 cells. We can detect the PERV sequences. In this case, we show 3 baboons here, and these are baboons before transplant and 90 days after transplant, and we see no evidence of PERV sequences on the lymphocytes. We are following this up and looking at multiple tissues throughout the baboons but that analysis is not yet complete.
[Slide]
We have looked, in this particular case, at longer surviving grafts. Again, 90 days post‑transplant with constant immunosuppression we see no evidence by PCR probe sequences on lymphocytes.
[Slide]
The conclusion then in terms of an anti‑PERV summary is that we really see no evidence of anti‑PERV antibody either in the normal human population or in swine practitioners. In the baboons we have looked at today, again, no evidence of anti‑PERV response in antibody‑based tests; and the same thing in terms of control and patient serum.
Clearly, we will continue to expand these studies to look at multiple tissues in baboons at multiple different time points with longer surviving xenografts, and we will continue to follow‑up the data. Thank you very much.
DR. AUCHINCLOSS:  Thank you very much. To conclude the morning's presentations, we will hear from Khazal Paradis, from Imutran, on retrospective patient studies.
Retrospective Patient Study ‑ Testing Strategy and Methods
DR. PARADIS:  Good morning. I first want to apologize that I am going to be presenting again just the patients that are participating in this study, which we have code names XEN 111, but I am not going to be presenting the results. I particularly want to apologize to those who have heard this apology several times.
[Slide]
XEN 111 is a study that had multiple testing labs, many collaborators, and has taken a lot of time to get everything together. The objectives of the study were to detect circulating endogenous porcine retrovirus or antibodies to PERV in subjects who had been in intimate contact to living porcine tissue. So the first aim was to search for evidence of transmission of PERV in those who had been in intimate contact.
[Slide]
The secondary aims were to search for horizontal transmission or human to human transmission of PERV in intimate contacts of patients who had been found to be positive for PERV by either antibody or PCR screen.
Then we also looked retrospectively to see if there were any unusual signs or symptoms in patients who had been found to be positive by PCR or antibody screen.
Finally, in those who had been found to be infected we would then look to see at the source pig line, the sequence of the PERV and compare it to the PERV that we would find in the patient.
[Slide]
The methods ‑‑ after a informed consent we obtained a short medical history and a questionnaire on exposure to pig tissue. Peripheral blood mononuclear cells and serum were prepared and aliquoted at the sites, frozen at minus 70 and then shipped on dry ice to the testing labs.
[Slide]
In terms of patient samples, we have 100 patients that come from St. Petersburg, Russia where they have a particular extracorporeal splenic perfusion for what they call immunotherapy. This is usually severe burns or cancer. These patients had their perfusion either on the same day as when we took the sample or up to almost ten years before.
We had 28 patients who were treated with the HepatAssist device that you heard Zorina Pitkin talk about earlier today. These patients come from the United States, France and Israel. We had 15 patients who had skin grafts up to 10 years ago, from Germany. Then there were 14 patients who had received pig islet cell transplants, and 9 of them were in association with a kidney transplant, and these patients have been immunosuppressed since. These come from Sweden and Auckland, New Zealand. We then have 2 patients who participated in an experiment with extracorporeal kidney perfusion. These come from Sweden. These lasted for 15 minutes and 65 minutes. Finally, we have 1 patient from Montreal, Canada who received extracorporeal perfusion through a whole pig liver for about 4.5 hours before having a successful liver transplant.
[Slide]
As an analogy to the microchimerism that we see in allotransplantatiion whereby cells from the transplant migrate and traffic through the recipient's body, and also because we knew that we had patients who were receiving extracorporeal splenic perfusion the day that we were to take the samples, we had to have a strategy to test for microchimerism.
You have already heard about this, but just to give you a refresher course, all pig cells contain PERV DNA, and on average you will see approximately 50 copies of PERV per cell. As you heard also from Gillian before, each pig cell contains approximately 2500 centromeric sequences so that if you did a ratio, you would do a ratio of PERV to centromeric copies and in a normal pig cell it would be approximately 50:2500. If you find a patient who has microchimerism and infection, there will then be more PERV than what you would expect for just a pig cell alone. Therefore, the ratio of PERV to centromeric would be treater than 50:2500, and indicative of infection. Of course, as you heard also, low‑level infection cannot be excluded in the presence of microchimerism using this method.
[Slide]
Here is our DNA testing strategy. PERV DNA was looked for by PCR, and this could be attributed to either a pig cell or a human cell. If you did not find any PERV DNA, then it would be considered no infection detected. If pig DNA was detected, we would then move on ‑‑ and this would be considered microchimerism if you had evidence of pig centromeric DNA, and this was done at GTI, or if you found pig mitochondrial DNA this would be microchimerism. If you found PERV DNA but you did not find any pig DNA, then the patient would be considered to be infected. If you had any evidence of microchimerism, then you would do the ratio which I just mentioned of PERV to pig centromere. Because of analogy to feline leukemia virus where the virus is sequestered in the salivary glands and then saliva can act as a reservoir, in patients who would be found to have microchimerism we went back and tried to obtain saliva to then test for PERV barriers in the saliva by RT‑PCR. If you found a patient had microchimerism, we then did the ratio. If the ratio was equivalent to what you would find in a pig cell the patient would be considered to have no infection. If the ratio was larger than what you would see in a pig cell, then the patient would be considered to be infected. For infected patients, we would then go back and test the body fluids for PERV, as well as test the close contacts which we define as persons living in the same household.
[Slide]
That was to test for PERV DNA, which would be testing for the latent type of PERV infection. To test for actual viral infection and looking for virions in the serum we went to testing by RT‑PCR. If the test was negative, then you would consider the patient to have no infection detected. If the RT‑PCR was positive, then you would consider that the patient was infected. Then we would test the bodily fluids by RT‑PCR as well as test the close contacts.
[Slide]
Finally, we have tested for the DNA and the RNA, and for a historical overview of the patient to see if they had been exposed to PERV virions we did the Western blot looking for antibodies to PERV. If it was negative, the patient would be considered seronegative. If it was positive, the patient would be considered seroreactive. For seroreactive patients we would then test the saliva by RT‑PCR as well as test the close contacts.
[Slide]
To give you a bit of more detail as to the actual tests ‑‑ you have heard about them already today, at GTI looking for DNA by PCR for PERV they were able to detect 1 PERV DNA copy/half a million human cells, and the false‑negative rate for 10 copies would be 0.03 percent.
At the CDC they did 2 PCRs. One was looking for PERV pol sequences, and they could detect down to 5 PERV copies/150,000 cells, and 1 PERV gag DNA copy/150,000 cells, with a false‑negative rate, based on empirical value, of less than 0.02 percent.
[Slide]
Finally, the microchimerism assay ‑‑ at GTI they used the pig centromeric assay, where they could detect 10 copies/half a million cells, and here the false‑positive rate was less 0.83 percent. At the CDC they used the pig mitochondrial assay where they were looking for 1 copy/150,000 cells, and the false‑positive rate here was less than 0.02 percent.
[Slide]
In terms of the other assays, the Western blot assay looking for antibodies was carried out at Q‑One Biotech. There they used a recombinant gag and if this was positive, they would then test against the whole virus looking for p30. At the CDC they used infected human 293 lysates looking for gag and p27.
For the RT‑PCR looking for virions in serum, the GTI was able to detect 400 particles/ml, with a false‑positive rate of less than 1 percent. At Q‑One, looking for PERV virions in the saliva, the sensitivity of that assay was 1000 particles/ml, with a false‑negative rate of less than 1.25 percent.
[Slide]
The interpretation of these assays are the following: No infection detected we be if we found no PERV DNA nor any PERV RNA in the patients. Microchimerism was defined as if ever we found any porcine genomic DNA in any of these patients. Infection with PERV was if we could find circulating virions in the serum and/or presence of viral DNA not which was not accounted for by microchimerism and/or if there was presence of virions in the saliva. Potential exposure to PERV would be if there was seroreactivity to any PERV antigens.
Now, these assays were quite complex. Although the study was designed two years ago, we have only completed the testing of the last five patients approximately ten days ago. We have had confirmatory testing by the CDC because we knew that this was a public health concern so that if anything had been of concern, this would be known to the CDC and reported appropriately.
We hope that over the summer we will have the report which will then be submitted to the regulatory authorities, and in particular to the FDA. Thank you.
DR. AUCHINCLOSS:  Thank you very much. Now, it is essentially 11:30 and our next formal presentation is scheduled for 1:50, and between now and then we have, I think, three things to do. We have questions from the committee to our various speakers; we have questions to the committee from the FDA; and we have lunch to have. To give you a sense of the schedule that I think we will follow here, we probably won't break for lunch quite at 11:50. That is only 20 minutes. I suspect we will go for about 45 minutes before we break for lunch. Then I won't give you a full hour for lunch, so we will be back here by one o'clock, or something. The discussion of the questions to and from I suspect will amalgamate into one process here, and I will try to make sure that over the course of the next essentially two hours we cover the issues that you have expressed to us. If I haven't by the end of that time, we will come back to them and make sure that we have.
So, what I am going to do is just initiate questions from our committee members, and what I will then try to do is capture topics and keep us focused on individual topics as we go so that it won't necessarily be one person and then another person, and one speaker and another speaker. We will go by topic. Just to get the process started here, I thought I would turn to Jonathan Allan because his list of questions is growing long, although I know lots of people have long lists, and also he is the first in the alphabet. Jonathan, do you want to start the questions for us?
Ques from the Committee
DR. ALLAN:  Yes, why not?  I have several questions but I will just start with the first speaker, Gillian. As a point of information, you were demonstrating that you were able to get microchimerism with the pig to baboon studies, in the primate studies that were performed. I really liked your real‑time PCR that you have done because it is very quantitative. So, one of the questions I had was were you able to quantitate exactly how many pig cells you were finding in the primates and over time, were you able to look to see whether the number of pig cells diminished or were stable?   The third part of that question is whether or not different organs gave you the potential of having more numbers of pig cells in terms of microchimerism?
DR. AUCHINCLOSS:  Is the question for Gillian Langford?
DR. ALLAN:  Yes, for Gillian, wherever she is.
DR. AUCHINCLOSS:  Will you come to the microphone, please?  We will be asking questions of all of our speakers. So, be prepared to step up to the microphone.
DR. LANGFORD:  In answer to the first part of your question, yes, we were able to quantitate the level and the number of pig cells, and we found that in most of the samples we looked at it varied from between 1‑20 pig cells per sample. We didn't really find a correlation between the tissue types that we analyzed and the level pig cells, although in some lymph nodes we did find higher levels of pig cells than in some of the higher tissues that we looked at but it wasn't universal throughout all the samples that we looked at.
DR. ALLAN:  Did it vary based on when you took the samples?
DR. LANGFORD:  All the samples that we analyzed were terminal autopsy samples so we don't have any serial samples taken from our primates. We are collecting serum at the moment to do a study to look for viral release but they were all terminal samples.
DR. ALLAN:  Which is another point for discussion, which is when you take the samples and when you test them. If you test them late after the transplants happen you may get a different result than you would get if you would take them two days after the transplant or a week after the transplant. So, those are things that one needs to consider. If you take it at the endpoint you may get a negative whereas the cells have already gone to tissues.
DR. AUCHINCLOSS:  Undoubtedly, that is true. What I would like to do is to take your question about the microchimerism and now focus on the microchimerism issue for a few minutes here, and then we will look for a new topic. All right, is the committee ‑‑ and I am looking for comments from committee members here ‑‑ is the committee satisfied that you can detect viral infection, should it occur, from microchimerism with the dilutional assays that are currently being used?  Marian?
DR. MICHAELS:  I think all of the speakers have actually pointed out that while it is able to detect infection versus microchimerism if you have a greater amount of PERV than you had anticipated based on the correlation of how much there should be of the pig cells versus the PERV to start with. I think low‑level infection isn't going to be able to be detected. I think everyone pointed that out.
DR. AUCHINCLOSS:  Yes, please?
DR. ONIONS:  First of all, I think all of the groups have presented developing assays that are very sensitive and well controlled, and I think that is to be congratulated on, the work they have done.
I think we are probably pushing these assays almost to the limit and I think this is going to raise some problems later on when we start to compare data from different groups. We are pushing the detection of microchimerism to a limit and we are pushing the detection of PERV to a limit. So, we are really asking a lot more of these assays then of conventional assays for HIV or for HTLV.
But my comment would be that I think that it is going to be important that each group actually validates in the background positive, let's say a 293 cell that contains two or three copies of PERV; that they then validate in those assay systems what level of infection can they detect above that. In other words, we actually do have real data, validated data of what the level of detection would be in various backgrounds of microchimerism because, in fact, some of these assays will have different backgrounds. They are all interesting approaches but they will have different backgrounds. For instance, if you do a cell dilution, that would give you potentially a very discrete signal but its sensitivity is going to be much lower than if you use a whole DNA sample, say. So, I think we need to have those data.
DR. AUCHINCLOSS:  Can you expand this for me, what do you mean when you said "we are pushing" these assays to the limit?
DR. ONIONS:  Well, it is the very issue of when you start to look at ratios when it is necessary. You either have to look at discrete cell populations or you have to look at ratios with some kind of approach that GTI has used. They are using excellent technology. They are using quantitative PCR technology, the best that is available at the moment. But you are inevitable left between a ratio between two results that are quantitative, each with their own level of variation. So, you are then always going to create a grey area where you are not sure whether you are looking at microchimerism alone or microchimerism in the background of infectivity. The same sort of arguments apply to the also excellent work done by the CDC. The same sorts of caveats apply.
So, I think what we have to do is actually to validate those systems to actually show in a real sense what infectivity they would pick up in a background of microchimerism.
DR. AUCHINCLOSS:  Okay, so there is a general comment that the assays that are currently being used would not detect a low level of infection in the presence of microchimerism, and you are suggesting that it would be nice if we could figure out exactly what that low level would be that we would miss.
DR. ONIONS:  It is not quite what I said. It is not an implied criticism. I think we are actually at the limit of what you can do.
DR. AUCHINCLOSS:  I didn't mean it as criticism.
DR. ONIONS:  All I am saying is that we need to know what level of infectivity we would pick up.
DR. COFFIN:  In theory at least it would be possible to improve the situation with some sort of sorting or separation technology. I wonder if any of the representatives would care to address whether they would consider that with the practical issues ‑‑
DR. AUCHINCLOSS:  So this is a question for either the committee or for the speakers, is there a different technology that would enable us to detect infection versus microchimerism perhaps by sorting cells ‑‑
DR. COFFIN:  Sorting cells, separating human from pig, for example.
DR. SALOMON: That is a great idea theoretically. The limitations of that, at least in looking at microchimerism in, say, the human transplant situations had been that you really are talking about very few cells in these tissue compartments. So the idea, let's say, of sorting 20 pig cells out of a large population ‑‑ and then what you want to do is analyze the tissue without the pig cells. Right?  I think that would probably be beyond the technology. Anyone could disagree with that but I would be skeptical.
DR. AUCHINCLOSS:  There are more comments on this possibility of other assays.
DR. VANDERPOOL:  I just want to ask a question, hopefully that we can keep in mind from the standpoint of where we came from and where we are going. We had a presentation to indicate that the last time we met ‑‑ certainly in October of '97 ‑‑ there was such concern over particles identifying endogenous porcine retroviruses as to put experiments on hold. Now, as I heard the speakers with my somewhat layman's ears, even though I am getting more and more educated by the week, I put stars by the presentations that were heartening in terms of former worries we had in terms of controlling and identifying infection.
Now, what I would like for the committee to address at some point is where are the worries, and how big are then?  I mean, do we really have worries?  And, if we have worries that there are infections that are being produced, what are they?  Of what nature?  How significant are they?  And, I totally agree that we should start with microchimerism, but I want to keep that larger perspective in mind. Where have we come now?  Are we in a place of greater comfort?  Of very good comfort?  Or, do we still need to worry and ask for more things?
DR. AUCHINCLOSS:  I think that is a fair comment. Let me go back to the big perspective. What I think we have heard from the morning is that a lot of data is beginning to accumulate that is essentially negative with respect to actual infection of humans having been exposed to pig tissue.
The issue that we are now addressing, over the next hour and a half or so, is how good is that data?  Can we trust the data that we have?  Are we interpreting it correctly, or are there other kinds of data that we should be looking at?  I think that is the issue that we are now addressing. The answer to your question is no. What we have learned so far reads negative. How good is that negative?
Back for a second now, I saw Jonathan shaking his head about there is no other assay that will detect infection in the setting of microchimerism except a dilutional approach.
DR. ALLAN:  Well, the context that I would like to look at is you have microchimerism, and the next part is the infection which is what assay ‑‑ RT‑PCR versus whatever. Then the third part of that is the antibody‑based assays ‑‑ are they good enough?  So, are the RNA‑based assays which detect virus, are they good enough from what we heard this morning?  The second part is are the antibody‑based assays good enough to detect ‑‑ from the negative results you are hearing today the question is are those negative results good enough?  Are they truly negative results?
DR. AUCHINCLOSS:  That is exactly the question. I want to come to the antibody issue for sure, but any other ideas?
DR. SIEGEL:  I didn't mean to interrupt but I did want to address the question and comment that Dan Salomon made a minute ago. With current sorting technology, if you have a rare population, say 1/1000 or even rarer, it can be very difficult to quantitate that population or to purify it because there will be in the general population other cells that will create noise and that will look like the rare population. So the purity of the rare population, if you were to try to sort it, may be very low. But it should not be particularly more difficult to remove such a population than to remove a more common population providing you have whole cells and you have antibodies that selectively can identify that population.
It seems to me that you may also remove a small number of cells that you didn't want to remove. That shouldn't probably create a problem in this context. It seems to me it is a technology that ought to be looked at. If you get less than 100 percent removal of contaminating porcine cells from a specimen the expectation may be that while you may not totally eliminate the problem you should see, if you were to get 90 percent or more removal, a significant shift in the ratio of, say, centromeric DNA to PERV DNA if there really is PERV infection and you are selectively removing the microchimerism.
So, I am not sure this is something that is feasible to do routinely but it certainly is a testable hypothesis that this can be useful. Much as David Onions was saying that we look at the sensitivity of the test to distinguish between microchimerism and infection, it seems it would be easy enough to also look as to whether that can be improved so that when samples are positive one might selectively study those samples, assuming they are whole cell samples as opposed to extracted DNA samples. One might then study those samples.
DR. CHAPMAN:  Just to clarify a point, my interpretation ‑‑ and I ask Onions and Allan and Coffin to correct me if they feel this is incorrect, but I think what you were trying to communicating was not that you think these particular assays that have been presented are being pushed to the limits but that all of the assays and approaches we are using are being pushed to the limits in terms of the competence of detection at the limit we are dealing with, and the confidence we can place in that determination between infection and microchimerism. And, barring a major conceptual or technological advance, we are probably struggling in an area in which our currently available technology, even with refinements on the present efforts, is not going to be able to give us a great deal more confidence than we have at present. Is that a proper interpretation of what you are saying?
DR. ONIONS:  I am not quite sure, but I will try to restate it. The limits of sensitivity and the limits of detection have been defined for these assays. So taking the limits of sensitivity as being usually lower, we know from the comments we have heard about quantitative PCR we can detect very few proviruses in the background of 105. I mean, it depends on which group you go to but we are talking about a few proviruses.
The routine limits of detection are usually put high to allow for statistical variation and various other factors. That is fine; that is understood. But these cannot be refined better than that with our current technology, in my view.
My problem I think is quite simply this one of we have this unusual problem of microchimerism. We have to relate a distinction between microchimerism and low‑level infection. Inevitably, however good your assay is, you are going to have this grey area. All I am really just saying is that I think it would be useful to try and actually validate exactly what we can pick up routinely as low‑level infection versus microchimerism. That is my comment.
My second comment would be that if that is all we are worried about, we are not worried about a great deal because if that is routinely the levels that we are going to be worried about, that is probably not a good sign. It means that we are not going to be hitting a major problem.
My caveat to all of that, of course, is that the kinds of work that have been done so far are not on the kinds of patients that we are going to be concerned about, which are the ones that are going to have immunosuppression and, on the whole, have not involved animals where we might not have complement lysis of virus because they are coming out of transgenic animals. With those two caveats, I think the data are very encouraging.
DR. AUCHINCLOSS:  Those are three really important points. If we get down to this level it would be kind of reassuring but we are not looking at the patients or the donor organs in the right combinations that really are the test ‑‑
DR. HIRSCH:  Or perhaps the tissues that you need to be looking at. I mean, we asked a couple of years ago that PBMCs be routinely tested, and everybody came here with presentations about negative PBMCs. On the other hand, we have heard that PBMCs may not be infectable, given the caveat that Dr. Onions said, that under certain circumstances it is conceivable they could be but, nevertheless, we have no data that they are. And, the question is should we be continuing to ask all the companies to rigorously look at PBMCs and be assured, perhaps falsely assured that represents no infection because you can't find it in PBMCs. If you are putting tissues in brains or you are using hepatocytes, then PBMCs, to me, would seem to be the wrong area to look at.
DR. AUCHINCLOSS:  This is going to become our next topic of conversation, which is what tissue should be sampled, but before we do that there is a speaker that wanted to be recognized that I haven't gotten to.
DR. GORDON:  Thank you. I would just like to ask a lay question ‑‑
DR. AUCHINCLOSS:  Just identification, if you could.
DR. GORDON:  Oh, I am sorry. My name is Allistair Gordon. I am with the Islet Foundation, in Toronto, Canada. The question I would like to ask is a lay question relating to the whole point of distinguishing between microchimerism and low‑level infection. If we have some results in which we have that ambiguous outcome, will the passage of time not resolve it?  In other words, will that low‑level infection not become a more unequivocal infection over time, especially in some of the patients that were tested that were immunosuppressed?  And, in the event that it remains indistinguishable from microchimerism, then does that tell us that we don't have a very virulent infectious agent?
DR. AUCHINCLOSS:  Anyone on the committee want to respond?  Jonathan?
DR. ALLAN:  I think that is a beautiful point, which is if you are trying to decide if it is low‑level infection either it will go negative, and that is okay, or it will increase, and that is not okay. If it increases or even if it doesn't increase, if you have active infection in the PBMCs or any tissues in the human you are going to get antibodies probably. So, even if you lose the detection system for molecular approaches you are still going to get the antibody approaches.
DR. AUCHINCLOSS:  And if it is really infection I want to know pretty quickly about it but, never mind, I think we have made the point here.
I promise we are going to come back to the antibody assay. We do want to come back to the antibody assay but right now the topic on the table is are we looking at the right tissues when we look at peripheral blood mononuclear cells?  Comments?
DR. HENEINE:  Walid Heneine, CDC. I want to come back to your original question, which is how much can we trust these negative data given the newly developed technologies and the limited validation we associate with them?
I just want to point out that the new information we have acquired when we have applied those new assays, for example, the data we showed on detection of viremia in pig plasma ‑‑ this is new information that we were not aware of when we applied RT‑PCR or reverse transcriptase testing to pig plasma and became aware that there are some free particles that are found in this compartment. Applicability to factors derived from pig plasma, porcine Factor VIII, when we used those techniques, again, we were able to define PERV in these factors.
So, given the data, one should not take lightly the results we are seeing, the negative results we are seeing. Unfortunately, we are dealing here with several limitations, for example, for the serology assay, and I know we are going to discussion this further, but the problems we have there seeing whether these assays can really detect antibodies in infected individuals is the fact that we don't have any infected person or animal that we can test.
DR. AUCHINCLOSS:  That we can use as a positive control.
DR. HENEINE:  Exactly. Unlike with an HIV‑infected person, we don't have a pool of infected people that we can use to evaluate the sensitivity of the assays of 99 percent, 95 percent or 50 percent. So, keep in mind those limitations that are inherent with us, and we have to live with them and move forward.
DR. HIRSCH:  Along that line, could I just ask you a question while you are still at the microphone?  You correctly say that there aren't any infected humans that we know of, and we have heard a lot about that non‑human primates probably aren't a very good model system, at least the baboons and other ones we have heard about. There was some data in our materials about cats and mink being perhaps more susceptible. Is anybody trying to develop models in these kinds of animals?
DR. HENEINE:  Yes, this is what John Coffin actually touched on in his talk. The discussion at the Banbury Center meeting was focused on really trying to develop an animal model where we can evaluate all these parameters that so far we are not aware of. But there are several ways one can approach that situation other than now looking at xenograft recipients in baboons, and whatever.
One question that was discussed was what if we go and inoculate high virus titers into baboons or other animal species trying to see whether a persistent infection can take place, and then study kinetics of viremia, and so forth.
DR. ONIONS:  Maybe I could just comment on his last point. I think he is right. I think when we look at the permissivity of a particular species for infection, it should always be borne in mind that the infection in vitro does not always mean you can infect in vivo. I think Karen, in her excellent summary of this meeting, pointed out that you can give chemografts into rat cells in vitro but it has not been possible to infect rats, as far as I am aware, in vivo.
On the other hand, we do have some primates now that Gillian Langford referred to, and both in collaboration with her and independently we have shown that rhesus cells can be infected. So, I think I think we need to look for a primate model. That might be worth investigating because, to my mind, it would be better to have a primate model because of all the other associated factors, including complement‑dependent lysis.
So, I think there are models that can be pursued, but I have some reservation about whether they are really going to give us the kind of data that would be helpful.
DR. AUCHINCLOSS:  We now have actually two topics on the table, which is fine. We will have more than that. One is which tissue do you want to sample in and now we have shifted over a little bit into which species do we want to look at and which species is worth looking at.
Can somebody summarize for me?  Yes, baboons can be infected with this virus; yes, rhesus cells can be but so far no animals?  Is that correct?  Does everybody agree that the baboon can be infected with this virus?
DR. HENEINE:  Baboon cells.
DR. AUCHINCLOSS:  Baboon cells, thank you. Chimpanzee, yes, also.
DR. HIRSCH:  Is there any evidence of baboon?
DR. ONIONS:  I am not aware of baboons being able to be infected but maybe someone else in the audience could correct me if that is the case.
DR. AUCHINCLOSS:  Sorry?
DR. ONIONS:  As far as I am aware nobody has shown infection of baboon cells in vitro. There adverse event data on the gorilla, there are data on the chimpanzee, and there are data on the rhesus that show that you can infect certain cells, but I would again add the qualification that in the case of the rhesus cells, in our hands, to do that we actually concentrated the virus very significantly before we actually infected the cells.
