1 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION CENTER FOR BIOLOGICS EVALUATION AND RESEARCH INTERNATIONAL ASSOCIATION FOR BIOLOGICALS NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES NATIONAL VACCINE PROGRAM OFFICE WORLD HEALTH ORGANIZATION + + + + + EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT + + + + + WORKSHOP + + + + Friday, 10 September 1999 The workshop took place in the Plaza Ballroom, DoubleTree Hotel, 1750 Rockville Pike, Rockville, MD, 20852, at 8:00 a.m., Regina Rabinovich, M.D. and Martin Myers, M.D., Session Chairs, presiding. 2 PRESENT: Regina Rabinovich, M.D. Session Chair Martin Myers, M.D. Session Chair David Onions, Ph.D. Panel Chair John Coffin, Ph.D. Panel Chair Philip Minor, Ph.D. Speaker James Robertson, Ph.D. Speaker Joerg Schuepbach, M.D. Speaker Jens Mayer, Ph.D. Speaker Thomas Broker, Ph.D. Speaker Neil Cashman, M.D. Speaker John Sedivy, Ph.D. Speaker Frits Fallaux, Ph.D. Speaker Michael Fried, Ph.D. Panelist Stephen Hughes, Ph.D. Panelist Johannes Loewer, M.D. Panelist Also Present: Gary Nabel 3 I N D E X SPEAKER PAGE Philip Minor, Ph.D. 4 James Robertson, Ph.D. 32 Jorg Schuepbach, M.D. 51 Jens Mayer, Ph.D. 66 Thomas Broker, Ph.D. 78 Neil Cashman, M.D. 102 Panel Discussion 129 Dr. Onions, Chair John Sedivy, Ph.D. 176 Frits Fallaux, Ph.D. 199 Panel Discussion 212 Dr. Coffin, Chair 4 1 P-R-O-C-E-E-D-I-N-G-S 2 8:04 A.M. 3 CHAIRPERSON RABINOVICH: Good morning. I 4 would like to welcome you back to Session 6, 5 Adventitious Viral Agents in Cell Substrates, and 6 congratulate all those that were here until 10:00 last 7 night for the latest part of the show, including Mr. 8 Harris, who put in a grueling 18-hour day in 9 yesterday, our visual aides person. 10 I would like to introduce the first 11 speaker, Dr. Phil Minor, from the National Institute 12 of Biological Standards and Control, who will be 13 giving us an introduction to adventitious agent 14 issues, both reviewing the past and current experience 15 with adventitious agent contamination of biologicals 16 in vaccines. 17 DR. MINOR: Thanks. Thanks very much. 18 Can I have the slide on, please, or do I just press it 19 here? 20 What I am going to do is to review firstly 21 all of biologicals, if you like, from an adventitious 22 agent point of view. So it won't just be vaccines. 23 In particular, I will be talking about the range of 24 source materials that people have used in preparing 25 biologicals. There will be a clear message that comes 5 1 out of that, which is that the more you use well- 2 characterized cells, the better. 3 I will also be talking about the SV40 4 story in some detail, which has been gone through a 5 number of times, but I will be going through it from 6 a particularly regulatory point of view because again, 7 there is a message there which says that if you get it 8 wrong, you will still be working on it 40 years later. 9 Finally, I will get onto the continuous 10 cell line bit right to the very end. 11 So there are a variety of source materials 12 that you can use if you are preparing biologicals. 13 They are sort of listed here, if you like. There are 14 biological materials which are made from whole 15 animals. That would include things like blood and 16 blood products. I will describe that in a moment. So 17 you can just go to a whole animal and take something 18 out and make your biological from that. 19 You can use your whole animal as a 20 substrate for growth. I will discuss that in the 21 context of things like influenza vaccines and the 22 like. 23 You can grow material on primary cells. 24 This was the main starting point for things like polio 25 vaccines in the early days, where the SV40 issue 6 1 arose. Finally, you can grow materials on well- 2 characterized cell preparation. 3 The further down the list you go on this 4 thing, probably the happier you are from the 5 adventitious agent point of view. 6 This shows some examples of contaminants 7 which have arisen when whole animals have been used as 8 source materials or the origin of the source material. 9 Most of these will be human rather than anything else, 10 but really an awful lot of the serious adventitious 11 agent problems that have arisen have arisen because of 12 materials sourced from whole animals or using pooled 13 preparation. 14 The first one on this list here is CJD, 15 Creutzfeldt Jakob Disease, which was transmitted by 16 growth hormone. The growth hormone was produced from 17 human cadaveric material. A very unpleasant disease. 18 It's almost impossible to detect the agent other than 19 by standing back and waiting for the incubation period 20 to go. 21 In France, there are still a large number 22 of cases coming through as a result of this. It may 23 well be that around 10 percent are recipients of 24 human-derived growth hormone, will actually wind up 25 going down with CJD in France. 7 1 Dura mater is another one. That should be 2 one T, not two Ts. Again, this has been shown to 3 transmit really quite readily when pooled materials 4 are actually used. Almost impossible to detect. 5 Clear, very, very serious kind of consequence of it. 6 Scrapie was first shown to be a 7 transmissible agent by the use of a TBE vaccine, which 8 was grown in the brains of sheep. TBE being tick- 9 borne encephalitis, which then transmitted scrapie to 10 a large number of the sheep that were actually 11 inoculated with it. So again, this is a whole animal 12 source material, if you like, that had quite serious 13 consequences, especially if you were a sheep. 14 Over the last 15 or 20 years or so, one of 15 the best examples of serious or disease-causing 16 transmissions of infectious agents has been through 17 human blood and blood-derived materials, clotting 18 factors in particular. In all of these things, the 19 entire alphabet soup of hepatitis viruses has been 20 transmitted by blood product. 21 In the early days at least, some of these 22 were really regarded as really a hazard, if you like, 23 of being a hemophiliac. So, for example, hepatitis C, 24 in the days when there was a non-A, non-B hepatitis, 25 it was really regarded as an inevitable consequence of 8 1 using factor 8 to treat hemophilia. I am not sure 2 that that is an acceptable way of actually doing 3 things any more. I am sure that hemophiliacs would 4 agree with that. B-19, paravirus B-19 is still 5 transmitted by clotting factors. 6 Finally, this one down at the bottom here 7 is a classic example of a transmission by a vaccine, 8 if you like, where hepatitis B was transmitted by 9 yellow fever vaccine back in the 1940s. The hepatitis 10 B actually came from the stabilizers of the albumin 11 that was actually put in there to keep it stable. 12 There is a story that Fred McCallum, who 13 is head of the Public Health Service in the United 14 Kingdom tells to the effect that he basically won the 15 war because he prevented Winston Churchill having a 16 yellow fever vaccine when he was going off to talk 17 with Stalin around 1944. 18 So most of the serious consequences really 19 come from whole animal source materials, if you like. 20 You can use whole animals as substrates. I'm using 21 the term "whole animals" in a fairly broad sense. 22 Eggs in the definition of the Animal Regulated Use Act 23 in the United Kingdom count as an animal because they 24 are embryonated. 25 For many years, rabies vaccines were 9 1 produced in mouse brain or sheep brain. They have 2 quite serious consequences, but not necessarily 3 associated with adventitial agents. You can get 4 encephalitis as a result of immune responses to the 5 non-invasic protein. 6 The Japanese encephalitis vaccine, which 7 is used for travelers in the United Kingdom, is still 8 made in mouse brain. So it's not an unusual source of 9 material, if you like. Smallpox for a long time was 10 made on the scarified flanks of calves. Like I said, 11 isn't any more. However, while these things seem 12 really quite primitive, in terms of how you make 13 vaccines nowadays, you still have a number of vaccines 14 that are made in eggs. Yellow fever is the classic 15 example, and influenza. 16 Yellow fever is not required to be grown 17 in avian leukosis-free eggs. The reason for that is 18 that there are a number of sites at which it was 19 manufactured throughout the world, where yellow fever 20 is a really very serious problem, such as Nigeria, for 21 example, South America, whatever, where SPF eggs, 22 avian leukosis-free eggs even, were really not freely 23 available. So yellow fever can in principle at least 24 be made in avian leukosis containing eggs, and in fact 25 is. I think there's no evidence that this has an 10 1 adverse consequence. But on the other hand, you 2 wouldn't necessarily want to have a virus in there 3 that you didn't know about. 4 Influenza is an actuated vaccine. Again, 5 it's not made on SPF eggs, that is, specified 6 pathogen-free eggs. They are avian leukosis free, but 7 they are not free of all the other variety of 8 pathogens that you would choose to screen for measles 9 vaccine production system, for example. 10 So even today then you have to bear in 11 mind that a large amount of vaccine that's made is 12 made on really quite crude materials, from an 13 adventitious agent point of view. It's not a trivial 14 usage. In fact, when you go through and consider what 15 vaccines are actually made on these days, they are 16 quite primitive, if you like, in some respects. 17 Primary cultures as been described 18 previously around here, are really cultures that are 19 made directly from the animal. So they are not one 20 pass. They are directly from the animal, if you like. 21 Here are a number of examples where agents are 22 actually being found or at least located in these kind 23 of cultures. 24 SV40 is one that I'm going to talk about 25 in some detail in a minute. This was in polio 11 1 vaccines in the 1950s and very early 1960s, probably, 2 a source from rhesus monkey kidney. Polio vaccines 3 are still made on monkey kidney, though they are not 4 usually on rhesus monkey kidney. It would be 5 cynomologous or something like that, for reasons which 6 I'll describe in a moment. 7 Nonetheless, a great deal of vaccine is 8 still made in primary monkey kidney cells. There are 9 reasons for that. There's a deep conservatism I think 10 about changing the vaccine production process if you 11 have a vaccine that works, largely because you are 12 dealing with a prophylactic material rather than a 13 therapeutic material. So you don't want to mess about 14 with anything if it's reasonably safe and effective. 15 I'll mention very briefly the defective 16 retrovirus story in chick embryos. I think Jim 17 Robertson will probably mention this in more detail, 18 but I will mention that just as I go by. Finally 19 recently, the FDA released a talk paper on a 20 preparation of urokinase, which is used in treating 21 the heart. This material was grown from primary 22 cultures made from aborted fetuses. I think it was 23 aborted fetuses or miscarriages, or whatever. There 24 were quite a variety of infectious agents were 25 actually found in this. I believe this one has now 12 1 been suspended. 2 The point is that there are still a large 3 number of materials which are made on really quite 4 basic culture systems, if you like, where adventitious 5 agents are a serious consideration, if you like. So 6 it's not all continuous cell lines versus the rest. 7 I mean there are -- most of the vaccines that are made 8 in the world probably come from other primary cultures 9 or eggs or things of that nature. 10 I will now talk about SV40. I'm sure in 11 this audience there are people who know far more about 12 SV40 than I do. But nonetheless, I'll talk about this 13 from what you might call the regulatory adventitious 14 agent point of view, if you like. 15 So it's a very common polyoma virus of old 16 world monkeys, and particularly rhesus macaques. The 17 difficulty with this was that when the rhesus macaque 18 monkeys are sacrificed and a primary monkey kidney 19 culture is made from him or her, as the case may be, 20 a silent infection is set up. So there is on evidence 21 of infection just by looking at the cultures. In 22 fact, these cultures can throw out as much SV40 as 23 they do polio, when you start infecting it with polio. 