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Simian Virus 40 (SV40):
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UNITED STATES OF AMERICA CBER-NCI-NICHD-NIP-NVPO SIMIAN VIRUS 40 (SV40): Tuesday, January 28, 1997
The workshop took place in the Natcher Auditorium, National Institutes of Health, Bethesda, Maryland, at 8:30 a.m., Andrew Lewis, Chair, presiding. PRESENT: TOM KELLY, Co-Chair
Panel-Audience Discussion
Session 4 Part 1: Mechanisms of SV40 Oncogenesis 1 SV40 Oncogenicity in Hamsters, SV40-Rodent Tumor Models as Paradigms of Human Disease: Transgenic Mouse Models, SV40 Transformation of Rodent Cells in Vitro, SV40 Transformation of Human Cells in Vitro, Experimental Tumor Induction by SV40 Transformed Cells, Session 4 Part 2: Mechanisms of SV40 Oncogenesis 2
SV40 DNA replication and Transformation Requires the DnaJ Chaperone Domain of Large T Antigen, SV40 Large T Antigen Functions as a DnaJ Molecular Chaperone: Implications for Tumorigenesis, SV40-IGF-1r Mechanisms: Studies in an SV40 Induced Hamster Mesothelioma Model, A Strategy for Assessing CTL Responses to SV40 T Antigen in Humans, Immunotherapy of SV40-Induced Tumors in Mice: A Model for Vaccine Development, Panel-Audience Discussion 3:
PROCEEDINGS DOCTOR LEVINE: Good morning. I'm Art Levine. I'm going to moderate this morning's panel session. We seem to have a somewhat smaller audience, possibly having shed the lawyers and the reporters, but we are down to science. For those of you who weren't here yesterday, I would like to take just a moment and summarize what I believe to have been the major conclusions from yesterday's meeting. First, SV40, at least in the form of its DNA, is or is not present in human tumors, and is or is not present in normal human tissues. And, we heard compelling data on both sides of that question, all from good labs, and I think that the question will only be resolved by an appropriately blinded study. The reason for the disagreement probably lies with the complexity of the PCR assay used to detect the SV40 footprint in modern times, and the sensitivity, and specificity and nuances of that assay. But, even if the SV40 footprint is in human cells, there was no evidence from the strong epidemiologic studies presented by Doctors Strickland and Olin that any apparent harm occurred as a consequence of the apparently massive exposure to SV40 in the early era of poliovirus vaccines. And, in fact, one might hypothesize that if SV40 were truly harmful for human beings, and it had been harmful right along as an endemic agent, then surely the rates of some cancers should have increased during the massive exposure to SV40 in the poliovirus vaccine. And, the fact that there was no increase in any rate that we could see, granted the epidemiologic studies aren't perfect, given that there was no increase in the signal rate of any individual tumor assayed, then one might comfortably say that SV40, in fact, is not a human pathogen. However, that leaves open the question of whether it's a human Commensal. Is this an agent that we live with, and that we've always lived with, independent of the poliovirus vaccine exposure. May I have the first slide, please? Okay. Well, just to rehearse some of the data on whether SV40 is an infectious agent for humans, there are a couple of studies I would like to call your attention to, and I think most of these are referenced in the bibliography that was handed out yesterday morning. Doctor Morris, who is here on our panel, instilled SV40 by the nasopharyngeal route in 1960, because a vaccine that had been constructed at that time devoted to the respiratory-cincitial virus was known to contain SV40. So, a series of volunteers was inoculated by the nasopharyngeal route, and they received a very high dose of SV40, about 104 tissue culture infectious doses. And, indeed, about 70 percent of those volunteers developed neutralizing antibody at a moderate titer, and, in fact, about 30 percent of those volunteers had SV40 recoverable from their throats about one or two weeks after the nasopharyngeal installation. So, we certainly know from this study that SV40 could infect humans. Secondly, as you heard abundantly yesterday, the oral live poliovirus vaccines in the early days contained high titer SV40, and yet, they induced no, or at least not detectable by the assays of that day, no neutralizing serum antibody, but many of these people did have positive cultures, up to about 30 percent of the recipients had positive stool cultures, for up to five weeks after they swallowed the poliovirus vaccine. So, it appears that by the oral route this virus, SV40, can colonize the human gut, but it doesn't cause a systemic infection. However, the injected or killed poliovirus vaccine, which still contained low titer SV40, since, as Doctor Hilleman pointed out, SV40 is more resistant to formalin than is the poliovirus vaccine, probably because of its double-stranded covalent close configuration, injected killed poliovirus vaccine induced high to moderate antibody titers against neutralizing antibodies against SV40 in about 20 to 25 percent of those people who were knowingly inoculated, and those antibodies were still present after three to ten years, depending upon the studies, and the same data were