DR. HIRSCH:  Did any of the sponsors say yes to baboon cells?
DR. ALLAN:  I mean, I think you have to consider whether or not, or how seriously people looked at baboon cells lines and how thoroughly they have investigated that, and I don't really know at this point.
DR. ONIONS:  I think Jonathan's point is very good ‑‑ perseverance, how much you use and whether you have luck, and it is probable that you could infect other primates.
DR. COFFIN:  It is also important to keep in mind in that kind of analysis that these cells lines are all different from one another. The cell lines used in humans in no way parallel the one that you might have from a baboon. You take what is on the shelf basically, unless you are looking at fresh tissue, which there hasn't been much result with. This whole effect could just be a difference between one cell line and another that might have nothing to do with the species of origin. So, we still have to keep that in mind when we are talking about negative results.
MR. BENEDI:  It is a non‑medical question, and maybe it is a silly one but I had it written down and you are touching on it now. The baboon and other non‑human primates that have been infected, are we not creating something that we haven't introduced to the human cells yet but have introduced to baboons that could go from the baboons to humans instead of pigs to humans?
DR. AUCHINCLOSS:  Your question, I guess, is by doing the baboon experiments are we setting up ‑‑
MR. BENEDI:  Are we creating something new that we haven't tested human cells with in the baboon that has been developed through the pig?
DR. AUCHINCLOSS:  I guess it would be theoretically possible but there are enough humans that have been exposed directly that the question has really been addressed even in the human directly.
DR. SACHS:  I think Dr. Coffin's point is extremely important. How many different cell lines of humans were looked at before some good ones that get infected easily by these viruses were found?  Was it quite a few?
DR. ONIONS:  Well, in our initial screening we did about 20 cells lines of which we got two infected.
DR. AUCHINCLOSS:  I think what we are hearing is that if you look hard enough for a cell line from any species you will find cell lines that can get infected under the proper circumstances in vitro with virus if you have enough virus. And, I think the importance of it is that the baboon, the rhesus, any other primate as an in vivo model is reasonable to be looking at in terms of screening to find out whether or not any infections occur, regardless of whether the data are identical on the cell lines.
Well, let's put it in practical terms, and the practical terms are that in non‑human primates you can do the experiment with transgenic organs; you can avoid complement; you can give them lots of immunosuppression; you can biopsy them at lots of different times; you can get lots of different samples from different places. So, you have all those things that you can do. But the disadvantage is that when you have done all the experiments and you get a negative result ‑‑
DR. SACHS:  Well, it is the same as saying two different humans. I don't see that there is any real difference. There is no evidence that there is a real difference between what is going to happen in a non‑human primate and what is going to happen in a human when exposed to a xenograft.
DR. AUCHINCLOSS:  So, in the face of a big negative, which is what we have, it is worth doing these experiments even though the result may be negative also.
DR. KASLOW:  I was just going to follow‑up on an earlier point from Dr. Wilson's presentation. You made it sound like, if I remember correctly, that human lung cells were the most favorable in terms of your infectability. Did I misunderstand that?
DR. WILSON:  The 293 cell line is actually in kidney.
DR. KASLOW:  I thought you mentioned lung cells as well when you were going over the various cell types that you had tried to infect or that somebody had tried to infect. Not true?   No?  Sorry.
DR. MICHAELS:  I think that was Gillian's comment, and Gillian correct me ‑‑ the porcine lung had more PERV. High levels of PERV came from the lung from the pig.
DR. KASLOW:  Thank you.
DR. HIRSCH:  I am not sure I agree with David's comments that a baboon model is necessarily as good as a human model, if I heard him right, and that just because you haven't infected baboon cells doesn't mean that the baboon can't be infected. I think the onus has to be to prove that the baboon can be infected. If the baboon can't be infected in vivo, then it is not a good model system.
DR. SACHS:  We have no evidence that a human can be infected in vivo.
DR. HIRSCH:  Well, I am not saying a human can but I am just saying that if you are trying to see what the effects are of pig endogenous retrovirus and using a baboon as a negative model, and you don't have to worry about it because you haven't seen it in a baboon, until you can show that a baboon cell or a baboon in vivo can really be infected then you can't say it is a good model.
DR. ALLAN:  That is the model though, the model is to determine whether or not that species can be infected or not. So, that is really the model. So, even when you get a negative ‑‑ that is why you are doing the experiment, to see whether the animal would be infected or not. So, it is reasonable to do that with high levels of virus in several different species. If you get a negative, like what we are seeing, it doesn't tell you that you are going to get a negative in humans but it gives you more information. So, I think we are saying the same thing; maybe we are not.
[Laughter]
DR. AUCHINCLOSS:  There are three or four hands up. Let me start over here.
DR. COFFIN:  I think in many respects the best animal model is the one that works, even if it is the mouse. I mean, you can get a lot of information on what could happen and what to look for in a virus infection with any species, any mammalian species into which you can get any infection at all. Obviously, you want to be as primate as possible, and you would like to learn that the infection can't go in certain species, but I think it is worth working right on down until one finds something that is infectable because of the amount of sort of basic information and information on what to look for in people that could be generated.
DR. HIRSCH:  John, wouldn't you say even though in vitro and in vivo are not necessarily parallel, but if you have data to show that pig viruses can easily infect cat cells that would be a model that you would be interested in looking at.
DR. COFFIN:  I would start there, yes. That would make, I would think, a sensible starting point. If that doesn't work you would just keep on going.
DR. COOPER:  This discussion about the model makes me think very closely because in vitro would be one thing to show that the cells could be infected but what we really want is an in vivo model and, as mentioned, we want a primate model. Now, a big problem with a primate model is that the primates, the Old World monkeys have these antibodies that destroy these pig tissues very quickly, and even though we have talked today about sort of long‑term follow‑up, we are actually talking about a very few cells surviving or we are following up an animal that has rejected its organ within a few days or weeks in most cases, often earlier than that.
Have we thought of looking at New World monkeys which do not have antibodies against pigs?  They have the sam gal expression on their tissues. So, the whole business of antibody complement‑mediated rejection would be overcome, and if we could show that you could infect a New World monkey cell in vitro, then you would have an in vivo model for a least putting in cells. New World monkey happen to be rather small, but at least you could put in pig cells in large numbers and see if they became infected.
We know that this would be rather like a concordant xenograft with the antibody problem, and we know that in other models of concordant xenografts in primates, for example monkey to baboon, with the standard immunosuppression of today we can have survival of over a year. So, we would then have follow‑up of living pig cells in a primate for more than a year, which would give us some idea perhaps in vivo whether these monkeys can get infected.
DR. AUCHINCLOSS:  From the experts, is there enthusiasm for going to a species that doesn't have a lot of anti‑gal to do these experiments?
DR. COFFIN:  Well, as far as infection is concerned, I don't think it is such a serious problem in any species because one passage through a primate cell will take care of that. So, virus produced by 293 cells, for example, will now be the same as virus that was produced after one round in a human. So, as far as that issue goes ‑‑ there may be other perfectly good reasons for doing it but I don't think that necessarily is the best reason for it.
DR. LERCHE:  The last speaker covered the issue I was going to bring up, namely that with regard to many retroviruses non‑human primates are not created equal in terms of susceptibility to these things, and I think a systematic approach is necessary for doing this, and I think the New World species are worth looking at in the grander scheme of things. So, in the interest of time I will just leave it at that, but there are other primates that haven't been looked at that may offer at least some comparative model systems to gain some more information.
DR. CHAPMAN:  My point has been partially made but I will try to underline it with more clarity than I did the last time I spoke. As long as there is not in vitro evidence that you can infect baboon cell lines I would argue that it is foolish to put efforts into in vivo models using baboons to ask the question of whether PERV can cause infections.
Now, given that the baboon is a good model for other aspects of things people are going to be looking at, like the physiology of whether the organ will function and perhaps immunology ‑‑ I defer to people more expert on that than I, it is reasonable and it is probably prudent to continue to study baboons who are exposed to porcine xenografts for other reasons for evidence of infection because evidence of an active in vivo infection with PERV in the baboon would be helpful. But as long as you don't have in vitro evidence you can infect any baboon cell lines I would argue that we cannot put any confidence or any weight on the absence of in vivo evidence of PERV infection in baboons and make decisions of the safety of xenografts.
DR. ALLAN:  I wouldn't say it is foolish to go in vivo into a model system when you haven't ‑‑ I mean, how many baboon cell lines have been looked at?  The problem, as John pointed out, is that every cell line is different and if everybody has looked at one baboon cell line or three baboon cell lines from one animal it makes it very difficult.
I agree with you that negative data is going to be very difficult to interpret in the baboon, but I think it is not that difficult to take a couple of baboons and bolus them with virus and see what you get, regardless of the in vitro data because it is worth doing.
DR. AUCHINCLOSS:  We are bumping up the problem of how you go about dealing with a bunch of big negatives. There is a comment down here and then a comment from the floor.
DR. PAUL:  I think there are numerous examples of viruses that don't grow in vitro and, yet, replicate in vivo. So, I would say that there is a merit in doing in vivo experiments. In addition, there is the possibility of developing standardized reagents for antibody assays.
DR. DINSMORE:  Jonathan Dinsmore, from Diacrin. I would agree with Dr. Coffin about if you are looking for an animal model you want one that is permissive for infection. I would directly answer Dr. Hirsch's question about the tropism of this virus. There is a basic biology of different cell types, different organ types that is very similar across species. So, you could look to see if it appears in PBMCs, whether it has the specific tropism for a specific organ because you can dissect that quite well in an animal model. And, I would actually go back to the work done with the cat virus that jumped dogs because there you know what the history is of that virus in cats. You also know what the history is in dogs, and you can see if it followed the same course. So, I would vote for looking for a permissive animal model to look at these viruses rather than continually generating negative data.
DR. SACHS:  The reason I asked how many cell lines had been looked for before you found some that got infected was exactly that issue. If all you are really looking for is to have a cell line that will show an infection, the evidence is that if you keep looking hard enough you will find it.
DR. HIRSCH:  Not necessarily. I mean, there is species specificity for a number of different viruses.
DR. ONIONS:  I concur with the last statement. For instance, feline virus has been very extensively looked at by all sorts of technologies, including serotype marking. For instance, say, in guinea pig there is not a receptor that that virus will use, whereas in closer related species there are receptors. So, you can't make that assumption.
Personally, before investing resources in this kind of experiments ‑‑ the ethics of using animals anyway, I would not do that until I knew that you could infect the cell in vitro. Infection in vitro does not necessarily mean you will get infection in vivo but at least it is a start.  For instance, now we know the rhesus can be infected in vitro, certain cells. That would be the species I would take as the species of choice to do the kind of bolus infection that has been suggested here already. That is, perhaps infecting those animals with high titer virus and 293 cells.
DR. SACHS:  Can I answer that?  I certainly agree with what you are saying. That could be, but I was just asking the question has it been done in a statistically meaningful way.
DR. ONIONS:  No.
DR. SACHS:  Has one looked at a similar number of baboon cell lines as the human?  If the answer is you have and there is a difference, then I would agree with it. But, if it hasn't been done, it is silly to be speculating that there is a species difference without having done that study. That is what I was asking.
DR. ALLAN:  The problem is that there are so few monkey cell lines to begin with and there is virtually baboon. We have generated some in my lab and we have sent them to Carolyn to look at, and maybe we will get some data on some of those cell lines. But the problem is deriving cell lines from monkeys. That is the problem.
DR. AUCHINCLOSS:  I am going to try a brief summary of this portion, the animal species to look at, in the following way: that we have a lot of negative data, and with negative data it is hard to have confidence. Some negative data is less reassuring than others because if you can't even infect the cell line in vitro, then you are really not impressed by the negative data. Therefore, most of these animal models should not receive targeted resources for this kind of experiment but, on the other hand, it would be equally foolish ‑‑ I think I heard everybody say ‑‑ not to look at those animals that were being used for experiments, anyway ‑‑ pig organs going into baboons, or whatever. But if there was one species amongst the non‑human primates ‑‑ I thought I heard rhesus as a species that you might use for further investigator of PERV in non‑human primates.
DR. VANDERPOOL:  I want to ask one question off of that good summary, and that is, we said we were reassured by the data we have heard, and then we said there are two caveats. Number one, we are not looking at the patients and, number two, the donor organs that are going to be put in these patients. Then we shifted to this animal model discussion. Are we saying by your comment that this committee feels that an animal model should be looked at as a prerequisite to clinical trials, or are we saying something else?
DR. AUCHINCLOSS:  That is a good question because it is the ultimate question that the FDA will be asking, is this a requirement?
DR. COFFIN:  I don't think it should be a requirement, but I absolutely think it should be done in parallel. First, did I hear you say that all data are equal but some are more equal than others?
[Laughter]
DR. AUCHINCLOSS:  All negative data are equal, and some are more equal.
DR. COFFIN:  I wanted to point out that retrovirology has actually become a fairly advanced anti‑retroviral. So, with a lot of viruses we have a very good understanding about what makes cell lines permissive or not. So, I would put a plug in here for also doing a lot of basic studies, like trying to identify the receptor for these viruses. Then we could find out very directly whether the receptor was present in the species or not and saves ourselves a lot of work. We could find out if permissivity or non‑permissivity at some other level, for example how efficiently the LTR works in certain kinds of cells ‑‑ all of that stuff is very well charted out with experience with HIV and MLV and lots and lots of other retroviruses, and I think deserves a lot of support.
DR. AUCHINCLOSS:  I think that is a really good point. One can separate from the FDA's point of view a requirement for testing ‑‑ I thought I saw around the table sort of a lot of people going no. So, I am going to suggest that that is the committee feeling, that is not something we are suggesting should be a company requirement. On the other hand, good basic research in this field should be.
Then I am going to suggest that we probably ought to break for lunch. But we still have a lot of questions to cover before 1:50. So, we are going to come back here at one o'clock and keep working as hard as we can. Okay?
[Whereupon, at 12:16 p.m., the proceedings were recessed, to be resumed at 1:00 p.m.]

A F T E R N O O N  P R O C E E D I N G S
[1:10 p.m.]
DR. AUCHINCLOSS:  We will get started and resume our conversation at the table here with contributions from the floor or from the sponsors, with questions to the sponsors if the committee members like.
We have a 1:50 talk scheduled.  That speaker has a plane to catch afterward so we are going to be on schedule and starting at 1:50 and we will break this discussion, which is not to say that it will necessarily end.  We have another day's worth of meetings.
There are at least four questions that still remain on my list that we should be addressing including what tissue should be sampled, quality‑control issues, the development of an antibody assay and timing of sampling.  These are questions that have been put on the table and I am happy to hear any other issues that anybody wants to put on the table for us to discuss as well for advice to the FDA.
Let me go ahead and go back to the "what tissue" issue.  So far, sampling has been in peripheral‑blood lymphocytes or mononuclear cells.  Does anybody on the committee suggest to the FDA that we should, in fact, be looking at some other tissue.  I will open it there.
DR. SALOMON:  I think the idea of looking at peripheral‑blood mononuclear cells has got two problems.  One has already been demonstrated.  The other one is that, and Carolyn, please correct me if I am getting the spin on this wrong, but the actual data you presented when you looked at the pig PBL as a source for PERV, these were activated cells whereas a lot of these studies have been done without activating them.
In some cases, we are doing DNA PCR which would show integrated pro‑virus.  So that might be okay but, in other case, probably a better marker for infection would be either the release of infectious virions or messenger RNA at least by RT PCR.  In both cases, you might have to activate the cells.
I would also point out that other cell types have to be considered in terms of infectious profiles.  I think the cell that everyone keeps coming back to is UT93.  This is a human kidney epithelial cell line.  It would appear to have the receptor.  I think John Coffin made a key point.  We don't know the receptor yet.  I hope that that will come out in the next year or so.  I know there are a couple of groups working on it but, in the absence of that, pig cell lines that you know have it, if we are talking about epithelial cell lines having it, it would, I think, be remiss not to test at least several epithelial cells from primary tissues as part of a profile for looking for infection in animal models or in human patients.
DR. ALLAN:  We have the opportunity, if you are looking at the pig‑to‑monkey studies, you may not have in humans which is to sample lots of different tissues.  I think, obviously, everybody would suggest that you have to do that.
I don't think I am hearing you say that we shouldn't be doing DNA from PBMCs on patients.  We still should be doing it.  It is just that it is not going to give you all the answers.  But the thing is, even if you get a very small amount of positive results and it is like microchimerism or something that we have heard earlier, if you continue to sample a patient, the real information comes if that varies, if it, all of a sudden goes up, with or without any RNA.
So the RNA may be intermittent.  Depending on the type of retrovirus, you could only have intermittent viremias and you may miss them when you are sampling.
DR. SALOMON:  Or you could also activate cells, is my point, or stress cells.  You could do the same thing with epithelial cells.  I also point out with humans you can do kidney biopsies, you can do skin biopsies, you can do cell, corneal epithelial, lung biopsy.  Those are not horribly difficult things to do, actually.
DR. AUCHINCLOSS:  Can do, but should the FDA be asking for them?
DR. MICHAELS:  I actually agree but not completely to the degree that you are suggesting in terms of the other types of tissues.  I think that when the other types of tissues are available that the FDA should ask that those be sampled as well, but I think to ask for a lung biopsy or a kidney biopsy if there is not another reason to be doing it‑‑I don't think you were suggesting that.
DR. SALOMON:  I wasn't suggesting that.
DR. MICHAELS:  However, things such as the Diacrin studies where we have brain tissue available on the patient that had passed away that, perhaps, that tissue could be looked at if it hasn't been looked at already.
The same thing with patients that, perhaps, are on hepato‑assist devices and then go on to have an allotransplant and have the native liver removed, we could, then, look at those tissues or when the abdomen is opened, take some of the lymph nodes.
DR. ONIONS:  I am still a proponent of, actually, looking at peripheral‑blood mononuclear cells for several reasons.  One is the reason I adduced earlier and that is that, in animal systems, the root of infection of the cells in the peripheral blood is not necessarily through the cells in the peripheral blood, it is through a stem‑cell precursor that is miotopically active.  That is my first comment.
My second comment is that what we are really concerned at here is, to a large extent, a public‑health issue and, therefore, the other tissues that you might think of looking at or at least fluids, body fluids, that are produced by the patient.  Saliva is a very obvious choice because that is the known means of transmission of some of these viruses.
Whether or not you put that in as a routine is, perhaps, a matter of conjecture at the moment but, certainly, I think most of the people who consider the matter have that in their option program that if they have equivocal signals from peripheral blood, that those, at least are examined.
I am a clinician, but I am very cautious about overinvasive procedures on patients without any rationale for it.  So, for the moment, I think peripheral‑blood mononuclear cells are still one of the best sources we have to examine.
DR. HIRSCH:  I am a clinician and I certainly would second that, that to go on a wild goose chase to look at a variety of different specimens with no real rationale that it is going to be there doesn't make any sense.  I think if you are talking about putting tissue in brains, certainly looking at spinal fluid would be reasonable.  If you are doing kidney transplants, looking at urine might be reasonable.
But without a good rationale to look at saliva or lung biopsy or salivary‑gland biopsy, whatever, doesn't make a lot of sense to me.  I would certainly put most of my money looking at antibody and developing a good, reliable antibody test.  It sounds, from what we heard this morning, that people are well on their way to doing this.
If you can find no antibody in humans who have gotten various xenotransplants, I think going on a wild goose chase looking for lots of different specimens doesn't make much sense to me.
DR. ONIONS:  Could I just come back very briefly on that last one.  I agree almost word‑for‑word with that except one comment, one I keep coming back to, and that is we are working towards better antibody tests.  We have heard presentations from several groups about that and they will get better.
But I think we must always remember that the probability‑‑and it can only be a probability at the moment‑‑is that antibody in these groups of patients is likely to be indicative of exposure and possibly of recovery.  The patients you worry about are the ones that don't develop antibody because the cats that develop life‑long infections, the gibbons that develop life‑long infections, the primates that are infected with alphatrophic neuron leukemia virus that develop life‑long infections, don't develop antibody.  Those are the people that you are worried about.
But there, you get an unequivocal pattern of infection because you can pick up virus, the pattern of viremia, and you can pick up virus in all sorts of other sites including peripheral‑blood mononuclear cells.  So antibody is an important adjunct to the test we need, but you need all of these tests because, only by having each of those tests, can you put the patient into the correct profile.
DR. AUCHINCLOSS:  I want to come back to this point but just let me close off the discussion.  What I heard for the FDA was pretty resounding silence to the notion that there was some other tissue that you should be actively seeking.  Is that what you heard?
DR. SIEGEL:  I guess what I would ask about that hasn't been addressed in the last two minutes but was raised earlier would perhaps be a little more discussion about plasma.  It was noted earlier that some retroviruses have an early plasma‑viremic phase.  Obtaining plasma certainly doesn't add any move invasive nature than obtaining lymphocytes.
Should there be more routine screening of plasma?  Should there be more occasional screening or is plasma not a high‑yield place to be looking.
DR. AUCHINCLOSS:  So we are about to come to antibody assays but, before we go there, we are talking, now, about other tissues and we are on the question specifically of plasma.  What do the experts say?
DR. HIRSCH:  I would certainly go for plasma.  I think plasma, given what we have heard so far, makes at least as much sense as PBMCs because it reflects not only what is in PBMCs but what may be elsewhere.  Certainly, I would go for that.  It is an easy source of material.
DR. ALLAN:  It is also much more informative because you are looking for RNA expression so you are looking for virus expression.  So it is much more informative.
DR. ONIONS:  I would agree with Dr. Allan's last point.  I think you need all of these.  Again, I would just reiterate, I don't think any of these should be done in isolation; that is, you need to look in plasma for plasma viremia, you need to look for latent infection or probable latent infection in peripheral‑blood mononuclear cells and you need to look for antibody.
Each of those gives you a different piece of information which, in combination, gives you a good snapshot view of what that patient is‑‑the status of the patient at that time.
The comment I make about looking in plasma is that I mentioned earlier using RT PCR which you can do and we have done.  But that is probably not the ideal test where you are putting in, or where you are potentially putting it, solid organs because you will get potential leakage of messenger RNA from the cells and, whilst there are techniques for distinguishing between messenger RNA and virion RNA, that is not, perhaps, the best methodology.
The best methodology is probably using reverse transcriptase‑enhanced PCR, RT‑PCR, because that will detect virions down to at least down to 102 virions.  So we can detect low‑level viremias using that kind of technology.
DR. COFFIN:  David, I assume you meant PCR‑enhanced RT, not RT‑enhanced PCR.
DR. ONIONS:  So sorry.  Thank you, John.  That is exactly what I meant.
DR. AUCHINCLOSS:  Did the FDA get what they are saying?
DR. SALOMON:  One point I wanted to emphasize, though, in my comments, is that I think that, oftentimes, in these discussions we get so focussed on vascularized organ transplantation that we don't think enough about the implications of cellular and tissue transplantation.
I realize I set some people thinking that I meant that you should go around biopsying all those organs.  That was the last thing I meant.  For example, if you do pig‑islet transplants, which is right on the horizon, and you inject them into the portal vein, therefore embolizing the liver, then I don't think it is unreasonable to request a liver biopsy.
If you do porcine stem cells, hematopoietic stem cells, into a patient in the tolerance‑induction strategy which is not that far off, then I don't think it is unreasonable to do a bone‑marrow aspiration.  So that is actually what I was specifically referring to.
DR. ALLAN:  I wanted to come back to what David just said.  I want to make sure I understood what you are saying.  When you are asking for an RT‑based assay versus a PCR‑based assay, what you are saying, basically, is that it is more sensitive, therefore it is a first screen.  But you are not saying not to do the RT PCR because you need to know that what you are looking at is really a pig virus and not some other retrovirus.
DR. ONIONS:  Our RT PCR assays and the assay that Walid calls AMP RT‑‑in other words, a reverse‑transcriptase that has a PCR input‑‑those assays, in our hands, give very similar sensitivities.  In fact, if you actually take the limit of detection‑‑that is down to literally 10 virions‑‑but‑‑the limits of sensitivity you put higher because you need a reproducible assay.
So you still are talking there about 100 virions in either assay system.  I prefer to use the reverse‑transcriptase‑based assay because you can detect, I think, a message that is creeping back into plasma.  Because if you look for a housekeeping message, then certainly some of the primates that we have been involved in looking at, you can pick up those messages.
DR. ALLAN:  I understand that.  What I was getting at was that if you use an RT‑based assay, it is not telling you what virus you are really looking at.  So, to me, it is a first screen but you still have to go back and do something like RT PCR to know that it is a pig virus.
DR. ONIONS:  Absolutely.
DR. AUCHINCLOSS:  You are allowed to have some jet lag, Dr. Onions.  This is what, your third trip in six weeks to the United States to help us out?  We really appreciate your coming.
DR. HENEINE:  I think the point that David was trying to make regarding the RT PCR analysis of PERV RNA, that in the pig plasma testing, sometimes you also detect PERV RNA that is not particle‑associated that is coming from the cells.  That would interfere with the interpretation of the data.  Therefore, you should be aware of that limitation.
What we don't know of is whether this situation will be present in xenograft recipients, cellular xenograft recipients or maybe it will be present in organ xenograft recipients.  But I guess this is what he wanted to highlight, that there could be a limitation in that.  That is not to say that we need not use this technology for diagnosis.
Again, I fully support also the other point that we have technologies available to look for reverse‑transcriptase testing which are markers of particles.  And we could also use those.
DR. ONIONS:  You interpreted me accurately.  Thank you.
DR. COFFIN:  It is not real hard to centrifuge the plasma and see if what you are looking at is a particle or not.
DR. AUCHINCLOSS:  Any other comments on plasma?  I think I am hearing a loud "yes" there.
DR. DINSMORE:  Correct me if I am wrong here, but difference between an infectious endogenous virus and a noninfectious endogenous virus is just that it could go on and infect another cell.  So you could actually have an endogenous virus that has malfunctional envelope protein.  You would make perfectly good viral particles but they would be noninfectious.  That could be picked up in this sort of assay.  So, again, we have to be careful about those interpretations.
