PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
INTERNATIONAL ASSOCIATION FOR BIOLOGICALS
NATIONAL INSTITUTE OF ALLERGY AND
NATIONAL VACCINE PROGRAM OFFICE
WORLD HEALTH ORGANIZATION
EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES ON
CELL SUBSTRATES FOR VACCINE DEVELOPMENT
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.
Regina Rabinovich, M.D. Session
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.
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
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.
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
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 –
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
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 that you could translate from the messages that are clearly there. Together we believe that to establish these cell lines, whether you make them in the lab or whether nature has made them for you through cancer, you need to diminish the amount of cyclin E to achieve an equilibrium where you have enough cyclin E to support cellular replication, but not too much so that it's an unsuccessful high level. Thank you very much. (Applause.) Oh, I did want to acknowledge -- could I have the slides for one second -- a very, very large number of collaborators. I'll leave them up here. But we're very grateful for our own students and collaborators at UAB, Jeff Engler, Doug Seer, Sean Van Tine, Kim Towns. At Baylor, Wade Harper's lab, UNC, Jack Griffith's lab, who did the things with the HSP- 40, and at Stanford, Theresa Wong, and our collaborators at the Free University, who did a lot of work on some transcriptional control that we collaborated on for several years. Thanks.
DR. RUSSO: Hi. Carlo Russo, from Merck. Very interesting talk. I have a couple of questions. One is, how can you be sure that what you are sampling by PCR is really an infective virus, it's not just a transient presence with a virus due to the fact that you can't control sexual behavior, and perhaps the woman has just been exposed to a virus?
DR. RUSSO: I may have missed the data. Did
you show the types that are associated with high grade lesions, the HPV types?
I didn't see on the table.
DR. BROKER: Well the real problematic thing for any clinical management, either vaccination programs or small molecule drugs, is this absolutely exploding number of virus types. The one thing that I think is going to -- and I commented a day or two ago that in the U.S. alone today, there are over 250 to 300,000 people immuno-suppressed just due to organ transplants, steroid use, or bone marrow transplants or AIDS. So there is an immense reservoir of particularly high risk patients. Nonetheless, most of the diseases are still being caused by a handful of viruses like 16, 18, 52. So I think, at least the ones we have to worry about today, are still manageable in number.
AUDIENCE MEMBER: I would like to ask a little bit about cell substrates. Considering that hela has multiple HPV integrants, I guess, are any of those infectious?
DR. BROKER: No.
AUDIENCE MEMBER: Can you get them back and
make them infectious?
AUDIENCE MEMBER: What are they missing?
DR. BROKER: They are all truncated within E-2, at best. Although there are 30 to 50 copies, depending on the hela sub-1. All the integrated copies are truncated.
AUDIENCE MEMBER: Would that DNA be transforming, even not infectious?
DR. BROKER: They do contain the E-6 and E-7 genes. Expression of those genes, as shown by studies primarily in zur Hausen's lab, must be maintained or you no longer can cycle hela cells. That is antisense to E-6 and E-7 in hela makes them not cycle any more. So the driving force of hela is the overt expression of E-7.
DR. BROKER: I don't know that I would go that far. It's obviously wonderful as a producer of all sorts of biomedical products.
DR. MURPHY: I actually wanted to ask you the same question, but I would phrase it in a different way. (Laughter.)
DR. MURPHY: This is Murphy from NIAID. That is, do you see any reason why, you know, having an intimate knowledge of hela cells and human papilloma viruses, that hela cells should not be used as a substrate for making live attenuated virus vaccines?
DR. BROKER: I don't know of any evidence of these genes being transduced out or in any way posing a risk. I was going to save it for the panel, but it occurred to me last night, I had challenged one of last night's speakers about the use of psorilins as a cross-linking agent. It gives me great concern that it is a known carcinogen. However, Brian and I were talking. Brian van Tine and I were talking last night, and he reminded me that there are biotinylated psorilins. So for all the debates regarding how to remove contaminating DNA, one strategy in principle is throw in a biotinylated psorilin, cross link it, and pass the whole thing over avidin magnetic beads or batch subtraction of the DNA. So in fact, that strategy may actually help you deplete adventitious contaminants very, very readily. So it is an alternative at least.
AUDIENCE MEMBER: Can I just ask you about hela again? We learned last night that not every HPV, if there's 30 to 50 copies, are not all active, I mean in caski only one was active.
DR. BROKER: Yes.
AUDIENCE MEMBER: What is the state of hela? Are they –
DR. BROKER: Very, very few are active. We and Wade Harper's lab are both sequencing all the transcripts. This actually was done by Elizabeth Schwartz and others in zur Hausen's lab in 1985, and a variety of groups since then in Japan and elsewhere have looked at the expression loci in copies in hela. There appear to be three or four different transcripts made from different positional integrants, but the majority are silent. A few of them are active. But so far, all the ones that are active have truncated E-1s. They have the cyclin E binding motif, but they don't have their normal carboxy terminus.
CHAIRPERSON RABINOVICH: Thank you very much. Let's go on to Dr. Cashman. Thank you for being so patient. Transmissible spongiform encephalopathies: vaccine issues.
DR. CASHMAN: It worked. My friendly A/V guy explained how to do this. I am Neil Cashman. I am predominantly at the Center for Research and Neuro Degenerative Diseases at the University of Toronto. I have a special interest and a long-term research effort in the expression and function of the normal cellular isoform of the prion protein. I am also obliged to mention that I am the chief scientific officer one day a week of a little biotechnology company in Montreal called Caprion. I want to spend a few minutes talking about prions and prion disease. We had a speaker yesterday who said "and now for something completely different." Well, how does a genomeless infectious agent grab you?
Creuzfeldt-Jakob disease is the most common human prion disease that we run into. I do want to spend a few minutes talking about this so that we are all on the same page with regard to public health risks. Creuzfeldt-Jakob disease or CJD is basically a disease you wouldn't want to wish on your worst enemy. It is a completely untreatable uniformly fatal disease resulting in death within six to nine months of presentation. Survival over a year is recorded, but it is not very frequent. The presentation is usually that of a kind of Alzheimers-like syndrome, with problems in memory and intellectual function, but it can also present as a disorder of gait and balance as well. Most people have mild clonus, which is twitching of the muscles, sufficiently forceful to move a joint. Other features of the neuro-degenerative syndrome are reminiscent of other neuro-degenerative diseases like Lou Gehrig's disease and Parkinson's disease. Basically it's like having every neuro-degenerative disease at once, telescoped into an unmercifully short period of decline. Fortunately, it is rare. Sporadic Creuzfeldt-Jakob disease occurs at about one per million populations per year. Also, somewhat fortunately, it's not a disease of children. The average incidence of CJD is in the 60s. There are three recognized forms of CJD. The most common being sporadic. This is a spontaneous onset of CJD in an individual for which we have no clue why they have developed it. There are familial variants, which seems to be passed as an autosomal dominant in families. That constitutes about 15 percent of the cases of human prion disease that occur. There are iatrogenic prion diseases, which are caused essentially by treatments and surgeries, well-meaning, but nonetheless transmitting the disease. Of course the transmissible spongiform encephalopathy that even my kids know is bovine spongiform encephalopathy, or so-called mad cow disease.
Since the early 1980s, this disease has affected about 200,000 cattle in the U.K. and Republic of Ireland, and a few hundred across continental Europe. About 2 million cattle have been killed in an attempt to stem the epidemic. This culling, as well as change in policies, such as feeding ruminant to ruminant -- we turned cattle into neo-cannibals -- is resulting in a rapid decline of new cases, predictions being that the epidemic in cattle may be essentially stamped out in the early part of the new millennium. I won't even say the new century. I'll say the new millennium. Unfortunately, this disease is unique, unlike every other known naturally occurring prion disease. It doesn't seem to obey species barriers, or at least obeys them to a much lesser degree. There is an outbreak of feline spongiform encephalopathy in house cats. There is spongiform encephalopathy in zoo animals, including primates. The primates that we are most concerned about are also vulnerable to this disease. To date, 44 people have developed a new variant of Creuzfeldt-Jakob disease, which is clinically and pathologically distinct from classical CJD. The statisticians predict there will be somewhere between a few hundred and maybe 80,000 cases. This does not include the chicken little predictions of the extent of the epidemic. The disease unfortunately seems to strike the young. There have been teenagers involved.
It is a relatively slower progression than classical CJD. There are clinical features that are distinctive, but I won't bore you with them this talk. The pathology is also absolutely distinct, including a preter natural accumulation of PrP Sc, which is this abnormal amyloid protein that's been linked to infectivity. This occurs both in the brain and in peripheral lymphoid tissues. Well, before we leave the clinical stuff about CJD and prion disease, I want to kind of set the stage with a sobering statistic, which is there is iatrogenic transmission of this group of diseases. Considering the penetrants and the young age of vaccinees, this is a scary possibility. This would dwarf every other iatrogenic transmission known to date. In humans, basically a few hundred cases have been attributed to iatrogenic transmission, from hormones extracted from cadaver pituitaries, from dura mater transplantation, which is the tough lining of the brain. But incredibly, the largest iatrogenic transmission known to date, also the first documented, was that in passage with a vaccine, which was a vaccine for looping ill of sheep. Formal and inactivated brain preparations passed sheep scrapie to about 1,000 sheep. So hopefully this will not be a pattern with human vaccines. Well here is the prion hypothesis. This has gone from being an object of ridicule to the middle of the road interpretation of prion infectivity. It has been sanctioned by the Nobel Prize committee, garnering the prize for Stanley Prussiner, the investigator whose ferocious work with this group of disorders and with this agent has given him, in my opinion, a well-deserved Nobel Prize. The basic tenants of the prion hypothesis are that there's a normal cellular protein, which is called PrPC, which has been cloned and recognized. It's expressed by just about every organism down to drosophila. It is a very old gene. It's incredibly well conserved in evolution. It is predominantly alpha helical in secondary structure. Now this normal cellular protein can adopt an alternate confirmation, which is rich in beta sheet structure. When this protein is in this alternate confirmation, it acquires many unique physical chemical properties. It becomes partially protease resistant. It tends to aggregate. It's very poorly soluble. Plus, it then seems to act as a catalyst for recruiting more confirmational copies of itself. Now whether this occurs by a kind of enzymatic confirmase activity or whether this is kind of a biological crystallization phenomenon is being actively investigated. But it is clear that this abnormal confirmation isoform of the protein, called PrP Sc for scrapie, is capable of recruiting more confirmational copies of itself from the normal cellular isoform. So onto vaccines. There are some concerns about vaccines. I will mention three areas that need to be considered.
I will dwell most of the time on cell substrates, which is nice of me considering this is a cell substrate meeting. I will also talk briefly about potential prion infectivity coming over in media supplements for those cells, and in excipients, which are compounds used to stabilize vaccines in their final formulation. In this case, luck seems to be at least partially on our side, because it's not easy to infect cells in vitro. It is possible to infect primary neuro cultures. It is even possible to infect neuronal cell lines neural blastoma. But there is not much infectivity, and basically each successful infection of a cell line is worth a publication or 10.This may be due to the fact that cell lines have very little PrPC, which is the precursor for PrP Sc. The conversion of the protein from PrPC to PrP Sc forms occurs at the surface or a post- surface compartment. So in general, cell surface abundance of the protein correlates with infectivity. Most cell lines in my own laboratory, including hela, express no more than one-tenth of the amount of cell surface PrPC that a primary neuron does. It has also been thought that the very act of cell division itself can kind of sterilize a culture because cell division can out pace the relatively slow conversion and processing of PrP Sc. So if you have a couple units of infectivity, they get progressively diluted by having huge numbers of cells that bear no infectivity.
Finally, those cells may die, the infected cells. There is also a poorly quanitifiable role for cell biology, which I put in quotation marks here. Things that we really cannot quantify at this point, like proper trafficking, post-translational modifications of PrPC that are important in conversion, and even sub-cellular distribution. The protein seems to accumulate in this glycosal phosphatindinol rafs at the cell surface. Some cell lines don't seem to support these sort of rafs. Now to make some kind of estimate about the spontaneous development of prion infectivity in a cell culture, especially a vaccine cell culture that may have hundreds of trillions of cells, I am going to back up and try to explore some assumptions about the spontaneous development of prion disease in humans, which is the species for which we have the best numbers. According to the prion hypothesis, an occasional accidental mis-folding of PrPC to PrP Sc is what triggers the recruitment process which proceeds on an exponential basis. Each molecule that's converted converts to more, da, da, da, da, da, da, which happens on a post-translational level. No genome involved. "Look, ma, no genome." But sporadic disease in humans is incredibly rare, one per million people per year. Humans have something on the order of 100 billion neurons.