true of the adenovirus vaccines. Next slide, please. So, that brings us to the question of whether SV40 is endemic in human populations. Well, several studies shown on this slide have demonstrated that close contact with rhesus monkeys or people working with monkey tissues in the laboratory have neutralizing antibodies and variable titer to SV40 in as many as 50 to 60 percent of those who have such contact. And, those studies were not only from this country, but the Soviet Union. Incidentally, one thing that was not mentioned yesterday is that there was a massive exposure to SV40 in the Soviet Union, in the early poliovirus vaccine era. To the best of my knowledge, those people were never followed up, or at least not followed up for a long period of time, but I'm told that access to their records still exist somewhere in whatever that part of the Soviet is currently called. Another study done by Brown, et. al., in 1975, looked at isolated populations. These included the Papua New Guineans and the Yorubas, Alaskan Eskimos, Brazilian Indians and so forth, who had no exposure to any vaccine and no exposure to monkeys, and Brown, et. al., found that in people in these populations who were seronegative for the BK virus, they, nonetheless, had low titer neutralizing antibody to SV40 in about five percent of those people, but in those who were BK positive in about 35 percent, suggesting cross reactivity, at least in that assay, which was the plaque reduction assay, between BK and SV40, but it was in the direction of BK spilling over onto SV40, rather than the other way around. Individuals who had been bled before 1954, in other words before the poliovirus vaccine or who were born after 1963, in other words after the SV40 contaminated polio vaccine had been taken from the market, in those two populations neutralizing antibody to SV40 existed in about four percent, four to five percent, mostly in low titer. Individuals receiving the polio vaccine during the period of SV40 contamination, 1955 to 1962, had antibody to SV40, serum antibody to SV40, in about 20 percent, perhaps, 25 percent, and Doctor Shah will say more about that. These are his data. So, there's no question that the incidence of infection with SV40 went up as a consequence of the contaminate poliovirus vaccine, but then it went down again, apparently, to what it had been before the poliovirus vaccines. Next slide, please. Now, finally, various groups have been tested for neutralizing antibody to SV40 capsid antigens by the ELISA assay, the enzyme linked immunosorbant assay, which is quite sensitive and reasonably specific, and here there are some interesting data. One paper by Zimmerman and Geissler from 1983, and another by Geissler, et. al., in 1985, these are fairly obscure papers, but important. These workers, first of all, looked at 51 medical students at the University of Wisconsin in Madison who had been bled in November of 1952, considerably before the poliovirus vaccine era, and they found that 12 percent of this population, in fact, were positive for neutralizing antibodies to SV40. Then, they looked at an entirely different group of people in Germany, who were born between 1959 and 1961, and found that 24 percent of them had positive sera. This is consistent with Doctor Shah's data, and those born after 1962, when contaminated vaccine was no longer on the market, went back down to about 13 percent. So, you can see that the rates of infection were the same before the contaminated vaccine and after the contaminated vaccine, these are higher than Doctor Shah's rates, but it is a more sensitive assay. And, finally, as with the plaque reduction assay, workers in the laboratory, with high-intensity SV40 exposure, had about a 55 percent carriage rate of SV40 neutralizing antibodies, and, again, that was consistent with the other data that I showed you by the plaque reduction assay. Cancer patients were looked at also, they had quite a variable incidence of neutralizing antibodies, between ten and 30 percent. Now, there's additional data about SV40 isolated from human tissues before the polio vaccine era. Two patients with progressive multi focal leukoencephalopathy, a disease we heard about yesterday known to be associated with the JC virus, and born in 1915 and 1933, had SV40 isolated from their brains by techniques that are still reliable. And, one patient wit metastatic melanoma born in 1894, so there was no question of exposure to the vaccine, had SV40 isolated both from tumor tissue and from pleural exudate. This is a very good study by Soriano, et. al., reported in Nature in 1974, and this was the virus itself, in addition to expressivity of T-antigen and neutralizing antibodies. May I have the next slide, please? That brings us to the next question by way of background, which is the following, is SV40 neutralizing activity in human sera explained purely by cross reactivity to the capsid antigens of BK and JC. Well, we know that JC, BK and SV40 all share capsid antigens, but in every assay that I was able to examine homologous reactivity was at least 100 fold greater than heterologous activity, and that was true by plaque reduction, fluorescent antibodies or immunoelectron microscopy assays, and these assays are known to be less specific