DR. COFFIN:  But, in most of the models, we know retrovirus as the production of those is very low by comparison because you get so much amplification on infection of other cells.  It is not at all clear whether you actually see that in any retrovirus in an animal.
DR. AUCHINCLOSS:  Okay.  I think it is time to move on to the question of do we now have an antibody assay which, last time we met, was the big missing piece.  Everybody said that when we had it, we would feel much more comfortable.
Let me come back to the point that David Onions was making that patients with persistent infection may not‑‑but that is not, necessarily, the issue that we‑‑at this point, we are still asking the question, can infection occur and so, even if you miss 80 percent of the people who are infected, it is still an excellent way of determining the answer to that question.
Is that a fair statement?
DR. ONIONS:  That is exactly a fair statement.  Could I maybe make a comment about the serologic assays.  There are several people, groups, systems, out there.  We have heard from the Nextran group, from John Logan, from Walid, from work carried out on behalf of Immutran using recombinant P30.  There are a number of approaches and I think all of these are interesting approaches.
I think one comment I have to make about them at the moment, and I think it is a criticism that Jonathan Allan has raised and I think it is a well‑made one, and that is that it relates to this problem that we don't have a patient population‑‑somebody else may‑‑that we know we can look at the patient population and follow it through.
We don't have that so the interpreting the significance of the data is difficult.  The second comment is that, at the moment, we are looking at reactivities to maybe one or two viral proteins‑‑sorry; we are looking frequently to inactivity to one viral protein instead of P30, although we are usually following up with, say, whole viral proteins as a sort of an adjunct.
Normally, when looking at serological responses, for instance, the FDA would require in an HIV test or an HTLV1 test, that you have reactivity to at least two viral proteins.  I think those kinds of criteria are going to become necessary to be sure that we are looking at true serological responses to virulence and not to crossreactive antibody.
What we can be certain is that, at least I am confident from my own data, that there are patients out there, people out there, with antibody that recognize epitope on recombinant P30.  Whether or not that is antibody elicited in response to a virus, I cannot say and I do not know.
What I think you, therefore, need is, as a minimum criteria, response to two viral proteins to be sure that you are actually looking at a response to a virus‑induced protein response.
DR. SIEGEL:  I have a question about that comment.  This issue of crossreactive antibodies has come up before.  In this particular case, at least as we move prospectively from here, we know, unlike in the diagnosis of many diseases, the absolute time of exposure and we are able to obtain pre‑exposure serum.
Shouldn't the existence of pre‑exposure serum give you a pretty good handle as to whether it is a cross‑reactive antibody or a newly arisen antibody?
DR. ONIONS:  Absolutely.  That is a very good point.  Unfortunately, in the particular patients that have been positive, those sera do not exist.
DR. SIEGEL:  But prospectively, obviously‑‑
DR. ONIONS:  Absolutely.  No, no; that would begin to sort itself out.  I absolutely agree.
DR. AUCHINCLOSS:  For my sake, can you elaborate a little bit more about‑‑there are people out there with reactivity to at least one epitope on P30.  They were people who received pig tissue or they were people who you just screened in the population?
DR. ONIONS:  We have a group of people from different studies who was to have antibody to P30, and we are confident they have antibodies to P30, because we can show the purity of the antigen by mass spectrometry.  We can also show you in the Sofigen system that the actual moiety that is binding to the P30 has the mass spec characteristics of human IgG.
So, I think, to that level we are confident there is antibody in these people but recognize an epitope on P30.  But, again, I will restate that we do not know the incidence of that antibody.
DR. AUCHINCLOSS:  But they were people who received pig tissue?
DR. ONIONS:  Yes; they were.
DR. AUCHINCLOSS:  Do you find any of the rest of us that have this antibody?
DR. ONIONS:  That is an interesting question.  In a validation study‑‑I have to be very careful what I say here if it gives away something, in the validation study, we did look and we did find two reactors.  Those reactors were exposed, at it turned out, to porcine material.
DR. AUCHINCLOSS:  But most of us are.
DR. ONIONS:  Yes; they were exposed in a very particular way.
DR. AUCHINCLOSS:  Antibodies?
DR. ALLAN:  I wanted to just add a little to what David said which is if you are looking‑‑and I think people have done a very good job of developing antibody‑based assays in monoclonal antibodies and recombinant antigens.  If you look at the established assays, either for HIV1 or HTLV, which are two human pathogens that are retroviruses, you typically need to have more than one antigen in your assay system to validate it.
There are a lot of problems with using one antigen.  If you look at the HIV system, you look at SIVAGM which is a virus found in African green monkeys, those monkeys don't may antibodies to gag, not that you wouldn't make antibodies to PERV, but it is possible that you might not make antibodies to one particular antigen or that it is not as sensitive as another antigen.
I don't know that it is that difficult.  It may be‑‑to produce enough virus to make either a whole virus assay, Western Blot or ELISA.  That is typically what is used for HIV.  With HTLV, it is a little different, because it is a cell‑associate virus.  But I would suggest that you try and stay away from those assays in which you use cell lysates because of the dirty nature of those things.
If you have a choice, and I think you have been developing them, but whole virus assays.
DR. ONIONS:  I absolutely agree with you.  I didn't want to compare one assay against another.  I don't think that is very productive.  But my own view is exactly yours, that you don't use cell lysates.
What we do is we use recombinant P30 as a cell lysate, but we do as a secondary is we use whole virus that has been sucrose‑banded so this is just virulence, not cell lysates.  So it is equivalent to looking at all the viral proteins in that secondary Western blot.
DR. ALLAN:  But should that be the first‑‑
DR. ONIONS:  I am inclined to agree with you, actually.  Yes; I think it probably should be.
DR. HENEINE:  The data we have speak to the fact that cell lysates are good antigens and there are high‑level detectable antigens there and are seroreactive with antisera, the antisera we use and with diluted antisera.  So, from these data, I am not convinced that these are not adequate antigens.
DR. ALLAN:  The only thing I would say is we don't have any positives, so you really can't know how good your assay is because you don't have a positive.
DR. HENEINE:  That is problem we are all faced with.  You don't have a group of 100 infected people where you can use their sera and define criteria for positivity like we do with HIV and
DR. AUCHINCLOSS:  This is the theme of the meeting; how confident are you with negative data when you can't find any positives?
DR. ONIONS:  Let me just address this question.  I think this is going to become a real issue.  There are very good labs out there with slightly different assays and I think you will find, as in all new areas of research, there are going to be differences of results.  That is inevitable.
One point I would make earlier, that I didn't make earlier, about the PCR‑type assays is that because people are pushing these to high sensitivity, we are going to get into stochastic things where you are almost at the Poissonian distribution of whether one sample taken from a patient is positive and then the second is, maybe, negative.  So those kinds of conflicts are going to occur.
There are also going to be some problems over different methodologies and whether they be for serology or for PCR.  What might be very useful for all of the groups involved is if we had a set of standards and those standards can be multiple.  They could be, for instance‑‑they might have to be polyclonal antibody raised against virion.  They might be antibodies raised in a primate against recombinant P30 as a standard‑‑such the we have a number of standards that can be used between us for inter‑lab comparison.
I think that is going to become necessary to get some feel for the sensitivity and specificity of some of the assays that are around because I do see that as a problem that is emerging.  But I think there is good will on all sides and I think that can be sorted out.
DR. AUCHINCLOSS:  I want to take that comment to go in two directions.  The first is the question of what the FDA should actually require of potential sponsors.  Are the antibody tests sufficiently well‑developed, useful and available that that should now become a part of a requirement for any sponsor coming to the FDA, that they can track their patients with some form of antibody assay.
DR. ALLAN:  It looks to me as though we are in the development phase of our antibody‑based assays and there needs to be some direction in terms of what we are really going to use to validate the assay and what assay it is going to be.  It is going to be an anti‑gag assay.
DR. AUCHINCLOSS:  That was the crucial word, and I saw nods around the table.  But if the FDA wants to hear more about this‑‑what I heard was still in the development stage and not at the point where antibody‑‑but, now, let's come to the second direction that I wanted to go which is actually part of a larger question that Dan was bringing up which is quality control and sharing of tissues and assays between the different potential sponsors which is a potentially tricky area.
What can the FDA do to foster interactions between the different companies, get good things to happen so that the assays are done the same way, that we know each company is doing this assay as well.  Is this an issue that you want to go into a little bit here?
DR. SIEGEL:  I think that as long as the committee is advising that we not require the assays, it is somewhat of a moot issue as to how we can get the companies to do the same assay.
DR. AUCHINCLOSS:  But what I am hearing the committee say is that the assay is not well enough developed to be a requirement at this time but still a high priority to develop antibody assays that are of good quality and that standardization between the different groups is important.
DR. ONIONS:  I wonder if I could take a little bit‑‑slightly reply to Jonathan Allan and slightly to extend the comment you just have made, Hugh.  Jonathan Allan is right.  I don't think we yet have assays that we can totally rely on because first of all I think we will find that there are going to be some differences between groups.  That is one issue.
The second issue, I absolutely do agree with him that a good serological assay should have a system for detecting more than one viral protein.  I would suggest, however, that, perhaps because of the speed of presentations, that has, perhaps, gone by the board.  The assays that have been used by most groups actually do detect antibody responses to multiple virus proteins.  That is the first comment.
The second comment was they have actually been validated in the strict FDA sense of the word validation; that is, against sera from patients who are clinically normal, from patients with HIV infections, patients with HTLV infections.  So those kinds of formal validations have been conducted.
We have also heard from John Logan and his group that pig veterinarians who have been exposed to virus who are doing very similar study at the moment in collaboration with a group in The Netherlands.
So that kind of work is ongoing and, I think, very near completion.  So I accept the partial criticism but I think it is only a partial one.  I think, of the point of view of standardization, that is a very simple thing to do.  I am very happy to hand out recombinant P30 to other people.  That is not a problem.  I think what we need are a few standard sera that we could use between labs.  That is the usual way of doing the kind of interlab comparison.
DR. KASLOW:  That is exactly what I was going to suggest.  At the very least, what you would have is a common panel that are available to everybody who is developing an assay so they can compare internally what they are doing and externally.
DR. WILSON:  I was wondering if we could just back up a little bit and ask that same question about looking for plasma viremia since I think I would value the committee's comments on whether or not assays for that type of analysis are better standardized and further along in terms of development.
DR. ONIONS:  Could I just comment because‑‑Walid expressed more elegantly than I did what I was trying to say.  Both Walid and I see the same kinds of data as has Jimmy Langford and that is that if you look in the plasma of pigs, you will find‑‑by RT PCR, you can certainly find RNA of PERV.
But the problem is if you also look for a control RNA, globin, you will find that so that you cannot distinguish whether that is really associated with a virus or whether that is just because you are looking at cell breakdown.  So that is probably in the context of the whole solid‑organ transplant into a patient not the methodology of choice for the initial detection.  Probably that ought to be based on a particle‑associated assay which John Coffin has emphasized is using reverse‑transcriptase.
Using very sensitive PERV‑type assays, you can do down to 102 or even lower in terms of the number of virions you can detect and we are sure of that because we can take virus particles actually and count them under EM and take the dilutions out.  You can validate that that is the number you can detect.
DR. WILSON:  But I guess the question you had asked regarding antibody assays was whether or not the FDA should require that and whether or not the same question would apply in terms of, for example, looking for RTX in these sensitive assays.
DR. ONIONS:  My position is very simple.  It has not changed for two years.  I actually think that if you are trying to determine the status of a patient, that you really need three separate pieces of information and those are the peripheral‑blood mononuclear cells PCR‑positive, is there evidence of plasma viremia by an appropriate assay which could be infectivity if we had a good one, and we don't, so the next best assay, in my view, would be PERV.  And the third criteria are the patients antibody positive.
So I think if you have those three sets of data, those are the data that are likely, in the most cost‑effective fashion, to give you a picture of the status of that patient.
DR. ALLAN:  From my perspective, there was some suggestion from, I think Jay, that said, "Well, I guess we are not going to require an antibody‑based assay."  I would step back and say maybe not the assay that some people are using, but I think that you do need to have an antibody‑based assay.
The only reason we didn't really push it last year was because there wasn't anything developed.  Now there is something developed.  So I think that you can require antibody‑based assays.  The only problem, then, is which assays.
DR. SIEGEL:  I don't think I was expressing what my thoughts were.  I think I was expressing what I heard Hugh say, that it is not something the FDA should‑‑
DR. ALLAN:  I would say just opposite.
DR. AUCHINCLOSS:  I didn't mean it to be‑‑I meant it to be sort of a trial balloon to see what the committee was going‑‑
DR. SIEGEL:  So you have some consensus on that.
DR. HIRSCH:  I would think the FDA should require, certainly, the collection of the specimens and testing by an appropriate assay as soon as a group determines there is an appropriate assay.
DR. VANDERPOOL:  I agree with the comments of Richard and Martin and a number of you.  Isn't there agreement on this issue of required standardized assay.  If so, I am not sure the committee, at this point, can spend its time determining which one that is but could, quite easily, get together a group of very experienced individuals who are using different types of assays and determine which of those is the best for the FDA to require.  Perhaps you would want more than one.
But I am not sure that this group can decide which one that would be that would give the best information as a requirement, not that others couldn't also be used by different parties.
DR. HENEINE:  Again, we are dealing with investigational assays that we have some experience with.  We don't have experience enough that the FDA can approve them for screening the blood supply like some HIV HTLV tests, but I think we can learn much by using those in these prospectively done clinical trials.
Imagine if you could be able to demonstrate seroconversion in a patient and persistent antibody production using those assays.  That would be very informative to know the status of the infection in that patient.
Regarding viremia, too.  We have two tools, like David was saying.  You could look at PERV RNA by RT PCR and you could look genetically for reverse‑transcriptase activity.  Again, if we can demonstrate the presence of the particles using these two tools, why not?  This is evidence of productive release of virions.
So we were not where we are today, where we were a year ago.  These investigational tools are available and we would like them to be validated more.  Unfortunately, we cannot.  All that we have is a panel of maybe five to ten antisera that are produced in several labs.  What we could do is pool these antisera so people can exchange and test sensitivities and specificities of these assays.
Regarding just one comment with David, the RT PCR PERV RNA, I don't think we need to discount its utility because of the potential problem with mRNA of PERV of cellular origin.  We don't have, yet, evidence that in people that are exposed to vascularized tissues that we will encounter that problem like we do in some pig plasma.
So, again, we will keep that thing open until we have that evidence that PERV RNA can be limited by that limitation.
DR. ONIONS:  I agree with you.
DR. DINSMORE:  I would like just like to make one more comment about the viruses in follow up with you.  If you have a xenotropic virus, it could be a reservoir of pig cells that are producing it and you would have viremia that  would not be infective.  So, therefore, I would encourage any of these assays to be coupled with something which shows infectivity, some form of infectivity assay.
That goes along with the antibody base test, too, because one has to know how to interpret the data that one gets and you should put some thought into how to interpret the results and what tests should be tied together.
Finally, I don't see why there has to be one antibody test so long as you have a protocol for validating anyone's given assay.  You could have ten different assays all validated to the same criteria and they would all be viable assays.  So I don't think you necessarily have to have every company using the same antibody.
DR. SIEGEL:  I guess somebody put to the FDA whether we had a role in providing a standard or helping validate them.  I wonder, though, given that we don't have any animal or human model with infection, what is the standard that one would provide to validate such assays?
DR. SALOMON:  I have been concerned about that all morning; really, you have ten different companies with three different, four different, assays, maybe more.  We all know that when we set up these assays in our laboratories, we never do them exactly the same way.  We have also all been at meetings where someone has taken a very similar assay and gotten very different results.
So it makes me concerned.  I can handle that in a scientific meeting, but I have more difficulty when that is part of a regulatory process, the end of which is at least a perception by the public that there is a risk here.  So I don't think that the FDA, at least this is my personal opinion, now‑‑I don't think that you can avoid this issue, Jay.
I think you are going to have to establish validity and that validity may change from this year until next year as the thing evolves.  I would finish by saying that I am sitting here thinking that maybe, after the first of the year, we ought to be getting a group of us together and having a workshop where people would agree‑‑there is a precedent for this, and that is the MHCHLA international workshops.  This has worked very well for defining validity of tests from typing.  I don't see why we can't do something similar like that collegially in PERV.
DR. SIEGEL:  I am not trying to avoid the issue.  I can visualize well how one might establish standards and help validate and cross‑validate tests, say, for viral DNA or viral RNA.  It is just hard to imagine how to do that for antisera when we don't know what the antiserum of an infected animal would look like, what antigens it would respond to and what sorts of antibodies, because we don't‑‑I mean, what is the positive control.
DR. CHAPMAN:  Not a comment, but a question.  It sounds to me like what is being laid out here, or I will ask it as a question‑‑is what is being laid out here actually an argument that the best approach and the fastest progress would come not from having FDA identify and sanction one serologic assay as the best for testing but rather an environment in which multiple assays were tested against the same specimens repetitively which would be either a requirement for testing in one lab and confirmatory testing in a second lab, or a panel of assay testing, until we get to the point where we have better development of the field.
DR. AUCHINCLOSS:  I don't know that this is a fact but I imagine it is a fact.  One of the concerns that companies have is letting their tissues out or their samples out to somebody else for fear that somebody else comes up with a positive that they don't have a chance to validate before it suddenly is public news.
Are you suggesting that it be a requirement that everybody make their serum from their patients available to everybody else so that these kinds of assays can be done‑‑
DR. CHAPMAN:  I don't know that I would want to suggest that because I haven't had time to think through all the implications.  But I think, from a practical point of view, the situation is that we have multiple people or entities with experience in developing diagnostic assays who are developing serologic assays.
Those assays have been validated, different assays to different extents, but reasonable well considering that you are working in the field where we have no known positives against which to test them.  Ideally, you would want to test each assay against a couple of thousand known positive infected humans and a couple of thousand known negative infected humans.
Instead, we are working in a setting where you can test them against presumed negative people and a variety of somewhat artificial positive controls.  And you can also assume, the limits of science and humanity being what they are, you are going to have false‑positive and false‑negative results and cross‑reactive results.
One way to clarify the significance of results in any assay may be to test multiple assays on the same sample and then use the discrepancies in results to target your further R&D activities on any or all of the assays.
DR. ONIONS:  Just a comment.  I think Louisa's last point is exactly the one.  I don't think this is a heavy‑duty issue.  I think it is a normal thing that you do in developing assays, and that is that you exchange reagents and compare results.  It is a very simple thing.
DR. DINSMORE:  Again, the foundation of scientific investigation is that it can be repeated in multiple labs by multiple methods.  So, in fact, all of these different assays, I think, rather than providing weakness show strength in that, by many different assays, by everyone's coming up with the same results and, therefore, it is less likely to be due to an artifact in one given lab's hands.
DR. AUCHINCLOSS:  Jay, what are you hearing from this?  Are you getting the information that you want or should we phrase the question differently?
DR. SIEGEL:  I certainly concur with the committee that there is a need for standards, standardization and for validation of sensitivity.  It came up before, I guess, with Dr. Onions' comment about the sensitivity of detecting virus against microchimerism.  Standards are very important for comparing across assays.
The FDA has played a substantial role both in in‑house testing and distribution of standards, for example, for HIV test that is for the blood supply.  There is no question that that is valid.  As to whether we have the resources and wherewithal to play such a role for this particular virus is something that we need to check into.  But I certainly hear the committee saying it ought to be done and I agree.
DR. PAUL:  I think would should give some thought to some virus neutralization which may be a very simple and an old method but, nevertheless, if there are samples that come up to be questionable, at least to look at virus neutralization as a backup test.
DR. MICHAELS:  Not to put David on the spot, but I was just curious with you all, the discussion going on, on the samples that you have on patients, and I know all the qualifications that you have made in terms of not sure if it
is crossreactive, have you sent it to any other groups that have, perhaps, different assays to have them look at it as well, yet, or is that something you are considering doing?
DR. ONIONS:  I have two completely different sets of samples from different origins that are positive.  One set has been compared with another laboratory.  The other set has not.  I would rather not comment further at the moment partly because some of this data is about to be published or submitted for publication and the other set of data, again, is under a sponsor who doesn't, at the moment, wish to go further with it.
DR. VANDERPOOL:  Just a quick comment.  I think that we let the genie out of the bag when we encouraged the FDA to have all those who are doing xenotransplant research to develop assays of various kinds for testing.  Now, the genie that got out of the bag was different people have done different things, developed different tests and surely have proprietary interests in quite a number of things done.
I think, in the spirit of what Louisa said, the question is for the people to get together, not necessarily choose just one thing, but at least find uniformity of test results between the various assays that are being set forward.
It may mean that one or two would be shown to be inferior.  But plurality is certainly possible within the general framework of being able to cross‑test between the various assays that have been developed at our urging.
DR. NOGUCHI:  I think what we still would like the committee to really give a strong signal on‑‑we have heard that, ideally, there should be three types of assays; peripheral‑blood mononuclear cells, serology and plasma.  But the real question is we required everybody to develop the PCR assays and we wanted everybody to develop the antibody assays.
Are you now ready to say we need to have everybody doing the antibody assays or not, because I think we heard a little divergence of opinion here.  We certainly can make it so that every clinical trial from this point on needs both the PCR and the antibody assay, not specifying the particular type of antibody assay, or are we truly still in a developmental field.
Basically, is what we are doing adequate requirements for continuing a trial at this point or do we need more based on what we now know?
DR. AUCHINCLOSS:  I thought that the answer I heard was bank serum don't require an assay because the assay is still developmental, or in the developmental stage, but push everybody to develop it together.
DR. COFFIN:  I would take a slightly stronger position that I think studies should continue to be done.  I think companies should still be required to continue to participate in this development of antibody assays.  I would be very uncomfortable right now just telling them to bank sera against something in the future.  I think continued research and progression‑‑
DR. AUCHINCLOSS:  I put it that way to kind of provoke a response from the committee.
DR. ALLAN:  It seems like most everyone has an antibody‑based assay because they have been told to develop them.
DR. AUCHINCLOSS:  Everybody should have an antibody‑based assay that they are working on.
DR. ALLAN:  Or should use one through whoever.  There are companies, I think, that are developed, not just Q‑One Biotech but other companies that have developed these types of antibody‑based assays.  So I think you can require the clinical trials to use antibody‑based assays.
Obviously, you can't tell them what assay they have to use, but it is just like with HIV when AIDS first came around.  Whatever we have got available, let's use it.  Let's not wait because you know what happened when people waited to test assays.  It can backfire.  So whatever resource you have available, I think you should use it.
DR. HIRSCH:  But the smartest thing, I think, would be, as was done with HIV, that you use the contemporary assay but you also bank material so that when you have a better assay come along‑‑
DR. LERCHE:  I would just like to second the comments of John Allan and John Coffin and probably others.  I think the antibody testing should become an active, ongoing component of follow up.  I notice this morning, in one of the presentations, that there was a fairly large body of patient samples or a population of patients that were not monitored by serology, at least in the presentation, and that represents a loss of a lot of information.
I think the more experience, the more numbers of patients that we can look at with these assays, the faster we will get to where we want to be.
DR. AUCHINCLOSS:  Phil, I am glad you got us to clarify that because I think what we just heard in the last several comments was a little bit different from what I thought I had heard before.
DR. NOGUCHI:  No; thank you for that because that has been a very critical reason for bringing this up before the committee.
DR. AUCHINCLOSS:  We may need to come back to some of it, but I did promise our next speaker that, at 1:50, she would be allowed to get going and make her plane.  Dr. Khabbaz from the CDC.
Guest Presentation
Nipah Virus Outbreak, Malaysia
DR. KHABBAZ:  Good afternoon.
[Slide.]
My talk is an informational talk so members of the committee can relax.  There are no questions and no vote.  In fact, if any of you is a subscriber to ProMed, you might even want to consider taking a nap because you have probably heard it all.
What I have been asked to cover is the most recent emerging viral zoonotic infection we have been involved in or are assisting in investigating and that is the nipah virus outbreak in Malaysia.  It has affected a large number of humans and pigs in that country.
[Slide.]
Before I talk about the nipah virus investigation, to frame it, I thought I would take you back to 1994 where a cousin of nipah virus, basically what we call now the hendra virus, first appeared.  This virus caused an explosive outbreak of respiratory infections in horses, in race horses, in Brisbane.  Thirteen horses died.  Twenty became infected.  Two humans became infected including this gentleman here, a trainer of horses, who was reported to actually have put his arm all the way in the mouth of an infected ill mare to try to help relieve respiratory secretions.  He became infected and died.
The title here suggests that the virus mutated.  That outbreak made headlines.  The virus did not mutate.  It was a new virus.  What we have learned since then about this virus is initially it was called equine morbillivirus.  Morphologically, unlimited sequence information suggested it may be a morbillivirus, but it is not.  It and nipah seem to members of a new genus of paramyxoviruses I will show you.
In addition to the explosive outbreak in 1994 in Brisbane, a year later, was discovered another smaller cluster, if you will, Mackay.  Two horses had become infected and one person who had handled tissue from‑‑actually assisted in an autopsy of one of the horses developed mild meningitis and recovered and, a year later, went on to develop meningoencephalitis and died.  Hendra virus was recovered from his brain so a latent infection.
[Slide.]
The Australians have done quite a bit with hendra virus.  They have been able to experimentally infect horses and cats, guinea pigs and black fruit bats.  With horses and cats, they were able to reproduce a respiratory infection and these animals experimentally infected, died.  Guinea pigs, a generalized infection, also fatal, clinically ill and fatal.
In fruit bats, it was a latent subclinical infection.  Nevertheless, in all these animals that were experimentally infected, pathology showed endothelial‑cell tropism and formation of syncytia in blood vessels.
The Australians have also been successful in identifying the natural host for this virus, fruit bats, flying foxes.  Four species of flying foxes appear to be infected in the wild.  About 40 percent of some of these flying foxes have antibodies to this virus and the virus has been isolated from fruit bats and sequences are identical to virus from infected horses and people.  The virus belongs to the paramyxo family of viruses.
[Slide.]
This is the natural habitat for fruit bats in Australia.  I will show you two‑‑
[Slide.]
This is the spectacled flying fox, one of four species that harbors hendra virus.
[Slide.]
This is the grey‑headed flying fox.  The other two are the red‑hooded and the black flying fox.
[Slide.]