24 So you wind up with a culture that's just stiff with 25 adventitious agent which you really don't want. 13 1 It's able to transform non simian cells in 2 vitro, and it can be tumorigenic if you have the right 3 kind of animal that you put it into. Between 55 and 4 62, probably at least a third of all the vaccines that 5 were made on these kind of cultures, because they were 6 pooled and the like, were almost certainly 7 contaminated with SV40. It wasn't a trivial 8 contamination. It was really quite a serious 9 contamination. 10 Because it was mainly an activated polio 11 vaccine, there wouldn't have been that much live SV40 12 in it perhaps. But SV40 is more resistant to formalin 13 than polio is. So almost everybody who received the 14 shot of inactivated polio in the 1950s, which would 15 include me, would have received live SV40 in some form 16 or another. 17 So the concern is really summarized here, 18 which is basically that everybody, I mean this is my 19 own take on it, that everybody -- I mean you can argue 20 that it might not have been sort of everybody, but I 21 think it probably was. But almost everybody who 22 received the full course of polio vaccine between 1955 23 and 1965, also got live SV40 stuck into them. That's 24 millions of people basically. 25 There were epidemiological studies that 14 1 were done at the time which really didn't cause much 2 concern, but they can all be criticized. Some of the 3 studies were really quite short-term, about two or 4 three years or so, looking to see if there were cancer 5 effects basically, as a result of SV40. It may be 6 that two or three years is not enough to actually find 7 such an effect, if it actually exists. 8 The longest which was assumed was over a 9 period of about 19 years. Most of the individuals 10 involved in that study would have been oral polio 11 vaccine recipients rather than inactivated polio 12 vaccine recipients. So they have had it by mouth 13 rather than by injection. Again, you could argue that 14 that might not be the right cohort to actually be 15 looking at. 16 So while the studies were reassuring, the 17 most reassuring thing was that there was no sudden 18 surge of cancers that you can actually trace back to 19 polio vaccine usage in the United States or in Europe 20 where these things were used in a big way. So it 21 really did seem that in the long term, over about 19 22 to 20 years, there was no real cause for alarm. 23 However, in 1992, Michaili Carboni and 24 colleagues and others, a number of others, including 25 Janet Butelle down in Texas and the like, identified 15 1 SV40 sequences which were present in a variety of 2 relatively rare tumors. So mysathelia, which is the 3 asbestos tumor, osteosarcomas, pendymonas, actually 4 the young chorioid plexus tumors of children, these 5 sequences do appear to be genuine SV40 sequences. 6 Where they come from is really not quite 7 clear. Part of the argument was that you could get 8 similar types of tumors in experimental animals, like 9 hamsters. I think that is probably the only example 10 where a hamster is cited as a good model for a human 11 being perhaps. But who knows? In fact, this might 12 actually be an argument that this has got nothing to 13 do with it. 14 So the question then arises as to where 15 did the SV40 sequences come from. Of course the 16 classic response really would have been it must have 17 come from the polio vaccine because why not? 18 Now SV40 was discovered around 1961 or 19 1962 or thereabouts, 1960 perhaps. Directly it was 20 discouraged. There were precautions put in place to 21 exclude it from polio vaccines, because it was known 22 to be a tumor kind of virus, if you like. These were 23 the kind of things that were put in place. They are 24 listed in WHO requirements from about 1962 onwards. 25 They reached their final fully flowered form, if you 16 1 like, by about 1965. A number of countries certainly 2 had put this in place before that. 3 The first thing you can do is to use 4 seronegative animals as the source of cells. So you 5 can use animals that have no evidence of SV40 6 infection as your source. That really is something 7 which is now very firmly in place, which manufacturers 8 now do. 9 The second thing is, you remember I said 10 that it was the rhesus macaques with the problems. 11 The problem was that the cell cultures didn't show any 12 sign of having defect, when they were actually 13 infected with SV40. What you can do then is you can 14 use species, such as cynomolgus or pattus monkeys, 15 where the primary monkey kidney culture cell, when 16 infected with SV40, will actually wrinkle up and die 17 on you. So at least you know you have got something 18 nasty and you can throw it away. 19 Finally, you can test your control of 20 production cultures for SV40 by the same kind of 21 procedure. That is why using sesetral cells to see if 22 anything comes through. 23 Around the period that this was taking 24 place, wild caught monkeys were being used extensively 25 in vaccine production. Up to a half of the cultures 17 1 would have been thrown away because of adventitious 2 agent contamination, mainly foamy virus, but certainly 3 other things as well. I think that just illustrates 4 the kind of lack of control, if you like, over the 5 source materials that was going on, and the extent to 6 which adventitious agents are really a serious problem 7 in finding monkey kidney cultures or primary cultures 8 in general. 9 An alternative way of doing this is to 10 actually use a validated cell bank. Certainly many 11 manufacturers use MRC5, and Mary of course used vira 12 cells, as we heard last night. 13 Nonetheless, a significant, if indeed not 14 a large proportion of the world's supply of polio 15 vaccine is still made on primary monkey kidney cells, 16 which should really fit this kind of criteria for 17 excluding SV40. 18 One of the questions that then arises is 19 were these precautions good enough? What we did at 20 NIBSC, because we happened to have about 150-odd 21 batches of vaccine archived from the years, was to go 22 back and look at them by PCR. PCR of course is the 23 cat's pajamas. It's really the best technique that 24 anybody ever invented in terms of sensitivity. It's 25 probably about as good as infectivity, at least in our 18 1 hands anyway. 2 But nonetheless, we went back and we 3 looked by PCR at 133 preparations of polio vaccine 4 which had been used in the United Kingdom between 1966 5 and about 1997. What we had done was looked at all 6 batches of vaccine which had been used since 1980, and 7 all of those were free of SV40 sequences. So that 8 gives you some reassurance that these precautions were 9 actually appropriate. 10 In fact, the only preparation which had 11 any SV40 sequence in it at all was a seed virus which 12 was used by a manufacturer for making vaccine from. 13 The amount that was in there was around two logs worth 14 of genome as opposed to seven logs of genomes in a 15 really full-fledged infected preparation. So there 16 wasn't that much in there. The manufacturer had also 17 treated this stuff with toluidine blue, which is 18 supposed to kill of SV40. This was done on the advice 19 of Albert Sabin back in 1960-something or other. 20 But nonetheless, it does seem to me that 21 it's rather a foolish thing to have a seed that's got 22 SV40 sequences in it at all. I think the WHO 23 requirements have now been changed so the seed has to 24 be checked to see if it does have SV40 sequences in 25 it or not. 19 1 This particular seed was not infectious 2 SV40. We did some quite serious studies on it, like 3 transvecting the DNA into cells to see if it would 4 work, infecting monkeys with it to see if we could 5 actually get seroconversion. There was no 6 seroconversion. So there was no infectious virus 7 there that we could actually detect. But nonetheless, 8 the seed did have material in it. 9 If on the other hand you look at materials 10 from around the 1960s or from other parts of the world 11 a little bit later than that, you can pick up SV40 12 sequences quite easily. So the method would have 13 picked it up had it been there. 14 So our conclusion from that was then that 15 really as soon as these kind of precautions were put 16 in place, no SV40 would have been present in all polio 17 vaccines used, at least in the United Kingdom and I 18 would guess in the United States as well, because it's 19 after the same kind of precautions were put in place. 20 So the precautions were adequate. Which means that 21 SV40 exposure of the population through polio vaccines 22 would have stopped around 1962. 23 So what you then have is the problem of 24 the chorioid plexus and appendinoma tumors, which 25 occur in children who are around two years of age or 20 1 maybe less. You have to say well how did they get a 2 hold of the SV40 sequences? One possibility, which is 3 mooted with some enthusiasm is that maybe you are 4 getting passage of SV40 from parents who did receive 5 the SV40 contaminated polio vaccine to their children. 6 So how this stuff gets around is quite important. 7 One of the things that we have been 8 involved in is doing serological surveys of 9 populations to see who has got SV40 antibodies and who 10 hasn't. It is about a five percent seropositivity by 11 the assay that we're using at least. It seems to peak 12 at around age 10 or thereabouts, and doesn't arise 13 after that. 14 So what you could argue then is that you 15 are seeing vertical transmission from parents down to 16 their children. What you could also argue is that you 17 are not picking up SV40 specific antibodies at all, 18 and they could be other human polyomas like the BK or 19 the JC, and it's cross-reacting antibodies that we're 20 picking up. I think that is still a thing that needs 21 to be resolved. This is how we were trying to resolve 22 it. 23 We have access to a number of sinomorgous 24 breeding colonies. One of them at least is absolutely 25 riddled with SV40. It's chronically infected. They 21 1 are all infected basically. 2 So this is just four examples of this 3 particular colony. There's about another 50 or so. 4 This happens all the time. The mothers here are 5 highly sera positive to SV40, all of them. What 6 happens is that the mother and the baby stay together 7 for about six months until the baby is weaned. Then 8 the babies are taken off, no longer being babies of 9 course. They are all banged up together in one 10 gigantic sort of teenage squabbling colony. 11 At the time of weaning, the babies are 12 uniformly negative. So despite the fact they have 13 been on the mother for six months, they have not sera 14 converted to SV40. Almost immediately you bang them 15 up together, or at least within about a month or so, 16 they sera convert. So we actually have a sera 17 conversion panel here, if you like, with about 50 or 18 100 or so sera, where the babies actually were 19 seronegative and then become seropositive. 20 My view on this is probably that the 21 babies don't get infected until you bang them up 22 together. But it may be that they are infected, but 23 they are just not seropositive. So what we have to do 24 here is to fish out the virus from these animals here, 25 and see if it looks like the mother's virus or if it 22 1 looks like the other babies' virus. 2 The point about this long story which I 3 have just been telling you about SV40 is that SV40 was 4 a problem between 1955 and 1962, and it's now 1999, 5 and we still don't really know what was going on. So 6 if you actually make a mistake, it's really quite 7 serious. It may keep you occupied for the rest of 8 your working life. 9 One last quick thing or two last slides 10 here. One is about reverse transcriptase of vaccines. 11 Dr. Schuepbach will be talking later and Jim Robertson 12 will be talking in a moment about detection of reverse 13 transcriptase in chicken cell grown vaccines, such as 14 flu or yellow fever or measles, mumps, rubella. 