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http://www.fda.gov/cber/minutes/0907evolv.txt 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.
PRESENT:
Regina Rabinovich, M.D. Session
Chair
I N D E X
P-R-O-C-E-E-D-I-N-G-S 8:04 A.M. DR. MINOR: Thanks. Thanks very much. Can I have the slide on, please, or do I just press it here? What I am going to do is to review firstly all of biologicals, if you like, from an adventitious agent point of view. So it won't just be vaccines. In particular, I will be talking about the range of source materials that people have used in preparing biologicals. There will be a clear message that comes out of that, which is that the more you use well-characterized cells, the better. I will also be talking about the SV40 story in some detail, which has been gone through a number of times, but I will be going through it from a particularly regulatory point of view because again, there is a message there which says that if you get it wrong, you will still be working on it 40 years later. Finally, I will get onto the continuous cell line bit right to the very end. So there are a variety of source materials that you can use if you are preparing biologicals. They are sort of listed here, if you like. There are biological materials which are made from whole animals. That would include things like blood and blood products. I will describe that in a moment. So you can just go to a whole animal and take something out and make your biological from that. You can use your whole animal as a substrate for growth. I will discuss that in the context of things like influenza vaccines and the like. You can grow material on primary cells. This was the main starting point for things like polio vaccines in the early days, where the SV40 issue arose. Finally, you can grow materials on well- characterized cell preparation. The further down the list you go on this thing, probably the happier you are from the adventitious agent point of view. This shows some examples of contaminants which have arisen when whole animals have been used as source materials or the origin of the source material. Most of these will be human rather than anything else, but really an awful lot of the serious adventitious agent problems that have arisen have arisen because of materials sourced from whole animals or using pooled preparation. The first one on this list here is CJD,Creutzfeldt Jakob Disease, which was transmitted by growth hormone. The growth hormone was produced from human cadaveric material. A very unpleasant disease. It's almost impossible to detect the agent other than by standing back and waiting for the incubation period to go. In France, there are still a large number of cases coming through as a result of this. It may well be that around 10 percent are recipients of human-derived growth hormone, will actually wind up going down with CJD in France. Dura mater is another one. That should be one T, not two Ts. Again, this has been shown to transmit really quite readily when pooled materials are actually used. Almost impossible to detect. Clear, very, very serious kind of consequence of it. Scrapie was first shown to be a transmissible agent by the use of a TBE vaccine, which was grown in the brains of sheep. TBE being tick-borne encephalitis, which then transmitted scrapie to a large number of the sheep that were actually inoculated with it. So again, this is a whole animal source material, if you like, that had quite serious consequences, especially if you were a sheep.
There is a story that Fred McCallum, who is
head of the Public Health Service in the United Kingdom tells to the effect that
he basically won the war because he prevented Winston Churchill having a yellow
fever vaccine when he was going off to talk with Stalin around 1944. So most of
the serious consequences really come from whole animal source materials, if you
like. You can use whole animals as substrates. I'm using the term "whole
animals" in a fairly broad sense.
Eggs in the definition of the Animal Regulated Use Act in the United Kingdom
count as an animal because they are embryonated. For many years, rabies
vaccines were produced in mouse brain or sheep brain. They have quite serious
consequences, but not necessarily associated with adventitial agents. You can
get encephalitis as a result of immune responses to the non-invasic protein.
The Japanese encephalitis vaccine, which is used for travelers in the United
Kingdom, is still made in mouse brain. So it's not an unusual source of
material, if you like. Smallpox for a long time was made on the scarified flanks
of calves. Like I said, isn't any more. However, while these things seem really
quite primitive, in terms of how you make vaccines nowadays, you still have a
number of vaccines that are made in eggs. Yellow fever is the classic example,
and influenza. Yellow fever is not required to be grown in avian leukosis-free
eggs. The reason for that is that there are a number of sites at which it
was manufactured throughout the world, where yellow fever is a really very
serious problem, such as Nigeria, for example, South America, whatever, where
SPF eggs,avian leukosis-free eggs even, were really not freely available. So
yellow fever can in principle at least be made in avian leukosis containing
eggs, and in fact is. I think there's no evidence that this has an adverse
consequence. But on the other hand, you wouldn't necessarily want to
have a virus in there that you didn't know about. SV40 is one that I'm going to talk about in some detail in a minute. This was in polio vaccines in the 1950s and very early 1960s, probably, a source from rhesus monkey kidney. Polio vaccines are still made on monkey kidney, though they are not usually on rhesus monkey kidney. It would be cynomologous or something like that, for reasons which I'll describe in a moment. Nonetheless, a great deal of vaccine is still made in primary monkey kidney cells. There are reasons for that. There's a deep conservatism I think about changing the vaccine production process if you have a vaccine that works, largely because you are dealing with a prophylactic material rather than a therapeutic material. So you don't want to mess about with anything if it's reasonably safe and effective. I'll mention very briefly the defective retrovirus story in chick embryos. I think Jim Robertson will probably mention this in more detail, but I will mention that just as I go by. Finally recently, the FDA released a talk paper on a preparation of urokinase, which is used in treating the heart. This material was grown from primary cultures made from aborted fetuses. I think it was aborted fetuses or miscarriages, or whatever. There were quite a variety of infectious agents were actually found in this. I believe this one has now been suspended. The point is that there are still a large number of materials which are made on really quite basic culture systems, if you like, where adventitious agents are a serious consideration, if you like. So it's not all continuous cell lines versus the rest. I mean there are -- most of the vaccines that are made in the world probably come from other primary cultures or eggs or things of that nature. I will now talk about SV40. I'm sure in this audience there are people who know far more about SV40 than I do. But nonetheless, I'll talk about this from what you might call the regulatory adventitious agent point of view, if you like. So it's a very common polyoma virus of old world monkeys, and particularly rhesus macaques. The difficulty with this was that when the rhesus macaque monkeys are sacrificed and a primary monkey kidney culture is made from him or her, as the case may be,a silent infection is set up. So there is on evidence of infection just by looking at the cultures. In fact, these cultures can throw out as much SV40 as they do polio, when you start infecting it with polio. So you wind up with a culture that's just stiff with adventitious agent which you really don't want. It's able to transform non simian cells in vitro, and it can be tumorigenic if you have the right kind of animal that you put it into. Between 55 and 62, probably at least a third of all the vaccines that were made on these kind of cultures, because they were pooled and the like, were almost certainly contaminated with SV40. It wasn't a trivial contamination. It was really quite a serious contamination. Because it was mainly an activated polio vaccine, there wouldn't have been that much live SV40 in it perhaps. But SV40 is more resistant to formalin than polio is. So almost everybody who received the shot of inactivated polio in the 1950s, which would include me, would have received live SV40 in some form or another. So the concern is really summarized here, which is basically that everybody, I mean this is my own take on it, that everybody -- I mean you can argue that it might not have been sort of everybody, but I think it probably was. But almost everybody who received the full course of polio vaccine between 1955 and 1965, also got live SV40 stuck into them. That's millions of people basically. There were epidemiological studies that were done at the time which really didn't cause much concern, but they can all be criticized. Some of the studies were really quite short-term, about two or three years or so, looking to see if there were cancer effects basically, as a result of SV40. It may be that two or three years is not enough to actually find such an effect, if it actually exists. The longest which was assumed was over a period of about 19 years. Most of the individuals involved in that study would have been oral polio vaccine recipients rather than inactivated polio vaccine recipients. So they have had it by mouth rather than by injection. Again, you could argue that that might not be the right cohort to actually be looking at. So while the studies were reassuring, the most reassuring thing was that there was no sudden surge of cancers that you can actually trace back to polio vaccine usage in the United States or in Europe where these things were used in a big way. So it really did seem that in the long term, over about 19 to 20 years, there was no real cause for alarm.