Now we forward to Malaysia, 1998, 1999.  I put up this map to try to orient you to where the action in Malaysia occurred.  The outbreak appears to have started in this area.  This is Ipoh.  This is 200 kilometers north of the capital, Kuala Lampur, here, the State of Perak.  Infections in pigs and humans appear to have started here, as I will tell you.
A cluster was recognized in the fall of 1998.  In December of 1998, another cluster of infections both of illnesses in pigs and humans was recognized in Sikamat.  It is a town in the State of Negeri Sembilan.  The largest cluster was actually near a town of Bukit Palandok here in the State of Negeri Sembilan.
I don't know if you can appreciate these blue marks.  There have been clusters and cases and illnesses in other parts.  The Bukit Palandok area has the largest concentration of pig farms in Malaysia.  There have also been cases of human illness in an abattoir worker in Singapore who had handled pigs imported from Malaysia, not recognized to be ill pigs.
[Slide.]
In terms of the chronology, now, retrospectively, what we have been able to reconstruct is that‑‑the story starts in January, 1997 so more than a year before a cluster of illnesses were recognized, the patient, a pig farmer, in Ipoh, developed encephalitis and survived.  Basically, that person is IgG positive for hendra and nipah and the viruses cross‑react and, between the standard, clearly have been fatal and nonfatal encephalitic clinical illnesses, going back, reports of illnesses in pigs.
October, '98 seems to have been an amplification in pigs.  A lot of pig disease and human disease recognized and the initial thought was that this was Japanese encephalitis.  Some diagnostic testing in Malaysia and Japan suggested that some of these illnesses may have been Japanese encephalitis.
As I mentioned, the outbreak moved to the Negeri Sembilan area.  I will show you an epicurve and you can appreciate the number of human cases there.
There were some things that didn't fit with this all being Japanese encephalitis in that predominantly illnesses in humans were in adult male pig farmers, mostly, primarily, Chinese ethnic.  Why would a vectorborne infection select adult male of one ethnic group is not completely consistent with what we know of JE.
Also, the deaths in pigs was inconsistent.  So, in March, basically, Dr. Chua, working in Dr. Ken Lam's lab at Malaya in KL got some specimens, brains and CSF material, from ill patients and attempted cell culture and were able to isolate the virus.  Actually, the virus was easy to grow in a number of different cell lines and produced syncytia.
Dr. Chua, by mid‑March, had done a couple of things there and, by mid‑March, came to the CDC, first came to Ft. Collins and brought with him serum samples and tissue‑culture material.  Basically, the JE serology was negative in 12 out of 13 fatal cases.
[Slide.]
He contacted us in Atlanta and arrived the next day with samples.  EM was suggesting a paramyxovirus.  I have to say, in hindsight, there was a good investment following the hendra virus for us to work with the Australians and get some samples and develop some reagents.  Hendra is considered at BSL‑4 agent and so the work was done in our BSL‑4 lab by Dr. Ksiazak and others.
So we had some reagents and were able to stain the  slide that Dr. Chua had brought and, basically, reacted for IFA to hendra.  Also, we had some PCR primers and they amplified a paramyxal sequence.  Immunohistochemistry using hyperimmune antibodies to hendra lit up as well in tissue samples.
We notified the Malaysian officials and they requested that we assist in investigating and helping control this outbreak.  And then the sequence the next day, we had sequence information that this was hendra‑like.  But, as I will show you, it is not exactly hendra, actually.  It is 25, 30 percent nucleotide different from hendra virus.
Serology; I mentioned that 12 of 13 negative for JE.  They turned out to be positive by an ELISA capture assay, IgM positive.  The Singapore cluster, as I mentioned, we also got samples and confirmed the same nipah virus.  The viral isolate from Singapore and Malaysia was the same.
[Slide.]
This is to show you a nucleocapsid of this virus, negative strain, by EM.  The virologists amongst you can appreciate, if you can see that, basically EM on the tissue culture showing here particles with a nucleocapsid.  And, in here, nucleocapsids budding on the plasma membrane tissue culture.
[Slide.]
This shows a comparison of the genome of hendra and Ipoh morbilli and parainfluenza and nipah and hendra as I will show you on a tree constructed by Paul Rotha and Bill Bellini in our division appears to be 25 to 30 percent, as I said, about average nucleotide difference.  They have sequenced, I think, by now, a large part of all the nuclear protein and another of other genomic areas as well.
[Slide.]
This is a family tree.  This is a genetic tree based on the nuclear protein sequences.  The nipah and hendra virus here appear to be on one branch of a tree and the suggestion is that they may represent a new genus.  They are close but different from a morbilli virus and the other viruses.
[Slide.]
This is the epicurve.  This is the outbreak cluster started in 1998 in Ipoh area.  These are the human cases.  You can see here the large numbers of cases in Palandok, in green.  The total number of human cases were 258.  This is the official number of cases.  There have been some cases, a handful of cases after that, additional to the 258 and there have been 101 deaths, over 40 percent case fatalities.
[Slide.]
What is this disease like in humans?  For the most part, patients presented with febrile encephalitis, fever, headaches, myalgia, within 24 to 48 hours of presentation to hospital as they progressed to coma and needed respiratory assistance.  Pathologically, it appears to be a multisystem involvement, and I will show you some of that, with vasculitis and syncytial giant cells.
It is noteworthy that the respiratory symptoms have been generally uncommon in patients in Malaysia.  I will have to add that of the 11 abattoir workers seen in Singapore, there was one fatality.  So, clearly, the case fatality there is different than whether it has anything to do with different exposure to the virus.  We don't know, but two of the 11 were respiratory and were pneumonia.
In the brain, what you see is a diffuse foci of necrosis and neuronal regeneration, and this is of brain or from a fatal case, and I don't know if you can appreciate the area of foci of necrosis.
[Slide.]
This is, again, showing that.
[Slide.]
This is the giant syncytia cell with immunohistochemistry staining in red, here, for the hendra/nipah virus.
[Slide.]
This is from immunohistochemistry on brain tissue.  Basically, the antigen is found in neuronal cells, glial cells and other‑‑endothelial cells.
[Slide.]
This is a blood vessel and you can see the staining.  Endothelial cells.
[Slide.]
This is to point out that, as I said, it is multisystem.  And renal tubules have stained with antigen as well, shown here.
[Slide.]
You concern yourself with animals and pigs.  This is what is the story in pigs.  This is one of the farm stalls in Malaysia.  You can see that pigs are kept at large concentration in stalls.  In pigs, the presentation is different in that there appears to be a significant respiratory component.
Pigs are described here with rapid, labored breathing.  A characteristic explosive nonproductive cough was described.  "Very loud," is what we are told.  Pigs developed neurologic changes as well, lethargy and aggressive behavior and some focal symptoms as well.
[Slide.]
In terms of the pathology, it is primarily a pulmonary pathology and viral antigens have been seen in the epithelial cells lining the upper airway.  But it is, by no way, only pulmonary in that it is also renal, heart, brain and other tissue.
[Slide.]
This is showing a bronchi, trachea, of a pig, again showing there is some free vascular filtrate as well.
[Slide.]
Same here.  Red is the‑‑it shows the slide that shows you immunohistochemistry lighting up of the antigen.
[Slide.]
In terms of the epidemiologic investigations that were carried out, one concern was whether this viral infection could transmit from person to person.  The concern was with the health‑care workers and pathologists and clinical microbiologists.  Investigation of this group was undertaken.  I think over 300 people were tested.  There were no cases of illnesses in any health‑care worker or pathologist or laboratorian.
In terms of the serologic testing, I think I can say with confidence, there is no evidence of acute infection but some of this testing is being repeated with nipah‑specific tissue at this point.
The case‑control investigations, looking for specific, clearly predominantly over 80 percent were pig farmers, also people exposed to pigs and abattoir workers in other areas, looking to see what specific exposure to pigs may be associated with this illness.
The cases; over 65 fatal cases are positive for nipah in tissue and others.  I will say that another of well, or control, pig farmers enrolled here turn out to be positive and some of them have had minor illnesses suggesting that the spectrum of disease with this virus may be broader than suggested by the encephalitic fatal illnesses.
We have also investigated other groups exposed to pigs, abattoir workers, soldiers, veterinary health‑care workers and others.  I mentioned the investigation of encephalitis cases in Perak and a number of the sera are still being run.  So I don't have the results on those.  I would just say that we saw fatal illnesses in soldiers involved in culling of pigs.  They ran out of protective gear so some of this was done without protective gear.
There have been a couple, maybe a handful, of cases with no documented exposure to pigs.
[Slide.]
In terms of control measures, again, killing of pigs makes the headline but that was part of a number of efforts.  One was basically to try to prevent movement of pigs from farms with illnesses to others.  That, apparently, turned out to be very hard.  There was quite a bit of illicit traffic of pigs and a farmer who had illnesses would try to recover rather than let the authorities know that you had illnesses.
Also, try to educate farmers and others in terms of protective gear and protected ways of exposure to pigs was undertaken.
[Slide.]
But the most sensational, and I think a major part was with killing pigs, culling as we refer to it.   Basically, what was done is an area of about five kilometers around the area of farms with activities was kind of defined and quarantined.  Within that five‑kilometer area, all pigs were killed.
[Slide.]
This is just to show you some of the massive killing of pigs.  Holes were dug and pigs were just walked into it and shot, as you will see here.
[Slide.]
This is some of the protective gear used.
[Slide.]
As I said, they ran out of it and so this was probably the better of the pictures I am showing, and covered with lime.  I have to say that the killing of pigs was effective and that it did, as I showed you on the epicurve, brought us down on the curve of illnesses.
[Slide.]
This is the killing of pigs.  Over, I think we are told, 2 million is the number of pigs in Malaysia before.  And half of those pigs have been killed.  The efforts have not stopped there and cannot stop there.  What the Malaysians have instituted is a surveillance system of pigs going to abattoirs and surveillance systems of farms where they test a limited number of pigs every 90 days and farms that have, I think, more than three infected pigs with serologic testing, then they cull the pigs and offer compensation for the pigs and also surveillance for human illnesses and activity.
I have mentioned a couple of foci as being two or three farms with illnesses in pigs and humans.  We think it is because of the traffic of pigs outside of those areas.
[Slide.]
An important aspect and question that came up was whether the virus has infected other animal species.  The reports were that yes, dogs and cats and other animals were dying.  And you can see this is pigs kept and dogs running around close‑by.  Some of these ill animals were tested.  Indeed, the virus has infected dogs and cats.
I think in one area, half of the dogs tested were positive.  I think the good news is there has been some testing, some systematic testing, done moving away in a concentric fashion from areas with illnesses in pigs and humans.  As you move out, the number of infected pigs‑‑I think beyond ten kilometers‑‑you don't see any ill animals.  So that is reassuring that at least that is not more widespread.
[Slide.]
There are a number of unanswered questions at this point.  I have listed some of them here.  Why and how did this outbreak start is not clear at all.  The reservoir for this virus, is it a bat?  I think preliminary evidence suggests that it may be a bat.  Some number of fruit bats, flying foxes, were collected by our Australian colleagues who participated in this investigation.
Preliminary serologic testing suggests that some of these bats may have low‑level antibody to nipah and viral isolation and sequencing will close the loop on this one, but that is very possibly the case.  Why did it start?  There were some fires reported in parts of Malaysia.  Could that have affected roosting of bats and moved them closer to pig farms?  Unclear.
Is it safe to return to business as usual?  I think I have shown you data that it is spilling into other animals and the fact that movement of pigs outside to suggest that no, and an effort to surveillance and continuing to control this is important.
I think the Malaysians I know are pursuing this very seriously.
[Slide.]
There are a number of other important unanswered questions.  One is the question of persistent infection.  It brings to mind the hendra experience where one person developed fatal illness a year after infection.  And so a long‑term follow up of the infected person is important.  	The question of latent infection, persistent chronic infection in pigs has not been answered at this point.
[Slide.]
This is the remnant.  This is what remains of pig farms in some areas.  I put this slide to remind me to emphasize to you that this outbreak has taken a huge toll not just in terms of human lives and morbidity and mortality and deaths of pigs, but also an economic impact on pig farming and on Malaysia, and to also tell you that there is significant concern throughout the area outside of Malaysia in terms of if the bats turn out to be the reservoir, they are not restricted to one country and efforts to set up surveillance and survey pigs and others will be undertaken.
Finally, I think‑‑and I should have made a slide here of acknowledgements to tell you that the work that I presented is the work of too many people to show here.  At the CDC, in our division, I would like to let you know that the pathology is the work of Dr. Sherif Zakil.  Dr. Bill Bellini and Paul Rotha did the molecular work and Drs. Pierre Rollin and Tom Ksiazak and others were involved in the viral isolation, in the serologic testing and isolation from the pigs and other animals and humans as well.
We had a large number of people in Malaysia and the team was lead by Dr. Tom Ksiazak who actually spent more than two months there and I know just left Malaysia this weekend, wasn't back in Atlanta when I left yesterday.  And also the Malaysian Ministry of Heath, the hospital staff who spent quite a bit of time involved in doing these studies and gathering that information and control efforts.
The veterinary authorities in Malaysia, as well, the University of Malaya.  Also Singapore.  I mentioned the cases there but we did have a team there go into a case‑control study.  Preliminarily, it looks like their exposure to urine and excreta may be associated with cases but, also, the Singapore authorities contributed to this as well as the Australian animal labs.  They had a team there and they were experienced with the hendra virus and the bat work was crucial.
I think I will stop there and entertain questions. 	DR. AUCHINCLOSS:  Thank you very much.  To bring us back to work of the committee today, other than the implication that we don't want the nipah virus in our potential donor pigs, connect this outbreak to this discussion.  What are the implications, or are there implications, to the infectious risks of a xenotransplantation and FDA policy?
DR. KHABBAZ:  I think this is a prime example of an emerging viral infection.  Three months ago, we had no idea that this virus existed.  It is not primarily a virus of pigs in that we think that the host is a different agent.
In one way, it illustrates the need for vigilance because these agents are there and cross species.  So I think it is vigilance.  It is sobering to see the impact, the number of illnesses and mortality both in pigs and humans.
In terms of implications of disease outside of the area, I don't think I can comment on that.  Until you start looking, you don't know but, clearly, if the bat is the reservoir, that host species is restricted to that part of the world.
DR. AUCHINCLOSS:  Let me put the question to the committee perhaps in a slightly different way.  To my way of thinking, when I listen to stories like this, I say, "So why are we interested in xenotransplantation?  These kinds of events occur in nature.  Xenotransplantation isn't the problem."  Is that an implication or is that a way of looking at this that is reasonable, or is that nuts?
DR. CHAPMAN:  There are two things I have heard over and over through this whole xeno discussion.  One is we have lived with domestic pigs in very close contact for thousands, if not millions, of years and, therefore, we know there are risks and they don't pose much of a risk to us.  I think Dr. Khabbaz did a very nice job of outlining the cautionary note in this about new infections.
The other side of this; yes, this is an explosive new infection.  It is an experiment of nature and it occurred in a part of the world that is relatively isolated from here.  But I think what it underlines to me is the significance and the importance of the husbandry requirements outlined in our PHS guideline in terms of the importance placed on raising animals, even animals we think we know well, that may serve a sources of xenografts in closed colonies with barriers to prevent just this sort of introduction of infection from vectors we may not be able to anticipate on the basis of what we know about the past history of the husbandry of the animals.
DR. KHABBAZ:  Actually, to follow up on Louisa, I will tell you that during this investigation, it came to our attention a couple of cases in this country and one elsewhere of Malaysians who have traveled there who had an encephalitic illness.  There was quite a bit of concern and they turned out not to be nipah virus.
But preliminary information from some of these cases, people had worked in pig farms but did not live there suggested more than two weeks, possibly, of an incubation period.  So with travel an with exposure, I think the scenario of this possibly having expanded beyond Malaysia is not too far‑fetched.
DR. COFFIN:  A perhaps subtle point, but like retroviruses, I would expect paramyxoviruses to be subject to the same kind of an activation by antisugar antibodies as human serum.  That seems to be not an absolute barrier to infection in a case like this.  It seems not to be a very good barrier at all.
DR. ONIONS:  Could I just make a general point?  First of all, I want to congratulate everyone involved in this work.  It has been an excellent to a really important outbreak.  But my comment is these are not the things that concern me from xeno, apart from the facetious comment that keep fruit bats out of your herd, the real issue is that the viruses that we really need to worry about are certainly new viruses that could, in fact, but don't, necessarily, have a very big impact, perhaps no impact on the pig population and have a long latent period in people, those are the viruses that we need to worry about and, by definition, we don't know what they are.
I would also caution‑‑I agree with Louisa's comments‑‑there are viruses out there we know very little about.  I reiterate a story I presented, and I am embarrassed about it because we haven't got any further with it, but there are closed colonies of dogs in which we have found a virus and others have found a virus here in North America which looks like a virus called desoxyvirus.  The only other known host of a desoxyvirus is the wart hog and occasionally gets into pigs.
We don't know where this virus came from.  We don't know why it is there.  But, clearly, in animal populations, if you look very carefully, there are viruses there that we just don't know anything about.  I think it is those kinds of cautions that I am concerned about.
These are spectacular but they are not really a risk for xenotransplantation.
DR. PAUL:  I think this is an excellent example just because zoonotic are already demonstrated, but nipah virus infection in Malaysia and hendra virus in Australia, those are only two examples that got a lot of publicity.  And yet, if you look at the swine industry in the last year and a half, swine hepatitis E virus, circovirus and there are a number of viruses that we don't know anything about, as Dr. Onions mentioned.
And we also don't have very good diagnostics.  We don't know what they do in pigs.  They may be totally nonpathogens.  They may not pose much of a risk to people.  Nevertheless, I think we need to be vigilant and support the studies to better understand what risks they pose.
Again, some of the studies mentioned earlier this morning, with the liver transplantation, are we testing for hepatitis E virus, swine hepatitis E virus?
DR. AUCHINCLOSS:  We come back to the FDA implications, however, Louisa's point was this kind of event emphasizes the importance of the breeding in captivity in closed herds kinds of requirements that were in the draft FDA guidelines and those seem to us to be appropriate.  Is that the right implication to take from this message?
Thank you very much.
COMMITTEE DISCUSSION
I want to return, for ten minutes, before we take a break for some coffee to one unanswered question from the morning session.  I suspect we will come back to the morning session questions yet some more in different ways.  When we return from coffee, we will have about an hours' worth of presentations and then end with committee discussion on the afternoon's questions to and from the committee.
The question that I think we have not answered from your morning list was the second of the two which was what if somebody comes up with a positive.  Let me phrase that in two different ways.  What if somebody comes up with a patient who is positive but the question was put to me, what if somebody in an animal system came up with a positive transfer of a PERV to a cat, for example?
So you could answer either of those two questions.  If I recall, the way we answered that question a year ago it seems to me was if you get a positive, call us back and we will talk about it.  I am a little bit inclined to come up with the same answer right now, but let me see what the committee as a whole thinks.
DR. WILSON:  Can you define what you are going to call "positive?"  I guess we should define what we want to call "positive" first.
DR. AUCHINCLOSS:  That is a very good point.  Could you do it for us?  What is positive?
DR. ALLAN:  Positive is a sustained PCR‑positive signal above microchimerism, RNA, antibody‑positive.  Any of those.
DR. ONIONS:  You can go in criteria.  Again, if we talk about the gamma retroviruses, the majority of animals exposed to exogenous retroviruses recover from infection, so they are not like HIV where, if you are infected, as far as we know, with very few exceptions, you are infected for life.  So there is a difference.  That is the first thing.
So if I had a patient that was antibody‑positive and there was no other criterion of infection in that patient, I would not be too concerned.  There would be evidence accepting the assays.  There would be seroconversion of the evidence of exposure to the virus.  So that might not be of tremendous concern.
If there was‑‑I don't know who is going to disagree with me‑‑if there is evidence of PCR positivity in peripheral blood cells that was sustained, that is another order of concern.  But if the patient was not shedding virus or did not have plasma viremia, whilst that patient was certainly of concern and, certainly, perhaps, at risk of developing some disease, it is less of a concern than the next one which is where that patient has plasma viremia because that patient potentially, it seems to me, is capable of transmitting that virus onwards.
And you want to be very cautious about whether the virus has been secreted in the body fluids.  So I think there is an order of positivity.
DR. MICHAELS:  Could I just add a proposal and it could be shot down.  If we find a patient that has gone through one of the procedures of porcine xenotransplantation and we feel that there is a true positive that, at that point in time, we would want further samples from that patient.  So we found a positive in the peripheral‑blood lymphocytes.
We would want to say, "Hold on.  Let's go back.  Let's get some more samples from this patient, repeat the peripheral blood lymphocytes, look at the plasma, look at serum, look at the saliva, look at other samples which I think some of the groups have put down in their algorithms of how they would approach this.
I think a reasonable approach is should be say to the FDA that we should also, at that point in time, say, let's put a little bit of a hold on until we can analyze those samples at that point in time and maybe look at the close contacts as well.  Would that be a reasonable approach?
DR. VANDERPOOL:  My questions go beyond some of what we have been talking about in terms of just testing and keeping it in house.  I am not sure we need the scare, but it is worth, in the light of this presentation, to raise the possibility that a patient with a xenotransplant organ would not only become positively infected but be pathogenic and possibly have a serious enough condition that could be passed on to others.
The question would be with respect to an experimental protocol, what would happen, like restricted sexual activity to quarantine, need to be put in any sort of consent form.  It seems to me the possibility of that is distant enough for that not to happen, but one of the scary things on this slide is to see all the pigs herded into a pit and shot.
We are not going to do that to humans, but what would we need to do if some egregious infection happened to occur.  I think it is at least worth considering without scaring the wits out of ourselves.
DR. ALLAN:  I want to get back to the antibody‑based assays‑‑
DR. AUCHINCLOSS:  Antibody‑positive but otherwise negative patients.
DR. ALLAN:  Yes.  I feel much more strongly in the case when you an antibody‑positive individual or an animal that that is a red flag, that is a situation when you have to stop and say, "What is going on?"
I know, in my lab, what we have done, we have taken an HIV‑infected cell line and IV injected three monkeys.  They don't seroconvert.  Those cells are dumping virus and you are not getting an antibody response.  So, many times, the antibody response is in response to an active infection in the recipient cells even if it isn't.  In most cases it is, so if you get an antibody response, you have got to stop and wait and see what is going on.
It may be that they have cleared the viral infection.  It may be that, but I am not going to assume that.
DR. ONIONS:  I don't wish to be misunderstood.  First of all, I don't necessarily disagree.  I think any of these positives is an indicator that is going to take a very clear look, maybe caution, maybe start.  I am not dissenting from anybody.  All I was trying to say is that I do think that, again, one has to do things by analogy at the moment and my analogies are not one just going to HIV.  They are the gamma retroviruses.
My only comment is that, in fact, if you go and look at 80 percent of street cats, 80 percent of street cats have got antibody to FELV.  But they are not, at that moment, at least the majority, not actively infected by that virus.
So, taking that analogy, it is of concern.  It means exposure.  It means the virus has been expressed in that patient.  That is clearly a matter of concern but, to my mind, it is not as great a concern as a patient who has got persistent PCR positivity, persistent infection in their white blood cells.
That is not as great a concern as the next level up when these patients are viremic.  They have got plasma viremia and they are possibly also shedding virus.  It just seems to me there is a hierarchy of concern.  This is not to say that any of them are not of concern, but it is a hierarchy.
DR. COFFIN:  There are two issues of significance.  Is that patient who has evidence of having had or having presently persisting virus replication.  I should point out, David, the presence of antibodies may mean the virus has gone underground but it almost certainly means it was replicating.
In the case of many oncogenesis models, it could be some time after that that the actual disease appears.  But, as far as pathogenesis in that patient, I think probably most of us would agree there is very little you can do but watch them very closely.  As far as that goes, that is what you do.  You watch them.  You take as many samples as you can and get as much information on the natural history and possible progression of disease later on.
The issue of greater significance, of course, is where that patient is capable of transmitting virus.  It is to learn to do everything possible to learn whether that patient is transmitting virus to somebody.  But that is not easy.
We have been through, I think, discussions on how you can follow up on contacts and whether you can do that voluntarily.  It probably, almost certainly, has to be voluntary on the part of contacts and so on.  Even in the case of HIV, examination for virus in saliva and semen and other bodily secretions is not very revealing as to what is going on for transmission.
The levels are low here and they are low there, but transmitted one way and not the other.  So it is not going to be easy without being able to do sort of significant contact tracing and that is going to be very, very hard to do.
DR. HENEINE:  This is to second basically what John has said.  I think it is very difficult to answer the question that you asked to define the criteria of infection, of PERV infection, in a human because, given the current knowledge, we cannot define those markers.
Or, what we can do is use those tools, diagnostic tools, available to us and then, longitudinally, follow up these patients that show any sign of positivity, either seropositivity, serologic or molecular.
Beyond that, it is speculation.  It is extrapolation from the FELV system.  John would argue that, no, we see things different in the HIV or HTLV systems.  So we really can't define those markers today.
DR. AUCHINCLOSS:  I think everybody would agree, we don't know the meaning of the implications of positive.  But the implications for the FDA are probably hold and get their experts back.
MS. MEYERS:  I am impressed by Novartis' protocol because they had contingency plans on what to do with the person if they are found positive and the fact that they can transmit it.  I am sure that all of this information is going to have to go into an informed consent so that people in the future who go through any type of procedure are going to know that if they are positive and if there is any doubt, they may be isolated.  Their spouse and their family may be tested, et cetera.
But what about the people now who have already gone through it.  They didn't give permission to be isolated.  The fact is, if you find them shedding virus and they are contagious, obviously, they should be isolated.  If it is totally voluntary, and they don't want to be, what are you going to do?
DR. CHAPMAN:  I read this question a little bit differently than the way I think it is being discussed.  I am not sure what the FDA wanted from it, but, perhaps, it is worth framing the way I read it and seeing if that is the response you wanted.
There has been discussion, if a person is identified as positive, as infected, what sorts of labor studies could be done or epidemiologic studies or contact tracing.  You are bringing up the issue of what sort of isolation precaution should be put onto someone and that would, in part, be dependent on what the laboratory studies shows about presence of virus and different secretions and, also, the law because I will say we have discussed, in our Public Health‑‑within the PHS in our meetings these issues when people bring up, from time to time, the issue of quarantine.