So one can make the kind of interesting calculation that a productive infection arising from a single neuron, you need about 10 to the 17th neurons, 100 million neurons across a million people in order to develop spontaneous scrapie. But of course that's not the only way one could potentially develop spontaneous CJD. Discovered by familial CJD and familial prion diseases, there are mutations within the open reading frame of the prion protein that apparently predispose to this accidental misfolding, such that somebody with a prion protein mutation that actually results in an amino acid substitution is basically guaranteed of developing the disease over the course of a lifetime if he or she lives long enough. So could this occur in vitro? Could certain cells in vitro acquire a somatic mutation which is then propagated to infect an entire culture, again, on a post-translational level? Well, let's run some numbers on this one. The mutation rate in man is about one per billion basepairs per year. I thank Dr. Kazazian for yesterday for pointing me to this reference. Thank you very much. The prion protein open reading frame is really less than 1,000 base pairs. It's a relatively small protein that's all contained in one exon. This gives rise to a kind of pseudo calculation that a cell can develop a mutant prion protein gene, a cell in vivo, and a human can develop a mutant prion protein gene in about one out of a million cells per year, if you take one out of a billion and multiply it by 1,000, that's one out of a million. This, just as an aside, this gives a rise to a kind of startling calculation that all of us in the audience are generating about 100,000 prion mutants in our brain per year. However, there must be a safety factor here because the rate of prion disease arising from somatic mutation cannot exceed the calculation of prion disease arising from individual neurons that we just went through in the last slide, which is 10 to the 17th neurons per year. So incredibly, somatic mutation is a seriously flawed non-efficient process for producing prion disease. Something on the order of one out of 100 billion mutations are productive of infection. This may give us some comfort when we turn to the in vitro scenario. So let's talk about cell substrates. Is it possible that spontaneous prion infectivity could arise de novo in a culture? I told you that substrate cells have usually less than one-tenth PrPC than neurons. So if we run these calculations, by misfolding one substrate cell per 10 to the 18th years, and I think that is a quadrillion or something like that, it's certainly comfortably larger than the projected age of the universe. Substrate cells, however, are less genomically stable than primary neurons. If one says that there's say 1,000-fold greater rate instead of one out of a billion basepairs, one out of a million basepairs can be mutated per year in a substrate cell, this gives rise to a calculation suggesting that you need 10 to the 14th substrate cells per year in order to have one productive, i.e. spontaneous infectivity arising in a culture. Now this number looks incredibly large, but when we think about the numbers we heard last night about the production of polio virus from vero cells, according to my calculations, 300 million cells are used per year to generate all those vaccine lots. Ten to the 14th is only 100 -- sorry, 300 trillion, and 10 to the 14th is only 100 trillion. Did I get that right? Please forgive me and correct me if I didn't get it right.
So perhaps it is possible, considering the enormous scale of substrate cell culture, that prion infectivity could arise through somatic mutation in a substrate cell, and could contaminate a vaccine destined for human beings. Well, there are some things to talk about with this model. If this is so, how come we haven't seen any vaccine transmissions yet? One of the factors is that very few human cell line vaccines have. gone into humans, certainly not on a scale of vero cells or primary cells that are used for culture. Another unsettling thing is that if indeed there is somatic mutation in a culture of human substrate cells, would we ever detect it? We are talking about something that would occur in one out of a million cells or even one out of a thousand cells, would ever be able to find by PCR or SSEP or anything you could think of, a mutation at this level. So aside from substrate cells, I did want to touch upon a few potential sources of infectivity.
The media coming in contact with substrate cells are potentially carrying prion infectivity. Bovine serum, fetal calf serum, and newborn calf serum is used as a supplement for proteins and growth factors and hormones for most cell lines. Some cell lines are also supported by human serum albumin. I am not aware of a vaccine cell line that's supported in this manner, but many recombinant proteins are supported with human serum albumin. There's also potential prion infectivity in excipients, this last compound that's added to the preparation to keep it stable before use. Many childhood vaccines are stabilized with pig skin gelatin. Pigs don't seem to be a species which spontaneously develop prion disease or a species that can catch prion disease via the oral route, although deliberate intracranial injection of BSE infectivity can produce a prion disorder. Human serum albumin is also an excipient in measles, mumps, rubella, and rabies vaccines. I would like to spend just a few seconds talking about human plasma proteins before I close, and give my final advice, such as it is. Human serum albumin of course comes from humans. Of course it's a plasma fractionation product. There has been a great deal of work trying to identify potential risks of transmission of CJD from human to human through blood or blood products. Suffice it to say that population studies, case control studies, and cohort studies have proven universally negative. There is no documented incidence of human CJD, classical CJD being passed through blood or blood products. There are of course case reports of people getting a transfusion and developing CJD, but one should not expect that transfusion or administration of a blood product is protective against CJD. The incidence of CJD in the transfused or treated population is the same as in the non-transfused or treated population. However, we're in more difficult territory with variant CJD. This is, I told you, an unusual agent. It seems to cross species lines with impunity. There are other features that are quite scary with regard to human blood and human plasma products, including albumin that might be used as an excipient. The agent starts of course in the periphery through oral exposure, suggesting a prionemia. There is a huge accumulation of PrP Sc, our only surrogate for infectivity, our only biochemical surrogate for infectivity, in not only brain, but in lymphoid tissue. The agent itself has odd properties. It is stable across species. It may in fact be specialized or selected. I realize that these terms are not often applied to a protein. I'm borrowing terms from agents that contain a genome. It suggests that this agent may be more virulent, especially with regard to peripheral exposure. In other words, one unit of classical CJD infectivity will not cause disease when injected intramuscularly. One unit of variant CJD infectivity may very well. There is no data. Another thing that has been noted is the young age of onset of variant CJD. This has been attributed to kids eating hamburgers and all kinds of weird meat products. But it could also be attributed to host factors which would promote infectivity in the young. Since vaccinees are usually young, we have to take this in mind. But our greatest risk factor here is that this is a new disease and we have no data. So how do we minimize the risks from vaccine transmission? Basically there's three ways that I can think of. I’m sure that other people can think of more. It is
important to add prion validation to the list of agents and microbes which are tested for in vaccine lots. This could be done two ways. The biochemical marker of infectivity is PrP Sc. This technology is in evolution. It appears that capillary electrophoresis, some types of optimized immuno blotting, and even ELISAs are reaching the point where one unit of infectivity will be detectible. Another important way of assay for infectivity is called the bio assay in the field, in which selected samples are injected into a species which is capable of supporting that infectivity. That would be non-human primates, and again, a technology in evolution, transgenics engineered to express human and perhaps bovine PrP. There is also the possibility of trying to prophylax cultures, substrate cultures with chemical agents.
This is also in evolution, but the classic molecule in this regard is congo red, which not only seems to bind to PrP Sc, but seems to dissolve infectivity in vitro. New discoveries out of Byron Kohe's lab that tetrapyrrole, including porphyrins and phthalocyanines, can block infectivity. Perhaps some of these compounds can be utilized at appropriate concentrations to use as a kind of antibiotic for substrate cultures. Finally, the slam dunk in this area would be to develop a cell line that lacks a prion protein gene. The prion infectivity, whatever the hell it is, seems to be absolutely dependent on the presence and expression of PrPC. So if one were able to ablate the prion gene out of a substrate cell, that didn't come with 300 other bad pathogens, this may be a strategy of obviating any prion infectivity in vaccines. So I would like to summarize by saying that it is possible, although not favorable, for substrate cells to be infected with prions. It is possible, considering the huge bulk of cells that are cultured, 300 million a year for one vaccine, that prion infectivity could potentially emerge by misfolding and/or somatic mutation in vitro. I will note that prion components and excipients may contain prion infectivity. Although this is an old story with regard to classical CJD, we don't have the information for the BSC variant CJD agent. We should worry, at least for the time being. The remedies for this are selective sourcing, avoid animals and people that could potentially be brewing prion infection, biological manipulation in vitro, including anti-prion agents, and maybe ablating the prion gene, and then validate, validate, validate. Prion infectivity should be added to the list of infectivities that are excluded in vaccine lots. I thank you for your attention, and I would be glad to answer any questions.
DR. KRAUSE: Phil Krause, FDA. In keeping with the idea that one presumably wants to find cell
substrates which carry the least risk, I guess
if one presumes that tumor cells have a greater risk of genomic instability than
non-tumor cells, are you then implying that there's a greater sort of
spontaneous mutation than prion risk from tumor cells than for instance primary
or diploid cells?
DR. CASHMAN: That's a good question. I guess this is basically not quantifiable. If one takes a rock solid cell that enjoys all kinds of DNA repair mechanisms then yes, that is less likely to give rise to the mutation in the prion protein gene. One area which should be investigated, I'm realizing from this meeting, is to take some cell lines and look at 1,000 clones a piece and see if any of the prion copies have acquired mutations. So this would be a piece of data that we could use to actually discuss this issue. Right now, I don't have any.
DR. ONIONS: David Onions. Could I just ask
the converse of the question that you posed for vaccine substrates, which is I
think one that has been discussed. That is, the idea of knocking out the PrP
gene. We know that PrP mice are viable, so it looks like perhaps an interesting
way to go. But can you also engineer cells over expressing normal PrP and use
those as substrates for infectivity? You mentioned that one of the problems was
the low level of PrPC in most of the cell lines you have used. Can you not
over-engineer cells so that they become susceptible?
CHAIRPERSON RABINOVICH: Modifier genes.
DR. CASHMAN: Modifier genes, yes. Modifier
genes have of course been proposed from animal studies of infectibility and
experimental scrapie. Dr. Prussiner has hypothesized a protein X, which may be
a receptor or may be a chaperon that somehow modifies susceptibility of an
animal to prion diseases. There is also a protein Y that Dr. Prussiner has
hypothesized. I agree with you from the bottom of my heart that there will be
modifier genes affecting susceptibility to prion diseases and the propagation
of prion infectivity in vitro. But we don't know what they are yet. At the
crude operational level of being able to infect cells, yes. We can infect cells
in vitro. So at least some of those modifiers have to be there. Did that make
DR. HAYFLICK: Hayflick, UCSF. I was intrigued by your observation that the species barrier for prion transmission is less, is reduced between non-human primates and humans, which would raise some additional concern about the use of primary tissue, and particular, primary monkey kidney tissue for the production of human virus vaccines, because contrary to popular belief, that tissue and any primary tissue does not contain -- and I'll use primary monkey kidney as an example, only cells that are derived from a particular part of the kidney.
A primary monkey kidney culture consists of an enormous variety of differentiated cell types that compose the vascular system and neurons. So that monkey neurons do play a part in the production of polio virus, for example, derived from monkey kidney. So that I think that it's important to mention that neurons are not only a part of brain tissue in considering various cell substrates. Also I was wondering whether there's some reason why you omitted the mention of trypsin as potential source in your discussion of substrates or media supplements for prion transmission. I didn't see trypsin as a component. Was there some reason for that omission?
DR. CASHMAN: Just blanking out. So thank you for adding that to the list.
DR. CASHMAN: Say it again. I'm sorry.
DR. HAYFLICK: Normal human cell populations have been used for the production of about three-quarters of a billion doses of human virus vaccine world wide, but these are not continuously propagable abnormal heteroploid cell populations. These are normal finite lifetime cell populations.
DR. CASHMAN: So these are cell strains?
DR. HAYFLICK: Yes, as I defined them. I realize there's a problem in understanding these terms.
DR. CASHMAN: Which vaccines?
DR. HAYFLICK: Virtually all pediatric vaccines, polio, rubella, mumps, measles, rabies, adenovirus, some rhinovirus vaccines, are all produced on a semi-continuous human diploid cell strain like WI38 or MRC5.
DR. CASHMAN: Thank you.
CHAIRPERSON RABINOVICH: I think you better clarify.
DR. LEWIS: Yes. To my knowledge, there are really no nerve cells in the kidney. The nerve cells lie on the spinal cord and porosises go down there. I don't believe there are any nerve cells in the kidney. Even if they are, once the nerve cell is differentiated, they basically do not grow on tissue culture. I think that needs to be corrected.
CHAIRPERSON RABINOVICH: Okay. I would like to move rapidly to bring the panel members up to the podium, and to invite Dr. Onions to come over and run things.
DR. ONIONS: I notice that we now have actually 45 seconds for discussion according to the program. (Laughter.) Brilliant as this panel are, I don't think they could do that. So could I have some guidance on when you would like to close this panel session? Perhaps somebody could give me some guidance.
CHAIRPERSON RABINOVICH: Forty five minutes.
DR. ONIONS: Forty five minutes. Thank you very much. Okay. We were charged in this panel to answer a number of questions. I'll come to those and try and go through and cover the areas that the panel will discuss. I would be very grateful for as much participation from the audience as possible. I thought it was just useful to pick up two strands that I think came out of some of the comments yesterday. One I think that's important to make, and that is that vaccine production is a very pragmatic process, and that once there have been lots of theoretical objections to particular cell substrates, particularly autogenic cell substrates, there are very practical reasons for the use of cell substrates that might be immortalized or neoplastic from a new generation of vaccines. I don't think we should lose sight of that. There are very practical reasons in scale up and use that I think we should bear in mind to produce therapeutic vaccines. The second concern was that came out, and perhaps didn't get enough airing as it should have done, and that is there clearly is a trend in society at the moment about concern in safety of vaccines. That perhaps therefore focuses particularly on the item we are going to discuss today, which is the possibility of adventitious agents. The question that the FDA asked us to evaluate, or at least one of the first questions, and they would like the panel to take a look at is do neoplastic cells represent the greater equivalent or lesser risk for the presence of adventitious agents than primary cells, diploid cells, or non chunogenic continuous cell lines. I am not sure, given my previous comments, whether I think necessarily that you can answer that in a simple sense, but does anyone in the panel want to sort of pick that one up first of all? What I could perhaps do is to prompt people, is perhaps to put up those which is just my suggestion, of some of the factors that might influence the risk of adventitious agent testing in a variety of cell substrates.
am not sure whether they will be necessarily better than human diploid cells or anything else if we even get a decent banking system going. It seems to me that when you get to that kind of stage, it's the concern about how you find what's in there rather than anything else. I think the actual extent to which you can characterize them is clearly to me, it's very similar.