However,
in 1992, Michaili Carboni and colleagues and others, a number of others,
including Janet Butelle down in Texas and the like, identified SV40 sequences
which were present in a variety of relatively rare tumors. So mysathelia, which
is the asbestos tumor, osteosarcomas, pendymonas, actually the young chorioid
plexus tumors of children, these sequences do appear to be genuine SV40
sequences. Where they come from is really not quite clear. Part of the argument
was that you could get similar types of tumors in experimental animals, like
hamsters. I think that is probably the only example where a hamster is cited as
a good model for a human being perhaps. But who knows?
In fact, this might actually be an argument that this has got nothing to do with
it. So the question then arises as to where did the SV40 sequences come from.
Of course the classic response really would have been it must have come from the
polio vaccine because why not? Now SV40 was discovered around 1961 or 1962 or
thereabouts, 1960 perhaps. Directly it was discouraged. There were precautions
put in place to exclude it from polio vaccines, because it was known to be a
tumor kind of virus, if you like. These were the kind of things that were put
in place. They are listed in WHO requirements from about 1962 onwards. They
reached their final fully flowered form, if you like, by about 1965. A number
of countries certainly had put this in place before that. The first thing you
can do is to use seronegative animals as the source of cells. So you can use
animals that have no evidence of SV40 infection as your source. That really is
something which is now very firmly in place, which manufacturers now do. The
second thing is, you remember I said that it was the rhesus macaques with the
problems. The problem was that the cell cultures didn't show any sign of having
defect, when they were actually infected with SV40. What you can do then is you
can use species, such as cynomolgus or pattus monkeys, where the primary monkey
kidney culture cell, when infected with SV40, will actually wrinkle up and die
on you. So at least you know you have got something nasty and you can throw it
away. So what you then have is the problem of the chorioid plexus and appendinoma tumors, which occur in children who are around two years of age or maybe less. You have to say well how did they get a hold of the SV40 sequences? One possibility, which is mooted with some enthusiasm, is that maybe you are getting passage of SV40 from parents who did receive the SV40 contaminated polio vaccine to their children. So how this stuff gets around is quite important. One of the things that we have been involved in is doing serological surveys of populations to see who has got SV40 antibodies and who hasn't. It is about a five percent seropositivity by the assay that we're using at least. It seems to peak at around age 10 or thereabouts, and doesn't arise after that. So what you could argue then is that you are seeing vertical transmission from parents down to their children. What you could also argue is that you are not picking up SV40 specific antibodies at all, and they could be other human polyomas like the BK or the JC, and it's cross-reacting antibodies that we're picking up. I think that is still a thing that needs to be resolved. This is how we were trying to resolve it. We have access to a number of sinomorgous breeding colonies. One of them at least is absolutely riddled with SV40. It's chronically infected. They are all infected basically. So this is just four examples of this particular colony. There's about another 50 or so. This happens all the time. The mothers here are highly sera positive to SV40, all of them. What happens is that the mother and the baby stay together for about six months until the baby is weaned. Then the babies are taken off, no longer being babies of course. They are all banged up together in one gigantic sort of teenage squabbling colony. At the time of weaning, the babies are uniformly negative. So despite the fact they have been on the mother for six months, they have not sera converted to SV40. Almost immediately you bang them up together, or at least within about a month or so,they sera convert. So we actually have a sera conversion panel here, if you like, with about 50 or 18 100 or so sera, where the babies actually were seronegative and then become seropositive. My view on this is probably that the babies don't get infected until you bang them up together. But it may be that they are infected, but they are just not seropositive. So what we have to do here is to fish out the virus from these animals here, and see if it looks like the mother's virus or if it looks like the other babies' virus. The point about this long story which I have just been telling you about SV40 is that SV40 was a problem between 1955 and 1962, and it's now 1999, and we still don't really know what was going on. So if you actually make a mistake, it's really quite serious. It may keep you occupied for the rest of your working life. One last quick thing or two last slides here. One is about reverse transcriptase of vaccines. Dr. Schuepbach will be talking later and Jim Robertson will be talking in a moment about detection of reverse transcriptase in chicken cell grown vaccines, such as flu or yellow fever or measles, mumps, rubella. This appears to be due to the presence of defective non-infectious particles. There are sequences from EAV and ALV both in these things, a ratio of about nine to one as I understand it. It does seem to me that you are not really quite sure what the AV sequence is in there and what ALV sequence is in there. It's probably going to vary from chicken to chicken in so far as these chickens have not been bred. In other words, every egg is a new experiment. You are really not quite sure what you are dealing with in that. I think that is quite an unfortunate position to be in. I'm not sure how you control it. Finally, this is my last slide, and this has to do with characterized cells. The issues that I have been dealing with really have been to do with primary cells and primary cell problems where the virus comes in direct from the animal origin. I