15 This appears to be due to the presence of 16 defective non-infectious particles. There are 17 sequences from EAV and ALV both in these things, a 18 ratio of about nine to one as I understand it. It 19 does seem to me that you are not really quite sure 20 what the AV sequence is in there and what ALV sequence 21 is in there. It's probably going to vary from chicken 22 to chicken in so far as these chickens have not been 23 bred. In other words, every egg is a new experiment. 24 You are really not quite sure what you are dealing 25 with in that. I think that is quite an unfortunate 23 1 position to be in. I'm not sure how you control it. 2 Finally, this is my last slide, and this 3 has to do with characterized cells. The issues that 4 I have been dealing with really have been to do with 5 primary cells and primary cell problems where the 6 virus comes in direct from the animal origin. I think 7 there is no doubt in my mind that that's the main 8 source of concern in terms of human health. 9 Nonetheless, there are clearly problems 10 which also arise with characterized cells and the 11 continuous cell lines, in particular. We have some 12 down here. 13 Now the regulatory authorities in the room 14 will be well aware of a large number of other examples 15 of this type which don't actually get published. I 16 think that's not so good. I think this stuff really 17 should be out there in the public literature. But 18 nonetheless, these are the ones which are well known, 19 I think. 20 CHO viruses, CHO cells have defective 21 retroviruses. Manufacturers take a great deal of care 22 to actually get rid of them in the final product. So 23 they are endogenous. 24 There are examples of things like BVDV 25 contaminating cells which are growing in culture, and 24 1 also other bovine viruses contaminating cells in 2 culture, particularly when they are grown on a very 3 large scale. Whether or not that poses a hazard is 4 another matter, but clearly there must be methods in 5 place to actually detect them. 6 The classic example here was the minute 7 virus in mice, where the tpa had been grown in CHO 8 cells on a 10,000 litre stove essentially, and then 9 tiled up for an effect with minute virus of mice. Now 10 this was on the order of eight logs, as I understand 11 it, of virus per mil, and yet a 10,000 litre fermenter 12 culture. This is probably more minute virus of mice 13 in one place on the planet than has ever been the case 14 before. You might want to think how you actually get 15 rid of it actually. 16 This is a question of actually getting the 17 cells infected while they are actually burning in 18 culture. So while family cells are clearly a major 19 problem, and while whole animal sources, if you like, 20 are probably the biggest hazard which is likely to be 21 raised in terms of human health, biological aspects to 22 do with well characterized cell banks, where viruses 23 may be introduced from biological materials or they 24 may be introduced by mice walking across the top of 25 the fermenter or whatever, are nonetheless a 25 1 significant matter. It really is not totally clear 2 whether these things have an implication for human 3 health. But I think you would be wise to make sure 4 that they are not actually present. 5 That's where I stop. Thank you. 6 (Applause.) 7 CHAIRPERSON RABINOVICH: Please identify 8 yourself. 9 DR. COFFIN: John Coffin of Tufts. That 10 was a really nice summary actually, Phil. But some 11 caution might be called for in translating the results 12 of vertical transmission experiments from monkeys to 13 humans. 14 As far as we know, simian immuno 15 deficiency virus in monkey populations are not 16 transmitted vertically. Yet HIV-1 is transmitted with 17 reasonable efficiency vertically in human populations. 18 So there may be some underlying biological difference 19 that perhaps a very subtle one, that promotes this 20 kind of transmission in people, where you wouldn't see 21 it necessarily in monkey models. 22 DR. MINOR: Yes. I take you point. We 23 are doing the studies for two reasons. Firstly, to 24 look at natural transmission to monkey on the grounds 25 that it might be a model, although I take your point 26 1 entirely. But also to supply serum conversion panels 2 so that we can try and sort out specificity of 3 immunological reactions as well. I take your point 4 entirely. 5 DR. ONIONS: David Onions, Glasgow. 6 Phil, when people switched to cynomologous 7 monkeys, and I can see the reason because you can pick 8 up SV40. That's very clear. But how do you know that 9 at the same time, you have not invented a new problem, 10 that you have got another polyoma virus in that 11 species that you are not detecting. I mean has anyone 12 done redundant PCR to look? 13 DR. MINOR: I think I would choose to look 14 at a polyoma man to answer that question. Anybody? 15 DR. MAJOR: Gene Major, Bethesda. In the 16 monkeys that we have used for the human polyoma virus 17 studies, we have screened all those animals for the 18 conventional SV40, but not have addressed the question 19 of whether or not there's other additional polyoma 20 type viruses that are present there. 21 Clearly by this time, PCR technology has 22 advanced to the point that perhaps if something were 23 there whose sequences were somewhat similar to the 24 ones that are currently expressed in these animals, we 25 may have picked it up, but we certainly haven't found 27 1 anything yet. 2 DR. LEWIS: Phil, I take it that 3 seroconversion is by neutralizing antibody? 4 DR. MINOR: Sorry? Say that again. 5 DR. LEWIS: I take that seroconversion is 6 by neutralization? 7 DR. MINOR: That's right. Yes. 8 DR. LEWIS: Have you had any chance to 9 look at monkey breast milk to see how long they may be 10 treating antibodian, so that the newborns can be 11 passive immune? 12 DR. MINOR: Right. The answer is no. We 13 have discussed, for example, getting the urine out of 14 these monkeys after they are banged up together. I am 15 told that actually chasing them around the floor is 16 insuperable. I'm not sure you can actually pry a baby 17 monkey from her mother long enough to actually milk 18 her. It's a worthwhile question. I think we'll have 19 another go and see if we can do something about it. 20 There may be some resistance, however. 21 DR. BROKER: Tom Broker, UAB. I wanted to 22 follow up on that exact question. We are facing the 23 same problem with potential vertical transmission of 24 human papilloma viruses. I'll mention it later in my 25 own presentation, but briefly, it does appear there is 28 1 some protective immunity during nursing. On the other 2 hand, removal of an infant, say through adoption to 3 another family, is the highest risk factor for a child 4 acquiring laryngeal papillomatosis later in life. So 5 a two to three year delay. 6 DR. MINOR: So is the assumption then that 7 the infant is infected, but it's not infected properly 8 then? 9 DR. BROKER: It's infected vertically, 10 presumably perinatally, perhaps just before or during 11 delivery, but receive sufficient passive immunity by 12 nursing that gives it life long protection. 13 So what I would propose as a potential 14 experiment is to literally take the, if possible, take 15 the baby monkey immediately away from the mother, and 16 don't allow it to nurse, and then just have different 17 lengths of time of nursing to see if this onset of 18 seroconversion is affected by a timing mechanism. 19 Alternatively, don't ever let that baby monkey be 20 housed with other baby monkeys in the daycare center, 21 and keep it with the mother even if it's not nursing, 22 and see if it fails to seroconvert. 23 So the question is, is it getting infected 24 from its playmates. 25 DR. MINOR: That's right. 29 1 DR. BROKER: Or is it receiving a period 2 of important passive protection from the mom. 3 DR. MINOR: I figured we could do that by 4 looking at the actual strain of viruses the monkeys 5 get infected with. We have a number of different gang 6 rooms, if you like. If you get a different strain in 7 each gang room, but it's the same strain within a gang 8 room, then I think that will answer the same question. 9 You can also go back to the mother and see what kind 10 of strain she's got too. But it's a valid point. 11 AUDIENCE MEMBER: I would like to 12 reemphasize one of the important points that you made. 13 I know you didn't have time to expand on it, but I 14 think it is extremely important. That is the need for 15 those organizations who discover a new virus or some 16 contaminant, cell population used for vaccine 17 production or in a production run, to make that 18 publicly known. 19 I think that the declaration by Genentech, 20 who has published this information under their name, 21 that an NBM contamination occurred in a 10,000 22 fermenter is an act of great courage. I think that 23 that kind of courage, this declaration by other 24 companies in this field, is very necessary for the 25 health of this industry. 30 1 I understand from some of the remarks that 2 have been made that there are others that are known to 3 a small coterie of people here that have not been 4 publicly declared. I urge all of you to think about 5 this seriously because it can and will have a great 6 impact on this industry. Thank you. 7 DR. MINOR: I agree totally with that. It 8 does seem to me that sooner or later the information 9 will leak out. I think the industry looks very bad. 10 DR. VAN DER EB: Van der Eb, Leiden. Did 11 I understand it correctly that ferrisfaruses were 12 found in human embryo material that was used for 13 urokinase production? 14 DR. MINOR: I think the FDA can answer 15 this one better than me, yes. But I mean that was my 16 understanding of it. It's out on the net. 17 DR. VAN DER EB: But where does it come 18 from? 19 AUDIENCE MEMBER: I think it's a rea 20 virus. 21 DR. MINOR: It's various rea viruses, plus 22 others. 23 DR. VAN DER EB: I see. Okay. 24 DR. FRIED: Mike Fried. Was any of the 25 old vaccines from the 1960s that were contaminated, 31 1 were they PCR'd up to show that the virus was the same 2 as being found today? Because it's also possible that 3 we all have a latent SV40 type virus which likes to 4 grow in tumor cells, and that's why you find it. It's 5 a passenger. But I mean since there's polymorphisms 6 in the sequence, if you can go back to the 1960s and 7 then find out if it's the same thing that we find 8 today, it would be helpful. 9 DR. MINOR: We looked at, when you say the 10 1960s, I have to emphasize this is very early 60s. 11 Certainly the things that we have got which came out 12 positive weren't used in the UK, or they might have 13 been used somewhere else. 14 We had a Russian SV40 and we had an 15 American SV40, and we had an SV40 of unknown origin 16 all from the 1960s, and they were all different 17 basically. They were different from the 7-7-7, you 18 know, the cos kind of sequence as well. So they were 19 all unique basically, in terms of the region we were 20 looking at, which was C terminus of t antigen. 21 CHAIRPERSON RABINOVICH: Last question 22 please. 23 MS. MARCUS: Carole Marcus Sequora from 24 Bassey Consulting. 25 I just wanted to clarify that urokinase is 32 1 produced from cells. It's not aborted fetuses. It's 2 newborns who did not survive. Just for the record. 3 DR. MINOR: Thank you. 4 MS. MARCUS: It was rea virus. 5 DR. MINOR: I'm sorry about that. 6 CHAIRPERSON RABINOVICH: Thank you. Our 7 next speaker is Dr. Jim Robertson, speaking on 8 experiences with retroviruses in avian and mammalian 9 cell substrates. 10 DR. ROBERTSON: Good morning. For those 11 of you who don't know, NIBSC is CBER's cousin from 12 across the pond in the U.K. What I am going to do is 13 pick up where Phil left off and concentrate on the 14 retrovirus aspect of viral contamination. Initially 15 I will look at say biologicals in general, but 16 ultimately focusing down on the vaccine issues. 17 So I will begin with some direct 18 information regarding retrovirus situations with 19 biologicals. I will go onto look at how some of the 20 regulatory guidelines deal with the issue of 21 retroviruses. I will go into look at RTase testing, 22 which is a reasonably current them just now, and 23 finish up looking at the recent situation of the 24 finding of retroviral-like particles in avian cells. 