Without going into any details about the discussion, I will say what you can do to involuntary confine someone is limited by law and is defined in law and it varies from country to country.  And there are also ethical issues involved.
It is not clear to me that FDA wanted us to struggle with those things now.  My interpretation of this was‑‑what I thought you were asking was‑‑well, in my mind, I boiled it down to this.  What would be the criteria, what would you identify, that would raise the expectation that this would be an appropriate point for the FDA to call a clinical hold on all trials until certain issues were clarified.
DR. AUCHINCLOSS:  Louisa, I agree with you.  That was the way I was reading it.  I don't think we need get into the issue of precisely which samples you need from which patient.
DR. CHAPMAN:  Who would clarify them?  How do you clarify them?  How far you do you go?  That can be asked later.
DR. AUCHINCLOSS:  The question is where is the stop point on all trials that leads to an effective clinical hold here; is that correct?
DR. WILSON:  Yes; or, alternatively, whether a certain result may impact a single trial where that result is found versus all trials‑‑
DR. AUCHINCLOSS:  Of the three positives that have been brought up that one can imagine, is there any one of them that would either enable that or other trials to go forward while additional data was being gathered is, I think, the heart of the matter.  Is that not correct?  What I thought I was hearing from the group, but I don't think it has been explicitly expressed, is pretty much that if you get a positive anywhere, you are going to need to stop everything and get your experts together to look at it carefully.
DR. ALLAN:  I would say not.
DR. AUCHINCLOSS:  Is that too strong a statement?
DR. ALLAN:  Too strong.  We have already heard one case where there are two potential positives that look like microchimerism that disappear or are transient.
DR. AUCHINCLOSS:  I understand.  Now we are on to a question of what is really, really positive.
DR. ALLAN:  Not even the question‑‑and I believe that there is a strong possibility that those were microchimerism.  So even if we were sure that those were positive, I would still not say that we need to stop a particular study based on microchimerism.  We are really moving past that to something like antibodies.
DR. SALOMON:  The key thing here is, I think, what you are trying to say is, without defining it for the moment, I think that if it is positive, if we infect a patient, and we all agree that the patient is infected, details aside for the moment, then I think all studies should stop then.  And I feel strongly about that.
I think that part of the deal that we are making with the public to rationalize moving forward cautiously with clinical trials is that we are monitoring.  Therefore, when I said to Jay, you can't just tell them, "Don't worry; we are banking all the serum," that isn't going to work.
No; we are studying them.  As it gets better, then we will study the banked serum.  I don't care.  But we are doing it, an insane reason here.  If someone is really infected, you have got to stop all studies.  Then we can discuss.
MR. BENEDI:  From a recipient perspective and one whose immune systems is compromised, and so is Bill's, and we worry about every single day of even catching the common cold, when you talk about someone getting infected and then everybody watching and stopping and seeing, that patient is going to be gone very quickly.
If, in fact, that patient is infected positively, they won't be around very much longer, only because of the compromised immune situation that they are going to find themselves in with the medicines they are going to take so they reject the organ that has been placed in there.
DR. HIRSCH:  I think we are overstepping the bounds here by a long shot.  We don't know if an infected person with this virus will even get sick let alone be around in a few weeks.  So, it would seem to me, that it wouldn't surprise me at all if someone along the way, in one of the xenotransplants, becomes infected with an endogenous pig retrovirus.
You still have to weigh the pluses and the minuses.  If this has, by that time, proven to be a useful bridge technique for cardiac transplantation or for whatever, one infection with one pig endogenous virus, to me, doesn't mean you stop the whole program, but you watch that individual very closely and you monitor all the other people who have had this kind of procedure.
But to put an immediate stop on everything seems to be overkill, to me.
DR. ALLAN:  I would think that, since we are early on in clinical trials, that if you do get a positive, regardless of what trial it is, you really need to stop at that point.  Once you are into a place where you are into a therapeutic mode or into that, that is a different story.  But we are in an early stage of the clinical trials.  I think you really need to put a stop‑‑
DR. KASLOW:  I think we have a paradigm‑‑I don't know whether it is totally applicable‑‑and that is with most, certainly, large clinical trials, there is often a data and safety monitoring board that would have some certain trigger points available to them.  When those trigger points come into play, that group is convened to discuss whatever that evidence is and make a decision, at that time, on an ad hoc basis as to whether the trial should continue and what other things should take place at that time.
It seems to me like, in general terms, that is what we ought to be doing.
DR. AUCHINCLOSS:  I think that is exactly right.
DR. SIEGEL:  Or only have purview of the trial that they are authorized to monitor.
DR. KASLOW:  I understand, so that the analogy may not be complete.  On the other hand, we could create a variation of it in which, since there is a known number of trials going on and it all comes under a common rubric, you could make the rule that any or all of those trials, if there were any single event that led to that threshold, it would trigger the same convening of whatever group you decide to monitor.
DR. AUCHINCLOSS:  You did a clinical hold once before; right?  There is nothing that keeps you from doing that at any point; is that not true?
DR. SIEGEL:  It should be pointed out that, in Dr. Hirsch's comment, once there is a proven effective therapy and, presumably, if that is an FDA‑approved therapy, then the whole legal framework changes.  Those therapies can also be seized, market‑withdrawn, or whatever.  But it is a different situation from the IND investigative situation where the clinical hold is a relatively simply administrative measure whereby we can stop a clinical trial.
DR. AUCHINCLOSS:  I understand.  But we are a ways away from‑‑
DR. SIEGEL:  Exactly.
DR. AUCHINCLOSS:  I don't think that is necessary to get there.
DR. SIEGEL:  I am just saying where that is necessary in the experimental environment.
DR. AUCHINCLOSS:  I guess, to sort of characterize it as everything stops is not the way I would want to do it.  I thought the expression here is that that is the moment where you would your experts to look at the information that you obtain rapidly and size it up, that would be the thing you would be doing.
I am guessing that we are really not very far apart on this topic is what I really mean to say.
DR. SALOMON:  I just want to be clear.  I don't agree.  I think the minute you have a positive infected patient, then everything stops.  It is okay if, five minutes later, you get everyone together and you have this big discussion, et cetera.  That's fine.  But I think that is important in terms of maintaining the trust of the public in us.
DR. KASLOW:  What you need to define, then, is the sequence of events that leads up to the decision that that positive is there as well.
DR. ONIONS:  If I could just very briefly‑‑first of all, I still hold the view that there is this hierarchy of difference of positivity.  I would have, before this discussion started, and I think what is useful about this discussion, is that you don't stick to rigid views and I am listening very carefully.
My view before had been that‑‑first of all, one point.  This is a complex issue.  It is not quite straightforward when you say something is positive that that patient is infected.  For instance, if you find low‑level virus in the plasma, that virus might actually be coming from the donated organ and it might not have ever infected the human cells.
That might be true with a large solid organ transplant, for instance.  Similarly, antibody might be an antibody response device actually to that organ and, again, that patient's cells may never have been infected.  So there are all sorts of caveats here that would have to be investigated.  But I still hold to that hierarchy of positivity.
My own view had been that if the top two‑‑that is, if the patient was plasma viremic or the patient had PCR‑positive peripheral‑blood mononuclear cells, that certainly would be my criteria for a stop.  You stop clinical trials.  You hold.  You review the data and follow these patients until you knew what the resolution of those patients was.
My caveat would be on that, my hold, was that antibody would not necessarily have triggered that, in my view.  That might have initiated more intensive surveillance of that patient, certain body fluids, probably in contacts, all the usual more intensive kind of surveillance without necessarily putting a hold.
But I am listening and I am hearing both Dan and I am hearing Jonathan saying that that might not be good enough.  I begin to understand and, perhaps, respect that point of view.  So I wouldn't be unhappy with that view either.
DR. GORDON:  Al Gordon, the Islet Foundation.  I just wanted to comment on the suggestion that a single positive would bring the sword down and all clinical trials would stop.  I think it is important to realize that there is going to be a spectrum of activity in these clinical trials.
There will be vascularized whole organs with immunosuppression.  There will be cellular transplants with immunobarriers and no immunosuppression.  We also have assays that, as we know, are very prone to false positives and, therefore, in recognition of that propensity for false positives, it would really be an unwise decision to drop all clinical trials should one occur.
I think we are also dealing, as we know, with the chain of events.  So far, no animal has been infected with PERV.  So even the first requirement of infectivity has not been satisfied.  Does it cause disease?  We don't know.  There has never been an infection.  If it causes disease, can it be transmitted to other people?  Well, it has never caused disease.
Therefore, we are now concerned about something several steps along a highly improbable chain and some are proposing that we take very draconian actions today.  I just want to put it into perspective.
DR. AUCHINCLOSS:  I appreciate your perspective.  We will come back to some of those issues this afternoon.  I don't mean to be using the sword characterization here.
DR. VANDERPOOL:  I think our basic commitment to the public is to make sure, by every conceivable means possible, that nothing like this ever occurs.  Should a bad situation present itself, it seems to me you handle that not by an overall stoppage of all the trials, but by immediate and thorough review of the that particular situation.
It may have been due to the herd.  It may have been due to the type of compromised state the patient was in.  It may have been someone on the surgical team that made some sort of mistake.  Who knows what it would be.
So it strikes me that we are trying to cross too many bridges before we get to them.  But what we are giving is a very clear indication that we don't just act like the measures we are taking will ever keep anything like this from happening.  We go ahead and, as we are going, we allow ourselves thinking about worst‑case scenarios and have in our minds, and perhaps in policy, what we intend to do about that.
MS. MEYERS:  Could I respond to that quickly?  I think that if a patient accepts risk, no matter what that risk is, even death, that is fine.  It is up to the person who signs the paper and agrees to the risk.  But the public doesn't have to accept that risk.  If you have somebody with a transmissible virus getting on the bus, taking taxicabs, going to work and interacting with other people, then it is really up to the FDA to protect the public from what could possibly happen even if it is only one in a million chance.
The situation in Indonesia proves that maybe, if somebody had recognized what was happening with the first case, or the first five cases, so many hundreds of people wouldn't have died.
DR. COFFIN:  I was just going to weigh in on the point that I think this last comment points out how complicated the situation is going to be and I think basically supports the "call us back when it happens" approach to this, but be ready to put at least a very short hold, as you suggested, onto things while it gets sorted out but then be prepared to move as fast as possible to relieve that.
DR. AUCHINCLOSS:  I think that is right.  I would like to comment about too many bridges too quickly.  It is hard to speculate about all the possible events.
DR. CHAPMAN:  I wanted to try to bring some clarity to this discussion by stating a null hypothesis, if you will.  Instead of talking in terms of positive‑‑first of all, I am not sure if everybody is meaning the same thing when they a positive.  So, instead, I would like to state a hypothesis that, instead of taking about a positive, we talk about evidence strongly suggestive of an active infection of a human.
And how you decide what that is‑‑I don't care to talk about lab criteria.  That may be convening a panel of experts or a series of consultations, but at the point that you have evidence strongly suggestive an active infection of a human, then what action should the FDA take and, instead of talking in terms of everything coming to a halt or stop or something like that, let's talk in concrete terms.
It seems to me when my colleagues at the FDA are faced day‑to‑day with having to make a decision about intentionally allowing additional people to be placed at risk‑‑not an experiment of nature; intentionally allowing additional people to be placed at risk or not.
So the question, it seems to me, to them is not when do you call a global halt on the entire progress in this field, when do you stop all clinical trials.  The question is what are the criteria under which the responsible move would be to desist from intentionally placing additional people at risk until enough investigation had occurred to sort out the issues and the level of the risk, risk assessment and containment control and prevention, which is what we do every day.
DR. MIKELSON:  Thanks, Louisa.  I agree.  That was one of the comments I wanted to make.  I think the other one was the comment was made by a member of the public which points out that we still don't have a good animal model.  That was another issue that I would like to hear the committee discuss some more about because, as you pointed out, the lack of positive data in all of the animal models really puts this whole question of how do you decide what to do if you do get a positive, or some indication of infection, into doubt because this has moved down multiple steps.
We started out the morning asking for more indications of tests of earlier events in a potential infection cycle, asking for antibody tests.  And those are all sort of agreed.  But we still don't have any idea of‑‑we don't have a good animal model out there.
I also agree with Dan.  If there is any indication of an active infection, we should stop until it is analyzed.  But how do we proceed without a good animal model?  That is what I want to know.
DR. HIRSCH:  Even without an animal model, I agree with you that‑‑
DR. MIKELSON:  I don't want the people to be the animal model.
DR. HIRSCH:  But there are a few things that might be worthwhile.  One is we now have fourteen FDA‑approved antiretroviral drugs for other retroviruses.  In a good replicative in vitro system that various people in this room might have, you could test a number of drugs and at least have the vaguest idea of what drugs this virus might be sensitive to so that, if some untoward event came along, you would at least have a head start there.
DR. AUCHINCLOSS:  I am inclined to kind of lean on my colleagues to see if we might end the discussion, but Jonathan looks eager so I will let him go.
DR. ALLAN:  Just the one point about what Dan said which is the active infection that Louisa added to.  The reason I think Dan is‑‑I am not going to read your mind but the reason why Dan feels so strongly, and I agree with him, and it goes across all the clinical trials, is because if you get an active infection, it means that PERV infects humans.  That is the reason to stop, because you go, "Oh; my god.  It infects humans."  That is as simple as it can be.
DR. ONIONS:  Hugh, I don't want to prolong the discussion, and I don't want to disagree because, actually, my instinct with this thing is a degree of conservatism, but I think it is the definition active infection that I have a problem with because I think this is such a complex issue that actually defining whether these patients are actively infective is not straightforward.
I will reiterate it is quite possible you could have virus in the plasma from a solid‑organ transplant, from a large number of cells, I am talking about here, that is not active infection, that is actually virus coming out of the donated organ.
You could have an antibody response to virus producing that organ that has nothing to do with active infection but, by conventional criteria, you would call that an active infection.
DR. HIRSCH:  What is an active infection?  What do you mean by an active infection?
DR. ONIONS:  That is my concern with making‑‑
DR. AUCHINCLOSS:  In many ways, it reduces to that, doesn't it?  The FDA is really more likely to face a situation where they are looking at that and not knowing exactly what it means.  That doesn't, necessarily, put everything on hold.  It means it is time to talk to your experts just to evaluate the data.
And then there is Louisa's stipulation that if you really, truly knew what that data meant, that would be the indication for a hold.  I think that there are levels like that.
DR. CHAPMAN:  Or maybe until you know what that data means.
DR. SACHS:  I gather the definition of active infection is going to be very different, quantitatively or even qualitatively, from what we have been talking about all day so far; is that right, Louisa?  How do you define active infection?
DR. CHAPMAN:  I don't know.
DR. SACHS:  A replicating virus?
DR. SIEGEL:  The shift I was trying to get people to make was from talking about a positive which may mean a positive test result and, with the state of the art at this time, may mean a false positive test result, to talking about evidence of some sort of state in the recipient.
So I think it may be more productive to discuss whether people think if there is data that an appropriate consultation with appropriate people with appropriate expertise develops a consensus that this is reasonably suggestive that there is an infection in the human.
I don't know what that data is but would that be criteria for a hold, as opposed to if you get a positive antibody test, would that be criteria for a hold?
DR. AUCHINCLOSS:  My fault.  I kept talking when I said I was going to stop talking.  If I were the FDA, I would think that I had heard what I needed to hear on this subject, but what do you think?  What does the FDA think?
DR. SIEGEL:  It is very difficult, other than on a case‑by‑case basis, to determine when you have enough evidence to have a high enough level of suspicion.  My take on the issue, having heard everyone, though, is that there comes a level of suspicion where, even if there is uncertainty, it is time to stop treating additional patients until you both develop the certainty, relevant data, and have public discussion.
I see a lot of‑‑since the transcript never shows head nods, I will say that I see a lot of positive nods.  If that is what the committee means, I am comfortable moving on.
DR. AUCHINCLOSS:  One of the pleasures of coming down here and talking to you is that we don't deal with hypothetical questions.  We deal with real questions.  Here we are dealing with a hypothetical and it is very hard to be precise and specific.  But you have just characterized what I think people have been saying, there is a level of concern at which you stop.
One more comment and then we are going to break for coffee.
DR. WALTERS:  I think the data monitoring committee notion is a worthwhile one to come back to.  There are committees that do monitor multiple trials.  For example, in the National Institute of Allergy and Infectious Diseases, there are a couple of committees that monitor quite a large number of trials and if there were a subgroup of this group that could be designated on a standby basis if something untoward comes up, that group could be called into action very quickly and could give an outside reading.
DR. AUCHINCLOSS:  I think that is perfectly reasonable.
DR. SIEGEL:  We have the capacity to telephone any member of this committee for advice as information comes up.  Once you talk about groups, then we get into the laws about consulting advisory committees and what you can do in closed session and what you can do in public session.  We will have to look into that because to convene people without public notice is something that you would have to ask Gail about but isn't so easy to do.
DR. HIRSCH:  But he is right.  Certainly, the AIDS clinical trials group and those have standing data safety monitor boards not for single trials but for a number of‑‑
DR. SIEGEL:  So some organized group.
DR. AUCHINCLOSS:  Time for a coffee break.  We will come back here at 3:20 for an hour's worth of presentations.
[Break.]
DR. AUCHINCLOSS:  We will begin the second half of the afternoon session.  The schedule will be slightly different from what is on the printed agenda.  We will be starting with Eda Bloom with an introduction and review of recent policy developments.  Then we will have a break and go directly to Taylor Wang and then come back to an examination of risk posed by different types of xenotransplantation with Eda Bloom from the FDA.
So I am going to introduce Eda Bloom at this point.
II FDA Xenotransplantation Policy Development
FDA Perspective
DR. BLOOM:  Thank you to the committee and the audience.  We have heard a great deal this morning about recent developments in science that are relevant to xenotransplantation.  For the rest of the afternoon, we are going to discuss the translation of that science into regulatory policy.
[Slide.]
First I would like to go over the definitions that are current for xenotransplantation.  We are defining it, and I say "we" meaning FDA and the Public Health Service, as any procedure that involves the use of live cells, tissues or organs from a nonhuman animal source transplanted or implanted into a human or used for ex vivo contact with human body fluids, cells, tissues or organs that are subsequently given to a human recipient.
The corollary of that is that xenograft products are live cells, tissues or organs from a nonhuman animal source used for xenotransplantation.  A little later, we will get into some of implications of these definitions but, for now, we will just continue with a brief background of the development of xenotransplantation policy.
[Slide.]
In 1993, FDA published a document that was entitled The Application of Current Statutory Authorities to Human Somatic Cell‑Therapy Products and Gene‑Therapy Products.  Among the somatic‑therapy products used for human treatment were listed xenogeneic cells.  That was none too soon because the first xenograft product IND was submitted to FDA in 1994, the very next year.
It became clear very early on that the use of xenotransplantation raised a number of safety concerns that could be very problematic not only for the patient but also for the public at large; that is, the transmission of xenogeneic infectious disease to the patient and subsequent transmission to their close contacts and to the public.
[Slide.]
Immediately, there began a series of cooperative efforts among PHS agencies including CDC, NIH, HRSA and FDA.  But it was not, in fact, as my slide suggests, limited to PH.  The Department of Defense was involved, is involved.  The Department of Agriculture.  And so, in fact, there are a number of government agencies that have been cooperating in the development of xenotransplantation policy and issues.
In 1996, the Draft Public Health Service Guideline on Infectious Disease Issues and Xenotransplantation was published in the Federal Register.  Before, and also subsequent to, that, the FDA, as well as various other Public Health Service agencies and including as well private agencies and private foundations have held a number of public meetings.
These public meetings have enabled us to obtain public input and, in fact, today of course is another in a series of that in which we do hear the public and we do hear the discussions.	
The most recent document that has been published, and I will discuss this one in a little more detail, is entitled Guidance for Industry, Public Health Issues posed by the Use of Nonhuman Primates Xenografts in Humans.  That appeared in April of this year as a notice of availability and the document, itself, is available on the Internet.
This document is a good example of what happens as a result of a lot of scientific evidence that has accumulated and a lot of public input.
[Slide.]
The document was issued by FDA to address concerns specifically regarding nonhuman primates as sources of xenografts.  The infectious‑disease risks posed by nonhuman primate sources was obtained both from historical data and, of course, there was a recent publication that HIV‑1 most likely, if not definitively, was sourced from a chimpanzee.
But there were two other issues that really moved the publication of this article.  One is that the proximity of nonhuman primates to the feral or wild state is still very, very close and the various Public Health Service Guideline recommendations, as Dr. Chapman alluded to, would be very difficult to apply to nonhuman primates if not impossible at this time.
That also refers to the husbandry issues of nonhuman primates.  For these reasons, the FDA decided to go ahead and publish this document.  We discussed the document in very great detail with the other Public Health Service agencies who accepted the principles of the document prior to its publication.
[Slide.]
FDA concluded, regarding the use of nonhuman primate xenografts that health concerns within the scientific community and general public were raised.  The current data indicate that recipients, their close contacts and the public would be exposed to significant risk by the use of nonhuman primate xenografts.
It was further concluded that additional research and evaluation would be needed to obtain information to assess and reduce the risk posed by the use of such xenografts.
[Slide.]
We made three recommendations based on these conclusions.  The first was that an appropriate federal xenotransplantation advisory committee such as the Secretary's Advisory Committee on Xenotransplantation, which is currently under development within the Department of Health and Human Services, should address novel protocols and issues raised by the use of nonhuman primate xenografts in humans, that such a committee should conduct discussions including public discussion as appropriate and that the committee should make recommendations on the questions of whether and under what conditions the use of nonhuman primate xenografts would be appropriate in this country.
[Slide.]
The second recommendation is that clinical protocols proposing the use of nonhuman primate xenografts should not be submitted to FDA‑‑that is our recommendation‑‑until sufficient scientific information exists addressing the risks posed by such xenotransplants.  Consistent with particular regulations under the IND regulations, any protocol submission that does not adequately address these risks, does not justify the safety of nonhuman primate xenografts, is subject to clinical hold due to insufficient information to address the safety risks.
[Slide.]
Finally, at the current time, we believe that there is not sufficient information to assess these risks and we believe that it will be necessary for there to be public discussion harkening back to recommendation No. 1 before such issues can be adequately addressed.
At this point, I would like to stop this topic.  We are now going to proceed to the discussion of risks in transplantation.  Dr. Taylor Wang will make a presentation on a new kind of encapsulation.  Part of what FDA has to do is not, as you can tell from much of our discussion today, only deal with what is here and now but we have to be able to deal with what is happening.  And our policy must be able to be flexible and to be appropriate enough to deal with what is coming in down the pike, not just what we have at our door at the moment.
Dr. Wang, who is Centennial Professor at Vanderbilt University, has something to say about space‑age technology and encapsulation.
Guest Presentation: Immunoisolation Technology
DR. WANG:  Thank you, Eda, for the introduction.  	As Eda said, I came from a different background in more ways than one.  Actually, I am a physicist by training.  My colleagues have asked me the question why a self‑respecting physicist wants to get involved in this blood mess.  I will let you know the answer later.
[Slide.]
What I wanted to say is this is not a one‑man operation.  Actually, this is a team operation.
[Slide.]
As you can see, it is a very large team.  It consists of an interdisciplinary approach from physicists to fluid mechanics and material scientists and surgical research, molecular physics and polymer science and pathology technology.  That is at Vanderbilt University where we team up with the University of New Zealand and which their primary responsibility was looking at islets and the isolation of islets and function of islets and retrovirus is one of the things they are looking at.
[Slide.]
The technology we are talking about is not very new.  People have seen it before.  It is called immunoisolation.  Immunoisolation is a very elegant procedure.  It is very beautiful, very simple.  In this instance, you have a capsule encapsulating the islet inside.  The capsule is designed in such a way that its pore size is controlled in that manner.  The glucose can come in.  Insulin can go out, antibodies and lymphocytes.  It is a very simple picture, very elegant and very simple picture.
Up to this point, it has not worked as well as it could for some reasons.  One of the reasons is to make a second assumption.
[Slide.]
The assumption is that the pore size is very uniform and that it is defect free.  But it turns out that the reality is that they are not uniform in pore size and not defect free.  So, therefore, in this circumstance, different laboratories get different results.
Therefore, we have to look at how to overcome that problem, accept the fact that there is imperfection in the processing that nature gives us.
[Slide.]
What if the processing imperfection is this; immunoisolation devices assume almost uniform size but, in reality, the size is a Gaussian distribution.  Not only a Gaussian distribution, people always assume‑‑say, this is the cutoff so we assume anything above that size does not come in.  As they say, well, it is only a very small tail so, therefore, maybe you can get away with it.
[Slide.]
But in reality, if you really look carefully, that is not the picture you should be looking at.  You should not look at the pore size density.  What you should look at is essentially the surface area that has a larger pore that allows the immune system to come in.
So, in other words, you can have 10 million pores that don't allow your immune system to come in.  You have one bloody big hole, and everything comes in so it doesn't do you any good.  So the pore size; density is not the question.  It is, rather, total surface area is the control.
[Slide.]
Therefore, the physical picture you end up having is actually what we call a barrier model or entrapment model.  The membrane serves as a finite thickness that the immune system will actually try to come in.  It will come in part of the way.  It will get stopped and eventually it can wiggle its way in and you can calculate the time involved to allow that to happen.
[Slide.]
As I say, my background is in physics.  The first thing I would do, I would write an equation down.  I was told this is not a community that favors the equation, so I will just show you the equation to try to tell you what is the significance the equation is telling you about.
[Slide.]
In the immunoisolation system, really, it is a random‑walk model.  It is telling you, essentially, how long the system will take to get into the capsule.  In other words, you have the immune system outside the wall.  How long will it take to get in?  The primary thing I want to emphasize is the d2 of R2‑‑that is, the thickness squared versus the pore size squared.
[Slide.]
This is important.  The reason it is very important is for mass transport, it so happens the R and d are flipped essentially in order.  Now, instead of d2 of R2 it is R over d.  That means there is are two requirements, or a dichotomy.  What is good for immunoisolation is lousy for mass transport.  What is good for mass transport is lousy for immunoisolation.
Fortunately, when we are looking at those equations, we can see they have different dependence, they have different R and d dependence.  By having different dependence, there is one advantage, that you can manipulate that way to optimize both of them which underlines the fact you must be able to independently control all the parameters of the view of the capsule.