think there is no doubt in my mind that that's the main source of concern in terms of human health. Nonetheless, there are clearly problems which also arise with characterized cells and the continuous cell lines, in particular. We have some down here. Now the regulatory authorities in the room will be well aware of a large number of other examples of this type which don't actually get published. I think that's not so good. I think this stuff really should be out there in the public literature. But nonetheless, these are the ones which are well known, I think. CHO viruses, CHO cells have defective retroviruses. Manufacturers take a great deal of care to actually get rid of them in the final product. So they are endogenous. Here are examples of things like BVDV contaminating cells which are growing in culture, and also other bovine viruses contaminating cells in culture, particularly when they are grown on a very large scale. Whether or not that poses a hazard is another matter, but clearly there must be methods in place to actually detect them. The classic example here was the minute virus in mice, where the tpa had been grown in CHO cells on a 10,000 litre stove essentially, and then tiled up for an effect with minute virus of mice. Now this was on the order of eight logs, as I understand it, of virus per mil, and yet a 10,000 litre fermenter culture. This is probably more minute virus of mice in one place on the planet than has ever been the case before. You might want to think how you actually get rid of it actually. This is a question of actually getting the cells infected while they are actually burning in culture. So while family cells are clearly a major problem, and while whole animal sources, if you like, are probably the biggest hazard which is likely to be raised in terms of human health, biological aspects to do with well characterized cell banks, where viruses may be introduced from biological materials or they may be introduced by mice walking across the top of the fermenter or whatever, are nonetheless a significant matter. It really is not totally clear whether these things have an implication for human health. But I think you would be wise to make sure that they are not actually present. That's where I stop. Thank you.
(Applause.)
DR. COFFIN: John Coffin of Tufts. That was a
really nice summary actually, Phil. But some caution might be called for in
translating the results of vertical transmission experiments from monkeys to
humans. As far as we know, simian immunodeficiency virus in monkey populations
are not transmitted vertically. Yet HIV-1 is transmitted with reasonable
efficiency vertically in human populations. So there may be some underlying
biological difference that perhaps a very subtle one, that promotes this kind of
transmission in people, where you wouldn't see it necessarily in monkey models. DR. ONIONS: David Onions, Glasgow. Phil, when people switched to cynomologous monkeys, and I can see the reason because you can pick up SV40. That's very clear. But how do you know that at the same time, you have not invented a new problem, that you have got another polyoma virus in that species that you are not detecting. I mean has anyone done redundant PCR to look?
DR. MINOR: I think I would choose to look at
a polyoma man to answer that question. Anybody?
DR. BROKER: Tom Broker, UAB. I wanted to follow up on that exact question. We are facing the same problem with potential vertical transmission of human papilloma viruses. I'll mention it later in my own presentation, but briefly, it does appear there is some protective immunity during nursing. On the other hand, removal of an infant, say through adoption to another family, is the highest risk factor for a child acquiring laryngeal papillomatosis later in life. So a two to three year delay.
DR. MINOR: So is the assumption then that the
infant is infected, but it's not infected properly then? DR. BROKER: Or is it receiving a period of important passive protection from the mom. DR. MINOR: I figured we could do that by looking at the actual strain of viruses the monkeys get infected with. We have a number of different gang rooms, if you like. If you get a different strain in each gang room, but it's the same strain within a gang room, then I think that will answer the same question. You can also go back to the mother and see what kind of strain she's got too. But it's a valid point. AUDIENCE MEMBER:I would like to reemphasize one of the important points that you made. I know you didn't have time to expand on it, but I think it is extremely important. That is the need for those organizations who discover a new virus or some contaminant, cell population used for vaccine production or in a production run, to make that publicly known.
I think that the declaration by Genentech,who
has published this information under their name, that an NBM contamination
occurred in a 10,000 fermenter is an act of great courage. I think that, that
kind of courage, this declaration by other companies in this field, is very
necessary for the health of this industry. I understand from some of the
remarks that have been made that there are others that are known to a small
coterie of people here that have not been publicly declared. I urge all of you
to think about this seriously because it can and will have a great impact
on this industry. Thank you.
DR. MINOR: I think the FDA can answer this one
better than me, yes. But I mean that was my understanding of it. It's out on
the net.
DR. FRIED: Mike Fried. Was any of the old
vaccines from the 1960s that were contaminated, were they PCR'd up to show that
the virus was the same as being found today? Because it's also possible that we
all have a latent SV40 type virus which likes to grow in tumor cells, and that's
why you find it. It's a passenger. But I mean since there's polymorphisms in
the sequence, if you can go back to the 1960s and then find out if it's the same
thing that we find today, it would be helpful.
MS. MARCUS: Carole Marcus Sequora from Bassey
Consulting. I just wanted to clarify that urokinase is produced from cells. It's
not aborted fetuses. It's newborns who did not survive. Just for the record.