than hemagglutination inhibition, CF immunodiffusion or immunoelectrophoresis, and there are abundant references in support of this notion that there is cross reactivity, but the homologous reactivity is very much greater than the heterologous activity. I would call your attention to one paper in particular, that of Penney, et. al., who did an immunoelectron microscopy and was able to directly visualize the interaction between the antibody and the capsid. Now, as I mentioned, Brown, et. al., in 1975, studied isolated populations, and found that 35 percent of those who had BK positive sera were also positive for SV40 in low titer, but five percent of the BK negative sera was also SV40 positive in moderate titer by plaque reduction, again suggesting some cross reactivity, mostly in the direction of BK and possibly JC spilling over onto SV40, but not the other way around. And, I also mentioned the study of Brown and Morris, in which they instilled the respiratory cincitial virus vaccine known to be contaminated with SV40 by the nasopharyngeal route, and then they went back and looked at that same sera from that same group a couple of years later and found antibody to the BK virus by hemagglutination inhibition, which is a sensitive assay. And, I would simply call your attention to the fact that the T-antigens of all these viruses are more highly related than are the capsid antigens. So, I think with that background at hand, we are now poised to hear at the beginning of this panel some formal comments from one or two people on the panel who have asked to speak, starting with Doctor Shah. After that, we'll move on to the questions that we have formulated for the panel to address. DOCTOR SHAH: I tried to discuss this question about the SV40 neutralizing antibodies yesterday in the review portion of my talk, but I did not have enough time to talk about it. So, could I have the first slide, please? This is a picture that I showed yesterday. It's in a temple where the monkeys live in close ecological contact with the human populations. In 1965-66, one of my major interests was to see if SV40 was transmitted to humans in this situation, and the stimulus for that was the SV40 exposure in the United States. And, as we discussed yesterday, in 1966-67 the follow-up period after the exposure of the human population was only three or four years, and it may take a long time for a virus-induced cancer to occur, so the rationale was that if SV40 is being transmitted to humans in India, and that is probably occurring for centuries, so if you studied the cancers in India you may get some sense of whether or not SV40 is involved in any human cancer. May I have the next slide, please? We did this study, and this was a summary of the antibody data from quite a large number of studies. This first line are the rhesus that were infected intra nasally, subcutaneously or orally, and after four to six weeks and 29 to 31 weeks these were the antibody titers. These tests were done, not by plaque neutralization, but by neutralization in test tubes. So, you always got these relatively high titers after experimental infection. These are naturally infected rhesus monkeys which were bled in India, and, again, the titers are in the same range. These are the individuals who received the SV40 subcutaneously. They received sometimes live SV40, but at the same time they received a great deal of inactivated SV40, so they were exposed to the antigen, inactivated antigen, in addition to the live virus, and they may have been exposed to the antigen alone a number of times without having the live virus. And, these are, again, they are not too far away from what are found in the monkeys, although the titers were somewhat lower, but in all instances they are sort of in the high range. And, these are the same people after three years, these are not my data, these are taken from literature, I think these are probably from Doctor Gerber's paper. And, these are Doctor Morris' data, and he's here, internasal inoculation of SV40, and, again, the titers tend to get lower as you see here. These are the oral polio vaccine, other people's data, no antibodies, and this is what we found in India, and only about five percent had low levels of antibodies, but look, these titers are extremely low, and quite different from what you see here. We thought for some time, can I have the next slide, please, we thought for some time that the infection in man may not be very efficient, so they are having a low level replication and low level of antibodies, at that time we were studying the animals, the serum specimens from North India, and this is where the rhesus monkey is distributed and where there is contact between the rhesus monkey and man, and we thought for some time that these are probably SV40 antibodies. We subsequently worked in South India, and where there is another macaque species, the bonnet macaque, or the macaque irradiata, which is free of SV40. There are many macaque species, of the 19 macaque species there are several which are not infected in nature. So, when we studied the human sera here, their pattern of antibodies was identical to what we had seen in North India. Can I have the next slide, please? So, what we concluded then, that the low levels of