If you can't, then you have no hope.  But if you are able to do that, you can control the mass transport, you can optimize the mass transport and, also, you can try to optimize essentially immunoisolation and also mechanical strength and plus a whole host of other things which I will not show you.
[Slide.]
But in able not to do that, one of the first things that you have to do is you have to make sure that the system you have chosen has allowed you to independently adjust the parameters.  Now, the current system you will be using is what we call a binary system.
The binary system has one drawback with everything tied to one chemical reaction.  With everything tied to one chemical reaction, therefore, when you adjust the one parameter for the benefit of one function, you ruin the other ones.  So, what you have to do, you have to become a multicomponent system.
[Slide.]
By having a multicomponent system, and how do you start to do it, you start to do bloody what we call grunt work.  What you do is you take all the matrices, all the possibilities, looking at all the reactions.  We spent two years looking at grunt work, we call it.  It is a 30 by 30 matrix.
[Slide.]
Not only is a 30 by 30 matrix, you are talking about they are dependent on molecular weight, depends on concentration, depends on pH.  So what you end up with every day, when you come to the lab, we have got a table full of trays and we just go through every day for two whole years.  	[Slide.]
Eventually, you get to the point you can find the system, a multicomponent system, that looks like it will do what you want it to do.
The drawback of the multicomponent systems is very complicated because not only do we have a primary reaction, there is a secondary, tertiary reaction.  But a nice aspect of this multicomponent system is if you can find out which reaction controls what, you can, in principle, fine‑tune one reaction to control one parameter at a time.
Therefore, for the first time, you can talk about optimization effect.
[Slide.]
In order to do the optimizing it is not just finding the polymer system.  You also have to optimize, essentially, the processing.  The process must be also optimized.  The process has two aspects; one transient effect and one the steady‑state effect.
The transient effect we call it the impact criteria.  The impact criteria is very simple.  What you started with is a droplet of one polymer with islets inside.  In order to enter the second medium, you have to go through, impact the surface and enter.
When it impacts the surface, what happens is the islets are slightly heavier so that the islets start to accelerate out to the wall.  Then there is a whole bunch of criteria that you have to be satisfied and I am not going to bore you will all the equations.
If you want to know it, I will show it to you.
[Slide.]
This is the equation.  But I am not going to derive it for you at this time.
[Slide.]
But I will show you the results of that.  The results are that you can see it, that if you control what we call the center mechanism, you actually can let the droplet inside start off‑center and make it very concentric.
There are two reasons why it is very important to make it very concentric.  One is because the concentric wall thickness guarantees essentially the behavior of the property because any type of weakness in the one spot is basically the weakness of the capsule.
The second thing is you want to be able to keep it concentric.  You want to be able to keep the islets getting away from the wall because, during the processing, you do not want the islets exposed to the chemical reaction.
The second thing is you do not want the islet to be very close to the wall because, when it is very close to the wall, they actually might influence the growth of the membrane.  So those are the things which you have to do.
[Slide.]
Then, in a certain sense, if you do it right, you know what you are doing, this thing will show you essentially the system can be reasonably adjusted in a certain time.  This is the process that you have to be able to control.  You must be able to control this thing so that, therefore, the islets and the membranes are very uniform and then you can start the formation.
[Slide.]
Another thing which you have to be very careful about is during the formation process, the convective flow is involved.  Any time you have convective flow involved, what ends up happening is the two poles will tend to have a different characteristic than the rest of the membrane.  That is something you cannot live with because that becomes a weak spot in the system.
What you do is, in order to do all those, this is, in a certain sense, what I will say what I learned from sort of a space flight.  What you do‑‑this is a very simple‑minded picture.  What it says is this; in order to do this properly and control everything you want to control properly, you let the drop come in falling down through the medium, engulfed by the medium.
The medium and the drop fall in together.  You do not have any relative velocity.  Without any relative velocity, therefore, you don't have imbalanced forces on the droplet.  Not only do you not have imbalanced forces on the droplet, the growth has become isotropic because it is through the diffusion process growth.  So it is radially inward.
Let me show you what I have done.
[Slide.]
If you don't do that, this is what the capsule looks like.  They have got tails.  They have got wiggles.  They have got surface, everything.  So it is not really as pretty as what you like.
[Slide.]
But if you do what you are supposed to do, you know what you are doing, and you use the processing, this is what the capsule looks like.  Each one is the same as the one before.  Today you make this one.  Tomorrow you make the same one.  And that is the process control we are talking about.
In order for this thing to work, you are talking about you have got to transfer 2 million capsules into a human body.  And you cannot have a weak spot.  Therefore, what you have to do, your process must be controlled in a manner that every day, when you are making it, it will come out the same.
[Slide.]
At this point, what we call‑‑you start a smoking test.  We are not talking cigarette smoking but we are talking about‑‑from a physics and engineering point, a smoking test means, let me take a preliminary capsule.  Let's see how this works.  It is not a final product, but what we see is we can control the mechanical strength.  One of the problems with mechanical strengths, you want the thing to have reasonable longevity inside.
You not only want reasonable longevity, you want is that the capsule stays alive longer than the islet stays.  So, therefore, things eventually will fall and what will end up happening is that islet died before the capsule falls apart.  Therefore, nothing from the islet will come out.  Therefore, the mechanical strength very much has to be controlled.  In principle, we can control the two in the parameter space.
[Slide.]
Another thing you have to control is immunoisolation.  This is very important with regard to pore size.  What you do is you find out one of the concentrations allows us to change essentially the pore porosity, the pore size, independent from everything else.  By allowing to do this, I can basically set how long I want the immunoisolation time for preventing the immune system to enter the capsule.
The third thing, of course, the thing works.  It is strong enough to hold it.  It protects it, but the question is does it function.
[Slide.]
The question has to be asked, essentially, does the islet still survive its function inside the capsule.  That is, in a sense, tied to the mass transport effect.  What we show here is the perifusion data and it looks reasonably well and even after six months‑‑this is actually nine‑month data.  After nine months, if we retrieve it from the animal, the system is still working reasonably well.
[Slide.]
How is the biocompatibility, the question is.  Biocompatibility, for instance, you can transplant those things in an empty capsule into the animal.  This one is for mice.  And it seems to do very well.  What we find out, however, this biocompatibility, as you go up in the animal, the biocompatibility requirement becomes more and more stringent.
For C54 mice, the normal mice, we almost can slap it together and it will work.  Now, the question is NOD mice which is essentially the autoimmune system has‑‑it turns out to be much more selective.  You can see for the same parameter space which I just showed you for the last one, the two ends are no longer working.  The center part still works.
[Slide.]
Another thing which you have to look at with the biocompatibility question is the surface characteristics.  You have to be able to control the surface characteristics, how smooth this thing is and all those parameters.  With all that, then you can say, "All right; how does it really look after putting it in an animal."
[Slide.]
This was retrieved from the animal about nine months after.  The islet was still working.  The perifusion data we showed you before has come from this drawing.  The islet is reasonably clean.  This, obviously, I will say that some one here is not looking so good so it is not in the picture.
[Slide.]
This is what we look at essentially from a rat islet transferred into the NOD mice and we are able to control the permeability.
[Slide.]
Now, more important for you guys, porcine islet into the mice, NOD mice.  Essentially, it is still working fine so even with a very big specie difference‑‑mice and the pig are fairly far apart.
[Slide.]
So I would basically to draw a conclusion, essentially.  The immunoisolation works.  It works in a certain sense.  The immunoisolation will do what we design it to do.  The question of whether it will really do what you guys want to do in the long run has to be tested.  That is something we are working on.
We are in the process.  We have put it in the large dog, in a dog trial, and the data looks very nice.  So right now we are doing an optimization effect; that is, we know there are a little things that we want to tidy up so now we are no longer talking about a smoking test.  We are talking about a real trial, so we are tidying up the parameter space.  We are making it a little bit smoother on the surface.
We make the membrane binding mechanics a little stronger.  We will make the materials a little purer.  All those little things we can do.  We are hoping that, in time, soon, we can get FDA's advice and approval so we can start to proceed.
One other thing which I want to emphasize, Al Gordon reminded me to emphasize, this approach does not require immunosuppression drugs if it works properly.  Obviously, if it works properly, it doesn't require.  Up to this, all the data is without immunosuppression drugs.
One thing that is it a very much a tradeoff study essentially because there are so many dichotomy requirements.  This is the tradeoff.  The most important is because they are very host‑specific.  On the C57, the tradeoff is much easier.  In NOD mice, it is a little tighter.  The dog gives me a different requirement and I wouldn't be surprised when we finally go to the human we still have to fine‑tune it.
But, as long as we can allow this ability to fine‑tune the system, I think that is the criteria we have to be able to, that we can allow us to do.  Then I think we have a good chance.
This capsule has a finite lifetime.  It is not going to say you transplant once for the next eighty years and you are going to be free.  It is almost like‑‑the way we look at it, this system, the capsule, should work about a year to a year and a half.  The islet probably works a little bit less than that but we would design the islet to work a little bit less than that.
So the islet essentially will die for various‑‑whatever the reason, because immunoisolation has a finite time, as I said.  The islet will die and the capsule eventually disappear and the capsule would just basically‑‑the body would somehow dispense of it.
What you do is you end up having to replenish on a regular basis.  The scenario we are talking about essentially you will transplant once and then, every six months, you will come and have booster shots.  However, by having these booster shots, you will be able to live in a sort of, hopefully, a semi‑normal life.
So this is the approach that we have been talking about and we have been talking to FDA.  There is no guarantee that absolutely that this is‑‑but I think it has a good potential to do a lot things that we want to do.
Thank you.
DR. AUCHINCLOSS:  Thank you.  Let's just have a moment or two for specific questions relative to this presentation.  And then I think what we are going to do is hold the open public hearing or at least see if there are questions or comments or presentations that people want to make from the floor, and then we will group, after the remainder of the FDA presentation, the entire committee discussion together.
DR. ONIONS:  I enjoyed your presentation very much.  But I wasn't quite sure what you meant at the end when you said that you could design the capsule such that the islet cell died first and then the capsule was, in some way, disposed of by the body.  Clearly, the concern would be if the capsule integrity is destroyed while the islet cells are still alive.
So what do you know about the process at the end, there?
DR. WANG:  For instance, we have been doing some of sort of in vitro studies right now.  What we find is we put the islets in a medium in the incubator, the capsule, encapsule the islet.  What we look at essentially is what is the mechanism that causes the capsule to break.
What we find out is essentially about the fifth month, we have got 5 percent of the capsule starts to disintegrate.  95 percent fine, 5 percent.  So we start looking at what was the reason the 5 percent started to disintegrate.  It turned out to be that really what you see it‑‑I am a physicist and you see this very often.   What happens is the inclusion problem.
When we encapsulate the islets, some loose cells get floating around the place.  The loose cells can be included in the membrane.  Apparently, when they are included in the membrane, in time, the cell will die and become a void.  That void causes the membrane to break.  That is one of the reasons.
The second reason that sometimes happens is that the material is not pure enough.  Then you can stimulate essentially a certain inflammation and that is what happens.  So those are the things that we know how to deal with and we are looking at.  Now we are getting materials.  It is coming down to what we call a 5EU per cc.
Before that, in a smoking test, it was 60,000 per cc.  So, even the 60,000, we didn't see major problems.  We see some residual inflammation probably, some low‑grade inflammation.  So we believe if we scale down, we can get away with that problem.  Inclusion is just something that you have to do it properly.
DR. AUCHINCLOSS:  Other questions?
DR. GORDON:  Dr. Wang, I am just wondering what is  the volume of 2 million capsules and where do you put them?
DR. WANG:  Actually, each capsule is less than a millimeter in diameter.  So we are looking at about 100Êcc's in volume, a little less than 100 cc's.  We put it into the peritoneal cavity.  That is where the experiment has been done up to this point.
DR. AUCHINCLOSS:  Thank you very much.
What I would like to do at this point is to declare an open public hearing.
Open Public Hearing
We have had one advanced request for time from Dr. Michael Schmoeckel.  I believe he is not actually here at this point but, if he is, can he identify himself?  When he arrives, I, of course, will make time for him to give his five‑minute presentation which may, in fact, turn out to be tomorrow morning depending on how our discussion goes and the time of his arrival.
Are there any other presentations from the public that we do not know about at this point?
MS. STEWART:  Sue Stewart with Genzyme Corporation.  We wish to make a statement about the nonhuman primate guidance on the standard definition of xenografts and xenotransplantation.
The definition used in the Draft Document for Use of Nonhuman Primate Xenografts in humans published in April of 1999 for the use of those materials.  If there is a decision to modify the definition to encompass other products that come in contact with nonhuman animal materials during the production process, special care should be taken to avoid impacting products where the product or the nonhuman contact material can be characterized to a level that eliminates concern for infectious agents' transmission.
Currently, there are many products which are being used to treat human subjects where this is the case.  These include live‑virus vaccines, the radiated cancer vaccines, cellular products that use feeder layers in their production and gene therapies using living cells expressing viral vectors.
Care also needs to be taken in an approach that would broadly define xenografts and then apply a tiered level of regulatory compliance based on perceived or actual risk.  We agree that a risk‑based model regulating xenografts has merit within the current class of products as defined in the 1996 PHS guidance and for nonhuman primate material as outlined in the April '99 FDA document.
However, a broader definition which would incorporate products not currently defined as xenografts could potentially make these products unavailable to patients in countries which have banned the use of any xeno material based on their classification as xenografts not on their level of risk.
DR. AUCHINCLOSS:  Comments or questions on this presentation?  Now, because the open public hearing was originally on the agenda for 5:30, I will try and make note of that and see if anybody else shows up at 5:30 for a presentation or we can reopen tomorrow morning with an open public hearing including Dr. Schmoeckel.  We want to include everybody who does with to speak but I just didn't want to have these dangling at the end of the open committee discussion that we will be having.  It looked like it would fall awkwardly at that point.
MR. MORE:  I am Alan More with Primedica Corporation.  I just wanted to kind of echo a comment that was just made and that was in defining what xeno products are.  We do have to be careful about the way that we approach them in a testing sense.  I think if what I heard Sue say was that cells or cell lines that are living cells are going to be considered xeno.
That has an entirely different safety approach.  So I do want to echo care being taken in terms of how these products are defined.
DR. ONIONS:  Could I just make a comment.  I think Alan More's comment is very pertinent.  If I remember correctly, and I could be wrong, and there are colleagues here from GTI in the audience that can correct me, as I understand it, the GTI protocol involved using packaging cell lines in the treatment of glioblastoma.  This was, in the United Kingdom, views as a xenotransplantation as well as a gene‑therapy trial which complicated their life, I'm sure.
So I assume here, in the United States, that that was regarded purely as a gene‑therapy application not as a xenotransplantation which I assume sort of encompasses the kinds of things you are hinting about.
MR. MORE:  Right.  I think that is a great example because there are some very well‑defined principles in approaching that type of therapy as opposed to approaching the concerns that we have regarding xenotransplant or tissue transplants where we don't have control over the cells.
DR. AUCHINCLOSS:  Anybody from the FDA want to comment on this distinction?  It is not necessary.
DR. SIEGEL:  I think we are right at the focus of topic II which is whether the distinctions between cell lines or vascular organs or distinctions between species, between ex vivo and in vivo exposure, between barrier exposure and not, how they should impact what safety measures should be taken.
The points are raised and I think we should move on and then discuss them in the full context.
DR. AUCHINCLOSS:  Then I will, at this point, end the open public hearing unless there is any other speaker.  I would ask Eda Bloom to resume the FDA presentation and then we will return to committee discussion.
FDA Perspective
DR. BLOOM:  Thank you, again.  In my last presentation of policy, I think that I probably put my spacer in the wrong place because there seem to be a couple of slides that I missed.  There is a slide missing.
What I also wanted to mention was that, in the realm of policy, many of you are aware of the 1996 PHS guideline.  That guideline also, as you are aware, is currently under revision.  I just wanted to not miss mentioning that the revised document is likely to address a number of issues.
The first slide, which appears to be missing or not dropping, is the idea of informed consent, the idea of FDA taking regulatory authority, the idea of the sponsor having the ultimate responsibility for safety of the trial.  	[Slide.]
In addition to those, the new guideline is likely to address safety on animal husbandry and pre‑transplant infectious‑disease screening to a greater extent than the earlier one, development of diagnostic assays and methodologies, maintenance of healthcare records, both for the source animal and for the patient and biosafety precautions.
[Slide.]
So, as far as the policy is concerned, the published policy papers include the 1996 PHS guideline for which a revision is in progress and the FDA document on nonhuman primate.
[Slide.]
Now we will move on to the examination of risk posed by different types of xenotransplantation for which Genzyme has given us a terrific introduction and I thank you.
[Slide.]
Again, with the definition that is raising concern.  At this point, I would like to go into it with a little bit more thoroughness, especially with the line that the ex vivo contact with human body‑fluids, cells or tissues or organs with xenografts, and those human body fluids, cells, tissues or organs that are subsequently given back to a human recipient or given to a human recipient.
It is important to note here that what needs to be given back to the human recipient would be human body fluids, cells, tissues or organs, not, for example, supernatant.
[Slide.]
However, there are a number of concerns for things that are still exposed ex vivo to xenograft‑type products and the concerns were what caused us to include the ex vivo exposure in the definition.  The potential for transmission of zoonoses or other xenogeneic infectious agents could, in fact, come through ex vivo exposure whether it be extracorporeal perfusion or co‑culture.
For example, there are techniques in which fertilization and early embryonic development are done on a monolayer of nonhuman primate feeder cells.  The reason for including all nonhuman animals rather than just limiting to mammals or to vertebrates is because cross‑species infectivity of viruses cannot always be predicted.
So, therefore, we have made the definition of xenotransplantation intentionally large.  This does pose problems for us and for everyone as far as how, then, do we apply the recommendations that were set forth, for example, in the PHS 1996 guideline to promote safety of such products as much as possible.
[Slide.]
The implication of the definition is that all sponsors of xenograft products should consider these recommendations and that complete implementation of approaches to risk control, however, may not be appropriate for all of the products.  What we want to do this afternoon is initiate the public discussion on the relative risks of certain classes of xenograft products which might be more easy to control the transmission of infectious disease.
[Slide.]
Thus, there is a spectrum, or we believe there may be a spectrum‑‑we would like to discuss this‑‑of xenografts as far as what kinds of risks they pose.  For example, would brief ex vivo exposure to a well‑characterized cell line such as an insect cell line, for example, pose the same risk as the permanent implantation of a whole organ from a wild caught animal.
[Slide.]
We believe there are a number of factors with potential impact on the risk of xenotransplantation.  Some of these are product‑related including the species of source animal.  For example, would a primate xenograft pose the same risk‑‑and I say fruit flys here because we actually have a protocol in which CTL are produced to treat melanoma patients and those CTL are produced by contact ex vivo with antigen‑presenting cells that are derived from the drosophila cell line.  That would fall under our current definition of xenotransplantation.
How about ex vivo exposure versus in vivo exposure?  For example, again, would exposure to a feeder layer such as antigen‑presenting cells or other feeder layer cause the same concern as a kidney graft that is implanted?  Again, the issue that was brought up by our public input, cell line versus fresh tissue.
Can a cell line that can be characterized and screened be considered of less risk than fresh cells‑‑that is, fresh cells, for example, of the same dose.  Again, that leads us into dose.  We have heard a number of presentations today in which the number of cells from a xenogeneic source animal might have been a few million.
Tomorrow, we will hear about suggestions of implantation of whole organs which would be severalfold more than that.  Do such transplants pose differential risks?
[Slide.]
Other product‑related factors that might impact on risk would be whether a graft is temporary versus durable.  So if you have, for example, an ex vivo exposure to a extracorporeal perfusion liver‑assist device as a bridge for transplantation, or whether you have ex vivo exposure to a whole organ, or whether you have a xenograft that is actually in place in the person intended to be permanent.
We have heard a couple of examples of the kinds of barriers; capsulation, those that are currently in use and ones that could be in use.  Do barriers provide sufficient protection that that might impact on the potential xenogeneic infection that could be transmitted from a xenograft?
[Slide.]
In addition to the product‑related characteristics that we need to consider, there are also patient‑related characteristics that had impact or that may have impact on whether or not a xenograft poses serious, or more serious risks.  For example, if a patient has strong immunosuppressive therapy, does that, then, predispose that patient and that patient's close contacts to a greater risk than someone who may, say, just receive a xenograft that might be either only temporary, if the patient may be either temporarily immunosuppressed or may be using blocking antibody or something that would be less of an effect on the patient's entire immune system.
Another issue that we want to consider would be whether the patient population would be one that one could be assured would be a compliant population that would come back for screens, that would come back for follow‑up exams and that would be available or willing to undergo whatever may be necessary should an infection occur.
[Slide.]
The current PHS recommendations for clinical trials in xenotransplantation has many different thrusts in order to apply known procedures to minimize the possibility of xenogeneic infection being transmitted.  These include the composition of the xenotransplantation team which would include everything from the veterinarian to a surgeon to the clinicians to the laboratory; the clinical transplantation site; protocol review by the IRBs, by FDA, by a federal committee; informed‑consent procedures specific for xenotransplantation; and procurement sources.  Certainly, we have to wonder whether it is important to have a closed herd of drosophila and if you can trace back to the original source of a mouse cell line.
The source animal facilities, again, present the same kinds of issues; pretransplant screening, preclinical studies and assay validation.  Now that might be something that‑‑should that have a sliding scale?  Maybe not.
[Slide.]
The current PHS recommendations also include, again, the herd and colony screening and surveillance; certain criteria for source‑animal qualification; the screening of the graft, itself, for infectious agents; source animal archives and records which may be maintained for many years; surveillance of the recipient which could happen for many years; infection‑control practices for the healthcare workers, for the close contacts; and a database maintenance for, again, many, many years.
[Slide.]
So what questions we will have for the committee are‑‑we have a spectrum, or possibly a spectrum.  We would like for you to comment on whether we really have a spectrum of risk posed by different kinds and different classes of xenografts and, if we believe that, then how this spectrum might actually be used for the application of the recommendations in the PHS guidelines.
DR. AUCHINCLOSS:  With that, I think we will have discussion and then go to the questions.  But, actually, I am going to confuse you still further.  I am actually going to reopen the open public hearing, because Dr. Schmoeckel is now here, and offer him the opportunity to make is five‑minute presentation.
Dr. Schmoeckel?
Open Public Hearing
DR. SCHMOECKEL:  Good afternoon.
[Slide.]
Mr. Chairman, ladies and gentlemen, on behalf of the Munich Xenotransplantation Research Group, I would like to give a brief presentation of our initial experience on pig‑to‑baboon orthotopic heart transplantation.
[Slide.]
We performed two series of experiments due to the requirements of the German regulating authorities.  We had to perform a feasibility study which comprised four non‑transgenic pig hearts that were transplanted into baboons.  This was a feasibility study which means that these animals were not allowed to survive long‑term but had to be sacrificed on the table after weaning them off cardiopulmonary bypass.
In a second series of experiments, we transplanted hDAF transgenic pig hearts provided by Imutran Novartis, into immunosuppressed baboons.
[Slide.]
As donors in our first series of experiments, we used normal landrace piglets at a body weight of between 13 and 14 kilograms.  The hearts were preserved with iced Celsior cardioplegic solution and the ischemic time was about 3.5 hours.
[Slide.]
As recipients, we had baboons, adult baboon, between 17 and 26 kilograms and orthotopic heart transplantation according to the technique of Lower and Shumway was performed which means that the native hearts were removed before the hearts were placed in situ.
In three experiments, we performed perioperative immunoadsorption for the depletion of preformed natural antibodies.  One experiment served as a control and no immunoadsorption was performed.
[Slide.]
Immunoadsorption consisted of the Ig‑Therasorb column.  The blood of the recipients was divided into plasma and cellular components.  The plasma was then directed to the Ig‑Therasorb column which contains f‑coupled polyclonal sheep antibodies against human IgM, IgG and IgA.  The depleted plasma was then reinfused into the animals and we used a total of four cycles per experiment.
[Slide.]
The outcome was that, in all three cases in immunoadsorption, we were able to wean the animal off extracorporeal circulation, after 100 minutes, 11 hours and 21 hours.  In each case, it was a deliberation termination of the experiment.  ECG showed normal sinus rhythm.  No ST‑segment elevation.  Echocardiography showed a normal pump function, an ejection fraction of 65 percent and a fractional shortening of 32 percent.
Invasive hemodynamic measuring showed a normal cardiac output of 1.9 meters per minute.  Histology confirmed that there was no hyperacute rejection.  In our control experiment, the graft failed after 29 minutes.  Of course, we were unable to wean this animal off cardiopulmonary bypass and histology, indeed, confirmed all signs of hyperacute rejection.
[Slide.]
This graph shows you the immunuadsorption procedure.  These are the hemagglutinating anti‑pig antibodies.  In our three experiments with immunoadsorption‑‑that is the black line‑‑you can see that we were able to deplete the antibodies below a critical deadline of a titer of 1 in 64 while, in our control experiment, the antibodies were presumably absorbed on the graft and led to hyperacute rejection and graft failure.
[Slide.]
Now, to our second series of experiments.  We used hDAF transgenic piglets provided by Imuntran‑Novartis.  Again, these hearts were preserved with Celsior cardioplegic solution and after an ischemic time of 160 minutes, they were reperfused in the recipient.
[Slide.]
Recipients were, again, baboons, adult baboons of a weight between 17 and 32 kilograms and we performed the same orthotopic heart transplantation according to Lower and Shumway.  These baboons were immunosuppressed with cyclophosphamide induction therapy from day ‑1 until day 4 and a maintenance therapy consisting of a triple‑drug immunosuppression with cyclosporine A, mycophenolate infected with ERL, and steroids.
[Slide.]
Rejection monitoring in the post‑operative period consisted of daily assessment of hemagglutinating anti‑pig antibodies, a daily ECG and echocardiography and physical examination of the recipients.
[Slide.]
This is the outcome of our first four experiments.  In the first experiment, the graft failed, indeed, half an hour after reperfusion.  We think that this is due to a technical failure because, during the procedure, it seemed that there was a nonperfusion of the transplanted heart.  But, at this stage, we, in fact, can't differentiate this possible ischemia‑reperfusion injury from hyperacute rejection.