CHAIRPERSON RABINOVICH: Thank you. Our next
speaker is Dr. Jim Robertson, speaking on experiences with retroviruses in avian
and mammalian cell substrates. The types of sources of fluids which have been negative for reverse transcriptase are listed here. Some species are not positive, turkey and duck cultures, quite a range of human cell lines. Simian rabbits have been tested and found to be negative. So the clear source of this RTase that was being picked up in the vaccines is quite clearly of chicken aviano origin. We would want to look at -- I should add that this RTase was known at the time to be particles associated and appears in the supernate of the cells. We are going to look at this particle to see if it would pick up any infectivity. In all, we looked at 10 different cell lines, mainly human, but including rabbits and turkey. Over 21 tests and 116 passages. In each case, in every test and at every passage level, the cultures were negative for reverse transcriptase activity. There's absolutely no indication that this particle is infectious. Since then, CBER and CDC have also come up with similar data, including use of PBMCs. No infectivity associated with these RTase containing particles. Where might these be coming from? Presumably they are derived from endogenous retroviral-like elements in the chicken. The information to date regarding such elements in the chicken genome are quite well characterized EV loci, which are related to the avian chosis virus family, and more recently discovered about 10 years ago, EAV-0 family, which is an older element than EV, and then older still, ART-CH and CH-1 elements. The information at the time and pretty much where it still exists is that we knew that there's a line of chickens which was negative for EV. It had been eliminated from the genome. This line of chickens, the culture fluids were positive for RTase. So we knew that it had to be at least one of these elements producing RT activity. At the same time, you couldn't eliminate the fact that EV might also be producing RT activity. The best bet was EAV-0, given the sequence information that was present at the time. More recently, in the last year or two, Joerg Schuepbach's laboratory has produced a good evidence for the presence of EAV-0 derived RNA associated with the RT particles secreted from CEF cells, and then this year, Walid Heneine, CDC, also produced the presence of EAV and ALV RNA.
CHAIRPERSON RABINOVICH:Thank you, Dr.Robertson. Any questions?
AUDIENCE MEMBER: Just a comment. For known
endogenous avian retroviruses or exogenous avian retroviruses, of the cell
lines, of the cells that you tested for infectivity, only the turkey cells would
have given a positive result. I would urge for avian-derived -- urge the use of
those cells, and a PERT assay is a sensitive readout, for detection of perhaps
unknown agents in these vaccines, end products, as being the most sensitive, at
least for avian-derived.
DR. MYERS: Martin Myers from National Vaccine Program. As I sit and count the number of immunizations that various populations receive with these particles in it, repeated immunizations with it, I wonder if there is any data on sero responsiveness in longitudinal –
DR.ROBERTSON:Yes. MS. SHEETS: Hello. I'm Becky Sheets from FDA. How would you recommend that avian-derived products be tested for retroviruses? EM is not very sensitive. The conventional test is often inhibited by the allantoic fluid, and therefore, is not necessarily a valid test. How would you recommend, if you don't use a PCR-based RT des?
MS. SHEETS: When you said infectivity test, were you talking about those specific for ALV or were you talking about general tests to detect any kind of retrovirus?
DR. ROBERTSON: Well, it would have to be an
avian retrovirus if one is performing the assay on chick cells. Propagating the
material, the test material in chick cells but using either RTase in general
or an ELISA specific for ALV test for increased presence of either RT or for the
presence of ALV antigen.
DR. SCHUEPBACH: We agree that there is no
sequence homology on the nucleic acid and on the protein label, but these are
the results which we found. We have to find an explanation for them. We don't
have at the moment. DR. SCHUEPBACH: May I have the first slide, please? Okay, from previous remarks, I heard that I was expected to talk about these avian retroviruses as well, but actually I was asked to talk about the induction activation of occult viral agents. So I will just have a few remarks on this other stuff. So occult viral agents are agents you don't detect or at least do not easily detect. They may include two groups: a group that includes known agents which are present at two low concentrations for easy detection. The reason for these may be latency; The other group consists of unknown agents. Since we do not have good detection methods for these, they may be present at low or also at higher concentration. Viruses known for their latency or various types of the herpes virus, true, they are latent in various types of non-permissive cells such as neurons cells, monocytes, PBLs, and others. They are activated from these latent stages by various kinds of stimulation of their host cells by differentiation, agents by the differentiation of precursor cells, to more mature cells. Again, by other activating agents. Other viruses could be considered in addition to the herpes viruses include the adeno viruses, the adeno-associated virus and the pathyloma and polyoma viruses of which we heard yesterday, and will hear more in a subsequent talk. Regarding the RNA viruses, I might discuss the measle viruses and of course the retroviruses. When we look at the mechanisms by which we can activate these various viruses, it is mostly by activation of their host cells, by cell stimulation, by induction of cell differentiation of these cells, and then by co-cultivation with cells which are permissive for replication. Now since we have different viruses and host cells systems, these methods vary greatly among the different viruses. If you have unknown viruses, you really don't know what to do. So the effect of such activation would be that from a lonely latently infected cell, by inducement of replication, a virus would spread throughout the culture, resulting in virus gene amplification in production of viral proteins. So this would make of course the detection easy. You might also have some pathogenicity which is easy to detect. However, our goal is actually not detecting any possible virus that might be present. The principal goal is to provide a virus production system which is free of such agents. It is suggested here the easiest way of achieving this is actually cellular cloning. Because if you have an agent that is present in only a minority of the cells, the chances that you derive a clone that is free of these agents is very high. If by chance you hit an infected cell, the descendants of that cell will all carry along the virus and of course then we come into a situation which makes detection of unknown viruses and also known viruses much easier because either all of the cells will be infected or none at all. So cellular cloning, if we hit an infected cell, has actually a viral gene amplification effect which is comparable to virus induction activation if it's successful. Most importantly, it is a procedure that works for all the latent viruses except endogenous retroviruses, but these are present anyway in all of the cells. So going on to the detection methods for these agents, let's first talk about known viruses. Since all the cells will be infected, we actually do not need the most sensitive procedures. We do not need procedures that detect the single viral copy. What we need is broadly reactive methods which go detect all the different members of a certain virus group. So I think techniques, old-fashioned techniques like hybridization techniques on the low stringency or if we want to use PCR or nucleic acid based methods, we should take care that we take a lot of different probes, use data generated primus, multiplex PCR and so on. Of course in addition, we should also do the classical methods, doing cell activation and co-cultivation as permissive cells, the routine detection methods of broadly reactive antibodies which detect all the different members. For those who think that what I have told so far is rubbish, and