antibodies were detected on a small proportion of human sera, we could not ascribe them to SV40 infection because prevalence in North India was similar to that in South India, and subsequent studies were, I mean especially the study by Brown, Sih and Babacek, which Doctor Levine referred to in the isolated populations, and that study suggested that the antibodies might be the cross reaction might -- the low level of SV40 antibodies may be due to cross reactivity with BKV antibodies. Now, if I remember correctly, they did not have at that time access to JCV antigens, so there was a portion they could not explain, I think Doctor Levine said that about five percent of the BKV negative sera also had antibodies, but then the JCV was not looked for. So, it may be that this might be cross reactivity with the non-viruses. Actually, we had proposed several years before JCV and BKV were identified that there are, perhaps, cross-reacting human viruses which are responsible for these antibodies. So, most of the data, most of the serological data that we had, could be, perhaps, explained as a result of cross reactivity, but there were some isolated instances which we could not explain, one which Doctor Levine referred to. One of the two cases of PML that was reported from Hopkins as due to -- well, they identified SV40 in the brain had extremely high titers of antibodies, high just like the sera from experimentally infected monkeys, and in serum specimens that Doctor Roh gave us many years ago, which were his collections for some other reason, we found something like four or five human sera, and this is all documented so I won't go into detail about that, of people who were bled, for example, in 1952, and in some instances there was more than one serum from the same person so we could check both samples, there were titers that seemed to be too high to be this cross reactive antibodies, but we never really completely solved that question. I would like to show on our transparency if I may -- I think this is the last slide, yes, last night we were discussing in a group about what this virus might be which might be circulating in the human communities, I don't, myself, conceive that, but supposing it is there, what would be its property, what would be its characteristics? And, Doctor Butel and I came up with these characteristics and, perhaps, most people would agree with them or might, perhaps, challenge some of it, one, that the virus that has been found in the mesotheliomas, the osteosarcomas, and in ependymomas is SV40 itself. It is not BKV or JCV, and it may be -- it is, for all intents and purposes, the monkey virus, the SV40, so we are really look for SV40, and it is not a question of BKV or JCV being misclassified as SV40. The second question was that originally was introduced, now it is circulating independently of the polio vaccine contamination, because all the ependymoma patients, most of the osteosarcoma patients, and, perhaps, some of the mesothelioma patients, were not exposed to the polio which was contaminated. So, they must have occurred, the virus seems to be circulating independently of the initial exposure, initial non-exposure, and you think of two possibilities. One, that is has always been there, even before the polio episode, and the polio vaccine may have simply increased the amount, or that the polio vaccine introduced it into the human population and then it is now circulating independently. As I think I said before, this is not easy to conceive, because these viruses are very highly species specific, they don't really cross boundaries, their genetic make-up is not like that of the RNA viruses. But anyway, the data suggests that it is independent of polio vaccine contamination, it is circulating. And, the third characteristic we thought one might ascribe to is that it is capable of being transmitted very early in life, and this is the data from the ependymoma cases, where the children were two, three, four years old when they had cancers, so that the infection must have occurred at birth or very soon after birth, perhaps, in utero even. And so, this then suggests that it may be either transplacental transmission or in some other way. Now, transplacental transmission would only occur, I think, if the mother was having a primary SV40 infection with a large amount of virus, a large amount of virus in the blood viremia. So, one would think that the mothers would have very high antibody response if they were capable of transmitting the virus to the fetus, and yesterday there was a suggestion from the floor as to what happened to the parents, did the mother show evidence of infection? And, one of the ways one could follow in order to clarify all these issues is to study the individual cases of these tumors from an epidemiologic point of view, in the families, in the surroundings, and see if you can find any evidence for this naturally separating SV40, and preferably isolate the virus itself, relying not so much on presence of antibodies. And, it was an attempt to do that sort of thing that we had looked at these 165 urines from transplantions, not transplantations, but HIV infected patients, to look for this independently circulating virus, but the contacts of the patients who have these tumors, I think