25 So to begin with, here is a short list of 33 1 the incidence of retrovirus contamination found in 2 biologicals in general, not just vaccines. I have 3 sub-divided these into two groups here. You see this 4 upper half here, this is where we have in the past had 5 overt adventitious contamination by a retrovirus of a 6 biological. For instance, being mentioned earlier, 7 ALV, that causes virus in yellow fever vaccine by 8 virtue of producing the vaccine in embryonated eggs 9 infected with the virus. The other one that was 10 mentioned earlier by Phil, HIV and blood products. 11 The bottom half here is a quite, somewhat 12 separate type of contamination. In fact, you might 13 find it equaler to call it contamination or not. 14 Certainly these are not adventitious situations. 15 These are situations in which an endogenous 16 retroviral-like particle is present in the 17 manufacturing process. 18 In the first instance here, it's 19 established that murine hybridomas used in the 20 manufacture of monoclonal antibodies produced, 21 secretes C type particles. These have been tested in 22 a variety of other mammalian, including human cell 23 lines, and generally are not infectious. 24 The titre can be very high for these types 25 of particles. You can get 10 to the sixth particles 34 1 per mil. I've even seen 11 particles per mil in one 2 instance. So you can have a very high burden of 3 direct viral particles. 4 It is also well established that CHO 5 cells, which are used for producing 6 biopharmaceuticals, secretes C type particles. You 7 also get intertestinal type particles from these 8 cells. These are probably much more characterized, a 9 bit more work has gone into describing the particles 10 from CHO cells, sequence information from the 11 endogenous elements within the CHO genome, which is 12 producing these particles, give some ideas as to why 13 they are defective. The reading frames are 14 incomplete. There are stop signals. So you don't get 15 a proper infectious virus from these endogenous 16 elements. 17 The latter type is the only type here 18 that's dealing with vaccines, even dry vaccines 19 produce either an ovo or cef cells. I'll come back to 20 that in a few minutes. 21 From a regulation point of view, how do we 22 deal with virus contamination and retrovirus 23 contamination? There are a couple of guidelines I 24 would like to bring to your attention. The first one 25 here is an ICH guideline, which looks at viral safety 35 1 evaluation. Admittedly it is only for biotech 2 products. The scope of the guideline does comment 3 that this is not, this guideline is not applicable to 4 vaccines. But I think it is worth looking at what it 5 says about virus contamination. 6 Within the document, it describes five 7 different cases of potential contamination, starting 8 from the most desirable case, where you don't have a 9 virus present in the process in any way, down to the 10 worst scenario where you know you've got a virus, but 11 you haven't a clue what it is. 12 The guideline goes on to state what is 13 acceptable and what is not acceptable in the 14 manufacturing process. The only two cases which are 15 generally acceptable of the first two cases, Case A, 16 where you have got no virus, and Case B, where you 17 have got a non-pathogenic retrovirus. The other cases 18 are only exceptional. Generally you don't want one at 19 all. The manufacturing is not allowed when you have 20 got a virus contamination. 21 So for Case B, really what you have here 22 is a murine retrovirus is probably the only 23 contaminant acceptable in the bulk harvest. If you 24 remember this guideline is applicable only to 25 recombinant products and not to vaccines in general, 36 1 and these recombinant products are highly purified. 2 The other guideline is the WHO 3 requirements which came out recently for use of animal 4 cells as in vitro substrates. That does include 5 vaccine production. 6 When it comes to testing for retroviruses, 7 this guideline has several -- many other guidelines in 8 the past have indicated, the types of assays being 9 used for retroviruses, specific infectivity assays, 10 electron microscopy and transcriptase assays, are the 11 three general approaches for checking for retrovirus 12 contamination. 13 There may be the use of specific antigen 14 detection as is in some particular cases, but these 15 are the generally recognized methods of going about 16 picking up retros. 17 If I can concentrate now on the RTase 18 assays. The traditional type of assay involves 19 incorporating a nucleotide precursor, a labeled 20 precursor of some kind into an assay using a rather 21 synthetic type of template. Then more recently of 22 course we have the PERTs, PB RT, AMP RT type of 23 assays, which includes a PCR amplification step, with 24 the result that these type of assays are incredibly 25 more sensitive than the more, as I can call it, 37 1 traditional type of assay, and what is often quoted as 2 up to a million fold times more sensitive by virtue of 3 incorporating a polymerase chain reaction. 4 Now using this type of assay, the cat was 5 set among the pigeons. When this paper came out, I 6 might even say that the fox was set amongst the 7 chicken coop. Detection of reverse transcripted 8 activity in live attenuated virus vaccines. This 9 quite naturally caused a bit of concern as to what was 10 going on here. The vaccines indicated, the one common 11 feature was that ovine produced in eggs of some kind, 12 measles vaccine out of CEF primed cultures, similarly 13 mumps. Yellow fever and influenza in ovo. But not 14 measles vaccine out of human diploid cells or rubella 15 vaccine out of human diploid cells. So the common 16 link here seemed to be the CEF, the chicken source 17 used in the production of the vaccine. 18 We joined in the boat here and started 19 looking at this issue. Every type of hen fluid that 20 we have looked at, CF fluids or an type fluid from a 21 variety of different strains of hen have all been 22 positive in the assay for reverse transcriptase. 23 Summarily, quail, jungle fowl, and pheasants are 24 positive. 25 The types of sources of fluids which have 38 1 been negative for reverse transcriptase are listed 2 here. Some species are not positive, turkey and duck 3 cultures, quite a range of human cell lines. Simian 4 rabbits have been tested and found to be negative. So 5 the clear source of this RTase that was being picked 6 up in the vaccines is quite clearly of chicken avian 7 origin. 8 We would want to look at -- I should add 9 that this RTase was known at the time to be particles 10 associated and appears in the supernate of the cells. 11 We are going to look at this particle to see if it 12 would pick up any infectivity. In all, we looked at 13 10 different cell lines, mainly human, but including 14 rabbits and turkey. Over 21 tests and 116 passages. 15 In each case, in every test and at every 16 passage level, the cultures were negative for reverse 17 transcriptase activity. There's absolutely no 18 indication that this particle is infectious. Since 19 then, CBER and CDC have also come up with similar 20 data, including use of PBMCs. No infectivity 21 associated with these RTase containing particles. 22 Where might these be coming from? 23 Presumably they are derived from endogenous 24 retroviral-like elements in the chicken. The 25 information to date regarding such elements in the 39 1 chicken genome are quite well characterized EV loci, 2 which are related to the avian chosis virus family, 3 and more recently discovered about 10 years ago, EAV-0 4 family, which is an older element than EV, and then 5 older still, ART-CH and CH-1 elements. 6 The information at the time and pretty 7 much where it still exists is that we knew that 8 there's a line of chickens which was negative for EV. 9 It had been eliminated from the genome. This line of 10 chickens, the culture fluids were positive for RTase. 11 So we knew that it had to be at least one of these 12 elements producing RT activity. At the same time, you 13 couldn't eliminate the fact that EV might also be 14 producing RT activity. The best bet was EAV-0, given 15 the sequence information that was present at the time. 16 More recently, in the last year or two, 17 Joerg Schuepbach's laboratory has produced a good 18 evidence for the presence of EAV-0 derived RNA 19 associated with the RT particles secreted from CEF 20 cells, and then this year, Walid Heneine, CDC, also 21 produced the presence of EAV and ALV RNA. When I say 22 ALV, I mean derived from the EV loci and not exogenous 23 ALV contaminating RNA. 24 So what can happen here retrovirus-like 25 particles, defective particles being produced from 40 1 endogenous elements both from EV and EAV-0 family of 2 endogenous elements. The presence of the RNA and 3 relsconstrictase in a particulate fraction leads one 4 to come to the conclusion that we have retroviral like 5 particles in the CF fluids of the chick cells, which 6 is present in the vaccines measles and mumps. 7 The absence of infectivity in the current 8 genetic information, sequence information that we have 9 on EV loci and the EAV-0 family of endogenous elements 10 strongly indicates that these particles are defective 11 viral particles. The only question mark that remains 12 from the regulation point of view, but also scientific 13 point of view, the possibility of pseudotype formation 14 during vaccine manufacture. The current evidence 15 suggests the particles that are defective in the 16 envelope-like protein and so there's a particulate of 17 pseudotype formation with the glycoprotein of vaccine 18 viruses being grown in the CF cells. 19 So to summarize a couple of these issues 20 then, from the practical point of view, testing for 21 reverse transcriptase as an indicator of retroviral 22 contamination, these assays are evolving, changing all 23 the time. One has to take into consideration the 24 strength of the assay and the validity of the assay. 25 There may be different requirements within an assay 41 1 for different sources of RT. It may be necessary to 2 use some other sort of method to asses the 3 significance of any RT detected because we know that 4 RT activity can derive from other enzymes. Telomerase 5 is or DNA polymerase, cellular DNA polymerase is. 6 These features are not specific to the more recent 7 sensitive type of assays involving PCR, the parents, 8 and the PBRT. These features were also factors that 9 had to be considered in the more traditional types of 10 assays. 11 It is often quoted that the RT levels in 12 chick cells is very low, given that it was detected by 13 a very, very sensitive assay, and has not been 14 detected by the more traditional type of assays. 15 Certainly some preliminary data that I have got 16 suggests that it is not quite as low as we first 17 thought. Really this RT activity in chick cells and 18 ultimately in vaccines is only just below the level of 19 detection of the more traditional type of assay. In 20 fact, this was a relatively novel phenomenon 21 discovered just a few years ago. It was in fact first 22 reported 20 years ago in the late 1970s by Berne and 23 Hofschneider at the Max Planck Institute in Munich. 24 They reported the presence of a novel type of RT 25 enzyme in chick embryos and in chick cells. That was 42 1 in the days before PCR. 2 So the level -- I certainly believe the 3 level of RT and the level of these particles is 4 actually quite high in chick cell fluid. Ultimately 5 I think what we have to do is look into the need for 6 standards, standard materials in some way to assess on 7 a quantitative basis the level of RT activity in chick 8 cells, in measles vaccine, in mumps vaccine, in order 9 to come up with some meaningful conclusions regarding 10 it. 11 So to look at the RT issues from a 12 regulatory point of view, a couple of comments I would 13 make. First, that these are state of the art 14 technologies. When these highly sensitive assays 15 first came about, it posed very useful from a research 16 point of view to what use are they in a routine 17 manufacturing validated type of assay. 