that we actually do need very sensitive methods, I offer the mega PCR, which has also been named catcher PCR by others. The purpose of this method is to take very rare sequences among a very high background of DNA or RNA. So here we convert the samples of up to 500, maybe even 1 milligram of DNA or respectively RNA. The principle is very simple. We use biotinylated capture probes which bind to these sequences inquest. We isolate these complexes on coded beads, wash the rest of the DNA away, and them amplify these by PCR with primus which are located outside of this capture probe.The advantage of this is that we absolutely do have no carry over because the amplicons are selected against when we do the capturing. It is this type of test which I would actually like to have been seen when testing in the question of xenotransplantation where the PERV sequences can be found in humans which have received pork material. I think this will be the test, to test these questions. Now using this method, it's actually very sensitive. You can detect a single copy here of HIV DNA. We still have double positive signal, is about one copy. This serial dilution was done in the proper range here. The fact that in these two, three last dilutions only one of the two duplicates was positive clearly demonstrates that we are in a Poisson distribution. So we can detect the single copy with this method in 100 microgram. DNA, we have actually demonstrated that there's 95 percent probability we can detect three double standard HIV copies in 100 micrograms of DNA. So now going on to the exclusion of unknown viruses, and I will talk about retroviruses later, we can actually use the same procedures as I have already described previously. We just have to take care that we really have broadly reactive methods. This is true for molecular based tests as well as for the more classical procedures. Now coming to retrovirus detection, of course also of cell cloning, here we have two situations, the exogenous retrovirus may not be present in none of the cells or in all of the cells. The endogenous retroviruses were always present in all of the cells.The known exogenous retroviruses are detectible by tests for conserved sequences. Of course you might also use universal pool primers for unknown retroviruses -- because of the endogenous retroviruses. Not all of which, or very few of which are actually harmful. So I think it is better at this time to switch from the analysis of cells to the analysis of particles. This is best done by the PERT assay which has been mentioned before by several speakers. Now when we devised this test in 1992, we devised it as an anti-family of related tests which would have in common that reversed inscriptase present in a sample would be used to create from a template primer combination and nucleic acid that is to be unamplified. Now in most instances, this will simply be the cDNA. There are other possibilities as well. You can take any nucleic amplification procedure, not just PCR. You may also use ligase chain reaction or NASPA or you can make use of auto replicated DNAs or RNAs in order to generate amplification product, which can then be assayed by different methods. So since we have provided for all these different methods already in 1992, we do not think that it is necessary to invent new names for these current assays. Now this test is actually very sensitive. This experiment in comparison to classical RT assay. It occurred as six to seven orders of magnitude more sensitive, and in a direct comparison with -- in the case of HIV, where we compared the method with RT PCR, detecting one copy of cDNA, we had the same dilution endpoints for two different samples. Actually as others, we can detect only a few particles in the case of HIV. We believe that in some cases we can detect even less than one particle. Now this is one of the theories taken from the Joerg Koenig paper in 1996, where we demonstrated that the measles vaccines, the mumps vaccines, the yellow fever vaccines, and the MMR vaccines all contain activity which is about three orders of magnitude higher than the background here on other vaccines, and were negative. Now in order to identify the viruses behind these activities, we along with the PERT assay, developed the method for the identification of unknown retroviruses. It is based on three properties of old retroviruses, namely, that they all are polyadenylated, that R sequences are repeated at both ends, and that cDNA synthesis has started here at the primer, binding site, and that for primers, tRNAase are used and the use of such tRNAase is actually very much restricted among the various retroviruses. For example, is just four PRNA primer equivalence. You can start cDNA synthesis for all exogenous retroviruses known today. So what we do is that we bind the retroviral RNA to poly t coated beads. Then we start here, the synthesis of the cDNA with one of the various t RNA primers, synthesizing the strongest of DNA. Then adding a tail here, and then with anchoredTCR, we can amplify this sequence and submit the sequencing directly.
Actually this method has also been used by the
group of Dr. Loewer at the Paul Ehrlich Institute,2 and even published before
us. But we have somehow optimized this procedure, so in general we need less
than one-thousand RNA sequences, sometimes as few as 20 or 40, 50, in order to
generate this sequence here. As soon as you have it, you actually know whether
you are dealing with a retrovirus or not. When you deal with a retrovirus, you
have to R sequence and then you can check with the other anchored PCR. Where
there is R here, it's repeated at the three prime end. If it is, you can then
amplify the entire genome with a little bit of luck by long PCR. So this is what
we use to identify this EIV-O sequence. We have also done some other work. For
example, we investigated the NIH 323 cell line. This was negative by convention
RT tests, but positive by PERT assay. We had a nice band in sucrose, and then
radiant. Using this procedure which we call parar, we identified 23 different
products, 15 of these were actually retroviral sequences from four different
groups. Three of them were unknown sequences, at least at that time. So far we
have not further characterized these sequences, but this is still awaiting. Now
staying with retroviruses, as Dr. Coffin pointed out yesterday, sometimes if you
have a cell line here, you are dealing with melanoma cell lines which were found
to be highly percipated by PERT assay. We analyzed what was in there. It turns
out to be endogenous murine leukemia virus, and later we were told that these
cell lines have actually been passaged in mice. If you have low titres of
activity, then that becomes a little bit more complicated. This is the analysis
of primary samples from a patient with MC cor cultures. No actually not cor
cultures, just cor cultures which were found lowly positive in the PERT assay
with activity in the order of two, maybe three times above background. Here the
patterns is a little bit more complicated. You have here a small peak that
might correspond in density to ritualized particles. This one might correspond
to cor particles. You have another identified -- unidentified peak here. It
will certainly be a challenge to find out what this stuff is. Next, please.
This is another example of a primary culture where we have a very short peak at
the higher density. This might be for particles, could be a different
retrovirus, a different virus, or just a subcellular particles containing some
cell or enzymes. Now you will say that this test of course detects only
retroviruses that are released. We are also worried about retroviruses that are
inside the cells, so stimulation may be necessary. Actually I think one
important question is or one possibility is that actually the vaccine virus we
would like to produce in such a cell might activate latent proviruses. So I
think it is important that we actually do not just test the virus production
systems while uninfected, but also when this seed virus has been added, and then
we harvest the virus. Now in some cases, as in the measles virus or so, this
has proven very easy. We had quite a good specificity. But in other cases, it
might be more difficult as indicated in this example, where we tested a vaccine,
experimental vaccinia, recombinant vaccinia virus vaccine against melanoma.