they would be a very rich source for investigating this. And, these are very rare tumors, but I think you can get to them if the resources are mobilized. Before I came here, I spoke to Doctor Grossman at Hopkins, who is the head of a tumor bank, a brain tumor bank, and a consortium of ten institutions where they look at brain cancers, and there are three such consortia in the United States. Now, he had, in his freezer, six ependymoma cases, one choroid plexus papilloma, he said these are rare tumors but you will find them within the ten institutions, there will be one, or two, or three patients currently undergoing treatment. If you want to reach these people, look at the families, look at the patients for evidence of these viruses, I think it would be a big thing. And, I think such a study can only be done by CDC or NIH, it would be beyond the capacity of individual workers to do such a study. Thank you. DOCTOR LEVINE: Are there any other members of the panel who would like to make a statement before we continue? Okay, then if not, may have the next slide, please, or my next slide? While we are waiting for the slide to go on, I would like to invite the members of the audience to take part in this discussion. I think it should be uninhibited and informal, and I thought yesterday we heard some superb comments and data from the floor, and my expectation is that this morning will be equally robust. Okay. So, here we pose some questions, and I would like the panel to begin to address them. We have talked about all of these questions already to some extent, but now we will focus more, and, again, I'd invite the audience to address these questions as well. What are the current data that suggest that SV40 is an endemic agent in the human population? How can SV40, which supposedly replicates poorly in human cells, spread as an infectious agent in humans? What additional data are needed to determine whether SV40 is endemic or not? And, what are plausible sources of human exposure to SV40, for example, vaccines, primates, humans themselves, or unrecognized recombinants. So, I need a member of the panel to lead off this discussion. Doctor Butel? DOCTOR BUTEL: I'll just comment that we have been doing some sera epidemiology assays using a plaque reduction test, looking for the presence of SV40 neutralizing antibody in various groups of people. In general, the numbers that we're finding agree with Doctor Shah's published data. If the group of people are of an age where they probably received a contaminated polio vaccine, we are finding neutralizing antibodies in, roughly, 20 percent of those people. In older people, that most probably did not get a vaccine, we are also finding antibody more in the neighborhood of maybe ten percent of those people. In younger people born after 1963, we are finding neutralizing antibody anywhere from two percent to ten percent, depending on the group that we are looking at. The titers are not too high, more like the natural infection data that Keerti just showed. They range from a neutralizing titer of one to 20, we've had some that are one to 320, and I guess I'm not persuaded that all of this can be explained by cross reactivity with BK or JC antibody. We've tried to grapple with this, and I would like to hear suggestions as to how to absolutely prove this, but we looked at the BK antibody titer in a group of people that were positive for SV40 neutralizing antibody, and compared that with a group that were negative for SV40 neutralizing antibody, to see if the one group had very high BK titers and the other group were low, and that is not what we found. We found a range of BK titers in both groups, and so the SV40 neutralizing antibody did not correspond to having a high BK antibody titer. So, we would like some suggestions as to how to maybe absolutely prove that this is SV40 neutralizing antibody that we are finding. DOCTOR LEVINE: Doctor Frisque? DOCTOR FRISQUE: I don't have an answer for you, but I think I have a related -- closely related question, which is I think the data which were nicely reviewed, years to corroborate this by Doctor Shah and Doctor Levine, certainly provide tantalizing evidence that SV40 exists in the human population, perhaps, was enhanced by the poliovirus experience. My problem is, is that the data that have been summarized and published certainly don't need meet my standards for current rigorous sero epidemiology. As many of you know, I come particularly from the HIV perspective, and the two-tailed issues of sensitivity and specificity just can't be addressed, I don't think, in the algorithm of applying a single assay, particularly, with a virus that has a relatively complex lifestyle, that involves both replication of structural antigens and then subsequent latent antigens that may or may not be picked up by a single assay. And, I think to move this field ahead, it seems to me as though, you know, a high sensitivity assay, followed by corroboration with a high specificity approach, is really what's needed, and that's certainly the state of the art in HIV and Hepatitis C virus and the specifics depend on the virus and, you know, how it manifests in the population. But, for example, if you take the Geissler paper, which I think is among the most tantalizing