DR. ONIONS: That would be a generally universal statement that primary cells are likely to be more difficult to characterize and therefore, if you can use a cell line, that is probably the way to go. But I think it's also worth making a countervailing point that there are still vaccine strains that are very successfully produced in primary cells. There are others that have been passed in primary cells and therefore change them into a cell substrate, the genetic stability of them. So there are nevertheless countervailing arguments. I think the statement is correct. That is, adventitious agent testing is clearly going to be more in the region –
DR. ROBERTSON: Another point which one could consider. Where neoplastic cells might be considered more susceptible than primary cell cultures, in that the primary cells are derived specifically for vaccine production. Whereas the neoplastic cells have probably been kicking around at least one, if not several laboratories, before being put into use as manufacturing of vaccines. Because of that, they may well have picked up something that you don't want to be there. Nothing to do with the cell type or the origin of the cell. So a virus of some different species all together which you really have got to check for. So if you are actually setting up a cell bank of a neoplastic cell, you shouldn't just be considering species of origin of that particular cell, whether it's porcine, human, murine whatever. You have really got to consider any virus under the sun. We know there have been instances of this happening. This morning there was the comment about I think it was a human endogenous retrovirus which was actually of murine origin, been picked up from passage somewhere.
DR. ONIONS: There is a good example in a commercial product. That of course was the Glaxo Wellcome's novalma cell line which was used to produce interferon, which in fact contained SNRV, and probably picked up SNRV in George Kahn's lab at some point during its history, I think was the general consensus. But clearly that was unknown and the whole system was used in the production for several years before it was realized that perhaps this was contaminated by an adventitious agent that you just would not expect in this cell line. So I think that is a very good point. There are issues about tumor cells. One of the things that occurs to me is that -- actually it does concern me quite a lot about adventitious agent testing. That is that it is rather traditional in character still. It is not very directed in terms of its specificity in looking for certain viruses. That is changing I think, but I think until recently that has been the case. For instance, we have known that in certain tumor cell lines, that viruses that we have only recognized in the last decade are certainly found. For instance, well HHV-6 isn't a transforming virus, but there are cell lines that carry HHV-6 that have been used in the lab for many years. The same is true for HHV-8, which is a transforming virus. So there is a concern that we may have cell substrates that are contaminated by other tumor viruses. Tom, would you like to pick up?
DR. BROKER: I think we have actually a wonderful opportunity for a so-called natural experiment. That is solid organ transplantation. It turns out, as we all know now, virtually all kinds of organs, not only the corneal we have just heard about, but kidney, liver, pancreas, part of the intestine,heart, lung, so forth, have all been transplanted. I think the opportunity is that the recipient is invariably immuno-suppressed until the transplant takes, and then they are slowly weaned off the drug like cyclosporin. Yet on other occasions, the transplant fails for one or another reason. One could go back into failed transplants to look for the reactivation of agents that came from all these different tumor or tissue types I mean. One example I could cite that we recently encountered in the course of our kidney transplant study is a pair of kidneys that went in from a five-year-old boy to a 19-year old female. Within a few days, the kidneys had completely become destroyed,necrotic. It turned out -- they suspected CMV infection, but it turned out to be adenovirus. The resumption, and I'm being completely hypothetical, is the five-year-old boy who had died in a bicycle accident, the donor, probably was in the age bracket where adeno was just a natural infection in his airway, and that these cells say from his tonsils or adenoids, which were in the midst of processing the adeno, became circulating, were in the kidneys, and the recipient female then acquired adeno-infected kidneys, and upon transplant to her, the virus reactivated and just wiped out the tissues. I might also say the different individual who received the boy's liver also lost the liver. So presumably these were entering through B cells that were in any of these remote organs. Nonetheless, the basic opportunity to look at organ recipients I think is the experiment to ask how much infectious agent is being transferred.
DR. RUSSO: Carlo Russo from Merck. I
think as you indicated, these patients are profoundly immune-suppressed.
Therefore, is going to be very difficult to assess where this agent came from.
In your case, it's very well possible that the woman was exposed to adeno
virus. Since she was immuno- suppressed, that's the reason why she got the
DR. ONIONS: Could I just actually take a backup actually? I was about to go back to the primary cell issue again. Phil gave I thought a wonderful presentation. I hadn't heard parts of this before. It actually started to worry me a little bit actually. To what extent do you need now to control the kinds of colonies of these particular primary colonies? I'm not sure, I mean I don't know what kind of testing goes on in these colonies for a range of adventitious agents. Can you maybe just comment on that? I mean are we dealing with inverted SPF animals?
DR. MINOR: Well with respect to the primate, you will certainly not. But they are increasingly heavily monitored. It depends very much on the manufacturer and how much monitoring they do. One manufacturer, for example, has only recently, well in the last four or five years I guess,started using colonies of monkeys that were monitored for foamy virus. The result of that is being revolutionary in terms of the number of cultures that you get surviving to production. You would have thought you might have started this a bit earlier perhaps. But you couldn't call them SPF, but they are increasingly closely monitored I think. Certainly some manufacturers have them more closely monitored than others. But one of the difficulties with the whole of adventitious agent business of course is you only really find what you are looking for. That's an ongoing problem. Things like chickens are a different matter. I mean I think this would establish what you need to do to make an SPF chicken colony. But primates are much more tricky.
DR. ONIONS: There are other cell substrates out there that are used, cells like primary hamster kidney cells in JV vaccines and various other things. So there are I think other vaccines out there that are going to come to attention because they use primary cells. I think we perhaps ought to start thinking of the kinds of procedures that are needed,like closed colonies and embryo derivation of these animals in some cases. Could I move to perhaps the third element. That is, we have heard a lot about retrovirus. Retroviruses always come back to focus when we deal with cell substrates. What is the panel and the group's feeling here in general about the concerns of using either immortalized cells or transformed cells, because frequently those -- well, that's not an accurate statement. Activation of transcription of endogenous genes is more frequent in such cells. Is that of concern or not a concern? Or do we have to go cell by cell, species by species, to answer that question? John, would you like to make a comment?
DR. PALLEY: That's a problem, in that you will
I guess you will have a hard time finding a human cell line that would not
express any human retrovirus, so I report that it's the case for HERV-K, the
special HERV-K family.
AUDIENCE MEMBER: Sounds good to me. But
just to push this point a little bit further on the HERV-K, you know, about
two-and-a-half years ago, there was a report in Cell, which is a better journal
than I usually publish in, which claimed that HERV-K env can act as a super
antigen that then stimulates diabetes mellitus in some people.
AUDIENCE MEMBER: Okay. I think it would be worth explaining how it's been modified. DR. ONIONS: I think the retraction is in fourth hit. So before you sort of put that out as a paradigm.
DR. LOEWER: I think the main point is that a couple of groups tried to repeat this data, and they were not able to repeat. So it seems not to be specific and even not effects on the T cell lines could be repeated. So let us depart from this idea.
DR. ONIONS: Can I maybe get your opinion? I really would like to get some feeling because we
have heard a lot, and it's scientifically really interesting by the expression of these human endogenous retroviruses. I think John has probably just summarized it. It looks like we're saying the list, this collected group here of retro, are saying that as far as we are aware, at the moment these are not of concern and uninfectious, and probably therefore there is not a great deal of point in looking for expression of these in cell substrates, the very pragmatic practical point. Would that be your opinion too, Johannes? Johannes is nodding. That's a "yes," I assume.
AUDIENCE MEMBER: From a research standpoint, it's absolutely worth pursuing to see if one can find these things eventually. But in terms of vaccine issues, I don't see how we could possibly deal with it now.
DR. SCHUEPBACH: Yes. I also would like to make a comment regarding that super antigen activity because we are coauthors in that paper. It is true that the presence of these sequences, RNA sequences in the serum we can not repeat, so it's not specific for IDDM patients. But to my knowledge, our data regarding the super antigen activity and the stimulation of VP cells has not been disputed by any other group. So that is still around. I think that the real important topic here is whether these endogenous viruses actually give rise to infectious particles. I believe that with the PERT assay, we actually can contribute very much to this question. I think, as I pointed out, of course the easiest thing is to test the super natants for reversed inscriptase activity. But I think with a little bit of additional work, it should also be possible to define that profile of RT activity and cellular, DNA polymerase activities along the different fractions of the sucrose gradient and tend to recognize any abnormal pattern that might be associated with infectious rate of viruses.
DR. HENEINE: David, I have a comment which could be redundant, but go ahead and say it. While thinking about all these questions from my simple mind, it looks like if you want to compare cell lines versus primary or diploid cells, the two questions that were raised is which ones transmit less adventitious agents or transmit less neoplasms to vaccine recipients. What we have heard so far about the mechanisms of the neoplasms, many of those are mediated by viruses or viral-like elements. So it looks to me that the majority of the concerns are rising from the adventitious agent group rather than from other elements. So therefore, in trying to make up our mind, based on the available data, which one is the more suitable substrate, maybe we can go very simply with a checkpoint list on these different cells, targets, which one we can test for the presence of these adventitious agents known, unknown, and which can be better monitored, which can be for practical reasons of culture as well, and make up our mind, rather than jumping right and left with different issues. If you can say cell lines, primary and diploid, and then go one by one, all these concerns that we have been talking about, and say which one is more suitable for each of these points so that we can conclude at the end. I mean it's one suggestion.
think at this point in time that you could dismiss the use of the PERT assay for looking for adventitious retroviruses or any type of retrovirus activity in there. So the question is going to be, when you find something, what do you do about it if the assay is positive?
DR. ONIONS: Could I just go to Arifa? She has been waiting very patiently. Then Steve.
DR. KHAN: Yes, thank you. I think it is
important to clarify the word "expression" in terms of human cell lines and
human cells. I think we all expect that there will be some RNA expression in
the human cells from endogenous retroviral sequences. However, I don't believe
that you are going to get particle production in the majority of the cells under
normal conditions. So therefore, I think the use of the PERT assay would be
very helpful to evaluate particle production from human cells, which can then
further be investigated for infectivity, as opposed to looking at RNA
expression, which I think you would find at some level in all human cells.
DR. HUGHES: I would argue that is with a
particular assay that has been tuned up to detect RT in a particular way. I
would be willing to wager that if we look hard enough, we could certainly find
evidence of particle production in any of these cells,simply because they are
full of endogenous viruses that -- I mean the very fact that there's obviously
expression is RNA present.
DR. ONIONS: My only caution about that is that you can make assumptions about affectivity that also are not true. Since Clive Patience is here –
DR. HUGHES: I think you have to do the test. I don't think you can make assumptions about it.
DR. ONIONS: Well, the point I was going to make actually was that Clive's group and our group showed that you could actually infect cells with PERV, yet those experiments have been done 20 years ago, and been done by very good people, including George Tadai, and were unable to show infectivity. It's just the techniques have changed slightly and we could get infectivity. So I think that you are making -- there is a straight yes or no about infectivity. That is not always the case with these retroviruses.
DR. SHEETS: Can I ask a very pragmatic question of Dr. Hughes?
DR. HUGHES: Sure.
DR. SHEETS: I'm Becky Sheets, FDA. What I hear you suggesting is that rather than testing for RT activity by a physical assay, as you called it, a PERT assay or a conventional RT assay, you think that it would be preferable to test vaccines for infectivity assays?
DR. HUGHES: The ability to transmit that RT assay to a reasonable recipient cell. I believe this is exactly what John –
DR. SHEETS: The pragmatic question is that would you do this testing lot by lot on vaccines? For instance, if you were making a vaccine in a primary cell substrate, for instance an egg, would you test each lot of vaccine or each batch of vaccine for infectivity assays? Then the really pragmatic part of it is, if you are making a flu vaccine, where the timing of production, the timing of testing, and the timing of lot release is very tight, would you recommend these infectivity assays on lot by lot for primary source?
DR. ONIONS: We're running out of time. Do you want to answer that? You have been asked a question, do you test lot by lot?
DR. HUGHES: Very simply, if we're talking avian systems, I think there are reasonable ways of determining that endogenous avian viruses are not infectious. My personal bias, and I mean it no more broadly than that, is in the case of avian viruses, as long as you carefully establish that the avian viruses that are present are not infectious for human, that that's not necessary. But that is my prejudice.