This was found highly positive by PERT. It had actually been produced by just
the lysing, the infected cells by ultrasonication. What we now find is here in
black, is the vaccinia virus DNA two peaks. We have here a major peak of RT
activity which does not coincide with the vaccinia virus peaks, and also is not
characteristic of retroviruses. So I think in this case, we can rule out the
presence of a retrovirus. Now it may also be interesting to find out whether
upon induction, viruses might come out. So this would add an increase of safety
to the vaccine. As retroviruses are regulated, you have the promoter in anti
sequences in the upstream LTR in the U3 region. Depending on the cell type,
activation state of the cell and the differentiation, you have various sets of
transcription favors interacting with these enhancer regions. In addition to
this balance of positive and negative transcription factors, you may have
positional effects as the chromatin structure or the DNA methylation. You may
now try to influence this balance by tipping it by either inducing mitosis cell
differentiation by substances that lock inhibitors or by alleviating the
negative positional effects, again by inducing mitosis or by inducing DNA de
methylation. The number of induces have been described in the past. The most
important ones are listed here at the top, allogenated pyrimidins, the
azacytidine, which only both of them working only in infected, cells. I will
not mention the others because of the lack of time. Now it depends a lot on the
virus whether azacytidine or the deoxy pyrimidine is preferable. For example, in
experiment in cell line where two types of different retroviruses are produced,
several type A particles here. The azacytidine is certainly better. But in
C-type particles, these cells produced IdUdr. Yes, the IdUdr is better. So you
might have to use a combination of these two drugs. So in conclusion, I think
induction activation certainly serves to amplify latent viruses for which
improved detection. I think it is more important that we early in the process
of selecting virus production systems be cloned B cells, and sub-cloned, because
this will amplify, because this really facilitates detection very much. In
consequence of this, we do not -- I think this is very important. We do not
need the most sensitive procedures. What we need is broadly reactive procedures
which will detect all the different agents. I also think that at the end, the
only important thing actually when dealing with adventitious agents, not just
with DNA, which might be infectious, is that the vaccine is free of these
contaminant viruses and for retroviruses I believe that this can be verified by
the PERV assay. Thank you. (Applause.)
tests are available and have been validated and that we understand the sensitivity of. So I guess in the context of thinking of highly conserved sequences to which we might develop primers that could detect a broad array of viruses, including some unknown related viruses, what can you say about the current state of the art? How good is that? How well has that been validated? Is that something which if we decided tomorrow we wanted to apply that to new vaccines producing neoplastic cells, we could simply say "let's do it" or is more work required?
What we also see in the gag experiments is
that there are additional env genes that are only on the almost intact. We have
here a protein that is about four kilodaltons more. So this actually could also
be considered as an intact reading frame. So we have several human chromosomes
that still contain gag and env genes. We have three chromosomes that contain
both intact gag and env genes, the chromosome 7, 19, and the Y chromosome. We
were interested whether these chromosomes or the intact genes on these
chromosomes are derived or located within one provirus or within several or
different positions within the particular chromosome. I would like to report or
tell you something about what we found out for the chromosome 7. We were using
for addressing that question, we were using a chromosome-specific, chromosome-7
specific cosmid library. We were screening for clones that contained both gag
and env sequences. What we finally found out, that we isolated the so far least
defective human endonuclease on chromosome 7. We were able to characterize the
proviral sequence within one cosmid clone that still has intact LTRs. So they
regulate to the elements. They are able to transcribe, as you will see. We
have an intact gag gene. We have an intact protease gene that protease is able
to cut itself from a gag protease, polymer precursor protein, and is furthermore
able to process encoded gag proteins. So it's typical retro-ized protease. We
know just from sequence comparison, one can deduce that the endonuclease within
the polymer genes also acted just by sequence comparison, no significant changes
compared to recently described active K in the nuclease. We have an intact env
gene. This intact env gene sequence has already been described by Johannes
Loewer's group as an MRA, which also shows that this sequence is actively
transcribed. So this is actually an expressed provirus. We have spliced on the
inceptors sides the corresponding position that would allow to splice an M on A,
and what we heard yesterday also, to splice an additional soft M RNA. What we
see is that this proviral sequence is only defective in the RT domain. It has a
single-based permutation within the YXDT motif. So very likely, this highly
important catalytic motif is -- so only in reverse transcription function this
probably missing from that proviral. Okay. We have here almost intact proviral
sequence. But now regarding infectivity, we had that already several times I
guess before. We have HERV-encoded retro of particles, several cell lines, even
in tissues, the placenta tissue for instance. We find HERV-RNA in these
particles. CHAIRPERSON RABINOVICH: I think we will hold questions at this point. We are going to take a 10-minute break now. We are going to come back and finish with the last two speakers. I need to figure out how to catch up time, and yet leave the time for the panel discussions. I ask you to do two things. Check-out time from the hotel is 12:00. You should know that. They have already called in a bunch of the taxis so that if you need taxi arrangement, please let them know so they can do that for you. Ten minutes we will start again. (Whereupon, the foregoing matter went off the record at 9:55 a.m. and went back on the record at 10:10 a.m.) CHAIRPERSON RABINOVICH: Is Dr. Broker here? Great. If you could take a seat please. The next speaker is Dr. Thomas Broker from the University of Alabama at Birmingham speaking on viral latency-papilloma virus model.