18 I think the time has come where yes, you 19 would say that these are state-of-the-art techniques 20 and can be and should be used for detecting the 21 presence of RT in your manufacturing process. However, 22 when it comes to, for example, chick cells, and until 23 we have a greater understanding of what the levels 24 might mean, and until standards are available, there 25 is really -- it is difficult to justify any 43 1 requirements to perform RT or PBRT assays on systems, 2 and basically here I am talking about chicks, which 3 inevitably will be positive. We know they are going 4 to be positive, that there's no great need to actually 5 require any manufacturer to do these assays. But 6 certainly there is a still a strong requirement to 7 provide evidence for freedom from retrovirus 8 contamination. This will have to derive from other 9 data. Thank you. 10 (Applause.) 11 CHAIRPERSON RABINOVICH: Thank you, Dr. 12 Robertson. 13 Any questions? 14 AUDIENCE MEMBER: Just a comment. For 15 known endogenous avian retroviruses or exogenous avian 16 retroviruses, of the cell lines, of the cells that you 17 tested for infectivity, only the turkey cells would 18 have given a positive result. I would urge for avian- 19 derived -- urge the use of those cells, and a PERT 20 assay is a sensitive readout, for detection of perhaps 21 unknown agents in these vaccines, end products, as 22 being the most sensitive, at least for avian-derived. 23 DR. ROBERTSON: Yes, yes. The turkey 24 cells are sensitive for rav, because it's virus, but 25 apparently not for the RTase. I have no idea, duck 44 1 cells are also negative, but I have no idea if duck 2 cells are susceptible to the -- 3 AUDIENCE MEMBER: They are not as good as 4 turkeys. Turkeys themselves actually are not 5 sensitive to all exogenous ALVs, but to most they are, 6 and all endogenous ones. 7 DR. ONIONS: David Onions. I really 8 enjoyed that, Jim. I just want to make a comment on 9 your comment about standards. I think as we heard 10 from Keith last night and what we're doing, and I'm 11 sure George is doing too, using the tac man technology 12 where you can actually quantitate the PCR product. 13 Then if you actually do EN counts of virus particles, 14 dilute these out, you can actually quantitate your 15 assay system and actually determine the number of 16 particles you can detect. 17 Now it seems to me that that is a useful 18 kind of standardization, and that you can then relate 19 that to if you like, a consistency of your starting 20 material, in this case the egg. 21 So I think in that case, applying those 22 techniques does have value, because it gives you a 23 kind of lock-to-lock consistency of your materials. 24 So that if something goes out of spec, then perhaps 25 something odd is going on in those materials. 45 1 DR. ROBERTSON: Whatever we want to 2 approach this standardization, one would have to do it 3 on a quantitative basis. I was sensitive earlier to 4 quote any actual figures, but to quote you some 5 figures that I have got so far, in one chick cell 6 preparation, the culture fluid, there was the 7 equivalent of the order, and this the first of 8 investigation, 10 to the 4 focus forming units of rav 9 in uninfected chick cell fluid. 10 A large current high level is when your 11 typical infection goes up to 10 to the 6th, I believe, 12 focus forming units. You are only talking about 104 13 drop, lower value. So you are not far away. It is 14 going to be difficult. If you have got an overt 15 contamination going up to 10 to the 6th, I think that 16 will be quite clear on a quantitative basis. But once 17 you drop down a bit, it is difficult to say whether 18 you have got an infection or whether it's just 19 background level of endogenous RT-derived activity 20 that you are picking up. 21 DR. MYERS: Martin Myers from National 22 Vaccine Program. As I sit and count the number of 23 immunizations that various populations receive with 24 these particles in it, repeated immunizations with it, 25 I wonder if there is any data on sero responsiveness 46 1 in longitudinal -- 2 DR. ROBERTSON: Well the reason I am 3 looking over your shoulder, we have Walid Heneine from 4 CDC. I'm not sure if you are going to say something 5 along those lines, but serologically, there is no real 6 evidence for reaction to ALVs. Epidemiologically, 7 when it first came out, we also, not ourselves but 8 epidemiological colleagues, to provide information. 9 There is no evidence again, for any increase in the 10 incidence of childhood cancers since the onset of 11 measles, mumps vaccination. 12 Walid, were you going to say something? 13 DR. HENEINE: Yes, I have. Regarding 14 transmission risks, so far the data we have where we 15 have looked at the presence of antibodies to avian 16 leukosis virus by western lot, I'd say we developed as 17 well as presence of ALV sequences and EAV sequences in 18 the peripheral blood lymphocyte from vaccinated kids 19 as well as in plasma. So far, the results have been 20 all negative for both viruses. 21 I have just one comment regarding 22 referring to these viruses in general as defective. 23 My comment is that the evidence we have so far on 24 those we have studied in a couple of vaccines between 25 in Europe and in the U.S. suggests that those, for 47 1 example, ALVs we're dealing with could be defective 2 because they come from loci that have deletions. 3 However, this may not be true for all the contaminants 4 from it we might find in other vaccines, because these 5 contaminants reflect the particles expressed from 6 these loci in the different cell substrates that Phil 7 mentioned in his talk, that the nature of these 8 particles and their phenotypes would vary depending on 9 the presence of the particular loci in these 10 substrates. 11 So just a comment, not to generalize that 12 we always should expect to have defective particles. 13 We might or we might not in certain cases. 14 DR. ROBERTSON: Yes. 15 DR. SCHUEPBACH: Joerg Schuepbach from 16 Zurich. I would also like to make a comment regarding 17 seroconversion. We have done two vaccination studies, 18 one with yellow fever vaccines, where about 120 19 individuals were tested actually for reactive 20 antibodies against HIV. The reason for this was that 21 in 1991, there have been reports that false positive 22 HIV reactions were found in people that have been 23 vaccinated against influenza. Influenza vaccine is 24 one of the vaccines which contains the EAV. 25 So we found that statistically highly 48 1 significant the vaccinees which have received the 2 vaccine which have the higher content, about 80 times 3 higher than the other vaccine which we used to use 4 too. That these patients actually had highly 5 significantly elevated antibodies to HIV 1 and 2, 6 although none of these actually became sero positive. 7 The serious bonds was highest in those individuals 8 that also had a history of measles vaccine. 9 In the second study, this was an influenza 10 vaccine where we tested two different brands, a split 11 vaccine and a rather crude vaccine. We also had a 12 response to HIV bond two in the third generation tests 13 in the vaccine which contained more in the crude 14 vaccine, which contained more of the EAV protein, and 15 again those individuals who had a history of yellow 16 fever vaccination had the highest type, the highest 17 increases. 18 We also tested yellow fever vaccinees by 19 PCR, RNA PCR and DNA PCR for EAV-0 sequences. We 20 found one out of 180 individuals in which both these 21 tests were positive as plasma on PBNC. At the moment, 22 we cannot exclude that this was the result of a 23 contamination, but we are working on that. So I think 24 that the matter is actually not as clear as has been 25 presented by others. 49 1 MS. SHEETS: Hello. I'm Becky Sheets from 2 FDA. How would you recommend that avian-derived 3 products be tested for retroviruses? EM is not very 4 sensitive. The conventional test is often inhibited 5 by the allantoic fluid, and therefore, is not 6 necessarily a valid test. How would you recommend, if 7 you don't use a PCR-based RT des? 8 DR. ROBERTSON: At the end of the day, it 9 would have to be an infectivity assay. There are also 10 some antigen, ELISAs for the viral antigen. I don't 11 have experience with those. I don't know the 12 sensitivity of them. Ultimately you are looking at an 13 infectivity assay which can be performed even on chick 14 cells, which are positive for RTase. One could assess 15 for after several passages on chick cells, looking at 16 an increase in RT activity or increase in antigen. 17 MS. SHEETS: When you said infectivity 18 test, were you talking about those specific for ALV or 19 were you talking about general tests to detect any 20 kind of retrovirus? 21 DR. ROBERTSON: Well, it would have to be 22 an avian retrovirus if one is performing the assay on 23 chick cells. Propagating the material, the test 24 material in chick cells but using either RTase in 25 general or an ELISA specific for ALV test for 50 1 increased presence of either RT or for the presence of 2 ALV antigen. 3 CHAIRPERSON RABINOVICH: Dr. Coffin, the 4 last question? 5 DR. COFFIN: Yes. I'd like to actually 6 address Dr. Schuepbach's comment. Did I understand 7 that you were basing your sero assays on the rationale 8 that there might be cross reactivity between ALV and 9 retro viruses in HIV? There is no rational basis for 10 that. There's virtually no amino acid the same 11 between those two viruses, except for some extremely 12 highly -- you know, three or four in pol and some 13 other places. Did you actually assay directly for 14 seroreactivity against ALV? It would have been a much 15 more straight-forward experiment. 16 DR. SCHUEPBACH: We agree that there is no 17 sequence homology on the nucleic acid and on the 18 protein label, but these are the results which we 19 found. We have to find an explanation for them. We 20 don't have at the moment. 21 CHAIRPERSON RABINOVICH: Thank you, Dr. 22 Robertson. 23 Our next speaker, Dr. Jorg Schuepbach, 24 from the Swiss National Center for Retrovirology. 25 Induction/activation and detection of occult viral 51 1 agents that are present in mammalian tissues. 2 DR. SCHUEPBACH: May I have the first 3 slide, please? Okay, from previous remarks, I heard 4 that I was expected to talk about these avian 5 retroviruses as well, but actually I was asked to talk 6 about the induction activation of occult viral agents. 7 So I will just have a few remarks on this other stuff. 8 So occult viral agents are agents you 9 don't detect or at least do not easily detect. They 10 may include two groups: a group that includes known 11 agents which are present at two low concentrations for 12 easy detection. The reason for these may be latency; 13 The other group consists of unknown agents. Since we 14 do not have good detection methods for these, they may 15 be present at low or also at higher concentration. 16 Viruses known for their latency or various 17 types of the herpes virus, true, they are latent in 18 various types of non-permissive cells such as neurons, 19 B cells, monocytes, PBLs, and others. They are 20 activated from these latent stages by various kinds of 21 stimulation of their host cells by differentiation, 22 agents by the differentiation of precursor cells, to 23 more mature cells. Again, by other activating agents. 24 Other viruses could be considered in 25 addition to the herpes viruses include the adeno 52 1 viruses, the adeno-associated virus and the pathyloma 2 and polyoma viruses of which we heard yesterday, and 3 will hear more in a subsequent talk. 4 Regarding the RNA viruses, I might discuss 5 the measle viruses and of course the retroviruses. 6 When we look at the mechanisms by which we 7 can activate these various viruses, it is mostly by 8 activation of their host cells, by cell stimulation, 9 by induction of cell differentiation of these cells, 10 and then by co-cultivation with cells which are 11 permissive for replication. 