DR. SHEETS: That's fine for SPF situations. I am asking this question because this is what sponsors ask FDA. So they want to know do we need to do this lot by lot, or if a cell bank, you can do a one-time characterization. Of a primary system, you can't do it that way.
DR. HUGHES: Use SPF chickens and don't ask. (Laughter.)
DR. LOEWER: I would like to make a comment to Dr. Hughes' comments. They are very sound in a scientific meaning, but they face regulatory problems, the main problem indeed. Regulatory authorities have to show that there is no infectivity and the proof of non-activity is always nearly impossible in a scientific sense. You will always find reasons to say you were not able to find infectivity. Look at HIV. If you would use MRC-5, for example, or a lot of other animal cells, you would never find infectivity of HIV. The same is true for many situations. So there a fundamental problem is to test for non-activity or noninfectivity.
DR. ONIONS: I would like to stop this because we are running out of time and there are other issues. I am going to take the Chairman's privilege and just say that I actually think you need multiple techniques, because I think as Johannes has just said, if you have complete infectivity, you will miss things as we would have missed cell lines producing ver. I think you really need to have a combination technique. So I think it's a belt and braces situation. That's a personal view. What I would like to move onto, is were asked by the FDA also to consider species of origin. I think really you end up in very general statements here. You can argue that if you are worried about adventitious agents, then clearly there are species barriers to the transmission of some agents. On the other hand, other agents do go across species barriers, sometimes in abortive replication. They can be very nasty. We of course know that herpes B going across species barriers is actually lethal. Ad 12 in hamsters is oncogenic. Equine herpes veras, which is an alpha herpes veras is oncogenic in hamsters. There are natural examples of cross-species transmission, the ovine herpes veras 2 is innocuous in sheep, but it kills cattle. So there are examples of these heterolic transmissions being worse than natural infections. Is there anything that we can say, the FDA have asked us, in a general statement about species of origin? My own view is I don't think you can, but does anyone want to make a statement?
DR. MINOR: I think sometimes it is better and sometimes it's worse. (Laughter.)
DR. ONIONS: Yes. That's exactly what I think. Thank you, Phil. I would like to drop the discussion now, because I think that sums it up. I'll turn the phrase back on the edge and say it's a case-by-case, it seems to me. I don't want to trespass really on yesterday's, but I think maybe just to come back to -- we're going to move onto assay systems in a second, but I think one of the issues we're coming around to in a second is latent viruses, because those seem to be the real concern. It may be worth just remembering some of the things that were partly discussed over the last two days. That is, that the complementation of defective viruses can occur. For instance, adenoarectus can be complemented by HPV in hela cells. We talked about psuedotype formation both today and yesterday. I think just I would like to make the point about pseudo formation. We talked a lot about retrovirus retrovirus pseudotype formation, but this can occur across viral species. For instance, paramyxovirus is rather badly, but they can, pseudo type retro viruses. So you could alter the host range when endogenous agent is expressed in your cells. Of course there are recombinants. Some of these recombinants, and we have got representatives who did the work here, interesting recombinants like SV-40 adnivos recombinants. One of my concerns, we'll come on in a second, I think is like polyoma viruses in cell substrates and that potential for interaction with other cells. So if we can take that as a kind of background, can we turn to a question of -- this had come up and was discussed by several candidates, unknown viruses. What are the potential candidates and what kind of systems do we use to try and go looking for those unknown viruses. Anyone want to comment on what we should be doing about novel cell substrates and you have got a virus there that you don't know anything about. What sort of technique should we be applying?
DR. ROBERTSON: This is potentially the most important door, also the most difficult to deal with. If you think back to what Phil was saying in the first talk this morning, all these instances of viral contamination, generally they occur with viruses unknown at the time, viruses expected, the presence of viruses in vaccines or biological preparations. Potentially is not something we're talking about today, it's not an endogenous virus or recombination between an endogenous virus, but something unknown that's going to leap up at us out of the dark. Of course almost impossible to deal with. But Joerg was saying this morning about this is what we should be looking out for, the unknown, and if possible using a more broadly reactive type of assay rather than highly specific type of assay to look for something. If we knew to look for something, that's fine, we can deal with it. It's what's not there which causes the problem.
agents are really much better if they are present at high concentration. So I don't know whether you accept that concept of cellular cloning in order to either get rid of these agents or to have them at the very high concentrations so that their detection is actually much easier. In the meantime, you can try to activate the host cells by all kinds of different agents. You do EM studies, you do serological studies, use broadly cross-reactive antibodies. I mean this is a wide field actually of methods you can employ. So I think using such an approach, we should actually be quite capable of detecting such agents.
DR. ROBERTSON: No.
DR. ONIONS: They might be useful in establishment of a master cell bank or something, you know, the first one. But I mean I would criticize, I don't think that current infective assays do pick up everything. I think that's the whole problem. I think, for instance, that it would miss -- well, polyem virus has perhaps been used.
DR. ONIONS: I agree. Can we move on, because I would like to just cover TSEs just before we have lunch time. I'm desperate for some lunch. If you have got something, sorry to inhibit you. If you can be brief.
AUDIENCE MEMBER: I agree completely with what
John says, but would add that if we are faced with a decision of whether to
approve the use of different types of cell substrates that are tumorigenic or
derived from tumors from which we don't know the mechanism of transformation, we
are faced with not only the question of should technology be applied, but is the
technology as it exists today and can be applied today, good enough to permit us
to say that it's okay to use these cells. So that is a very practical question
which perhaps could be answered.
DR. ONIONS: Okay. I would just like to finish up, because we heard a really I think important interesting talk from Neil. While perhaps the risk of spongiform encephalopathies in the kind of cell substrates we are concerned with is probably extremely remote, the consequences of being wrong about this issue are potentially devastating. So it is certainly worth cautious consideration.
Really I think Neil in his talk, already summed up these key issues about the possible origins, are the mutations spontaneous or infection, and the kinds of cell substrates of concern might be, it seems to me, are the neuronal cells. Since it's recently shown that in the peripheral ntroduction of TSEs, the B-lymphocyte might be important to carriage, then perhaps lymphoid cells, particularly B cells that are invariably used, and since we're looking at the possibility of using HIV and T cells, maybe that suddenly becomes an issue. Maybe we should be looking at lymphoid cells for the potential of there being spongiform encephalopathies. Which brings you back to the question that Neil finished with. That is, what should we do? It did strike me that one of the possibilities was that there are now very good, very interesting new cell lines being based on retinal cells, which we heard from Dr. van der Eb and others, which look very, very promising for the generation of anti-viral vectors. But as they have, as I understand it in theory, I mean just at the simplistic level, should not one thing be done and just sequence the PrP gene in that? The probability of having a key mutation seems to me extraordinarily remote. But then it's a relatively simple thing, cheap thing to do is to go and sequence the PrP gene. Is that something that we should do in that kind of a situation? Should we also do that in T lymphocytes? It's a trivial thing to do?
DR. CASHMAN: Okay. You can't cope with somatic mutation, I agree. But certainly one can cope with a mutation that's in every cell, yes.
DR. ONIONS: Neil touched on validation technology. I think that's important. I think there are new techniques for doing validation of TSE removal, but using a disrupted PrP protein. But I don't think that's going to be applicable to quite a lot of the processes that are used to produce vaccines at the moment. It is by technology products, but not to vaccines. I just wanted to touch on testing because I know that Neil has an interest in that area. It seems to me that we were sort of rather optimistic a couple of years ago, and indeed, there have been publications by Bruno Esch and others on specific antisera of PrP Sc. But those haven't held up. They actually pick up aggregated protein and not, as I understand it, strictly PrP Sc. We can use, and we have been using, treatment of protease followed by immuno blotting. It certainly works, but it isn't that sensitive. The problem, it seems to me, is that we really don't have a specific test that's an in vitro test. The only thing that you are left with, at the moment, it may change, but at the moment is animal inoculation. Would you like to comment?
CHAIRPERSON RABINOVICH: Thank you, Dr. Onions. There is a light repast outside for those that have been so patient. I would like to get everybody back in here in 15 minutes. For those of you who would like to avoid the wholesale garage sale that's going to go with your luggage, for those of you that haven't checked out of your room, I encourage you to complete that now. Thank you. (Whereupon, the foregoing matter went off the record at 12:33 p.m. and went back on the record at 12:53 p.m.)
CHAIRPERSON MYERS: Back to order. I would like to introduce a co-chair and a new person we are very pleased to have attending the meeting, Dr. Gary Nabel, who is the new Director of the Vaccine Research Center at NIH. So he is going to join me in chairing this session. I am probably going to disappear before the end of the session to make a plane. The first night I got here, as some will recall, I came in a little late. I ran into a couple of you in getting a beer because it was after the time of the close of the meeting. The discussion ensued as to what is a designer cell substrate. What do we mean by that? My first reaction to that of course was it's anything that I happened to have made. Clearly at this point in the meeting, it's not primary cells.
I suppose from a strictly semantic perspective, it would be a cell substrate created with specific characteristics. It could be immortal or not. But I think over the last couple of days, at least my thinking on this and for the purposes of this discussion, by a designer cell substrate, we mean a cell substrate of defined origin and with a defined pedigree. It is probably immortalized because it is likely to have been cloned. It will be validated as specific pathogen-free and at least specific pathogen sought and perhaps in certain circumstances, defined as non-infectious. For the purposes of the next discussion, we are really talking about immortalization. Jim McDougall, as you know, presented his paper yesterday. So we'll start this session with the first paper by Dr. John Sedivy from Brown University, who will talk about differences in the capacity to immortalize rodent, primate, and human cells by tissue culture passage or viral transformation.
DR. SEDIVY: Thanks very much for the invitation. I am sorry that Jim gave his talk yesterday because -- well, maybe it will jive all together. I was asked to give somewhat of a historical overview on the issues of replicated cellular senescence, and obviously the topic of cellular immortalization. So from a historical point of view then, this is the Hayflick phenomenon. This experiment has been performed in numerous labs and always with the same result. This happens to be an experiment in my lab. You will see a number of slides like this from me today. What we're plotting here is replicated lifespan, the doublings of the culture versus days. We see a culture growing and then reaching a non-proliferative plateau. This is what we define as senescence. Really the interesting point here is that the correlation here of this plateau is with the number of cell divisions as opposed to chronological time. The question that has been plaguing this field ever since its inception is well, is this really some type of a terrible artifact. I don't really want to get into this discussion. It really revolves around the issue of media and media artifacts, and have these really been adequately resolved today. I don't think they have, especially for some more specialized cell types. I think they have been pretty well resolved for keratinocytes, maybe breast epofelial cells, fibroblasts, et cetera. One really has to keep in mind that if one sees a culture that is slowly declining in its proliferation, this could simply mean that increasingly a larger and larger fraction of those cells are withdrawing from the cell cycle. This could be perfectly explained by inadequate culture conditions, such that eventually on the macroscopic scale, the culture has ceased proliferating. There are really three arguments that have been used historically to justify the claim that replicated senescence is a biologically interesting phenomena. Here we are plotting, again very simply, the mean-like span of a species versus fibroblasts replicated life span in tissue culture. As you can see, there is a rather striking correlation, such that animals that don't live for very long don't have cells that live for very long in tissue culture. The next phenomenon that one often sees cited is the age of the donor plotted against -- here is the age of the donor, and the remaining life span of the cells, in this case fibroblasts taken from that donor. As you can see, the points are all over the place. In fact, more recently, this view has been challenged by a recent paper in PNAS from Vince Cristofalos, who actually claims that this correlation doesn't exist. But if you read the literature, you will see this coming up over and over again. The one fact that seems to remain, at least to my knowledge, and that is if you look at these points down here, these are fibroblasts taken from individuals that suffer from premature aging syndromes. These are called progerias. Typically, these cells have a very short life span. So this really is the issue here. How do we differentiate between senescence, quiescence, and differentiation. I think that for the purpose of discussion today, this is really not a point of major interest, but for historical reasons I'll go through it rather quickly. Quiescence is defined as a reversible process. So what we are talking about here is essentially a cell cycle phenomenon. That is, we can have a culture that is cycling or contains a large fraction of cycling cells. Then these cells can withdraw into the quiescence state. Then when they are induced with the proper growth factors, and here of course the key phrase is what are the proper growth factors to elicit this phenomenon. At any rate, we are talking about a reversible process. Whereas senescence by definition is irreversible. So then of course the very interesting next question is how do we differentiate senescence from terminal differentiation. I don't really have answers here because in many cases, this is very difficult to do in many specialized cell types. What one would like to see in general is the absence of features that are characteristic for terminally differentiated cells. But this is not possible in many cases. So really this has given the impetus to a search for molecular events. So then if we pose the question are there molecular events that are unique to senescence versus quiescence versus differentiation, again, the picture is not very clear cut. I don't want you to absorb this whole slide. Suffice it to say that this is well, not all, but the major part of the regulatory circuitry in G-1. Here you see the D-type cyclance. CDK-4 and CDK-6 driving RB phosphorylation, which in turn drives the second phase, which is cyclin E production, activation of CDK-2. Of course there are a lot of modifying proteins here, CDK inhibitors, kineses that activate the basal CDK kinase, et cetera. Now this is an area that is receiving a lot of attention. The general theme, at least to me, it seems that there's a high degree of overlap between mechanisms that regulate quiescence, senescence, and differentiation. I don't think this is really surprising because all these three states are characterized by the absence of cell cycle progression. In most cases, by an arrest in the G-1 or a G-0 state. The one central theme is that the regulation of cyclin dependent kinase activity is necessary to achieve a physiological cell cycle arrest. In addition to the cyclins, which are the positive affecters, there is a number of CDK kinase inhibitors that have been shown to play a key role. The two major inhibitory pathways that act on this basal cell cycle machinery are the RB pathway, shown here, and also the pathway regulated by the tumor-suppressor protein P53. In both of these pathways, CDK kinase inhibitors have been shown to play key roles. So let me turn to the issue of immortalization. We all know that senescence can be overcome because quite obviously, there are many cell lines out there that are very immortal.