Our second survivor survived for 20 days and had to be sacrificed due to progressive anemia.  In fact, we were unable to transfuse these animals because we had no baboon blood available.
Our third baboon was sacrificed after 11 days.  In this case, we had anemia on the first post‑operative day and transfused the animal with human blood.  However, the animal developed renal failure on day 11 most probably due to hemolysis.
Our fourth experiment, actually, is still ongoing on day 8 today and the animal is still well and alive.
[Slide.]
Just briefly, a couple of functional data of our 20‑day survivor.  Again, ECG showed always sinus rhythm.  Echocardiography showed a normal cardiac function.  The ejection fraction was 69 percent the fractional shortening was 37 percent.  We had a minimum HB, as I already mentioned, of 4.4 g/dl on day 20.  This led to the termination of the experiment.
Due to the cyclophosphamide‑induction therapy, we had a very low white‑blood count on day 9, 0.2, which recovered to 2.0 again on day 20 and a minimum platelet count in day 13 of 19 which recovered again to 103.
[Slide.]
From this limited experience, I would like to draw the following conclusions.  Hyperacute rejection of non‑transgenic pig hearts can, indeed, be prevented by immunoadsorption and hDAF transgenic pig hearts are, indeed, able to sustain the life of an immunosuppressed baboon for up to three weeks at least in our experience.
Thank you very much for your attention.
DR. AUCHINCLOSS:  Thank you very much.  Any comments or questions?
Thank you very much.
Now we will close the open public hearing again.
Committee Discussion
The reason for this convoluted performance is that now the remainder of the day is just committee discussion which can involve questions of any of the people who have presented but also an effort to address the questions that have been posed to us by the FDA.
I am, frankly, perfectly content to have comments as we did this morning from the floor as well.  We basically have two things in the big picture to deal with.  One is this issue of the definition of xenotransplantation and I think we will start there.  And then we want to debate the value, usefulness, of the concept of relative risk.
Let me start, then, with the definition of xenotransplantation that the FDA has presented.  Is there anybody on the committee who wants to suggest some modification of that?
MS. MEYERS:  I want to ask, under that definition, would insulin from pigs or cows be considered xenotransplantation or the heart valve made from pig tissue.
DR. AUCHINCLOSS:  I think it says "live cells," does it not?  The definition; "any procedure that involves the use of live cells, tissues or organs."
MS. MEYERS:  So the heart valve is not live cells.
DR. NOGUCHI:  The key word is "live.":
DR. AUCHINCLOSS:  The heart valve is not live, the insulin is not a cell or a tissue.  Is that correct, FDA?
DR. NOGUCHI:  That's correct.  Heart valves are fixed and deactivated.
DR. AUCHINCLOSS:  Live cells, tissues or organs.
DR. ONIONS:  I think this just takes me back to that earlier point and it is related to the one that Alan More raised that this would imply that people using cell lines that have been grown in the laboratory and reviewed in the laboratory when put into a patient, for whatever reason, would then have to come up with a definition of xenotransplantation.  At least, I assume that is correct.
DR. AUCHINCLOSS:  I believe that FDA intends it to mean that; is that correct?
DR. NOGUCHI:  Yes.
DR. AUCHINCLOSS:  A cell line from a nonhuman source is xenotransplantation.
DR. ONIONS:  My only comment about that is that it is duplication of regulation.  I think that is a lot easier to do in the FDA where you have a much more defined structure.  I know that that did raise complications in other countries where conflicts between different regulatory bodies arise.  That probably isn't such a problem for the FDA where you sort of an overarching structure.
That is my only comment, that you might find something that is both a gene‑therapy protocol and it is also a xenotransplantation protocol.
DR. SIEGEL:  You are looking at the same people.
DR. AUCHINCLOSS:  It's them, either way.  My question was that the pig Factor VIII that we heard discussed this morning would not fall under this definition even though there is concern that PERV might be there.  Is that a problem and what do we do about that?
DR. BLOOM:  You are right.  That is a blood product but it is not a live cell or tissue so it would not fall under xenotransplantation.  But that doesn't mean that we wouldn't look at such products and take precautions for them.
DR. AUCHINCLOSS:  Okay.  So there are other ways of looking at those products and we don't have to consider them as part of our xenotransplantation.
DR. BLOOM:  That's correct.
DR. AUCHINCLOSS:  Does anybody want to modify the definition or are we content to push on to the bigger questions.
DR. SALOMON:  Can I ask just one question?  Then what is a vector, a retroviral vector, let's say?
DR. BLOOM:  That is a good question.  If a retroviral vector, let's say, is produced by a mouse cell line, it is ex vivo and then the vector, itself, is what is administered or the vector, itself, then is used to infect human cells ex vivo.  The mouse‑producer cell line, itself, under those circumstances, hasn't had direct contact with the human cells going back or with human body fluid going back so that would not be xenotransplantation.
However, if you do direct contact between the mouse‑producer cell line and the human cells, it would be.  If you implant the mouse cells that are producing the vector into the human, it would‑‑
DR. SALOMON:  So the vector is not alive.
DR. BLOOM:  Well, it is not considered a nonhuman animal.  We haven't gone down to viruses.
DR. ONIONS:  I am not trying to be awkward, but the very kinds of issues they use where people put packaging cell lines that are irradiated in contact with, say, CD34 stem cells, just for clarity, that would still come under the definition of xenotransplantation?
DR. BLOOM:  They are irradiated but they are still alive.
DR. ONIONS:  Yes; I appreciate that.  That's fine.
DR. AUCHINCLOSS:  That is xenotransplantation.
DR. SIEGEL:  That's right.  Under this definition.  We don't have any notion or belief that this definition or any definition captures those products that are most at risk and fails to capture those products‑‑and that all the products it excludes are less at risk.  There is a lot of discussion, then, do we have a narrow definition that only includes a transplantation of organs, or only transplantation or implantation of cells?  Or do we have a broader one.  We started out including ex vivo perfusion.
What is clear to us, and I should say, too, that as far as this definition goes, in part it is part of‑‑the next definition or the newest definition will be part of a PHS guideline.  It is not simply an FDA decision what the definition should be but what is clear to us is that, particularly with the broader definition‑‑at least it is clear to us and we are seeking your input, there exists a spectrum of different risks, maybe not a unidimensional spectrum, maybe not only high and low, but just lots of different types of risks.
It is hard to imagine applying all the same policies to all the different types of things we are talking about and we are looking for guidance as to how to cope with that.
DR. AUCHINCLOSS:  That is question 2; right?
DR. SIEGEL:  It is in all the questions.
DR. AUCHINCLOSS:  It is everything we are going to do from now on, I think.  So we are content with what we have here.  A definition is a definition, but there might be implications to different wording.
All right.  Let's move on this concept of relative risk and the particular implication‑‑correct me if I am misphrasing this‑‑is that if we could identify forms of xenotransplantation under this definition that were so unrisky that they would potentially not be subject to the guidelines that you are proposing, that would be an important thing to identify.
That is the real implication.  It is not that there would be some‑‑let me rephrase it.  The baboons, or the nonhuman primates, you basically said no to for the time being.  So you have taken one category of xenotransplantation and said, "We are not interested in that right now."
I didn't hear or sense in the questions that you gave us that you were looking for us to give you other examples of such risky xenotransplantation that we should put them in that category.  What I got from the series of questions you gave us was that you were looking for examples of xenotransplantation that would be the other way, so unrisky that they wouldn't necessarily have to face all of the same stringent‑‑
DR. SIEGEL:  Sort of.  Unfortunately, I don't see the question as nearly that simple.  I think that different factors are likely to predict different types of risks and it is not a question of something being so unrisky that nothing needs to be done but rather that, perhaps, by virtue of being a cell line or by virtue of being an invertebrate or by virtue of having a barrier, maybe we don't need to have, let's say if it is drosophila, a veterinarian on the team.  Or maybe you don't need to know what the grandparents of the origin of that drosophila cell line ate, for example, because the concerns about foods were, perhaps, more based on TSE issues and I don't know if drosophila carries TSE.
And the issues of who is deferred from blood donation or should tell all their sexual partners or should donate blood annually to a bank for the remainder of their life.  It is already late in the day and this is not going to be the beginning and the ending of these discussions‑‑it might be the beginning.  It won't be the end by any means.  But we are being faced with a lot of protocols and we are seeing a need to draw some distinctions and not to apply all the same rules, many of which were written with the thought of vascularized organ transplantation.
We are seeking further guidance as to which of these factors do or don't matter.  How much comfort should we take in the fact that it is a cell line or that it is transient or that it is only a few cells or which ones matter more or that it is an invertebrate and how do they matter.
DR. AUCHINCLOSS:  I am going to get the discussion going by suggesting the opposite point of view from the one that I think you have just come up with.  To me, in this situation of extraordinarily low risk, it is meaningless for us to try to quantify greater and lesser extraordinarily low risks.
There is no evidence that I have seen, and now we will come up with exceptions, but, in general, there is no evidence that I have seen that any particular form of xenotransplantation, whether it be with a barrier or cells or cell line or any of the things, frankly, that are mentioned in your list, that would lead me to say we can relax our guidelines.
There are clearly some exceptions to that.  In a cell line, probably, the grandparents of the origin of the cell line may not be as important but they become relatively trivial.  I think the concept is wrong.
DR. VANDERPOOL:  While you are confronting the concept, I want you to confront something more and that is the title of the document of this session of Xenotransplantation: Public Policy Development.  I notice, in our deliberations today and in the subject at hand that we are still dealing with risk.
That is great.  I think we ought to deal with risk.  But when it comes to moving xenotransplants to clinical trials, risk is one‑half of one‑third of the equation and that has to do with risk‑benefit assessment.  I think we need to keep in mind the ethics of xenotransplantation clinical trials involve a balancing, or at least a consideration, of what the risk‑benefit profile should be.
Tomorrow, we will see some protocols that, ipso facto, talk about benefit.  Risk can't be the only factor involved.  Benefit has to be the other side of that portion.
Secondly, to follow the Belmont report, respect for person is another factor in clinical trials, respect primarily through the process of informed consent which is daunting for xenotransplant clinical trials particularly any that would involve organ.  And the final issue is the issue of justice, who gets recruited under what situations and who gets the chance or who takes the chance.
I just want to preface my comment by saying, preface this discussion by making a fervent declaration that xenotransplantation public‑policy development should include additional things besides risk.
Perhaps this could be a division of tasks.  Perhaps the FDA is primarily concerned about risk and the NIH and the Office for the Protection of Research Risk will be concerned about the other features of the ethics of clinical trials.  But I want to register this because even though we may not talk about it now, when the time comes tomorrow to talk about the possibly proposed protocols, benefits, respect for persons, informed consent and justice will be part of the equation of that discussion.
DR. AUCHINCLOSS:  I agree entirely.  Thank you very much.  Tomorrow, we will be talking about some potential applications and benefit will become very much a factor in the discussion.
DR. VANDERPOOL:  Just to make sure what my question is, we are talking about policy development.  I don't know what all the FDA sees in terms of its policy development but, as we go through these documents, the first couple of introductory pages do have some of these issues, but if we look at the actual policy statements, they do dwell fundamentally on risk factors.
I just point that out, not to say that is a mistake but let's just be sure that we recognize what the focus so far on policy development within the FDA has been regarding xenotransplantation.
DR. ONIONS:  I wanted to slightly disagree with our chairman, with some reluctance, but I think‑‑
DR. AUCHINCLOSS:  Do you want to get onto the relative risk issue?
DR. ONIONS:  Yes; I was actually talking about your‑‑
DR. AUCHINCLOSS:  Let me just hold for half a second.  Someone was about to respond to Dr. Vanderpool.
DR. ONIONS:  Sure.  Sorry.  Of course.
DR. NOGUCHI:  Not to try to take this too far afield, but I think the reason that it is framed in this way is by no means does FDA defer or accede its role in deciding the ethical component of risk and benefits and further societal issues.  We are an integral part of that.
However, it is always the risks that come at us and that hammer us first in the face and so that is why we are trying to bring that as one component of the overall discussion of developing public policy.
You are right on target where the ethical considerations, by necessity, must be integrated in that.
DR. VANDERPOOL:  And, Phil, you and the other FDA persons here are the last people I would ever say you are neglecting something.  The point is that, as you think about policy development, will you see it as your purview to move beyond risk with a particular concern for other issues involving clinical trials.
It is a question I ask of you.  It is your decision, but you are so right.  I mean, I am not criticizing you for focusing on risk.  Someone has got to and you are doing it and we are doing it.  But will you move beyond risk, focus on risk or other matters is the question.
DR. SIEGEL:  That really has to be done on a case‑by‑case basis.  After all, we are not talking about benefits.  We are talking about potential benefits as none of these therapies are proven to have any benefit.  So it is hard to talk about the general principles.
As we debated a clinical hold, we heard a lot and took into account the fact that there were people dying of liver failure who felt that this device gave them their only chance.  We take that into account.  We would certainly deal with a heart‑transplant protocol differently from the xenotransplant protocol, say, to change hair color or something like that.
But it is a little bit hard to spell out or to get advice on what the general rules are.  It is much easier to talk about the specific rules such as we will do tomorrow in talking about specific applications.  We look at the scientific feasibility that has come into play.  At the first advisory committee meeting on xenotransplantation, there was a great deal of discussion on the baboon bone marrow as to the balance, does that have any chance of helping, was one of the big issues.
So, not only in the future, but I think as we go along, we certainly do agree and take those issues into account.
DR. AUCHINCLOSS:  Now, Dr. Onions?
DR. ONIONS:  I think Harold's point about benefit and risk is a very important question we would come back to.  But what I just wanted to do with this risk issue is I think I disagree in the sense that if you have a cell line produced from a clonal cell and produced as a multicell bank, such as many other biotechnology products are where you can extensively test that cell line, then that, to me, is likely to be intrinsicly safer than an organ from an animal.
However well‑controlled the cohort of animals going up to xenotransplantation is, you cannot have the same degree of definition of that product.  I would also suggest that the kinds of procedures that now come under the definition, like irradiation of those cells, add a further level of security.
The next level of security, slightly weaker, might be some form of encapsulation technology.  If you can validate that technology to show that it reduces virus egress, then, clearly, that would be safer.  But you have to validate it.
So you can go up the level and then you go the primary cells which you still might be able to do some testing on before they go into the patient.  So, again, that increases the level of security.  So I think there is a degree of gradation security.  I couldn't quantitate but I think there is a gradation.
DR. AUCHINCLOSS:  I have overstated my case to stimulate a little bit of discussion, but I will keep on overstating it a little bit longer.  Let's take the barrier device.  The barrier device, we are told, I think the numbers were that it reduced the risk of viral transmission by, what was it, five‑log or something like that?  It was big.  That is terrific.
But, again, if you have got a risk that is so small anyway and now you make it even smaller, does that really affect policy?  Do you approach your policy issues differently?  I don't see that you do.  I don't think you are any less careful about an islet transplant from pigs that are encapsulated from an islet transplant from pigs that are aren't.
DR. ONIONS:  I don't think it makes any difference to the kinds of criteria of surveillance, any of the criteria of informed consent, any of those issues.  I don't think it makes any difference.  All I am saying is I think those processes are probably intrinsically safer.
DR. AUCHINCLOSS:  I agree.  But my translation of the FDA questions was into an effective change in regulation.  There is no doubt in my mind that, scientifically, there are gradations of risk.  But do they affect what the FDA should do?  I can't find any.  When I say any, the cell lines is an example of where, yes‑‑
DR. ONIONS:  Clearly, the case of the cell lines because, in most cases, it is impossible to go back to the source animal so, very clearly, that must be an exception.  At least it would seem so.  It seems to me that the public has guidelines.  I mean, this is a remarkable document, a very good document, in that they try to encompass everything.
But it seems to me that the FDA is being excellent in terms of producing points to consider that are very specific about certain issues.  It seems to me, clearly, as we progress, then a points‑to‑consider document on porcine xenotransplantation might be well worth while because there is, clearly, where most of the activity is going to go on.  So you could see specific documents that relate to specific activities.
The odd balls, and they may not turn out to be odd balls, like using drosophila cells, can be dealt with on a case‑by‑case basis.  I don't see a great difficulty.
DR. ALLAN:  The only issues that I would think would impact relative risk in terms of what the FDA would do is if you were using, let's say, a whole organ from a monkey versus something that was as simple as injecting a few cells because what we talked about a year ago‑‑we talked about allowing certain clinical trials to go forward in thinking that whole‑organ transplants will weigh off in the future and we didn't have to worry about them.
So the idea, then, is if people are ready to do whole‑organ transplants into people, is that a significant enough risk they need to do something different or decide to allow that to happen on a different basis that you would allow what we have already‑‑
DR. AUCHINCLOSS:  At this point, the FDA has taken the nonhuman primate donors off the table.
DR. ALLAN:  No; I am just using that example.  But a pig organ.
DR. AUCHINCLOSS:  Let me ask you, do you see any difference in the guidelines of the regulations that the FDA should provide for pig cell transplants into the brain for Parkinson's compared to pig heart donors or kidney donors?  I don't.
DR. ALLAN:  That is the issue.  I think that is one of the questions‑‑maybe I am wrong, but that is one of the questions, a major part of this.
DR. AUCHINCLOSS:  That is the nature of the question.
DR. ALLAN:  Yes.
DR. AUCHINCLOSS:  Are there some kinds of xenotransplantation that should have different levels or regulation?  I just can't find the examples, and then we find the exceptions to my statement, like a cell line doesn't have to have its grandparents identified.
DR. WANG:  This is just a comment.  Let's take a hypothetical case.  Some day in the near future, we can prove to the scientific community's satisfaction encapsulation will work.  Say we have a great deal of confidence this will work for a long duration, say, will work for a year.
Encapsulation in many ways will prevent a retrovirus to leak out into the body.  If that can be proven, would the committee considering saying, all right; you might not have to go through all the pedigree of looking at the virus and looking at the islets, the history of the islets, for three generations for five generations.
I think what we are looking at is a certain amount of guidelines which say, can you relax a certain amount of requirements, not that you do not have control of the whole transplantation or the whole procedure, but the requirement relaxation, you can probably look at it as a function of degree of risk.
DR. AUCHINCLOSS:  The committee members can disagree with this statement, but I cannot imagine an encapsulation technology that would convince me that no virus could possibly escape because no encapsulation device would ever break allowing cells free.  I just cannot conceive of such a technology.  Does anybody want to disagree with that?
DR. PAUL:  I would concur with the chair on this.  I think, going back more generically, the guideline really, whether it tissue or whether it is a cell, and even cell line‑‑perhaps they pose a different degree of risk but they also pose a different risk.  For example, cell lines could be persistently infected with agents that we don't have identified yet.
Going back in time, we can come up with example after example.  There is one incidence in canine vaccines.  A vaccine was produced and distributed and used on thousands and thousands of dogs and then USDA isolated a blue‑tongue virus.  Blue‑tongue virus was not one of the agents that was required to be tested.
So I think, going back to circovirus, circovirus contaminates PK15 cell lines, PERV.  So I believe that going back to relaxing guidelines for encapsulation, the defects in manufacturing, you can validate all you want but there is always, in nature, an immune response and immunosuppression.  There are a number of uncontrollable factors.
So I really believe that we need to‑‑we may have different tests, or different, for example, cell lines.  The one advantage would be that you can have the cells with a particular lineage, very tested, well controlled in freezers or at least you can go back‑‑you don't have that luxury with the organ transplant.
On the other hand, the genetic lines of pigs could be well characterized.  But I really would recommend that we have similar guidelines regardless of whether it is tissue, cells, cell lines or encapsulation.
DR. ONIONS:  Could I just endorse that statement.  I absolutely 100 percent agree.  I have heard this sort of comment before that maybe we can get away with "dirtier pigs" if we use such‑and‑such a technique.  In my view, that is absolutely not the case for exactly the same reasons that have just been enunciated.
But I think there is another very good reason and that is, by using barrier conditions, we will be keeping out the things we probably don't know about and that may be in the cell lines.  I think circovirus is a very good example where we know that a wide number of porcine cells lines are infected by circovirus.  People really didn't realize it until quite recently.
And there may be other agents like this that we don't know about.  But, possibly, by having very high standards of pig husbandry, hysterectomy‑derived and so on, that we will keep out at least a proportion of those agents.
DR. WALTERS:  The first five criteria that the FDA laid out have to do with the cells, themselves, and the last two have to do with the patients.  The first of these is fairly straightforward and fits quite easily into a risk‑benefit framework, namely the degree of immunosuppression of the patient.
However, I would like to caution that the last one, which is behavioral factors, opens up a variety of very complicated issues that, I think, differ in kind from the first six.  And they really take us back to the early 60's and renal dialysis in Seattle in a committee that chose people on the basis of how upstanding they were in the community which led one commentator to say that the Pacific Northwest was no place for Henry David Thoreau with a pair of bad kidneys.
I do think that these issues are important.  They really get into an area that Harold Vanderpool has recommended that you open up in more detail and that is the question of justice or criteria for the selection of patients to participate in trials.
For example, should a candidate for xenotransplantation, in principle, be simultaneously a candidate for an allograft, or not necessarily.  Nothing in the policy guidelines that have been laid out thus far addresses that question.  Would you like all or some of the early xenotransplantation trials to be placebo‑controlled; for example, the introduction of neural tissue for the treatment of Parkinson's disease.
So there are issues‑‑if it is going to be a well‑rounded policy development, it really needs to go beyond the risk‑benefit question.
DR. SIEGEL:  Of course.  I think we address those issues in other ways and in other settings.  But I want to give a little background to this issue of behavioral factors because it is a complex one and one that has left us somewhat troubled.
There are obvious inherent dangers, as you point out, in how such a screening‑‑what it could mean in terms of justice and access.  But when we discussed before this committee‑‑or not this committee, the parent committee which was totally different members and probably the case of a baboon‑marrow transplantation in 1995, it was pointed out by several, including non‑committee members, the issue that it was important that it was important that such studies be conducted on somebody and in a population of people, potentially, who one could thing would be pretty reliable in terms of following up with recommendations regarding getting follow up medical care, regarding perhaps, if necessary, barrier precautions, regarding, if necessary, lifetime surveillance and blood sampling as recommended.
And then we were faced with discussion, for example, of the fact that patients with severe alcoholic liver disease and acute alcoholic hepatitis who may not be doing well may not be candidates for human livers and wouldn't it be great to do xenotransplantation in this population.
The question arose are these individuals that one can draw the same presumption about and should that be a factor in determining whether or not such a protocol is appropriate.
I am not sure I know the answers, but if you have any help with them, that would be useful.
DR. AUCHINCLOSS:  I think it is the most complicated question of them all.  It was on my list of exceptions to my general principle, but it is an exception that, again, goes the other way.  I do think that you can define a population of people under behavioral factors that would make them a more risky example of xenotransplantation from the point of view of the public's welfare for exactly the reasons that you indicate.
But that puts you in a terrible dilemma as far as the ethics of informed consent and the ethics of selecting people for trials.  I don't know what the right answer is but I suspect the answer is that there are some people who should be excluded from xenotransplantation at this stage.
DR. VANDERPOOL:  I completely agree with you on what you are saying about the degree to which these behavioral issues fall under the question of risk.  But as soon as you do that, as Dr. Walters has said, as soon as you start talking about risk and start moving into human behavioral risk, you move into the whole human arena of who is willing to take which risk, beyond physical or physiological risk, health risk.  What about psychological risk?
Or what about psychosocial risk of people who don't have the right support system?  They are not alcoholic.  I also would add my voice to an appeal to make this question of risk a broader‑‑the last factor needs to be made its own subset of issues in which justice and other factors are brought into that discussion in order to decide what to do.
I have one other quick point to make, and that is, on the surface, I don't see a problem with rating risk according to whole‑organ versus cellular, and so on.  I don't know why you would want to ask this this early.  It seems to me, with more scientific data, there is going to be, naturally, a time in which the person who comes in with an encapsulated cellular protocol shouldn't have to jump through all the same hoops as the person who comes in with a protocol for pig's heart transplants will.
But is it too early to be asking that question?  I can see why the urge would be there, but is it too early to ask the question?
DR. SIEGEL:  I think probably it is for encapsulation.  I am inclined to believe, as some have commented, that we are a long way from having validated them.  I just heard this morning about microchimerism resulting with a product where there was a barrier‑‑not encapsulation, but a barrier.  I heard about maybe a five‑log reduction but, nonetheless, viral transport in a product where there was a barrier.
So I think that one would want to have a lot of data about any barrier approach or encapsulation approach before taking much comfort in its protection.
A lot of things are happening already though that it may not be too early to look at.  I would look to the issue that Dr. Onions picked up first, the use of cell lines, the use of cell lines ex vivo for antigen presentation, for co‑culture, whatever.  There are a lot of particles that do that.
The application of the full extent of the guidelines would pose a substantial resource drain on companies and potentially on the federal government as well, for example, if we begin to, or plan on, archiving sero and tissue specimens on a regular basis on all patients who might have received their own lymphocytes that had antigens presented to them by well‑characterized drosophila or murine cell line ex vivo, or who might have received a certain tissue of that nature and the implications regarding the source animals so also large.
So it is not really too early because we could start conservatively but we could wind up actually creating problems if we do.
DR. AUCHINCLOSS:  Jay, I want to come back to the specific examples that you, obviously, have encountered so that we can talk about them directly with you.  But I had failed to recognize a comment from the floor.
DR. PITKIN:  Thank you.  Zorina Pitkin, Circe Biomedical.  We wish to make first a comment regarding the revision of the guidelines.  In particular, we feel that, in  the guidelines, should be recognized the use of cryopreservation, cryopreserved cells.  That allows for conclusive, comprehensive quality‑control testing prior to clinical use.
Certainly, the importance of use of clean animals, good husbandry practices and thorough testing is important and that should be applicable to all of the sponsors.  However, if the final product could be tested, then maybe the quarantine time or animal derivation or some of the testing that is currently applied to the animals of the source of the tissues could be applied to final product only.
Secondly, if I may just respond to the use of the system with the barrier, I was shown that there was at least a five‑log reduction where the highly loaded‑‑well, PK15‑‑that was shown to produce PERV in high volumes, that is when the five‑log reduction was shown.  However, the hepatocytes were shown not to produce infectious PERV.  So I think that should be taken into consideration.