12 Now since we have different viruses and 13 host cells systems, these methods vary greatly among 14 the different viruses. If you have unknown viruses, 15 you really don't know what to do. 16 So the effect of such activation would be 17 that from a lonely latently infected cell, by 18 inducement of replication, a virus would spread 19 throughout the culture, resulting in virus gene 20 amplification in production of viral proteins. So 21 this would make of course the detection easy. You 22 might also have some pathogenicity which is easy to 23 detect. 24 However, our goal is actually not 25 detecting any possible virus that might be present. 53 1 The principal goal is to provide a virus production 2 system which is free of such agents. It is suggested 3 here the easiest way of achieving this is actually 4 cellular cloning. Because if you have an agent that 5 is present in only a minority of the cells, the 6 chances that you derive a clone that is free of these 7 agents is very high. 8 If by chance you hit an infected cell, the 9 descendants of that cell will all carry along the 10 virus and of course then we come into a situation 11 which makes detection of unknown viruses and also 12 known viruses much easier because either all of the 13 cells will be infected or none at all. 14 So cellular cloning, if we hit an infected 15 cell, has actually a viral gene amplification effect 16 which is comparable to virus induction activation if 17 it's successful. Most importantly, it is a procedure 18 that works for all the latent viruses except 19 endogenous retroviruses, but these are present anyway 20 in all of the cells. 21 So going on to the detection methods for 22 these agents, let's first talk about known viruses. 23 Since all the cells will be infected, we actually do 24 not need the most sensitive procedures. We do not 25 need procedures that detect the single viral copy. 54 1 What we need is broadly reactive methods which go 2 detect all the different members of a certain virus 3 group. 4 So I think techniques, old-fashioned 5 techniques like hybridization techniques on the low 6 stringency or if we want to use PCR or nucleic acid 7 based methods, we should take care that we take a lot 8 of different probes, use data generated primus, 9 multiplex PCR and so on. 10 Of course in addition, we should also do 11 the classical methods, doing cell activation and co- 12 cultivation as permissive cells, the routine detection 13 methods of broadly reactive antibodies which detect 14 all the different members. 15 For those who think that what I have told 16 so far is rubbish, and that we actually do need very 17 sensitive methods, I offer the mega PCR, which has 18 also been named catcher PCR by others. The purpose of 19 this method is to take very rare sequences among a 20 very high background of DNA or RNA. So here we 21 convert the samples of up to 500, maybe even 1 22 milligram of DNA or respectively RNA. 23 The principle is very simple. We use 24 biotinylated capture probes which bind to these 25 sequences inquest. We isolate these complexes on 55 1 coded beads, wash the rest of the DNA away, and them 2 amplify these by PCR with primus which are located 3 outside of this capture probe. 4 The advantage of this is that we 5 absolutely do have no carry over because the amplicons 6 are selected against when we do the capturing. It is 7 this type of test which I would actually like to have 8 been seen when testing in the question of 9 xenotransplantation where the PERV sequences can be 10 found in humans which have received pork material. I 11 think this will be the test, to test these questions. 12 Now using this method, it's actually very 13 sensitive. You can detect a single copy here of HIV 14 DNA. We still have double positive signal, is about 15 one copy. This serial dilution was done in the proper 16 range here. The fact that in these two, three last 17 dilutions only one of the two duplicates was positive 18 clearly demonstrates that we are in a Poisson 19 distribution. So we can detect the single copy with 20 this method in 100 microgram. 21 DNA, we have actually demonstrated that 22 there's 95 percent probability we can detect three 23 double standard HIV copies in 100 micrograms of DNA. 24 So now going on to the exclusion of 25 unknown viruses, and I will talk about retroviruses 56 1 later, we can actually use the same procedures as I 2 have already described previously. We just have to 3 take care that we really have broadly reactive 4 methods. This is true for molecular based tests as 5 well as for the more classical procedures. 6 Now coming to retrovirus detection, of 7 course also of cell cloning, here we have two 8 situations, the exogenous retrovirus may not be 9 present in none of the cells or in all of the cells. 10 The endogenous retroviruses were always present in all 11 of the cells. The known exogenous retroviruses are 12 detectible by tests for conserved sequences. Of 13 course you might also use universal pool primers for 14 unknown retroviruses -- because of the endogenous 15 retroviruses. Not all of which, or very few of which 16 are actually harmful. 17 So I think it is better at this time to 18 switch from the analysis of cells to the analysis of 19 particles. This is best done by the PERT assay which 20 has been mentioned before by several speakers. 21 Now when we devised this test in 1992, we 22 devised it as an anti-family of related tests which 23 would have in common that reversed inscriptase present 24 in a sample would be used to create from a template 25 primer combination and nucleic acid that is to be 57 1 unamplified. 2 Now in most instances, this will simply be 3 the cDNA. There are other possibilities as well. You 4 can take any nucleic amplification procedure, not just 5 PCR. You may also use ligase chain reaction or NASPA 6 or you can make use of auto replicated DNAs or RNAs in 7 order to generate amplification product, which can 8 then be assayed by different methods. 9 So since we have provided for all these 10 different methods already in 1992, we do not think 11 that it is necessary to invent new names for these 12 current assays. 13 Now this test is actually very sensitive. 14 This experiment in comparison to classical RT assay. 15 It occurred as six to seven orders of magnitude more 16 sensitive, and in a direct comparison with -- in the 17 case of HIV, where we compared the method with RT PCR, 18 detecting one copy of cDNA, we had the same dilution 19 endpoints for two different samples. 20 Actually as others, we can detect only a 21 few particles in the case of HIV. We believe that in 22 some cases we can detect even less than one particle. 23 Now this is one of the theories taken from 24 the Joerg Koenig paper in 1996, where we demonstrated 25 that the measles vaccines, the mumps vaccines, the 58 1 yellow fever vaccines, and the MMR vaccines all 2 contain activity which is about three orders of 3 magnitude higher than the background here on other 4 vaccines, and were negative. 5 Now in order to identify the viruses 6 behind these activities, we along with the PERT assay, 7 developed the method for the identification of unknown 8 retroviruses. It is based on three properties of old 9 retroviruses, namely, that they all are 10 polyadenylated, that R sequences are repeated at both 11 ends, and that cDNA synthesis has started here at the 12 primer, binding site, and that for primers, tRNAase 13 are used and the use of such tRNAase is actually very 14 much restricted among the various retroviruses. For 15 example, is just four PRNA primer equivalence. You 16 can start cDNA synthesis for all exogenous 17 retroviruses known today. 18 So what we do is that we bind the 19 retroviral RNA to poly t coated beads. Then we start 20 here, the synthesis of the cDNA with one of the 21 various t RNA primers, synthesizing the strongest of 22 DNA. Then adding a tail here, and then with anchored 23 TCR, we can amplify this sequence and submit the 24 sequencing directly. 25 Actually this method has also been used by 59 1 the group of Dr. Loewer at the Paul Ehrlich Institute, 2 and even published before us. But we have somehow 3 optimized this procedure, so in general we need less 4 than one-thousand RNA sequences, sometimes as few as 5 20 or 40, 50, in order to generate this sequence here. 6 As soon as you have it, you actually know whether you 7 are dealing with a retrovirus or not. 8 When you deal with a retrovirus, you have 9 to R sequence and then you can check with the other 10 anchored PCR. Where there is R here, it's repeated at 11 the three prime end. If it is, you can then amplify 12 the entire genome with a little bit of luck by long 13 PCR. 14 So this is what we use to identify this 15 EIV-O sequence. We have also done some other work. 16 For example, we investigated the NIH 323 cell line. 17 This was negative by convention RT tests, but positive 18 by PERT assay. We had a nice band in sucrose, and 19 then radiant. Using this procedure which we call 20 parar, we identified 23 different products, 15 of 21 these were actually retroviral sequences from four 22 different groups. Three of them were unknown 23 sequences, at least at that time. So far we have not 24 further characterized these sequences, but this is 25 still awaiting. 60 1 Now staying with retroviruses, as Dr. 2 Coffin pointed out yesterday, sometimes if you have a 3 cell line here, you are dealing with melanoma cell 4 lines which were found to be highly percipated by PERT 5 assay. We analyzed what was in there. It turns out 6 to be endogenous murine leukemia virus, and later we 7 were told that these cell lines have actually been 8 passaged in mice. 9 If you have low titres of activity, then 10 that becomes a little bit more complicated. This is 11 the analysis of primary samples from a patient with MC 12 cor cultures. No actually not cor cultures, just cor 13 cultures which were found lowly positive in the PERT 14 assay with activity in the order of two, maybe three 15 times above background. 16 Here the patterns is a little bit more 17 complicated. You have here a small peak that might 18 correspond in density to ritualized particles. This 19 one might correspond to cor particles. You have 20 another identified -- unidentified peak here. It will 21 certainly be a challenge to find out what this stuff 22 is. 23 Next, please. This is another example of 24 a primary culture where we have a very short peak at 25 the higher density. This might be for particles, 61 1 could be a different retrovirus, a different virus, or 2 just a subcellular particles containing some cell or 3 enzymes. 4 Now you will say that this test of course 5 detects only retroviruses that are released. We are 6 also worried about retroviruses that are inside the 7 cells, so stimulation may be necessary. Actually I 8 think one important question is or one possibility is 9 that actually the vaccine virus we would like to 10 produce in such a cell might activate latent 11 proviruses. So I think it is important that we 12 actually do not just test the virus production systems 13 while uninfected, but also when this seed virus has 14 been added, and then we harvest the virus. 15 Now in some cases, as in the measles virus 16 or so, this has proven very easy. We had quite a good 17 specificity. But in other cases, it might be more 18 difficult as indicated in this example, where we 19 tested a vaccine, experimental vaccinia, recombinant 20 vaccinia virus vaccine against melanoma. This was 21 found highly positive by PERT. It had actually been 22 produced by just the lysing, the infected cells by 23 ultrasonication. 24 What we now find is here in black, is the 25 vaccinia virus DNA two peaks. We have here a major 62 1 peak of RT activity which does not coincide with the 2 vaccinia virus peaks, and also is not characteristic 3 of retroviruses. So I think in this case, we can rule 4 out the presence of a retrovirus. 5 Now it may also be interesting to find out 6 whether upon induction, viruses might come out. So 7 this would add an increase of safety to the vaccine. 