So in a very simplistic and general sense, we can think of cell culture in three broad categories. We can have primary cells or cell strains that have a limited life span and senescence after several passages. We have a category of cell lines that are immortal, not necessarily by the 3T3 protocol, but in general, they display the characteristics of unlimited lifespan, non-malignant phenotype, and in most cases by the ability to become quiescent. Finally, we have the large group of cell lines that are derived from either tumors or have been transformed by one process or another. These of course also have an unlimited lifespan, but they have a malignant phenotype as defined by one or more criteria. They also usually cannot become quiescent. This again is the Hayflick plot. What I am showing here is a rodent culture, mouse in this case, and human. This little bump on the curve in fact is senescence for a mouse fibroblast culture. So it's been known for a very long time that rodent cells can overcome senescence spontaneously. You can also see the great difference between the replicated lifespan in vitro of human cells that go on for a very long time. If this experiment here was continued, it would level off and you would see the typical Hayflick phenomenon. So the human plateau up here in fact is corresponding to this rather short plateau senescence in rodent cells. So the relatively low frequency of immortalization -- I should point out that this doesn't really seem like a low frequency, but on a per cell basis, it actually is an event that has a frequency of 10 to the minus 5, to 10 to the minus 6. It's just that the X axis is plotted in days here. The fact that this immortalization can be stimulated by mutagens has led to the hypothesis that this in fact is a mutational event in nature. This is supported by the existence of several viral genes, such as, and we have heard about them here, SV40 large T antigen, polyoma large T antigen, animal virus E1A, HPV E6 and E7, that can cause immortalization. In fact, when these genes are introduced into rodent cells, they are sufficient to cause immortalization in a single step. In other words, if you take a rodent culture and you put SV40 large T into those cells at this point, the curve would look like this. No apparent senescence under the right culture conditions. So what are these viral oncogenes doing to promote immortalization? Without going into a lot of detail, there is a large body of evidence that now indicates that these proteins interfere with the function of the P53 and/or RB growth inhibitory pathways. In agreement, there's a lot of data from knock-out mice now recently that has shown that the elimination by gene knock-out of a variety of negatively acting affecters can result in apparent one-step immortalization, as shown here for example. To date, embryo fibroblasts from strains deleted for P53, P16 inc 4A, P19 arf 1 in P21 cip 1 have displayed this apparent immortalization phenotype. So what happens in human cells? Normal human cells have never been observed to spontaneously immortalize. Senescent cultures do not give rise to sub-populations that resume proliferation as shown here. Treatment with mutagens has been shown to sporadically give rise to immortalized derivatives, but the frequency of these events is significantly lower than that in rodent cells. Let me now talk a little bit about the phenomenon of crisis. So what happens when we put, for example, SV40 large T or E1A into a human fibroblast? What we get instead of immortalization, is a phase of so-called extended lifespan. So here we see a primary cell, the initial proliferative phase. This is senescence or the Hayflick limit. The introduction of a viral oncogene is going to cause an extended lifespan for variable duration, typically in human fibroblasts of 20 to 30 divisions. Then one sees a second proliferative decline. This has been designated as crisis. Now this decline at the end of this extended lifespan which we call crisis, this word is somewhat ambiguous, because it has also been applied to rodent cells. These cells do not display a two- stage mortality process. So to distinguish more clearly between senescence and crisis, some groups have started to use the word "M1" for mortality stage one, and "M2" for mortality stage two. Senescence is different from crisis. These are not just the same proliferative decline. The main distinction is that cells in senescence or M1 are truly non-dividing. Whereas in crisis cultures, the apparent absence of proliferation on the macroscopic scale is actually the result of ongoing cell division combined with ongoing cell death. This is an experiment that was performed in my lab. What we show here is that elimination of the CDK inhibitor P21 in a pre-senescent normal human fibroblast causes an apparent extension of lifespan that is equivalent in magnitude to that elicited by SV40 large T antigen. So also in human cells now we have been able to do ablative intervention. That is eliminate the activity of certain negatively acting affecters and cause an apparent extension of lifespan. In terms of cell substrate design or the technology that would go into doing this, this was really strictly an aside, we have now developed methods -- these are really based on gene knockouts, homologies reculmination gene targeting, that can be used to delete entire genes, multiple genes in human cells, including normal human cells. So let me now turn to my last topic, which is the molecular clock of aging. I think probably this is where I am going to overlap with what Jim has already said. As I told you, there are some older observations that correlated entry into senescence with the lap cell division as opposed to chronological time. Quite a few years ago, this has led to the proposal for the existence of some sort of a molecular clock. Then one envisioned that the running down of this clock would generate a signal that triggered the senescence program. Then the expression, for example, SV40 large T could either prevent senescence by overriding a signal from this clock or by what I think is more likely now in light of new evidence, actually interfering with the senescence machinery itself. So as you know, the currently prevailing hypothesis is that the nature of the molecular clock is the attrition of telomeres. This is a slide by one of my dear friends, Chris Counter, who has fancifully imagined H-TERP, which is the catalytic sub-unit of human telomerase sitting here at the end of a chromosome end. So this is a telomere here. Then catalyzing the addition of the telomere heximer. You can see the telomerase RNA that acts as a template for that process right there. Germ cells and some key stem cells are known to express telomerase catalytic activity while the majority of somatic cells lack this activity. The estimation of telomere shortening for one generation is in human cells between 50 to 100 bay spares. So that's 50 to 100 bay spares per S phase. This correlates reasonably well with the average telomere length in a young human fibroblast of 18 to 20 kilobases and the length of 8 to 10 kilobases in the senescent fibroblast. I think it's an important observation that senescent cells in fact contain appreciable telomerase. So here we have a normal cell or a young cell. We get attribution of telomerase. At this point, the telomerase are maybe 8 to 10 kilobases in length. This generates a signal. If the cell is now driven into the extended lifespan phase, these telomeres will continue to erode because telomerase is not expressed in that state. Eventually one enters into a crisis which is caused by erosion at the end, genetic instability, et cetera, et cetera. It is really the nature of this signal that I think is one of the enduring mysteries of the field. One can really now beginning -- we can start to see the process as being composed of a clock, a signal, and then the senescence machinery itself which is most likely composed of the same players, CDK cyclin inhibitors, et cetera, et cetera, that are used in other types of responses such as differentiation 23 and quiescence. The linguistic definition of senescence is the state of being or the process of becoming old. This term has therefore been used to describe essentially any sort of age-related irreversible proliferative decline. In light of these new molecular insights, I prefer to use senescence in the more restrictive mechanistic sense to designate the response triggered in normal cells. I really believe that senescence is an active genetically programmed process that responds to an inductive signal. Perhaps telomere shortening, but that is not 100 percent clear. How the signal is generated is not really well understood. One can argue that the ensuing growth arrest has the obvious advantage of preventing the cell from becoming grossly genetically unstable. In contrast then, I think of crisis as an unphysiological state. You have to do something to the cell to drive it to this point, and that it leads eventually to the catastrophic breakdown of chromosome stability, which is caused by critical telomere shortening on many chromosome ends. So now this is really just a restatement of the two-stage mortality process. What I have added here now is telomere length in kilobases on the Y axis, the replicative age on the X axis. So here we have a cell in the beginning. If this happens to be a germ cell or a stem cell, it will maintain telomeres because it will express telomerase activity. Most somatic cells will start down the slippery slope of telomere attrition, eventually entering into a physiological state of growth arrest, through which they can be driven by either the expression of certain viral oncogene or the eblation of certain inhibitory pathways that are intrinsic to those cells. The cells then enter into extended lifespan. They continue to erode telomeres. They enter into a state of crisis, which is characterized by genomic instability. Finally, at this point, one can attain a truly immortalized derivative in the key step here, is the expression of telomerase catalytic activity. I should also point out that telomerase need not be expressed at the final step. It has been shown experimentally that telomerase can be artificially or experimentally activated anywhere along this line, and that that will lead in some cell types, not necessarily all cell types, to immortalization. However, I think the large body of evidence suggests that at least in vivo, and by this I mean during the natural development of malignancy, the activation of telomerase activity is a relatively late step. So if crisis doesn't exist in rodent cells, and bypass of senescence is sufficient for immortalization, how does telomerase become expressed in somatic cells, rodent somatic cells? The bottom line here seems to be that telomerase is not very strictly regulated in rodent cells and tissue. A variety of rodent tissues have been shown to express telomerase activity. Telomerase negative primary cultures often become telomerase positive over time even prior to reaching senescence. In contrast, telomerase appears to be regulated very stringently in human cells. Therefore, telomerase activation could occur in rodent cells that are undergoing immortalization either prior to or after the senescence bypassing event, and could easily occur in the subtle and gradual fashion so that no clearly apparent downturn in proliferative capacity of the ball culture would be observed. In other words, one step immortalization that one sees so often in rodent cells may in fact require two steps, the obvious step of senescence bypass and very likely a second step that may be very subtle, at least in rodent cultures. That is, of activating telomerase catalytic activity. So I think that is about as good a summary as I can think of in 20 minutes. I will be glad to entertain questions. Applause.)
AUDIENCE MEMBER: Bill Egan, from the FDA. When you immortalize cells, you know, after they go into crisis or whatever, what becomes the length of the telomere? Does it go back up to 20 kilobases? What maintains the length of that telomere at a fixed
DR. SEDIVY: That's a very good question.
AUDIENCE MEMBER: Why doesn't it become 30 or 40 kilobases.
DR. SEDIVY: In fact, it seems that excessive telomere length is not good, at least in human cells. It's been known for a long time that many spontaneously immortalized human cell lines which we love and honor like 293 and Hela, et cetera, et cetera, have very short telomeres. These telomeres can be maintained at a length of one to two KB. These cells seems to be perfectly happy with that. So I think it's more the maintenance of the telomere length rather than the absolute length of the telomere. If you artificially introduce telomerase catalytic subunits into fibroblasts, what one typically sees is that the best clones are ones that build up telomere length to about 8 to 10, 12 KB and then maintain it at that level. It seems to be a function of the expression level of the H-TERP gene, because if one does this experiment, you see cultures that very slowly erode their telomeres. They will eventually senesce. You see cultures that build up telomeres to maybe 20, 30 kilobase in length. That doesn't seem to be good for them because the rate of growth goes down. So really the best cultures are the ones that maintain at least in fibroblasts. So I think it's the maintenance rather than the absolute length.
The other explanation is that mouse cells don't senesce. They neither undergo crisis or they undergo senescence. In fact, that plateau that we are seeing during the immortalization is not senescence. It's a differentiation-like process. There are people, including myself, that given this kind of loose distinction between quiescence, differentiation, and senescence, would prefer to define senescence now as a process that is triggered by telomere erosion. Obviously when a mouse primary fibroblast culture undergoes senescence, it's not doing it, probably not doing it because it's receiving a telomere signal. I don't know if that is -- does that make sense?
DR. HUGHES: I had hoped you would comment on the mice themselves.
DR. SEDIVY: The mice themselves? What do you want to know about the mice themselves? They are alive.
DR. HUGHES: Yes, I know. But in the Greider experiment with telomerase knockout.
DR. SEDIVY: If you knock out telomerase in mice, it takes six organismal generations to observe lethality. Okay? What you see at each generation is that the average telomere length. So generation one, it's 50. Generation two, it's 40. Generation three, it's 30. If you take mouse and real fibroblasts at any one of these generations, they senesce in vitro on schedule. Is that what you wanted?
DR. HUGHES: (Inaudible.)
DR. SEDIVY: Well, I think I just offered you one explanation for that. That is that what we are calling senescence is not senescence. It's something caused by some insufficiency in the median that is in fact triggering a differentiation event. Actually, Jim McDougall and I also don't quite agree about what's happening in his keratinocyte cultures because what he is calling senescence, some keratinocyte biologists would prefer to call differentiation.
DR. NABEL: Okay. If we could move the questions along, maybe brief answers. Then we'll move onto the next speaker.