Some of the remarkable outcomes of the care with which we undertook the genotyping is the following. In the pre-transplant population, the prevalent types are those that are commonly seen in the general population as causing disease, namely HPV- 6, 11, and 16. Those types persist in those women who were pharmacologically immuno-suppressed. We see a scattering of other types, but the common types from prior studies are those that predominate in the renal transplant cohorts. In contrast, those women who are in various stages of immuno-deficiency as a result of AIDS, do not show the same genotype profiles. The only member in common is in fact most common of all genital HPVs, HPV-6. What we see instead are niche homologs of the common types. For example, HPV-45, as you are going to see, if a close relative of HPV-18, which is often cited as a common virus. But we don't see that in the AIDS cohort. HPV-52 is our most common virus. It is a close homolog of HPV-16, which we don't see amplifying in this cohort. Most notably are the ones that I indicated by stars, which are a very rare detection within the general population, but in fact are most common viruses in the AIDS cohorts. In particular, we have identified 13 new HPV types based on less than 75 percent sequence homology to each other or to any other known papilloma virus. They are all members of what has been designated group A-3, which appear to be an AIDS- defining subset of HPVs. These can be at least considered in the context of phylogenetic trees based on sequence alignments in the L1 region. So, for example, HPV-16, the main cause of cervical cancer in the world, is seen in the renal cohort, but a very close relative, 52, is seen in AIDS. Six and 11, that cause benign genital warts and laryngeal papillomas are here. One of the main groups coming up in AIDS is this group of cousins of these guys. HPV-70 is one of our most common types, as well as 45. They are in the HPV-18 family, but represent new members of this niche. The group I just mentioned, A-3, that is so commonly seen in AIDS, include our members jyn 2, 3, 4, all the way up to 13, MM8 and 61, 72, and 83. That cluster seems to be an AIDS defining group. The other ones that we have seen abundantly are 51 and 53 in this arm. Overall, in the Birmingham and generally Alabama population, every virus types seen with the star we have found one up to 23 times, indicating that we have universal presence and also detectibility of all of the known viruses within our immediate population. While this is up here, I also wanted to point out the very large huge group of epidermal dysplasia formus viruses that other labs have studied. Again, it is a very rare group of illnesses, in fact, only defined a few hundred times in all of medical history in terms of individual patients. However, there is this huge ramification of somewhat related, but clearly distinct genotypes that comprise the family or subgroup of viruses responsible for EV.It is known that these patients all have particular cell-mediated immune deficiencies. Again, suggesting that particular arms of the immune system are responsible for either containing or failing to contain different subgroups of the papilloma viruses. As we look at these women over a period of time through these six month or so samples, what we also find, and other labs have exactly the same results, is every time we sample, you may or may not see the type you saw before. It may switch. For instance, we have this patient who had 6 plus 16, and then 11 plus one that was minor and we couldn't tell, then jyn 2, and then type 40, and then we had a type 53, but the others disappeared. Everyone's experience in the field has been that the viruses rise above a detectibility threshold, stay there for a while, days or weeks or months, and then fall below detectibility, only to be replaced by a different HPV type. These are not new infections. They are basically cryptic or latent persistent infections that fluctuate in their levelsof replication and detectibility. Pretty much anybody is showing that flexibility. What I want to state at this moment before showing the correlation with disease may sound controversial, but I will stick by it. We have found a brand new HPV type for every 10 people that we have looked at. Philodelius and Ethel Michelle Diveres and zur Hausen and Shamen in European study of tutanius papilloma viruses have found a new papilloma virus for just about every other person they have looked at when they use the combination of nested PCR and DNA sequencing. Robbie Burke's group, Jill Polefski's group, have very comparable experiences looking at anal papillomas or female genital tract. It is my contention right now that instead of 80 HPV genotypes or 150 that have been officially named, that there probably are millions of variants, virtually a continuum. We feel that basically everybody has their own personal micro flora, that these are passively acquired or vertically acquired, not necessarily sexually, but certainly possibly sexually, and that they simply are part of the human condition as are microflora, just as we have microflora composed of bacteria and many other viruses, and that they basically are utterly ubiquitous. I will come back to that point in a moment. We did try to correlate the various other medical parameters in these cohorts, especially the AIDS cohort, with CD4 count, HIV virus load, other infecting known STDs like herpes, chlamydia, trichomonas, so forth. The one correlate that held up and not surprisingly at all, was that the degree of pap smear abnormality from normal, abnormal cells of unknown significance, low grade dysplagias or high grade dysplagias, is with CD4 count. The medians, these are all the people who had multiple infections, a high risk virus type, a low risk, no virus at all, and had either normal or these various abnormal pap smears. These bars here are the median CD4 count in each of these groups. The one place where we saw active disease, low and high grade dysplagia, these by median, is when people fell below the CD4 count of 200 cells per cubic millimeter. In summary of that data, we found that it's very very possible to have negative pap smears, but definitely have HPV infections. We feel these are people who have not yet reactivated long enough to have resulted in cytologic change as a result of infection. We have on the other hand, the people with overt disease by biopsy or by cytology, and the higher the grade lesion, the more likely it is to see either single infection or especially multiple virus types present within that patient at that time. So the more that we can detect the virus, that is, the more it has replicated or amplified throughout the population, the more cells that are shedding the virus in effect, the more likely we see disease. So to summarize this part of the talk, I feel that they are virtually ubiquitous. They are typically sub-clinical, persist in or latent infections. There are staggeringly large number of genotypes if we take the care to look. I might say that the reason these are typically not found is that people use generic cross-hybridizing probes or have cut off their probe sets. If you're not probing for something, you are not going to see it. Most of the viruses in this number 60, 70, 80 and above, are not even present within the commercial probe sets. So if you aren't probing, you are not going to see them, and you are going to get lower numbers.They can be found throughout the genital tract in 60 to 75 percent of the people that we have looked at who are admittedly good yielders, because they are immuno-compromised, but I think this simply represents the general infection in the population. They can be found in oral and esophageal mucosa. Utaneious types persist in hair follicles. There's a wonderful study from Amsterdam by Tershaget and Ingebor Boxman. She plucked hair follicles, both eyebrow hairs and pubic hair, and 60 to 70 percent of all people harbored EV viruses or other rare virus types in their hair