of them, it's the one that applied an ELISA technique of a high sensitivity, I don't see any corroboration that that proves that that's, in fact, SV40, and ELISA's, of course, are, you know, renowned to be prone to various kinds of cross reactivities that are uncharacterized. So, I would like suggestions as to how, you know, what would be needed to sort of move this ahead in the two-tailed front of a screening assay and then a corroboration assay. DOCTOR BUTEL: Well, let me respond to something you said. HIV serology is very different, I think, from what SV40 serology would be, because HIV has many more antigens, and in the case of SV40 we know there's the neutralizing antibody is directed against VP1, and there aren't other antigens that I think would be important in neutralization. And, I would like someone to explain what assay is better than a neutralization assay for identifying a specific antibody, where you have a plaque assay for the virus. I think it's more specific than CF or HI, or even an ELISA. DOCTOR LEVINE: We have some comments from the audience. Doctor Pass? DOCTOR PASS: If I can show a couple of slides that I just gave to the gentleman in the back. With the help of Paula Rizzo, when she was in my lab, because we were doing some hamster assays, we also set up an ELISA, not only to measure antibodies, serum antibodies to SV40 in the hamsters, but also hamsters, as an indirect ELISA. And, essentially, the question is, what do you compare your numbers to? So, we, essentially, have two baselines that we compared to in these patients. I felt that it was reasonable, this was before the blood and urine paper, that it would be reasonable to compare titers to cord sera, so we got cord sera and used that as a baseline, but also from patients who come to the NIH we took their blood and got sera and called the baseline the mean of those patient sera plus three standard errors. And, if the levels were then above that baseline, we would call it positive. And, the results are summarized here, I have the graphic results in the next slide, but this slide shows that if you compared a cord sera, both mesothelioma patients and normal volunteers, will have levels above cord sera, and that's not significant. But, if you then look at the levels above the baseline, which is comparing to general population I guess, you find a statistically significant increase in levels of antibodies to T-antigen, at least, SV40 T-antigen, in the sera in the mesothelioma patients. Next slide. DOCTOR LEVINE: Could I just ask a question? Did you use JC and BK antigens as controls? DOCTOR PASS: No. No. Again, the purpose of this was to see whether the mesothelioma population as a whole had some difference from a normal population with regard to neutralizing antibodies of T-antigen, and the data is graphically depicted here with the normal volunteers at the bottom and at the top. Very frankly, when we started to do this, I didn't know how much -- I didn't think there was much in the literature that really guided us, so we sort of just started from scratch and did it because we had the sera. DOCTOR LEVINE: Thank you. DOCTOR GOEDERT: Could I make a comment? I think as a first cut, I think that, you know, sort of diversifying the approach to detecting antibodies in this particular problem is probably an extremely valuable undertaking. You clearly need some kind of a confirmation or sorting out of the different virus reactivities that could be SV40 and could be other epitopes. DOCTOR URNOVITZ: May I add to that comment? I agree with the last speaker that -- I'm Howard Urnovitz with Chronic Illness Research Foundation, I was a manufacturer of one of the HIV test kits, and I think that as we went forward and looked into other bifluids in blood, we found a completely different distribution pattern of antibodies, and what we found out is the ELISA is a heck of a great screening test. The confirmation is even better to tell us which is the difference between false positives. We didn't go out of our way, because then we started to recognize our false positives were antibodies to human endogenous retro viruses, which was not HIV-1. And, the bottom line is, is the further we look the more we realize that all we could really get out of an antibody test is what most of the intended statements say on the manufacturer's test, is that it suggests an exposure to the virus. I mean, that's about as far as you can go with the data. So, I would respectfully submit that we may want to think that the antibody tests have given us great direction in which way to go to research, but I think I'm just cautioning the group that you may be over-interpreting the antibody data, and I think you need to move to more of a molecular biology approach. I just don't see how you can go any further with the antibody assays and conclude that it was an endemic. DOCTOR LEVINE: Anybody else from the panel? Doctor Dorries? DOCTOR DORRIES: We did ELISAs and HI tests on BK virus and JC virus, and I only can say that our homologous activity was clearly much higher than the heterologous activity. In HI tests, it was much better than in ELISAs, and I think then that indirect ELISAs might be even better. DOCTOR LEVINE: From the floor? DOCTOR OZER: Harvey Ozer from New Jersey, a