8 As retroviruses are regulated, you have the promoter 9 in anti sequences in the upstream LTR in the U3 10 region. Depending on the cell type, activation state 11 of the cell and the differentiation, you have various 12 sets of transcription favors interacting with this 13 enhancer regions. 14 In addition to this balance of positive 15 and negative transcription factors, you may have 16 positional effects as the chromatin structure or the 17 DNA methylation. You may now try to influence this 18 balance by tipping it by either inducing mitosis cell 19 differentiation by substances that lock inhibitors or 20 by alleviating the negative positional effects, again 21 by inducing mitosis or by inducing DNA de methylation. 22 The number of induces have been described 23 in the past. The most important ones are listed here 24 at the top, allogenated pyrimidins, the azacytidine, 25 which only both of them working only in infected 63 1 cells. I will not mention the others because of the 2 lack of time. 3 Now it depends a lot on the virus whether 4 azacytidine or the deoxy pyrimidine is preferable. 5 For example, in experiment in cell line where two 6 types of different retroviruses are produced, several 7 type A particles here. The azacytidine is certainly 8 better. But in C-type particles, these cells produced 9 IdUdr. Yes, the IdUdr is better. So you might have 10 to use a combination of these two drugs. 11 So in conclusion, I think induction 12 activation certainly serves to amplify latent viruses 13 for which improved detection. I think it is more 14 important that we early in the process of selecting 15 virus production systems be cloned B cells, and sub- 16 cloned, because this will amplify, because this really 17 facilitates detection very much. 18 In consequence of this, we do not -- I 19 think this is very important. We do not need the most 20 sensitive procedures. What we need is broadly 21 reactive procedures which will detect all the 22 different agents. 23 I also think that at the end, the only 24 important thing actually when dealing with 25 adventitious agents, not just with DNA, which might be 64 1 infectious, is that the vaccine is free of these 2 contaminant viruses and for retroviruses I believe 3 that this can be verified by the PERV assay. Thank 4 you. 5 (Applause.) 6 CHAIRPERSON RABINOVICH: We'll take just 7 a couple of questions because I would like to leave 8 the rest for the panel discussion. 9 DR. COFFIN: John Coffin. I would agree 10 that if you get preparations of vaccines that are 11 negative by all these assays, you can have a pretty 12 good level of confidence that they are not 13 contaminated with retro viruses. The problem is, if 14 you do these enough, it may well be that no vaccine 15 will pass these tests. 16 What I think is very important to add to 17 this would be one more level to your last slide. That 18 is an infectivity step. As in the example we saw 19 before when one perhaps collects a panel of cells or 20 cell lines which are pert negative, and there seems to 21 be reasonable numbers of those, and then test the 22 vaccine, the induced stuff and everything else by 23 infectivity and induction of pert activity on those 24 cells. 25 I think that would be a much more useful 65 1 and reliable test for the presence of viruses that 2 might be problematic than simply looking at the pert 3 activity in preparations with cell soups. 4 DR. SCHUEPBACH: Yes, I agree with you. 5 I actually thought that was included in those 6 conventional methods which I have listed for the known 7 viruses. Of course you should also do some studies 8 for retro viruses. 9 AUDIENCE MEMBER: You mentioned results 10 associated with particles from supernatants of primary 11 human materials. Did you try to find retro virus-like 12 sequences in these particles for para assay? 13 DR. SCHUEPBACH: Yes. These are very 14 recent results. We are in the process of doing that. 15 DR. KRAUSE: Phil Krause, FDA. One of the 16 issues in testing vaccine products is obviously what 17 tests are available and have been validated and that 18 we understand the sensitivity of. So I guess in the 19 context of thinking of highly conserved sequences to 20 which we might develop primers that could detect a 21 broad array of viruses, including some unknown related 22 viruses, what can you say about the current state of 23 the art? How good is that? How well has that been 24 validated? Is that something which if we decided 25 tomorrow we wanted to apply that to new vaccines 66 1 producing neoplastic cells, we could simply say "let's 2 do it" or is more work required? 3 DR. SCHUEPBACH: I'm actually not very 4 familiar with other viruses than retro viruses. But 5 I think these things, however they exist, should 6 clearly be developed. 7 CHAIRPERSON RABINOVICH: Thank you. We 8 will go onto our next speaker, Dr. Jens Mayer, from 9 the University of Pennsylvania. The status of HERV in 10 human cells. 11 DR. MAYER: Okay. My talk will deal with 12 -- can I have the first slide, please? Okay. My talk 13 will tell you something, I hope, about the status of 14 these human endogenous retrovirus regarding the coding 15 capacity and the expressions. Just again, it was 16 mentioned before already what is actually an 17 endogenous retro virus. HERV is created by the germs 18 of infection of an exogenous retro virus. This leads 19 to radical inheritance of this newly created virus 20 following generations. In the course of the 21 evolution, it will be also inherited to newly arising 22 species. 23 The human genome, like all mammal genomes, 24 and also some invertebrates, invertebrate genomes 25 where it has been shown, contains several families of 67 1 elements and so on. It has been estimated that about 2 one percent of the human genome of such retro origin. 3 These elements antiquated already several million 4 years ago through the genomes of human predecessor 5 species. Some present for at least 30 million years. 6 Some have been shown to be present for at least 40 7 million years. We have several indications of 8 different various families. So they were independent 9 of several exogenous retro viruses. Some of these 10 elements that are now present in the human genome 11 existed. Single copy, and some have copies, copy 12 numbers up to 1,000, per haploid genome. 13 But as I said, most of these sequences 14 were already present for a long time. Therefore were 15 targets for mutations. Most of these families then 16 became coding deficient or they do no longer encode 17 for retro R proteins. However, even if they are 18 coding deficient, many of these families are still 19 transcribed in several human tissues. Some have been 20 discolored just by virtue of their expression. 21 It also seems that the expression of these 22 sequences is regulated in certain tissues and tumors, 23 so we heard that there might be an deregulation of 24 families. It seems possible that that deregulation 25 mechanism is not present in certain tumor tissues. 68 1 Just a word regarding the nomenclature of 2 these sequences. The tRNA that was originally used in 3 the priming of the transcription process, the life 4 cycle of the exogenous vaporized, and according to the 5 amino acid and tRNA codes for, and this single code 6 for the amino acid stands dependent. This is just one 7 possible nomenclature of perts. It's still very 8 confusing. 9 I said that most retro viruses are coding, 10 HERVs are coding deficient. However, there are some 11 good described examples, especially some new examples 12 of coding in tact HERV sequences. At least there are 13 some in tact genes. We have already known for a long 14 time the so-called ERV-3 sequence that belongs to the 15 R-family. This, we agreed, pro-virus, or pro-virus 16 sequence encodes, and 1.9 KBN open reading frame. 17 That open reading frame is highly regulated to the 18 transformation of trophoplasts into sensitio- 19 trophoplasts in the placenta. So we have here clearly 20 an up-regulation during a developmental stage. 21 We have for instance, you have H-family 22 and we have about 1,000 copies of that H-family. 23 Among them are 100 copies that are still in tact 24 regarding the pro-virus structures. They have an LTR 25 gag pol env, LTR structure remaining 900 lack N gene. 69 1 There has also been reported that this HERV-H families 2 are expressed in various cell lines. We see the 3 highest expression for these elements has been 4 reported in cell lines that are derived from germ cell 5 tumors, and germ cell tumors I guess you will hear 6 some more about germ cell tumors later on. 7 Just this year, Lindeskog, Mark Lindeskog 8 reported the isolation of an intact HERV-H env gene. 9 So it is now clear that there is within the human 10 genome one intact HERV-H evn gene. It's not know so 11 far whether there are any among these many sequences, 12 whether there are any intact gag of pol sequences. 13 I would like to mention the new discovered 14 HERV-W family that has originally been reported, has 15 been isolated from retro virus by particles from 16 multiple sclerosis patients. It has also been 17 reported that these HERV-W sequences are up-regulated 18 in the placenta. Joni Blanc also reported this year 19 the isolation of an intact HERV-W in the genes. It is 20 also not known whether there are intact gag pol genes. 21 I would like to in the second part of my 22 talk, report about results for our family of clearly 23 outlines from our other HERV families in the coding 24 capacity. This is the so-called HERV-K HML-2 family. 25 This is quite complicated. 70 1 The human genome contains several families 2 that use lycine primer binding site or TRNA for primer 3 binding. They were named human MMTV-like sequences, 4 one through six. The family that we are talking about 5 is reported in more detail by Ono and co-workers and 6 the original sequence was the so-called HERV-K 10 7 sequence, which is by the new nomenclature is the 8 HERV-K HML-2 sequence. 9 We have reached about 25 to 50 copies of 10 that HERV family is present in old world monkeys, but 11 not in new world monkeys. One concludes that family 12 is present for at least 30 million years in the 13 genomes. 14 In the past, there have been reports of 15 isolation of the isolations of intact HERV HML-2 16 sequences. So there were reports about intact gag 17 sequence and intact protease sequence has been 18 reported, that is able to process that HERV-K gag 19 protein, intact pol sequences with RT activity, with 20 endonuclease activity, and have been reported and also 21 intact MRNA has been reported from the group from 22 Johannes Loewer. And also what we heard yesterday 23 evening, there is also an additional splicing product 24 from the N gene, the so-called C-ORF that still has a 25 rav-like function. 71 1 What is known already for a longer time is 2 that these particles or the cell lines are derived 3 from germs of tumors or typically testicular tumors of 4 the young man. These cell lines do produce with rav 5 particles. Boller and coworkers could show that these 6 particles are encoded by the HML-2 gag protein, 7 labeled antibodies, and recognized that gag protein. 8 If we look at patients suffering from germ 9 cell tumors, we also have some surprising results 10 regarding that HML-2 sequences. Namely, if we look at 11 the antibody status of these patients compared to 12 controls or other non-germ cell tumor types, we see 13 that mixed germ cell tumors and here especially, 14 seminomas, these patients have very high antibodies 15 directed against HERV-K gag and HERV-K N proteins. 16 These tumors or these antibody titres are already very 17 high if the tumor is clinically detected. From other 18 results, we also know that the precursors of these 19 tumors, the so-called carcinoma in situ, also 20 expresses already on the RNA level these HERV-K HML-2 21 sequences. 