AUDIENCE MEMBER: Alex van der Eb, Leiden. You just already answered, I think, my question, which was why do mice cells, mouse cells enter senescence while they have such long telomeres? In fact, you answered already part of that question. Do these cells that enter a so-called senescence have high levels of P21 or P16 or something like that?
DR. SEDIVY: Yes, they do. Yes, they do.
AUDIENCE MEMBER: So there is a signal then.
DR. SEDIVY: If you take a knockout mouse for P21 that doesn't undergo senescence. It just keeps going. But you know, that's what I was trying to say. That is that op regulation of P21 is not a molecular market for senescence. P21 is op regular because of oxidated stress, osmotic stress, differentiation signals. This is a very general machinery that is used to establish cell cycleresce. I, in fact, don't know of any molecular marker that is specific for senescence. This includes the famous senescence-specific betagalactocytis activity. You know, you see a lot of people staining cells, and they turn blue and they say it's senescence. Everybody knows if you put hydrogen peroxide on your cells, they turn blue as well.
AUDIENCE MEMBER: Just a brief comment for
those people who might be setting up assays that would be monitoring P21 sip.
We, as I showed, found that P21 is elevated in a subset of the HPB infected
cells. We did three other related assays. One was to look for P21 MRNA. It
turns out it's abundant in all differentiated cells. But there is a post-
translational control on the accumulation of P21. It turns out what happens is
that if there is not a signal that unscheduled in a synthesis is underway,
namely, abundant cyclin E, then proteosomes rapidly degrade the P21 that's
translated. When weput in proteosome inhibitors, P21 piled up in all cells and
all replication was blocked. We went on to ask one additional question. That
is, how does P21 actually block S phase or DNA synthesis. Unexpectantly, it had
nothing to do with blocking cyclin E activity. It turns out cyclin A, CDK-2 or
cyclin A CDC-2, can phosphorylate DNA preliminary cell and all these other
subunits Ishowed. The one thing cyclin A can't do is bind to PCNA. But when
the P21 sip piles up in these cells that have excessive cyclin E, the way the
P21 is actually functioning is by binding to the PCNA and blocking elongation,
oncogenes in cells at about the time they are
entering into senescence, especially rodent cells?
AUDIENCE MEMBER: What happens in spragues? I
mean are they different than muskulorattus? Do they have shorter telomeres
that they go through?
AUDIENCE MEMBER: So even though one has 60 KB and one has 2 KB?
DR. SEDIVY: I mean, you know, this kind of all argues that the length of telomeres has nothing to do with this plateau in mouse cells that we define as senescence. Okay? I think there's a result that's kind of floating around, which is also consistent with that. That is, we all know the wonderful experiment of expressing telomerase in human fibroblasts which causes immortalization. It doesn't do that in mouse cells, which also argues that the length of telomeres in mouse cells is not what is triggering this growth
DR. SEDIVY: Yes. Those are very old and classical experiments. In general of course, senescence is a dominant state.
DR. FALLAUX: First of all, I would like to thank your organization for inviting me here. The subject of my talk will be on the generation and characterization of new helper cell lines for the construction, provocation, and protection of recominance replication effective adenoviral vectors. In the past few years, the interest in vectors derived from human viruses. This is caused by the fact that from the many years of intensive fundamental research on human adenoviruses, it has been found that adenoviruses have several favorable characteristics, including high stability of variance. The variance is very easy to grow into pure with very high fibers. It has a very broad host range. Importantly, it has the capacity to transduce non milotic cells. This makes adenovirus a very potent gene therapy. It is known that it has very low kinisity, and there is there ample experience with adenoviruses as vaccines. This slide shows a schematic representation of the adenoviral genome. It is a double stranded linear DNA molecular of approximately 36 KD, carrying several genes, flanked by inverted herminal repeats. The genes are sub-divided in so- called early genes and in late genes, depending whether they are stressed early or late during the lytic infection. This slide shows you a scheme of the classical methods to construct the common adenoviruses. All urrently used adenoviruses carry a deletion in E-1. This renders the virus replication effective, and it also provides space to insert therapeutic genes. Now in the old days, we used to isolate the DNA from wild type adenoviruses at 5 or S-2. In purified DNA, and I just -- the restriction enzyme cla-1, which puts ones in area region one. We then purify the large fragment. In addition, it also needs the construction of an adaptor plasmid which carries the transcriptase unit, including geno-fenchfras, but also the left inverted herminal repeat and a part of the adenovirus sequence which is also present in the large fragment. Pro-construction of these two moleculars in so-called helper cells, and the helper cell is the 293 cell made by Frank Reim. Upon close inspection, another mination occurs, creating now the recombinant adenovirus, carrying the gene of interest at the position that we want. You can proficate these elongated viruses due to the fact that the helper cells complement the missing elong function. Now despite the encouraging results of things so far with the use of recombinant adenoviruses, there are also several problems associated with the use of such vectors. These problems include the growth infectivity range. That is, you do not only infect the target cells, but also non-target cells. This may cause pathogenicity. Also, the viruses are rather immunogenic. We only leave off E-1, and all the other viral genes are still present and can be expressed to low levels, resulting in numerous responses by the host, both humoral responses, antibodies, and cellular responses against new cells. The cells are killed and the therapeutic effect is lost within several weeks. Another issue is the occurrence of replication competent adenovirus, abbreviated RCA. I will focus on this topic. There are various sources of replication competent adenoviruses. In a sort of infection, during the production of viral, or as an earlier stage, or during the construction of the recombinant factor, especially when you use classical methods, if you use the large clavon fragments. If the digestion if not complete, you have RCA, namely the wild virus. It has also been shown recently that you can generate RCA by homologous recrimination because the factor and the helper cell carry adenovirus sequences that overlap. As a result, by homologous recrimination, you can get RCA. I will focus on this source of RCA. Well, how does it work, homologous recrimination resulting in RCA? This is a scheme of a typical elongated factor. This is a scheme of the integrated adenovirus sequences in the helper cells. The helper cell line is 293, and more recently, we made alternative cell line 911. Both helper cells carry the evon A, evon B in chorion regions. But in addition, they also carry sequences that enclose structural protein lines, downstream of evon B, and upstream of evon A, be left for determinal repeat. Those sequences are also present in the factors. So that a sequence overlap 5 prime and 3 prime of the therapeutic gene. As a result, you can get homologous recrimination by which the recominance virus now trades its therapeutic gene for region E-1, and becomes replication competent. Now what you can do about this is to avoid the sequence overlap. We'll come back to that later. Well there are only a few helper cell lines available when you work with recombinant adenoviruses. We are fortunately in our lab to have three of them, including the two in our free cell line, recently an iomosa line, and even more recently, the PER cell line. All three cell lines are obviously of human origin. They are all derived from primary diploid embryonic cells. 293 is derived from kidney cells, 911 from retinoblasts, and PER C-6 as well. Now when I started to work with recombinant adenoviruses in our lab, which is the lab of Professor van der Eb at the Leiden University, I used obviously 293 cells, and I met with some technical difficulties. Since we had a panel of adeno virus transformed human cells, including cells of kidney, lung, and retinoblasts, I decided to screen a panel of cells in order to find an alternative for 293. From this panel of cells, I selected one particular retinal cell line. We named it cell line 911. The reason for this name was to get the attention of our colleagues in the U.S.
(Laughter.) Well, the construct we used to make the 911 cell line is shown over here. It carries the adenovirus sera type 5 nucleotides 87 to 5,788, including evon A and evon B. Now I want to be short on the 911 cell line. The most important findings were that they performed very good in virus titrations. We also found that the virus use of 911 are up to three times higher with various viruses, also recombinant viruses, three times higher than obtained from 293. Some other characteristics of this cell line are that they express very high levels of evon A and evon B, are highly transfectable, which is important when you want to construct recombinant viruses at the classical method. The use of the viruses are very high, as told, and they perform very well in titration assays. So we concluded that 911 is a good alterative for 293. However, I have shown you the construct we used to generate the 911 cell line. We now have a situation which is similar to 293. Namely, and also in 911 cells, besides evon A and evon B encoding sequences, also sequences of the left inverted termin are repeat, and sequences in part encoding protein 9 are present. So again, there is overlap and you can create RCA. So what we decided to do is start all over again and make now the cell line in combination with a so-called matched vector, now sequence overlap. What we did was to make a so-called packaging construct carrying only the evon A and evon B encoding sequences in which evon A is driven by PGK, a heterologous promoter, and a heterologous poly and signal, and lay matched vectors that are deleted of exactly that elong region which is present in the packaging construct. Thus affording sequence overlap and thus eliminating homologous recrimination as a source of RCA. This shows you one of the packaging constructs we constructed. Present are adenovirus sera 5, sequences four, five line to 3,511. Those are only the evon A and evon B encoding sequences. Evon A is driven by the human PGK promoter. Evon B is under its natural promoter, and directly flanking the evon B stop codon as the polyadenylation signal derived from hepatitis B virus. Now before we decided to transfect in this vection construct into our retinoblasts, we decided to do some functional assays with this construct first.
You can see this in the second line. Transfected this plasmid to come in plasmid in evon B, and we did obtain reasonable amounts of foci. Obviously also the construct we used to generate 911 resulted in focus formation. Unfortunately, also when you are packaging construct, which we used to make the PER cells gave foci, thus indicating that the packaging construct allowed the functional expression of early region non-probenes. So then we went to the actual experiment, transfected the packaging construct into primary human diploid retinalblasts. We could establish seven clonal cell lines. We tested these clones for first, expression of evon A and evon B proteins. Now we found that all clones expressed very high levels of evon A in both 55 and 21 K evon B, when compared to 293 and 911. We also looked at vector use. We looked at three clones, clone 3, 5, and 6. As you can see, we found that the three PER clones tested exhibited similar use of recombinant viruses, compared to 293 and 911. Since PER C6 played the highest use, we decided to analyze this clone in further detail. Now of course the major issue for us was to test whether or not our approach to use the PER cells in combination with matched adenovirus vectors would reduce or even eliminate the generation of RCA. This testing has been performed at the enzyme. What I did was to amplify an RCA free master stock of a typical adenovirus vector and amplify it to 293 or PER C6. What I found, I can summarize it for you, is that amplification of 293 resulted in RCA positive vector batches in approximately 50 percent of the places. Now for a clinical setting, that means that you might consider to throw away half of what you had made. In the case of PER C6, fortunately in none of the batches amplified on PER C6, we were able to detect RCA, not even in a large scale production setting. So we concluded that our strategy to make a PER cell in combination with new matched vectors severely reduced, maybe even eliminated the RCA problem, at least by homologous recrimination. The next two slides summarize some of the other features of PER C6 cells that contain three to five copies of the packaging construct, very high levels of evon A and evon B, comparable to 293 and 911. Good use of the different vectors, also similar to the other two producer cells.
The cell line was very stable. We have now come over passage 250 actually. So far we have not detected RCA, and the list of productions with different vectors is still increasing. We have a master cell bank available for PER C6, also a working cell bank. Importantly, the PER cells were made on a GLP conditions, using certified U.S. bovine serum and trypsin. currently, InterGene is doing all kinds of tests which were necessary for the use in the chemical setting, including mycoplasma and sterility testing. In the academic lab, you can simply draw the cells to standard medium. Finally, I would like to thank all the people that are involved in this project. Number one, the PER cells were generated in the lab of Professor van der Eb at Leiden University, in the Applied Virology Group, supervised by Dr. Gugen. The packaging constructs were made by Edie von Frel of InterGene, and all the downstream processing, a lot of work is currently being performed by InterGene, supervised by Valeria in the Adenovirus group, supervised by Dr. von Laud. As I told, all the RCA testing was performed at Genzyme by Kathy Hay here. Thank you.
DR. FALLAUX: Actually we did not test that
yet. However, we did test this for the 911 cell line, the weakly tumorogen in
nude mice. So you might expect the PER cells would exhibit the same feature
with respect to that.
DR. SHEETS: Becky Sheets, FDA. I had a similar question, but I have a couple of other. Does the PER cell stay diploid or is it aneuploid? Also, the individual from whom you obtained the retina, did they have wild type RB genes or were they -- you know, was this someone that died of retinal blastoma?
DR. NABEL: John?
AUDIENCE MEMBER: 293 cells have become quite popular in the laboratory for reasons that have little to do with their ability to support adenovirus vector replication. Have you checked these other cells, for example, high levels of transvectibility in being a good host for other kinds of viruses and that kind of thing that make 293 cells so beloved by many virologists?
DR. FALLAUX: In fact, all adenovirus transformed cells are as highly transvectible as 293 cells.
DR. NABEL: Okay. If there are no further questions, then I think we can just proceed onto the panel discussion. John Coffin will chair that. If the panel members want to come forward and get started.