The second comment I would like to make is about the microchimerism with the use of the barrier.  It wasn't shown microchimerism as‑‑first of all, you have to define what microchimerism is and, in the case of the use of the system with a barrier, there was no microchimerism but I think further studies have to be shown whether or not it was just a DNA detection that was not detected further down.
Thank you.
DR. COFFIN:  I was going to get back to the cell line and the cell‑line issue.  We should be a little bit careful about thinking that this is actually very different from some of the things that we have been discussing here today.
For example, many mouse‑cell lines harbor infectious, endogenous xenotropic virus which, on co‑cultivation with human cells, are very likely to give quite a good infection of those cells doing exactly the same things, kinds of things, that we have been discussing for introduction of pig organs into people.
So one wants to be careful about sort of, in a blanket way, saying that these have some reduced risk on this‑‑
DR. SIEGEL:  I guess that is our thinking, in part, as to why we would include those cell lines.  But the thinking, also, is that, as opposed to a fresh organ, which you can do a certain amount of testing with, or a live animal that you can do a certain amount of testing with, with a cell line, you can do, and a sponsor potentially can do, rather extensive testing prior to administration.
That may obviate some of the concerns that we otherwise would have, for example, as to the source animals for that line, or as to even the extent of risk and risk‑control measures necessary within the clinical protocol.
DR. COFFIN:  It would certainly reduce the need to do it more than once but it probably has to be done at least once on a cell line and then adequate protection.  Most of the cell lines that are used are often ones that have been around for a long time and are used because they work well.  I think there is probably quite a bit of resistance, in general, to going back to well‑validated sources and actually, under GMP conditions, deriving brand‑new cell lines for use.
My guess is that most manufacturers are loath to do that when they have a cell line that is producing a product or a system that works well already.
DR. SIEGEL:  It might, paradoxically, decrease safety, I would think, to try to supplant well‑characterized cell lines with new ones.
DR. ONIONS:  I would just agree with John about the last point.  The point about using cell lines is that you can characterize them.  It would concern me if people were using certain murine cell lines because, clearly, in gene therapy, we spend a lot of time screening retroviral vectors for RCR.  Then, if you start putting in a cell line, you have subverted that whole process, it seems to me.  So that would concern me.
But I just wanted to pick up Zorina's point because I think it is a point that perhaps we didn't respond to, and I think it is important one and the point I was trying to make earlier.  Where you can characterize cells, that does seem to me to have an advantage in terms of risk evaluation.
Clearly, a cell line offers the greatest opportunity because it is clonal, it is derived from a mater cell bank and you can exclude things like using murine cell lines that express retrovirus.  In the case of a primary cell system, you still may have that opportunity and there is a limited opportunity, I think, for instance in the kind of work that Circe does where they can do some screening before those cells go into a patient.
That seems to me to be at a higher level than just putting a whole organ.  Therefore, you might reasonably, and it is something that maybe this committee should consider, defer some of the testing from the source animal to the cell line, itself.  That does not seem, to me, to be unreasonable intrinsically.
My final point is, however, that usually in regulation, we start tough and get weaker as you begin to work out what the problems are and where there are not problems.  I am not entirely convinced we have done that with xenotransplantation entirely.  We have spent a lot of time considering PERV and, like my colleague Prem over there, I have as many or more concerns about certain other viruses and certain of the protocols that people are considering.
Some people look for certain viruses.  Others don't.  There is a degree of consensus about certain viruses but there is not a uniformity here.  I think that, perhaps, more emphasis should now be placed on what should definitely be excluded in the source material that goes into a patient, whether that be excluded at the herd level or whether it be excluded at the cell‑line level.
I don't think we have yet had that detailed consensus on those viruses.
DR. WANG:  This is maybe because, as I say, my background is a little bit different from everybody on the committee.  You guys have been talking to do cell lines.  Basic source herding means an increase in the comfort zone and, therefore, in a certain sense, reduces the risk.
As you know, the risk factor is a series matter.  You reduce the risk here.  If you have a series of risks factor, then you can essentially multiply then and tell you the total risk.  Can you guys sort of have some idea what is the risk that is acceptable risk.  In the final package, what is the acceptable risk?  One in ten million?  Something quantifiable so, therefore, people like us, me, we can start thinking about it.
DR. NOGUCHI:  Let me try to address that particular issue because that comes up time and again with xeno, with gene therapy, and so forth.  If you want to take a crude example, it is our impression, and you would do this for human allo, you want it as sterile as possible when you start.  If it is infected, you do everything you can to not use that organ unless it is a life‑saving sort of thing.
I think we are taking that same approach here.  But, in terms of getting an actual numerical value, we will not do that because that is the wrong road to go.  What we are really saying here is, as a society, do we feel comfortable enough with the available data, because the data can always be improved as to what the actual risk really is.
Some of what we are talking about here is, for this particular issue, we broaden our definition of xeno.  We, the FDA, think that, perhaps, we have captured a few things that don't need the full panoply of full federal not only regulation but oversight.  As Jay has pointed out, if you are doing a tumor vaccine using drosophila cells, but you are going to have to archive not only that patient but every other patient, enroll them in a database, bring it to a national committee which has yet to be formed, that can seriously impede something that, perhaps, is not necessary for that class of product because we already have a lot of experience with that.
What we are saying here is, by far the bulk of what is captured under our new definition is still totally experimental.  We have no idea of risk.  When we don't know the level of risk, even if we put all these factors together, we are still talking an unknown unknown unknown.  And that could go any way you really want.
But I think, here, we are just really trying to struggle with the fact that is FDA in the right place with this definition for the current level of xenotransplantation recognizing that we are capturing some things that were already regulated but under a less full public oversight with full NIH and CDC participation.
FDA regulates all these things anyway.  So when we say can we take some things off the table here, it is not like they won't go through all the risk‑benefit evaluations.  Prem is absolutely right.  We know all about the problems with cell lines and unknown agents in them.  The SB40 is a classic example.
So we are not talking about, necessarily, less regulation.  We are talking about somewhat less public oversight.
DR. MICHAELS:  I was going to ask a question regarding that as well.  I was going to query if we knew enough about, say, the drosophila cell line that, perhaps, the nonvertebrate cell lines and tissues and organs and such might not have to go under this type of regulation.  I don't know the answer to that.  It was really a question to throw out.
DR. AUCHINCLOSS:  Is there anybody on the committee who would like to offer an opinion?  Are there categories of nonhuman animals from which cell lines would not need the scrutiny that we are suggesting in the pig cell lines?
DR. MICHAELS:  Or the mouse cell lines.
DR. AUCHINCLOSS:  Whoa.  Don't touch the mouse.  We know that is a bad one.
DR. MICHAELS:  Right.  That is what I am saying.  That is actually what I meant.  I think any of them that are mammalian derived should stay in the definition but I am just querying whether‑‑
DR. AUCHINCLOSS:  Nonmammalian cell lines; are they safe?
DR. PAUL:  Even a lot of nonmammalian cell lines use mammalian media supplements.  Like fetal calf serum; a number of examples of bovine virus diarrhea, contaminants in serum.  So I think that that is another factor.
DR. MICHAELS:  But you are not saying that they wouldn't be going under regulation, still.  It is just that you would not be storing the samples in the same fashion.
DR. SIEGEL:  We have lots of products that are made at various points in the presence of animal serum but we don't regulate them as xenotransplants.  But we sure do make sure that they don't get contaminated.
DR. SALOMON:  Can the virologists comment on insect cells?
DR. ONIONS:  An insect cell has some retroviral‑like elements in it which are quite interesting.  But if we take that up‑‑I think there is a degree of clarity which comes from Marian's posing the question.  It seems to me that mammalian cell lines, I think I would concur, would stay in because if you go to more obscure mammals than are currently used, then often those are not being evaluated thoroughly.
So, for that reason, I would keep them in.  Sub‑ mammalian, then, I think, perhaps, the same degree of rigor in terms of sampling and storage probably would not have to be present.
DR. SALOMON:  How about an avian cell line.  We already know about the resorting of influenza.  I always venture into the virology with caution.
DR. COFFIN:  You also venture into an area for other subcommittee meetings having to do with the question of reverse‑transcriptase‑containing particles by these cell lines in vaccine products.
DR. PAUL:  The question that I would have would be the number of insects that serve as vectors for viruses.  Really, the burden should be on the manufacturer to show that they are not a risk.  That is the approach that I would use.
DR. COFFIN:  Help Canada had a workshop I think about a year and a half ago‑‑I think it was November of '97‑‑as part of their national policy development.  There were some ideas that came up in an infectious disease workshop within that workshop that I did not anticipate and have not heard discussed in other forums but I think are relevant here.
There are several people here who were in that working group but I don't recall that any of the virologists here were in that working group.  So I would like to put them on the table for the virologists to discuss.  I am sort of switching topics here but there are two suggestions in here for the committee to discuss whether a temporary exposure, like a bridging xenograft, may pose less risk of infection than what is intended to be an endstage organ transplant, and the other is whether the bulk of the xenograft, like a large organ may pose more risk than a xenograft that consists of a few cells.
I think the intuitive assumption I have usually heard was the assumption that more is riskier and longer is riskier and the proposal that came out of the retrovirologists in that group and that I would like to hear the retrovirologists and other virologists here discuss was that, in fact, those may not be significant determinants of risk, that, in fact, a small number of cells in the host for a short duration but that are proliferating may be riskier, given what we know about retroviruses, HIV for example, needing activated cell lines to proliferate than a large bulk organ that stays in for years with inactive cells.
DR. COFFIN:  I would, in general, more or less agree with that position.  But I would also go with the principle of our chairman that, again, relative risks here are not necessarily, particularly in the context of where it is sort of the same experiment, really what is on the table, I think, in a sense.
I would certainly not automatically line up the risk.  In any case, if I were to asked to line up the risk of infection, I would not automatically do it with the bulk of the organ or, even, necessarily, the duration of the transplant because the risks that we are talking about also include other factors such as the risk of transmission subsequently.
So you have to also take into account the subsequent lifetime of the individual and things of that sort if you are going to take the overall risk altogether to help creating a transmissible agent.
DR. HIRSCH:  I think you can't make absolute conclusions but you can certainly say, from experience with other situations‑‑for example, HIV and transfusions and needle‑stick injuries‑‑that more is worse than less.
But, on the other hand, a very short exposure can transmit virus.  And we know, in the situation of scalp electrodes and CJ prions that it can just be a very momentary exposure and you can get transmission of these things.  So you can't make any absolute conclusions but, certainly, the principle of more is more likely and longer exposure is more likely, I think, are reasonable generalizations.
MS. MEYERS:  In discussing this question about risk, it is impossible for anybody to come up with any kind of a formula because you have all of those little problems called human diversity and human weaknesses and a whole other bunch of factors that can complicate the question.
So I agree with Leroy that the ethical questions including risk need to be handled very carefully but I am going to say this for the five‑hundredth time, FDA does not have even one bioethicist on staff.  It is not right that decisions about maybe excluding alcoholics, or whatever, should be made by the people at FDA who are basically scientists and really are not familiar enough with these types of ethical problems.
Another problem is that the IRBs, as we are learning in recent days, are not reliable.  I think that anybody's institution would be very pleased to say, "We are the first to do xenotransplantation in Cincinnati," and rubber stamp whatever protocol is put in front of them.
So I think that there are a lot of things here to worry about that are not scientific but are very, very important in the long run.
When it comes to how people interpret risk, healthy people interpret it much differently than sick people.  To healthy people, any risk is unacceptable.  They assume that every drug they take is going to be safe and they are shocked if they get a side effect.  So they are not willing to take any risk at all.  I hate to see the day when we find CDC, instead of running around in a jungle looking for pig viruses could be running around in a department store or a MacDonald's because everybody who ate in there got sick and got a pig virus and whose fault was that.
On the other hand, a person who is dying of heart disease or liver failure is going to take any risk.  So risk is very relative.  Until anybody here can promise me that there is no risk, the general healthy public is going to say that they don't want a person who has gotten transplant that has contained any animal virus before we know whether that virus is going to be safe in human beings, they won't want to be exposed to that person.
That, I think is the bottom line, because if anything goes wrong, FDA is going to be blamed for having caused a major disease.
DR. WALTERS:  I would like to come back to the behavioral factors one more time.  One of my concerns about the overtones of that point as it is currently phrased is that it could seem to exclude certain groups of patients who I don't think ought to be excluded.
There was mention of chronic substance abuse.  I would want to be sure that this doesn't apply to people who are recovered alcoholics and that they will not forever be stigmatized because of bad decisions that they made early in life.
Also, I think mild psychiatric disorders like depression or anxiety disorder ought not to be disqualifying even though they might complicate a person's participation a bit.  I think one has to be careful even not to exclude poor people who, perhaps, don't own a car and who might find it more difficult to get to a clinic on a regular basis for surveillance.
Maybe programs have to be a bit more proactive in helping patients get to the clinic if they find themselves in that kind of relative poverty.  So whatever is said about behavioral characteristics, I think has to be said with a great deal of sensitivity.
DR. SACHS:  I think one of the major problems in this discussion is the fact that it is impossible to separate a discussion of risk with a discussion of benefit.  It is only the ratio of risk and benefit that makes any sense.  If a procedure had no benefit, you wouldn't be willing to accept any risk.
On the other hand, if the procedure really had benefit to an enormous number of people, then I think people are willing to take a risk, even if it is exposing them to something that is not of direct benefit to them, because people do care about their fellow man.  But it has to be demonstrated that there is enough benefit.
I think the problem there is, at this point, we haven't gotten to that stage in the field of xenotransplantation.  Hopefully, we will get there but I think it is premature to start worrying so much about defining what the risk should be until we have a better handle on the benefit.
DR. AUCHINCLOSS:  I agree with you.  But the question will come up tomorrow morning in some of our discussions there.  Right now, I think the FDA has put a question to us.  Are there certain features of some tissues that have so little risk that they can relax their guidelines?  So far, we haven't come up with a whole lot to help them.
There are some comments, but I want to come back to see if we can find some things that help you.
MR. BENEDI:  I just wanted to touch in on the behavioral issue.  In my tenure of President of the largest transplant recipient organization in the country and in the world, really, I saw a lot of people die on the waiting list.  There are over 70,000 people waiting for transplants today.  That is why we are all here, to try to save lives.
But, having said that, I think, and I do take issue with the comment that patients will take more risk or any risk.  I think we have a responsibility, those that we benefit from this type of procedure, to the community and to society as a whole not to unleash something that we will regret later at the cost of just having our lives expanded for just a little period of time.
As far as behavioral criteria, I think it is essential.  We have it now.  There are profiles in every hospital of patients.  If they don't have the support mechanisms to take the medicine on a regular basis, to come to labs, those people are not transplanted.  Why would we do less for‑‑we really don't know what the outcomes are going to be.  I think there should be very strict criteria, behavior criteria.
As far as justice, the justice part of it comes to the society as a whole and not to the individual patient.
DR. ALLAN:  I just wanted to come back to what Louisa said about what the risks are depending on dose, organ type, that kind of a question.  I think it is important because we didn't really address it last time.  I think it is very difficult to address.
John Coffin was saying even if you had certain cells, if they are expressing a lot of virus, it can be worse.  The way I see it is if you are using a whole organ, you have a whole diversity of cell types which each could express a different type of virus.  They have a different potential for long‑term implantation, for migrating to other sites.
So I think there really is a difference and it may come to whole organs, whether they are ex vivo perfusions versus implantations.  So, in some sense, it is like a seat factor.  When I was here last year, and we were just talking about Parkinson's patients and putting cells into the brain, I am on the back of my seat.
When we start talking about ex vivo perfusion, I am starting to get to the front of my seat.  And then when we start talking about transplantation of whole organs into patients, I am really at the edge of my seat.  That is how I look at risk in this particular case.
But you are right.  It is really difficult to sort that out.
DR. SIEGEL:  Dr. Auchincloss, you put the question about nonmammalian or nonvertebrates to the committee.  Several times you talked about, Dr. Allan, the level of risk.  But part of the issue here is the type of risk.  Some of the procedures that are recommended in this guideline are recommended for particular risks because of particular risks.
So the notion, for example, of lifelong surveillance of a patient or of notifying sexual contacts or blood deferral may come from specific risks.  Lifelong surveillance, for example, arose in significant part because of concern about retroviral and herpes viral chronic risks.
Or the notion of understanding what several generations of the feed history of the donor animal arose from concerns about TSE‑type risks.
So the question, to kind of rephrase your question, one might ask, if one used a nonmammalian source, do we know enough about, say, insect cell lines that we are not concerned about latent infections, that we don't need to do lifelong monitoring of somebody, or we don't need to do maybe blood‑deferral monitoring of somebody who had an ex vivo exposure to insect lines.
I am not answering that question.  I am just kind of reposing your question because, frankly, I don't even know what the answer is.
DR. AUCHINCLOSS:  The more specific you are, the more chance I have of getting some responses from the committee.  I haven't heard very many.
DR. ONIONS:  Could I comment?  I think this is a very real issue.  I will modify my position to say nonvertebrates because, clearly, thinking about it, obviously chickens carry a particular subgroup of retrovirus, an exogenous one, that can infect mammalian cells, for instance.  They carry other viruses that affect mammalian cells.  So do fish, for that matter.
So if we go to nonvertebrate cells, then I assume that everyone there will be using cell lines.  Is that the case?
DR. SIEGEL:  So far, I don't know of anyone using fresh insect organs.
DR. ONIONS:  In my view, that comes back to the fact that you can characterize the cell lines so I would have a great deal of comfort about that in comparison to other aspects of xenotransplantation.
So it seems to me that if you have got a cell line of insect origin, then clearly, to me, lifelong monitoring and all these other issues are not real, provided the appropriate monitoring to exclude all the alpha virus and flavivirus and so on that can replicate in insect cells have been excluded.
But that is a standard kind of job that CBER would do and do very thoroughly.  So I don't see there is a necessity there to go into this prolonged patient monitoring and advice to contacts and all the rest of it.  That is more like a traditional biological product, in my mind.
DR. AUCHINCLOSS:  So a specific recommendation might be drop nonvertebrate cell lines from your screen.
DR. ONIONS:  Yes.
DR. AUCHINCLOSS:  I tried to state it again rather than look for reactions around the table.
DR. MICHAELS:  As long as they are well characterized.
DR. SIEGEL:  Again, the testing of the line, itself, is one type of control.  We have many controls.  Are we going to bank serum on the nurses who take care of the patients who receive lymphocytes that were exposed to drosophila cell lines ex vivo?
DR. COFFIN:  I should point out that many of us have, or virtually all of us have, been exposed to certain kinds of co‑culture and xenotransplantation with invertebrates with some frequency, actually.
DR. SIEGEL:  Insects bite.
DR. COFFIN:  Insects do bite and they take in and extrude blood.
DR. HIRSCH:  I was just thinking, do therapeutic maggots for wounds and leeches fall into the category of xenotransplant?
DR. VANDERPOOL:  It seems to me that one of the problems is you are asking us to opt for categories.  For every category like cells, there is someone here who can think of 40 reasons why they are especially dangerous.  But I take Jonathan Allen's comments very seriously and that is if you get a whole organ, it is going to be more dangerous than the liver perfusion experiments we have had presented  to us today.
So why couldn't you, instead of looking for categories, think of a wonderful English word like "ordinarily," and put that in your guidelines.  "Ordinarily, people will be followed for life, be autopsied at death," or whatever which gives you the possibility of making‑‑I mean, the FDA is not known to be the liberal Marxist organization in the United States.
We know you are conservative in terms of protection.  So it seems to me that you could use some kind of language that allows you to have discretion over the things you deem to be safe and do not have to be followed as thoroughly as others.
DR. SIEGEL:  We have that language.  It is inherent in the word "guideline."  These are guidelines.  They are not rules.  They are not regulations.  And they are not laws.  What we are seeking here and what we are receiving here is the guidance we need to determine‑‑what we must do when we have a guideline is, nonetheless, look at individual applications and see, either they did everything according to the guideline or they didn't but they may or may not still be acceptable from a safety perspective balanced against benefit and so forth, based on the fact that they have alternative approaches that are equally good.
And that is always possible with a guideline.  But, in order to do that well on a case‑by‑case basis, we need‑‑the feeling is that the fact that it is fewer cells or ex vivo or a cell line or an insect, how do those weigh into that?  I think we are getting some useful guidance in that.  Obviously, each case has to be looked at on its own merit.
DR. AUCHINCLOSS:  As I went through your list here.  Immunosuppression; no, it is not a factor.  Time of exposure; no.  Barrier encapsulation; no.  Dose of implant; no.  Cell line and maybe species source and behavioral factors as we talked about.  Those are the only places that I could find any reason to think that you could categorize risk sufficiently differently to warrant changes in public policy.
DR. SIEGEL:  Is that a reasonable sense of other committee members as well?
DR. ONIONS:  Yes, except I would, perhaps, just like to‑‑I concur with all of that except that I do feel that there is this possibility with primer cells where they can be characterized before they go in that you might alter the way in which the testing level‑‑whether you go to herd level or the cell level.  It doesn't mean that you don't have the same rigor, but you might alter where you do it.
DR. AUCHINCLOSS:  Clearly.  There are some very practical things that you don't do to a cell line that you do do to a live pig.
DR. CHAPMAN:  Two points I would like to put on the table.  One is I would like to amend your statement to say, on the basis of current knowledge, because it seems to me there are things you listed as not being reasons for determining a gradation of risk such as barriers which are, in fact, however, very easily readily addressed by preclinical experimentation which may define a body of knowledge which, then, may give us a basis for determining a gradation of risk.
The other point I would like to bring up.  Let me begin by acknowledging the tremendous respect I have for the depth and breadth of expertise represented on this committee and in this room.  But, these nonvertebrate arthropods that we are discussing as minimal risk are, in fact, such prolific vectors of human disease that there is a whole field of expertise of medical entomology.
Let me say the one course I took in it was one of the more difficult ones I ever took in my life, including organic chemistry and physics.  So I would humbly suggest that, the expertise of our attendance not withstanding, it might be wise to consult some medical entomologists on their vision of the risk posed by nonvertebrate cell lines before taking any action.
DR. AUCHINCLOSS:  Both are good points.
DR. GORDON:  I would just like to, if I could, throw my hat into the ethics ring and then probably run for cover.  But there has been discussion of benefits and risk.  I have heard expressed, maybe twice, the notion that the benefits were largely theoretical, speculative and potential.
But, in the case that I am most familiar with, I think it is well beyond that.  In the case of islet transplantation, we have known for the last seventy‑seven years that the product of pig islets, namely pig insulin, works to control blood sugar in humans and works very well.
Therefore, what we do know is that if we can keep pig islets alive in people, we will have cured diabetes.  On the risk side, there was an interesting irony expressed early on that, in order to prove the safety of xenotransplantation, we need a population of PERV‑infected animals or people to use as positive controls.
I won't even comment further on that, but, obviously, the fact that we don't have such a population‑‑in fact, we don't even have one single case‑‑I think, in itself, stands as very important evidence.
The final thing is on the notion of justice.  I think it is very discriminatory to allow extremely risky behavior for some people, risky behavior such as providing surgery to AIDS patients or allowing people to travel to countries where the ebola virus is endemic while we may restrict access to others where no in vivo risk has ever once been demonstrated.
So I don't think we can set a higher hurdle for this procedure than is generally accepted in medical procedures in general.
DR. AUCHINCLOSS:  Jay, I have been taking, under topic II, the discussion questions for the committee.  I have been taking them essentially as one big group, questions 2, 3 and 4.  We have been pushing it.  At this point, I have gotten what I can get.  Have you gotten what you can get?  Do you want to push the question in a different way to the committee?
DR. SIEGEL:  Well, I think that we have been discussing topics 1 through 5 and I am pleased with the discussion.  I would ask my colleagues here if they have other specific issues they think need more comment.
DR. NOGUCHI:  No.  In spite of the fact we have been going around, this is exactly the kind of advice we need.
DR. WOODLE:  Hugh, I just want to get back to the benefit issue.  I think several people have come very close to making a point.  I think David Sachs came very close.  I am not sure that‑‑I'm sure that you have sat down and thought about what the Brave New World would be when xenotransplants actually work and we are able to do these.
But the potential upside on this is so tremendous and we haven't had anybody actually stand up today and say what it might be like if xenos work.  Sure, we are going to have less deaths on the waiting list, but there is going to be a huge number of patients out there in whom, in the course of their disease, they can be transplanted earlier.
They will be in better condition.  They won't sit in the hospital for weeks deteriorating, being nutritionally depleted in a serious condition where we know the outcome of an allotransplant is going to be worse than it would if they could have been time electively.
The operations can be done in the morning when the team is rested.  No more night‑time operations.  No more operations under suboptimal conditions.  The organ should be in optimal condition, minimal storage times, no more human living donors, decreased risk of transmission of HCV, HBV, Epstein Barr virus.  Those are just a few of the things that I think, unless we sit back and think about what this Brave New World is going to be, we don't really know what the potential upside is.
DR. AUCHINCLOSS:  That is a perfect transition for me, Steve, because that I think brings us towards tomorrow's conversation about some potential early trials of xenotransplantation where risk‑benefit analysis clearly becomes part of the equation.
Before I close the meeting, let me, because on our agenda we had the open public hearing scheduled for 5:30, did anybody come in who wanted a chance for a formal open public hearing, just so I didn't miss somebody by the change in schedule.
Are there any other comments that anybody on the committee wants to make on these discussions this afternoon or questions from the FDA?  Otherwise, I would end today's meeting and reconvene tomorrow morning at 8 o'clock.
DR. PAUL:  I think this has been an excellent discussion.  I just would like‑‑there is one reason that I am on the advisory subcommittee and that is I bring an expertise in swine virology as a veterinarian.  It has been wonderful to get educated on endogenous retroviruses, but I think that we should, at some point, give some time and discussion for exogenous porcine viruses.
DR. AUCHINCLOSS:  To the other nonendogenous viruses.
DR. PAUL:  Right.  I think that is something that would be very beneficial.
DR. AUCHINCLOSS:  Barring any other comment, we will go ahead and end this meeting and tomorrow morning reconvene at 8 o'clock.
[Whereupon, at 5:40 p.m., the meeting was recessed, to be reconvened on Friday, June 4, 1999 at 8Êo'clock a.m.]
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