follicles. No disease, it's just part of the human condition. I believe they are vertically transmitted perinatally, mother to baby. Some of them are clearly pre-natal infections. As we know, there's long-term maintenance that requires viral replication in concert with host replication in the cell cycle. So what I would like to do now is tell you a little bit about a very unexpected observation we made in Hela cells. This goes back to last night's talk regarding the structure of the replication complex of HPVs. As you know, cyclin E is one of the key checkpoints or entries into S phase. Ectopic expression of cyclin E can speed up entry into S phase, and it can even bypass the need for some of the RB phosphorylation by cyclin D. It's simply one of the key steps that needs to follow the induction of the DNA replication enzymes. HPV E7, the viral oncogene that in fact binds RB and can help bypass that step, among the E2F enhenca protein regulated genes is cyclin E itself. In other words, HPV infection upregulates cyclin E. So we asked whether the induction of cyclin E is essential for the reactivation of unscheduled cellular DNA synthesis in the upper stratum of squamous cepathelium that differentiated caratinocyde. I'll just summarize that data. I got you to the point last night where the E1 diheximer, the double helicase held together by the HSP-70 cochaperone protein, is there. The next thing that loads in the study we did with Theresa Wong at Stanford, is the recruitment of the cellular DNA polymerase, and showed direct interactions between the helicase and the catalytic sub-unit of pol alpha, P-180, as well as its P-70 sub- unit. This was the first indication of what P-70 does in the four sub-unit complex of pol alpha, which includes two primary sub-units. The answer is, it brings the polymerase to the ora itself. The next thing that comes in is cyclin E, CDK-2 complex, that critical S phase entry point. AS a result of that, what happens is upon cyclin E finding an appropriately assembled pre-initiation complex, five target proteins are phosphorylated. They include: the E2 protein, which appears to be displaced by that event; in addition, P-70 helps displace E2. So the loading of this and the phosphorylation kicks this guy out. Secondly, E1 is phosphorylated. These two subunits of preliminary salpha that bind directly to E1 are phosphorylated. When all four of those have been successfully modified, the kinase phosphorylates cyclin E itself, which is displaced and degraded by ubiquitination. That enables the pre-initiation complex to convert to the elongation complex. In studies with Wade Harper and Jien-Ling Ma at Baylor, two things were done. The first is together we found that there's a cyclin binding motif that the amino terminal have at the E1 protein, which in fact is shared with a number of other things that bind the cyclin E. That motif involves an RXL. That is, an arginine something leucine motif right there. In addition, their candidate phosphorylation cites, the series of serine, serine, serine, and threonine, mutation of any of these, the motif or any of the target phosphorylation cites, diminishes the capacity of cyclin E to convert the pre-initiation complex to an initiation complex. So the functional requirement for phosphorylation has been verified. But keep in mind this location. We'll come back to it in a second. So we assumed that the consequence of upregulation of cyclin E by E7 gene expression would identify those cells that are capable of supporting papilloma replication. To our amazement, we found the opposition. This is our epithelial raft model. We have done the same in natural papilloma lesions. Here we monitored cyclin E expression, over expression in the tissue. Here's bromo deoxy uridine incorporation or PCNA upregulation. These are the match. What we found is the cells that had high cyclin E could not replicate. In fact, they are mutually exclusive with those capable of supporting DNA synthesis. Conversely, PCNA, which is upregulated by papilloma 7 and cyclin E do co-localize. But we see a number of cells where PCNA is present and there is no cyclin E. So we have a reciprocal pattern to what we expected. I am just going to very briefly tell you that P-21 cip, one of the inhibitors of cyclin D and cyclin E, is also upregulated by E-7 expression in natural condylomas or in our E-7 expression raft cultures. You can see those signals in the upper strata again. So we have P-21 upregulation, again, in a subset of cells. When we look in rafts or in natural papillomas, we see that those cells that have high P-21 are mutually exclusive from those capable of supporting either viral or cellular DNA synthesis. When we did the third pairwise combination and looked at cyclin E and P-21, we found perfect colocalization of those two. So ironically, the cells that have high cyclin E also have high P-21 and do not support replication. This was really perplexing, except we did know this inhibited that. But we assumed cyclin E was in the licensing factor for engaging in replication. So what we came to feel is the following model: that in the course of unscheduled DNA synthesis reactivation, if cyclin E appeared in the appropriate timing or sequence or amount, once a pre-initiation complex formed, you would successfully phosphorylate the target proteins, polymorases and E-1 and E-2 proteins, and successfully engage in elongation replication. Conversely, if too much cyclin E appeared and it appeared in an untimely fashion, its inhibitor, P-21, would recognize misassembled complex. They would cross stabilize. They would both pile up to high levels, and those would be defective in engaging in elongation. Now we put this all together by asking how does this play into the establishment of immortalized and transformed cells and cancers. What Wade Harper had found is that when he did pull-down assays with cyclin E to ask in hela cells what binds to cyclin E, almost all the things that came down in the assay was that E-1 protein from the resident HPB-18 in the cell lines. Up until that point, people had thought the E-1 gene was deleted from hela. In fact, it's present. The entire length of the E-1 gene is still present in hela. In fact, is expressed. Now the functional assay that our lab did was that we found that hela cell extracts could not support HPB replication in our cell-free system, that there was a missing factor in hela that the extract needed. We could put 293 cell extracts or any other cell line that we could find, they would easily complement papilloma replication in vitro. But anything from hela cHa caski or any other HPB transformed cell line could not support it. The upshot of the whole thing is that every papilloma transformed cell that we studied expressed a full length E-1 transcript, but in all cases, the transcript had an either frame shift or a stop code on partway through the gene or miss sensed mutations in this vicinity, so that in tact E-1 could not be made. But in all cases, it made the RXL portion that interacts with cyclin E. So we added a little cyclin E back to hela cell extracts and immediately restored full complementability to those extracts, establishing that the missing thing in hela cells was cyclin E. So our conclusion is the following. I think we feel very confident about it. In the process of immortalizing cells, either natural cancers or attempts at making cell lines, substrates in effect, it's good to have all the upregulation of DNA polymerase, topasomerases, PCNA and so forth, that help rapid cell cyclin. But the one thing that E-7 upregulates you don't want, is cyclin E. So what these natural experiments did, is figured out a way to sequester part, but not all, of the cyclin E by putting in retaining fragments of E-1,capable of mopping up that one product that's upregulated that you don't want to have. That is, cyclin E. Brian Van Tine, last night, also indicated there's evidence of some antisense in other papilloma lines like cHa caski, which would again, modulate the amount of E-1 |