DR. COFFIN: By my calculation, we are running almost exactly an hour late. A check with the board outside reveals that we really probably can not go much past 3:00 before we start to lose people quite seriously. So we probably should shoot for an hour in which we either have a lot or a little to do, depending on the will of the crowd and our host. This panel discussion actually as two functions. One is a discussion of the last topic covered. That is the designer cell substrates, two talks we heard today and the one yesterday. Then secondly, where we really earn the generous honoraria that FDA is paying us, where we try to summarize and hopefully answer some questions that might be useful to our host in terms of development of policy, ideas for further meetings, experimentation and so on. Tentatively we'll plan to sort of split the discussion half and half between these issues, but I think we can play that by ear as we go along. Again, I expect widespread audience participation, particularly since these are topics that I myself am not really actively working in and am familiar with. The questions that on the first part, on the designer cell substrates, that we were charged to address are summarized on this overhead. Before I turn it on, I want to apologize in advance for two things. One is my handwriting is very bad, so you are going to be subjected to that for a while. Secondly, I was given a rather blunt instrument to write with. Thirdly, of course I'm not well enough organized like some of the previous chairs who have prepared these ahead of time. So this is a paraphrase, I hope an accurate one, of the issues that were raised, that are raised in the points to consider. The first regarding designer cell substrates is the issue of whether cells that are derived by the kinds of defined means that we have seen, and we have seen the example of introduction of telomerase plus or minus oncogenes, viral oncogenes, or viral oncogenes alone whose function among other things was to stimulate or inactivate genes that are involved in senescence. Whether cells that are created in this way in fact offer significant safety, create safety issues relative to other cell lines, whether they offer advantages or disadvantages, whether we can go through the sort of defined risk algorithm that was given to us at the beginning of the meeting, to address these and anything else. So if we could get onto the first point here. Are there significant safety issues relative to tumor or neoplastic cell lines? In other words, uncharacterized, what we should call it perhaps, uncharacterized cell lines. Cell lines that have just been handed to us either in tumors or that have arisen by means we don't really understand very much about in culture. Would anybody on the panel care to –
DR. ONIONS: It's really a question of clarification from my point of view. But it seems to me that one of the advantages of cell lines that come from potential oncogenic background is they have some of the features from mass culture that industrity. That is, they can be grown in an anchorage-independent way. They can be grown in high density in fermenters. What's the position with telomerase immortalized? I assume actually they are mimicking much more the kinds of cell type that Dr. Hayflick would define as -- I forgot what he used, apologies – the first stage of cell strains. That probably do not have those particular phenotypic properties. Do you know what the stage of those cells are?
DR. SEDIVY: Well, you know, we have a really
limited experience. This game has only been played for a few months, maybe a
year in some privileged labs. I think what we're really talking about here
is proof of principle. In my lab, we're not interested in growing cells in
fermenters. We are interested in cell cycle progression. But I think if
somebody wanted to make a cell line that grows well in fermenters, I think it
may be a good idea to contemplate some of these new approaches.
DR. COFFIN: Want you to be more specific about what you are knocking out. Which genes have you knocked out?
DR. COFFIN: Any response to that? Do we feel in terms of this first issue that there are significant safety, differential safety issues of these kind of cells relative to the relatively uncharacterized lines?
DR. MINOR: From the point of view of the
infectious agent side of things, I mean I don't see much difference between,
you know, a brand new tumorigenic cell line that appears in your hand and one
that's actually being designed to actually appear like that. It seems to me
they are both uncharacterized and you would have to look at both of them very
carefully. I'm not sure that you have new infectious issues simply because you
designed it to be transformed.
DR. COFFIN: If you avoid serum, I think would be a highly desirable trait in vaccine production if one could engineer that. Obviously there are BSE issues, and that sort of raises what might be the tip of an iceberg. Is that a practical goal for production?
DR. FALLAUX: Actually, for the production, we
now can grow PER C6 in serum-free media in suspension.
DR. SEDIVY: You know, I am by no means expert on this, so I can, you know, basically restate what I think is already out in the literature. That is that you can definitely immortalize human fibroblasts, pre-senescence fibroblasts by putting H tert in. That's been shown now in a number of laboratories. You can also immortalize retinal pigmented epithelial cells as reported by the Texas group. I am not sure whether it's been published yet, but I have heard that you can immortalize T cells, CD8 positive peripheral lymphocytes. Jim McDougall says that you can not immortalize keratinocytes unless he said something different yesterday, in that you need to interfere with the RB pathway in addition to putting H tert. There's also some indication that breast epithelial cells may need an additional step to become immortalized. That's all I know at this time.
DR. SEDIVY: I don't think this work has been published, so I think I'm just telling you something that I heard at another meeting. So maybe we should just cool it. But I would presume that if they grew in suspension before tert, they would grow afterwards as well.
DR. COFFIN: Obviously these immortalized T
cells would be of great interest to people who are interested in growing
attenuated HIV vaccine.
DR. COFFIN: But when doing that, of course as soon as you start selecting for these additional characteristics, when you are introducing new and uncharacterized genetic changes.
DR. FRIED: Right.
DR. COFFIN: That these are less important than safety issues, than changes that might have led to immortalization in the first place.
DR. SEDIVY: You know, I think I would also like to second the point that was brought up earlier. That is that sure, we can always make the claim that we know exactly what we did to the cells ourselves, but we don't know what the cells have done on their own during those zillions of passages that they are growing in my lab. It's definitely being documented that H tert immortalized fibroblasts are karyotypically very stable. But if you passage them for long periods of time, you will find anemploy these. So I don't think that that's any different from any other established cell lines. So it's really, you know what you did, but
DR. COFFIN: But you don't know what happened.
To bring a point to this, if one is concerned about issues of what might happen
with DNA from the cells that was carried along, then it sounds like, it sounds
from what I'm hearing like there may not be a great deal of difference between
using these cells and using these kinds of cells as compared to using relatively
uncharacterized cells. Although there are very good reasons for making such
cell lines, that this particular issue may not be the most important one.
DR. ONIONS: Just as a general principle about whether it's useful or not to engineer cells rather than go out and select a transformed cell, a pre-existing transformed cell from a tumor, it does strike me that again, that it's under control and that you have a number of choices. The kinds of studies that PER C-6 has been involved in give you a very precise engineered system that's absolutely ideal for vector production. But it perhaps also highlights with respect to the mistake that was made. That is, that you had another possibility here where you could actually choose the cell. You could validate its origin. You could check the person. That is the other advantage of you being able to engineer materials, that you can actually pre-select the actual source of the material that you start with. hat would have been an advantage that was unfortunately missed in this particular case. That's not to undervalue the value of these cells, but it does seem to me that that's what engineering cells can give you. It gives you control at each stage of the process.
DR. ONIONS: It was -- I understand only too well. It's not at the end of the day a criticism of PER C6 success, which I think are excellent. But really just that where possible, that that should be done.
DR. COFFIN: Okay. So I think we have a consensus here that there's lots of useful things about such cells, but that we really don't' know whether they enhance any particular safety issue or not. I think that's a sort of at least some sort of closure on that particular point. The points we were asked to address also included the use of the defined risk algorithm that was mentioned at the beginning of the meeting, to evaluate these kinds of things. Andy Lewis had that on his slide, which I have asked him to put back on. This will also, I mean with this, we will sort of segway into the general discussion as well I think, because these are the issues. So the question is, can we go through and do this, and is it possible in this particular case, just using this as an example of this kind of procedure, to assess the level of risk posed by these issues, infectivity, infectious and so on, quantitatively. My own feeling right now is that we're no where near a position to do this, certainly for DNA issues. We might be able to put some sort of numbers on viral issues. It's a little hard to see exactly how because there's so many different ones, which could have a different contribution. But maybe we can get some further comments on these sort of issues from the panel.
DR. ONIONS: My only comment, and I understand why a defined risk approach was used, and it certainly makes you think. I mean that's one of its great virtues. I think one of the real intrinsic problems, that if you applied, you can give yourself a false sense of security. It would strike me that Phil's story about SV40, if it turns out that is the origin, SV40 in people, would have given you such a false sense of security, I think, because you might have come through that exercise in the 1960s. I'm not sure how you would have predicted that that agent was there a priori. So I'm not sure that you can give guarantees that 1 and 10 to the 6th dose is one-half X, if you don't know what X you are looking for.
DR. SEDIVY: Yes. I mean I broadly agree with that. I think it's worth trying to do some sort of numerical calculations, so long as you don't believe the numbers that you get out at the end of it. (Laughter.) Because I think one thing it will do is it will tell you where you think you are confident, on what stage of the process you are actually confident. Then you can actually question whether your confidence is misplaced or not. But I think if you come out with a number, I think you are asking for trouble.
DR. ONIONS: I think that's absolutely right. I was taken by Neil Cashman's risk assessment, quantitative risk assessment today. I think again, what it did, although I think he himself didn't believe certain the numbers at the end of the day, it makes you think about the process. I think that's fine and I think I would agree with that.
DR. HUGHES: One of the things that's true about the numbers that I've seen is no one has attempted to put what I guess I would call a confidence interval on the numbers. One of the things that makes me uncomfortable is that I think in some cases the uncertainty is as large as the number. I would feel a bit more comfortable with a calculation with which I'm fundamentally uncomfortable, if I had a better notion of how uncertain people were about the assumptions they were making in generating the numbers in the first place.
DR. ONIONS: I think that's what we're all saying, is I think I started it off by criticizing the whole approach. I think what Phil said is what Ithink. I think Dr. Lewis said the same thing. That is, don't believe the numbers. All it is is gives you a manner of approaching what are the issues, really. I think that's the way it should be treated. I agree. I don't think anyone should believe the numbers at the end of the day.
DR. HUGHES: I think it might help if when someone put down a number, they at least put down a range of numbers, and that would generate a range of confidence at the end. I think what people will see when they do that, is that the ability to define the confidence interval is going to expand when you multiply the numbers together. I think that act may in a sense help define how uncertain the number actually is.
DR. COFFIN: I think from a regulatory standpoint, what often happens is that the far end of the confidence interval on the worst possible side is taken, and then that's propagated through. You never see the other side.
DR. ONIONS: I don't want to just go into an academic discussion about risk assessment because I'm not really interested in it, in that formal sense, but there are two other approaches that are used. The engineering industry uses a form of analysis that doesn't do risk assessment like that. It actually looks for holes. It looks for what could go wrong. In a sense, that's really probably what we ought to be doing. Then there are four mechanisms of that kind of analysis. The other form of analysis is the one that has become fashionable in the U.K., which is this concept of the precautionary principle, which ultimately, it seems to me, means you don't ever do anything because you never know what might happen, which seems to me completely dumb.
DR. COFFIN: So we're voting against the precautionary principle. You can't be sure of anything, but you can be sure of that. Are there any other points anybody would like to make about this? One could say that this is a useful way to organize your thoughts on this subject, but shouldn't be taken as giving you either additional grounds or comfort or discouragement, unless you actually had a situation where you had measurable quantities. Are there any other issues or questions regarding the designer cell substrate issue that anybody wishes to raise?
DR. COFFIN: If we do that, we can turn off that slide, put up my next one, and then you can ask the question.
DR. SHEETS: There's one question.
AUDIENCE MEMBER: Jerry Sato from Merck. I understand the reluctance to put a number on something when you have such degree of lack of confidence and the assumptions that are going into it. But I do think that getting an order of magnitude of where we are is actually helpful in our thinking about what we feel comfortable going forward with or not. When you have a lack of confidence in each of those areas, you also have to ask the question, what are the chances that all of your assumptions are wrong? In other words, are two of them wrong, three of them wrong, five of them wrong, seven of them wrong? Because you have to put a degree of that's not likely to happen. So if you multiply the lower end of the confidence interval for all those things, then you will never do anything. But that's not the way it works in reality. So I think it would be useful for somebody from the engineering community, where they design bridges that aren't supposed to fall down and other things, to try and put a bit little more sophistication into this analysis, because in the end, somebody is going to ask our community, which is the regulatory community, the academic community, and the industrial community, for the number or at least what they thought the number was when they went forward with their act of faith. Because there is a certain amount of common sense that goes into it, which is the basis of the act of faith. Then there is whatever kind of quantitation we can put into it. It's the combination of those two things that I think we are going to have to reassure the general public about.
DR. HUGHES: I would recommend to you the book Strategy and Conscience by Anatol Rappaport, which attempts to deal with the issues having to do with what was called strategic thinking, in which you calculate, for example, the probability of some unlikely event, such as thermonuclear war. Mr. Rappaport does a very good job of making clear why doing the calculations when you don't have the proper data is in fact a very risky and misleading proposition.
AUDIENCE MEMBER: I guess it might be worth pointing out that there are, however, some cases where you clearly do have the proper data. Right? You know the sensitivity of a specific assay for a specific adventitious agent, if in fact you choose to figure out what it is. So you can actually answer, based on those kinds of questions, and based on that kind of data, the specific question of how sure are you that something isn't there. If a better assay comes along, and it's still negative, then you can say by how much more certain you are. So just because you can not come up with good estimates for some of the numbers, seems to me it would be crazy to throw the baby out with the bath water and claim then that you shouldn't attempt to come up with good numbers for those things that you can.