22 We were interested to see or to find out 23 where in the genome are these impact genes located 24 that are responsible or that cause finally the 25 production of these gag and env antibodies. As I 72 1 said, it has previously already been reported that 2 they are intact genes, but it was not possible because 3 of the high copy number of these sequences to isolate 4 or to at least chromosomally assign these intact 5 genes. We, therefore, tried to chromosomally assign 6 these intact sequences using a combination of the so- 7 called protein truncation test and using a 8 monochromosomal hybrid panels, or panel of human 9 rodent fusions, fusion cells. 10 We were able to show that there are at 11 least, still at least eight intact gag genes within 12 the human genome, and at least three intact env genes. 13 We did not publish that. There are also several 14 intact pol genes within the human genome. 15 I just want to show you how we got these 16 numbers. This is the protein truncation test that has 17 been described by Roest and coworkers in 1993. So it 18 was originally developed for the detection of APC gene 19 carriers that carry it, the APC gene. So the APC 20 lesion is characterized by trends or not completely 21 translated APC proteins. It is almost like the 3 22 prime terminus. 23 So we have three possibilities. One is 24 that both are intact, both genes are intact. The 25 carrier will carry one defective APC gene. The 73 1 defective person would carry the two defective genes. 2 The principle of the test is that the coding sequence 3 is PCI amplified, where the protochomo contains the T- 4 7 promoter and the translation initiation sequence. 5 So if this PCI product is then in vitro transcribed 6 and translated and impressed and radiolabeled amino 7 acid, electrophoresed, and then auto radiographed, you 8 will see according to the status of these donors that 9 you will have only full-length proteins, the carryover 10 also show an additional shortened protein and 11 defective people will only produce defective proteins. 12 We in principle used the same test because 13 we in principle have the same situation. We have some 14 defective gag genes within the genome. There must be 15 at least one gag or env gene because we have the 16 antibodies. So we put -- in principle used the same 17 test. 18 What we did was we are looking for the 19 presence of full-length gag genes or env genes on the 20 human chromosomes and then tested the PCI product we 21 got from the chromosomes for their coding capacity. 22 This is the result for the gag coding capacity. So 23 gag protein would result in a protein of about 73 24 kilodaltons. So these are controls that give the 25 respective proteins. 74 1 You see that there are several human 2 chromosomes that contain or produce a full-length 3 protein. There are eight human chromosomes that 4 contain at least one gag gene that contains four full- 5 length proteins. 6 I also would like to mention that we are 7 also able to demonstrate the defective gag genes if we 8 see here, these proteins that are just smaller than 9 expected. These are very likely the gag genes that 10 are defective. Stop codons within the coding 11 sequence. 12 We did the same for the HERV-K env genes. 13 We see here that three chromosomes produce a protein 14 of about 76 kilodaltons. These are the chromosomes 7, 15 19 in here on the chromosome. 16 What we also see in the gag experiments is 17 that there are additional env genes that are only on 18 the almost intact. We have here a protein that is 19 about four kilodaltons more. So this actually could 20 also be considered as an intact reading frame. 21 So we have several human chromosomes that 22 still contain gag and env genes. We have three 23 chromosomes that contain both intact gag and env 24 genes, the chromosome 7, 19, and the Y chromosome. We 25 were interested whether these chromosomes or the 75 1 intact genes on these chromosomes are derived or 2 located within one provirus or within several or 3 different positions within the particular chromosome. 4 I would like to report or tell you 5 something about what we found out for the chromosome 6 7. We were using for addressing that question, we 7 were using a chromosome-specific, chromosome-7 8 specific cosmid library. We were screening for clones 9 that contained both gag and env sequences. 10 What we finally found out, that we 11 isolated the so far least defective human endonuclease 12 on chromosome 7. We were able to characterize the 13 proviral sequence within one cosmid clone that still 14 has intact LTRs. So they regulate to the elements. 15 They are able to transcribe, as you will see. We have 16 an intact gag gene. We have an intact protease gene 17 that protease is able to cut itself from a gag 18 protease, polymer precursor protein, and is 19 furthermore able to process encoded gag proteins. So 20 it's typical retro-ized protease. 21 We know just from sequence comparison, one 22 can deduce that the endonuclease within the polymer 23 genes also acted just by sequence comparison, no 24 significant changes compared to recently described 25 active K in the nuclease. We have an intact env gene. 76 1 This intact env gene sequence has already been 2 described by Johannes Loewer's group as an MRA, which 3 also shows that this sequence is actively transcribed. 4 So this is actually an expressed provirus. 5 We have spliced on the inceptors sides the 6 corresponding position that would allow to splice an 7 M on A, and what we heard yesterday also, to splice an 8 additional soft M RNA. 9 What we see is that this proviral sequence 10 is only defective in the RT domain. It has a single- 11 based permutation within the YXDT motif. So very 12 likely, this highly important catalytic motif is -- so 13 only in reverse transcription function this probably 14 missing from that proviral. 15 Okay. We have here almost intact proviral 16 sequence. But now regarding infectivity, we had that 17 already several times I guess before. We have HERV- 18 encoded retro of particles, several cell lines, even 19 in tissues, the placenta tissue for instance. We find 20 HERV-RNA in these particles. 21 We have no infectivity so far shown for 22 any of these HERV sequences. We do not really know 23 why. There are several reasons that can be mentioned 24 for the HML-2 family. It has to be reported that the 25 env protein cannot be cleaved into the auto membrane 77 1 transmembrane domains. It is conceivable that they 2 are defective genomes that are packaged into these 3 particles, so only if they would be able to get a new 4 cell, they would only deliver defective genomes. 5 It is also not clear whether the 6 receptors, that they were once used by that, retro 7 families are still present and would still be used. 8 So what you should take home I guess is 9 that human endogenous are expressed in several tissues 10 tumor types that are highly up-regulated in certain 11 tumor types. Several HERV families are still able to 12 encode proteins, and among them, the HML-2 family that 13 still encodes all essential proteins. We have almost 14 intact HML-2 provirus within the human genome. Thank 15 you. 16 (Applause.) 17 CHAIRPERSON RABINOVICH: I think we will 18 hold questions at this point. We are going to take a 19 10-minute break now. We are going to come back and 20 finish with the last two speakers. 21 I need to figure out how to catch up time, 22 and yet leave the time for the panel discussions. I 23 ask you to do two things. Check-out time from the 24 hotel is 12:00. You should know that. They have 25 already called in a bunch of the taxis so that if you 78 1 need taxi arrangement, please let them know so they 2 can do that for you. Ten minutes we will start again. 3 (Whereupon, the foregoing matter went off 4 the record at 9:55 a.m. and went back on 5 the record at 10:10 a.m.) 6 CHAIRPERSON RABINOVICH: Is Dr. Broker 7 here? Great. 8 If you could take a seat please. The next 9 speaker is Dr. Thomas Broker from the University of 10 Alabama at Birmingham speaking on viral latency- 11 papilloma virus model. 12 DR. BROKER: Thank you very much. I would 13 like to deal with two subjects under this topic. The 14 first is a study of the prevalence of HPV in the 15 general population, and then following on Dr. Mayer's 16 pattern that you just heard, a study of some 17 endogenous sequences in papilloma virus transformed 18 cell lines with some surprising results. 19 We have done some inside 2 hybridization 20 studies of the expression of human papilloma viruses 21 in biopsies from women with HIV/AIDS who were 22 moderately immuno deficient. This is one example, but 23 fairly typical. 24 What you are seeing is a full thickness of 25 across the cervix. The various probes that we used 79 1 reveal the expression of one of the major early 2 transcripts of papilloma virus, the E4, E5. You are 3 seeing it here in bright field illumination and dark 4 field, matched pairs, basal layers right there. 5 As I indicated yesterday evening, 6 papilloma transcription is differentiation dependent 7 and occurs typically in the upper half of the skin. 8 E6, E7 messages, the delayed early oncogenes are hard 9 to see in bright field, but fairly easy to see in dark 10 field. You can see they follow a comparable 11 distribution. 12 The capsid component, L1 or L2, again, is 13 right at the very top of the last live layers of the 14 epithelium. Also to the point, the vegetative 15 amplification of viral DNA is in the upper half of the 16 epithelium. 17 Papilloma infections of the genital tract 18 in fact have been designated an official AIDS-defining 19 illness in the syndrome because of the significant 20 upregulation of HPV gene expression in women who have 21 AIDS or other immuno deficiencies. With that 22 knowledge in hand, and pictures like this, we 23 undertook the following study. 24 We decided to investigate the prevalence 25 of HPB in the population by focusing on immuno- 80 1 deficient groups. The three that we have chosen so 2 far are: women who are in enstay renal failure and in 3 need of a kidney, and most clearly ill; those then who 4 get a kidney and are pharmacologically immuno- 5 suppressed beyond their underlying illness; and those 6 with AIDS. 7 The strategy that we're using is an 8 extension of the techniques Steve Wolinsky and I 9 developed really 12 or 13 years ago when we first 10 proposed the use of degenerate primers for looking at 11 related genomes. The pair that's most commonly used 12 in the papilloma field is our original design called 13 MY911, but Louise Chao and I moved right next door. 14 We found that this region is a little too long to use 15 in form one fixed tissues, and this particular pair 16 has some wonderful restriction fragment polymorphisms 17 available that will allow us to do genotyping after 18 amplification. 19 So basically we start with the nested PCR 20 approach, outer primers and inner primers. The 21 starting material is cervico vaginal lavage, which 22 harvests cells from throughout the lower genital tract 23 of the women. We amplify and then we put it through 24 several different assays. Initially, agarose gel 25 electrophoresis to look for a 278-base amplimer. 81 1 Secondly, restriction fragment 2 polymorphisms which usually can tell us which genotype 3 is present. But if it's a pattern that we cannot 4 recognize, we will put it through sequencing. As you 5 are going to see, about half of the fragments that we 6 amplify we need to sequence. 7 The results of this study, I am going to 8 summarize. It's absolutely mind-boggling. Seventy 9 four percent of all women in the AIDS cohort have 10 clearly identifiable HPVs. We have managed to type 11 over 85 percent of these so far. Fifteen percent are 12 still under investigation. In more than half the 13 cases, the individuals yield multiple HPV types. 14 The study is longitudinal, and has been 15 going on for three-and-a-half years now. Many of the 16 members of the cohort have been sampled two up until 17 seven different times at six to 12 month intervals. 18 So that's our biggest cohort. 19 These are the renal transplant cohort. We 20 have statistically significant numbers. I would like 21 to point out that in instage renal failure, but no 22 pharmacologic suppression, about 59 percent of those 23 women have detectible HPV. Again, quite a few, a high 24 percentage have multiple infections. This carries 25 over to that portion of this group who go onto actual