DR. RABINOVICH: Gina Rabinovich, NIH. A non-regulator asking a question from experience learned this summer, in which we have been dealing with using a quantitative number, i.e. the numbers that the Federal agencies and the global agencies has set for acceptable limits of mercury, i.e. methyl mercury, and then trying to attempt to understand what those uncertainty factors mean for thimerosal and vaccine exposure im. The concern I have, and I think it has been heard, is that these numbers take on a life of their own. They become the standard against which things are measured. So that that kind of concern needs to be entered into, attempting to use the data when those data do exist, but understanding the limits to it.
DR. COFFIN: That's inherent in regulation, that things become standard.
DR. LEWIS: Lewis, FDA. To follow up a little
bit on what Phil was saying. I think one of the areas that we could approach
with some confidence is the ones who saw Keith Peden's data last night using the
tac man assay to detect JC, BK, and SV50 in human tissues. Now if someone comes
in with substrates they derive from a neuroblastoma, which we learned at the DNA
tumor virus meeting a year or so ago, is it's usually contaminated with BK
virus. We wanted to be sure that there was no BK virus in that substrate. Then
we could apply this assay with a fairly sufficient level of sophistication and
say with some certainty, based on the volumes and things that were tested, the
level at which that particular genome or that particular virus is absent. So I
think that we sort of view that as a possible starting point for a quantitative
approach. Now obviously you can't do that unless you know exactly the probes
and things that you are working with, and you define the limits of their ability
to detect things. But I think this is one of the sort of examples that was
going through our mind when we were thinking about how to do this. So you start
at the place where you might be able to generate some relevant data that's
meaningful. The other stuff will fall into place as we get better.
DR. COFFIN: We're leading into you, Becky.
DR. SHEETS: I'm patient.
DR. COOK: I'm sorry. Jim Cook. As I was
sitting there thinking about how you would describe these issues to a patient
or to a group who is asking you about the wisdom of using a vaccine, it seems
like in addition to trying to generate some logic about calculations of numbers
and risks, that every opportunity that you have, it would be worthwhile going
back to history and saying well, we have done virtually something like this
along the way ever since vaccines have been developed, and the experience with
this approach has been the following. So maybe there could be some real numbers
in a historical sense, used to color or give some more real meaning to these,
what are otherwise theoretical things, to help communicate this to the public,
as well as to provide some, a little bit more logic than just phenomenology to
the calculations that are being made now. So if history is used to color the
estimations that might be of some use.
DR. COOK: Say it again?
DR. COFFIN: How comforting is it to tell
patients that there are three or four cases of paralytic polio?
DR. COFFIN: That's clear in light of things in the movies, because lately we're doing a terrible job where these people show up with these anecdotal cases of somebody's child gets vaccinated, and then two months later is diagnosed with autism. It's automatically due to the vaccine.
DR. ONIONS: But I think history can also be a
dangerous thing. I mean the British government has been criticized, partly
justifiably, but I think partly unfairly for the problems of the way BSE problem
goes up. But the issues concerning public health were based on people were asked
what is the risk, what is the risk of the human population of the BSE outbreak,
when we had a few hundred cases of BSE in cattle. Well, there were only a few
hundred cases. The general assumption was, and it was a widely shared general
assumption by those who were informing the area, the people who worked on
scrapie and so on, well, scrapie has no evidence whatsoever of scrapie
transmission to man. We have been eating scrapie- infected sheep for
generations and it doesn't seem to have been transmitted to people. There is no
evidence of that whatsoever. The probable likelihood that BSE is of scrapie
origin that's gone through the rendering process, because we have never seen a
spontaneous spongiform encephalopathy in cattle, so that's probable origin
ipso facto, you know, there's no problem. It wasn't quite as glib as that
because actually within a year, all the controls on human food were in place and
so on and so forth. They were badly conducted, but they were theoretically in
place. So I think history can also be dangerous. I'm not sure that we can
always learn the right lesson.
DR. ONIONS: I'm sorry. You misunderstand. What you are saying, I absolutely agree.
DR. COFFIN: Do you want to continue this discussion or do you want to break into –
AUDIENCE MEMBER: Very similar aspect, although a little bit less scientific. As I perceive the discussion, of course there's this highly sophisticated, highly conscientious scientific community, and there's the general public on the other side.
The general public, to my perception, consists of at least two sub-groups. One group that is sort of generally benevolent and would believe scientists. But there is a very strong group that is not believing scientists. They are sort of using that as a political tool to attract attention. In our country, we have had this experience with the Green Party, that has become very influential in the European Union and maybe in other countries as well. Now one thing I also, since we're ending and coming to the end of this meeting, would like to raise, isn't it the responsibility of scientists also to do something to better educate the public? I know this is a utopian goal, but at least if we could increase enough of people in the general public who are educated or better educated in science and biology and biomedic issues, we would at least have a political community that might support scientific issues more valuable than we have had it so far. You know, in our country at least, every time when the Greens demanded to stop all biomedical, all gene technology research, and it course never happened. After they come to power, maybe they have different outlooks on life. But I think as a scientific community, unless we do something at least for the future, we might be in a very difficult situation to defend certain issues. If I confronted some of the violent ideologically pure Greens in our country, because the trick is, we have been discussing here, I'm sure they would say "Shut it down because this is unsafe, totally unsafe." So what I am trying to recommend is we have to do something to have more people in the general public who can appraise and can assess the difficulty and the uncertainty in any biological research. We can never get down this figure to 10 to the minus 80. So we have to raise understanding on the other side.
DR. SHEETS: So have we put oncogenic DNA to bed?
DR. COFFIN: No. I would like to use the sort
of summary -- I think that's in a sense, an overhanging issue. We have talked
about infectious risks and measurements and so on considerably today and in the
past few days. I think an overhanging issue is this oncogenic DNA issue
regarding the specific charge of the meeting, which is the use of tumor versus
other kinds of cells, tumor and neoplastic whatever, transformed cells versus
other kinds of cells as substrates for vaccine production. Although I think
many of us here, perhaps all that's here, feel this is not a risk to be really
concerned about in a scientific sense, I think many of us here might agree that
the issue is not completely put to bed in the sense that we can't put any real
good numbers on it. So now if you ask your question.
DR. HUGHES: I will answer in two different ways. I will give you my opinion, personal opinion, and then I will tell you what I think should be done, which is slightly different. Personally, and this would apply if you approached me to do something to myself, I am not concerned. However, it is my view that the data that we have, particularly for the consequences of putting DNA into animals, is not sufficient to satisfy me as a scientist. I am going to try to help my colleague, Dr. Coffin, and some of my colleagues at the FDA to try and organize a simple study that would be more satisfying to me. I think I would feel more comfortable if we had more data that was of the experiments done ongre a larger scale under more controlled conditions. I think that would give me a greater degree of comfort.
DR. COFFIN: For the same general reason?
DR. MINOR: Broadly speaking. I mean I think it's clearly a very, very complex issue about how you actually induce a tumor. So you do your 3T3 assays and you pick up H ras. Okay? I mean it's an artifact of 3T3s or was that just a question of how common H ras is. If you go and put your DNA in intravenously, is that the same as putting it in subcutaneously, for example? I mean if you put it in because it's been picked up by an envelope virus, is that going to make any -- will it be picked up by an envelope virus? Will that make any difference? I mean it seems to me that there are so many sort of loose ends to it that I don't think while there is no evidence that DNA is tumorigenic, and I buy that 100 percent, it doesn't seem to me that it's necessarily been dealt with properly. That is why I guess I am agreeing with what the previous two speakers said.
DR. LOEWER: So as I already have said, I personally believe that there's not a real big risk with purely oncogenated DNA. Purely oncogenated means three or five or six. But I realized that there is still, I believe, lack of experimental data. This was already mentioned by John Coffin, in saying that since 18 years, this question is on the table. Since 18 years, no additional experiments have been performed. I would like to propose that regulatory authorities, which are involved in regulation of these biologicals, the major ones, that we should sit together and to join the efforts and maybe decide on periments which can be done in the foreseeable timeframe. But I look forward to see what types of experiments John may recommend.
DR. FRIED: I think most of the evidence we
have so far, which is limited, says at least putting DNA into animals, we
haven't seen anything happen. We have only seen in NIH3T3 cells, and we now
know that there is a defense mechanism in the cell when it sees an oncogene.
That is this P, this arf, which is the alternative reading frame of P-16. So
it's like an immune system of the cell, specifically for oncogenes. The
radiation activation of P-53 is a different pathway. This turns on, and arf
activates P-53, and P-53 then closes itself to go through apoptosis. The only
positive things are in NIH3T3 cells. They are the classic cell where the arf
gene is inactivated. Probably that's why people have been using that for
years. They are very easy to transfect because maybe even transfection kills
the cells in terms of P-53. But that said, I would like to see a lot more
injection of DNA from different tumor lines into animals, and to really put it
DR. COFFIN: Of course you have enormous problems, including the fact that sarc is never seen. It's a human oncogene. For many years, the most popular viral model.
AUDIENCE MEMBER: I had a question. If given the unknowns, and given the data that was presented about hit and run DNA modification potentials, would the panel in the context of this type of vaccine development, and given the unknowns, give the vaccine to someone with a strong family history of malignancy or who was a cancer survivor who we know is at increased risk for a second cancer, if that was you or your family member?
DR. COFFIN: The question, to sort of focus
that a little bit, the question is whether we would consider there to be a
greater risk in certain sub-populations who might have sort of pre-activated oncogenes
or some other fact of predisposing.
DR. MINOR: It would also depend on what you
are trying to protect them against too, wouldn't it?
DR. MINOR: This is apart from the DNA issue?
DR. SHEETS: Well, in a continuous cell line,
certainly there are -- it may be aneuploid, but it's not tumorigenic in
animals. So you can comment if you'd like about whether you think the DNA is
DR. SHEETS: So you wouldn't suggest to make a live viral vaccine in vero cells?
DR. MINOR: I think it would depend on the live viral vaccine. I mean I think OPV clearly has been made in vero cells. You can scrub it clean. I think you can more or less destroy anything that's actually hanging on the end. I have more serious thoughts perhaps about things like a paramixo virus vaccine, because you couldn't clean it up so much perhaps.
DR. COFFIN: We are very fast losing our audience, so I think
DR. HUGHES: Isn't it partly the question do you know the life history of your cells as opposed to the state of the cells at the end?
DR. SHEETS: Vero cells are a bank that is well characterized. The reason for the question is that we have numerous live viral vaccines of the sort I described that are being proposed to be made in vero cells. Manufacturers prefer vero cells because one, they can be characterized. Two, you get a high yield. Three, they can be grown in the sorts of fermenter culture that you heard about.
DR. HUGHES: I'm not particularly bothered as long as I know that the sort of life history of the cell. But I think the question is, if you have a cell that's been in culture for a long time and has had a complicated culture history, do you know that history?
AUDIENCE MEMBER: I asked the last panel the same question. It comes down to the question really is the adventitious agent issue put to bed as well. Do we now have the assays in place that can easily be applied to validate the freedom from adventitious agents of these kinds of new cell lines? The answer that Dr. Broker gave in the last panel suggested that one could attempt to use DNA chips and things like that, which to my knowledge aren't assays that at least tomorrow I could go out and do on a cell line and give me some confidence. So my question to you is, sort of using the standard assays that you are all aware of, without developing further assays for this specific purpose, do we have enough information to be sure that these kinds of new cell lines are safe from the adventitious agent perspective?
DR. ONIONS: That's another unanswerable question, isn't it. I would just make the point that I think you have to adopt somewhere between good science and pragmatism. I mean you could theoretically go and do representation difference analysis on all these cell lines. Actually, I don't think it's possible because you don't usually have the partner. But theoretically you could do that. That's not really a practical solution. It does seem to me that we do know virus types that tend to be latent in cells, and that it's sensible to perhaps think of strategies of widening the brief of detecting those agents, because I'm not convinced that the kind of routine types of infecting -- infectability assays when they work are as sensitive as PCR, as just Phil pointed out. But I am not convinced that always the right infectability assay is present to actually detect certain agents. So that you are probably relying on a combination of things. Perhaps we do need to look at redundant PCR
DR. COFFIN: I would think the producers would have a big attraction, is set up the same assay and use it for everything.
DR. MINOR: I mean I think you could also argue
that you have used these assays for looking at human diploid cells and primary
cultures, and all that sort of stuff. Right? What's the difference in
principle in terms of adventitious agent contamination between those and the
cells you are looking at here? I am not sure there's much difference. But are
the concerns as big or as little.
DR. COFFIN: I would like to also second the thanks to the organizers for setting this up and bringing us here. DR. LEWIS: Yes. On behalf of the sponsors and those of us at CBER who worked on this, we really appreciate the effort that the session chairs, the panel chairs, and the speakers have put into this meeting. When you attempt to put something like this together, there's always a question of how it's going to turn out. I think the success that we have enjoyed here the past three days is a tribute to the work, an incredible amount of work, that has gone on on a very short period of time. I think that I was very concerned when we were trying to contact folks in May to do this by September. For those of you who rose to the 13 challenge, I can't thank you enough on behalf of the sponsors. With that in mind, I hope everybody has a great trip home. Get your papers in whenever you can. Thank you. (Whereupon, at 2:57 p.m., the proceedings were concluded.)