standard way of trying to discriminate cross reactions is to do competition assays. And, in fact, if there is antibody to SV40 and you are suspicious of BK, why not try to block the antibody to SV40 with BK virus. In fact, Keerti and I, many, many, many years ago, used an assay to verify the neutralization, which was, essentially, an immunoprecipitation of purified virus of SV40, which was radio labeled, and that was susceptible to competition assays. So, I think there are assays out there that people can do. They are not convenient for screening, and so I think the issue is, if you identify antibodies that you think are interesting, from patients that you think are interesting, to follow them up with research protocols. DOCTOR LEVINE: I'd just point out that Geissler actually did do a blocking test, but he probably did it with the wrong antigen, he used polyoma and a lambda protein, but not BK or JC. DOCTOR OZER: And, I would just conclude with the comment that we -- since it neutralizes SV40 virus, the one that Janet was talking about, it must be on the BK virus virion, and, therefore, so it's very clear what the competitor should be. DOCTOR LEVINE: Other comments from the panel? Doctor Oxman? DOCTOR OXMAN: I was going to say the same thing, that really if you cross absorb the five percent of the sera that are neutralizing in the plaque assay with both native and disrupted BK and JC, you'll either find you are absorbing those cross reacting antibodies or you've got some residual that's SV40 specific. DOCTOR LEVINE: From the back? AUDIENCE: I'd like to make some comments about some of the things Keerti Shah said. This business of cross reactivity is really an interesting problem, and I think we shouldn't forget other papova viruses, like LPV and that sort of thing, so maybe this is a time to rethink some of our strategies and think again about viruses like LPV and how they fit into this whole equation. The second thing, with respect to virus neutralization studies, I'd like to remind everybody about the Ashkenazi-Melnick experiment, I think it was '62, but they infected monkeys with SV40 and found out, number one, even with infected monkeys, using the techniques they had back then, it was hard to show and recover virus from some of the infected monkeys, and the antibody levels they saw in these monkeys in some cases didn't go over a one to ten dilution based on virus neutralization tests. So, the point is, depending on who is doing the experiments, you know, it's not really that different from what you see in monkeys and humans, in some cases. Also, if you use different species of monkeys, you might get somewhat different results. How do you compare, though, the results you get with a certain monkey and what you might see in humans? That's a very important question. DOCTOR LEVINE: Thank you. Anybody from the panel wish to comment? Okay. Yes? DOCTOR MINOR: Can I go back to something that Doctor Butel said about her sero studies? DOCTOR LEVINE: Sure. DOCTOR MINOR: If I understood it right, my name is Philip Minor, I'm from NIBSC in the United Kingdom, as I understood it, the people who are of an age to have been exposed to the -- vaccine were 20 percent seropositive, or thereabout. Were those highest titers or lowish titers? I mean, my difficulty is that they were exposed at least 30 years ago probably, and this is an awful long time for an antibody to persist, if it's, you know, just straightforward antibodies you saw. Can you say a bit more about the titers and how long it was since the exposure and so on? DOCTOR BUTEL: The titers, in general, were low, one to 20, one to 40. The exposures, presumably, were at the time when the contaminated polio vaccine was given, although, there's always the possibility that if SV40 is circulating in the human population they might have been exposed again. Many of these sera were collected, oh, ten, 15 years ago, they've been in storage. DOCTOR LEVINE: I think in Joe Fraumeni's study he looked at antibodies shortly after the contaminated vaccine and followed that group initially for four years, and then I think there was a subsequent follow-up at ten years. And, if I remember his data correctly, he found high titers initially, and they fell to what he called a moderate level thereafter. But, Doctor Morris, you were of that era, can you comment? DOCTOR MORRIS: Yes. The titers that occurred in about one third of the volunteers were relatively low, one to ten, one to 20. DOCTOR LEVINE: Thank you. Doctor Shah? DOCTOR SHAH: Yes. I think in the antibodies that occurred as a result of SV40 contaminated polio vaccines were a good bit higher than what we saw in the human population, and I believe they were followed for at least 13 years, without any marked reduction of titer. And, there was a controversy at the time that the antibodies are persisting, so there must be live virus there, on the other hand, whether the mammary cells can persist. I think that controversy was not resolved, but the antibody titers did not decrease, at least not markedly, for at least 13 years. DOCTOR LEVINE: But, we should go back and look at Joe's data, because he did, I think, have a careful record, but I couldn't find it in the publications of what the titers actually were on given patients. DOCTOR SHAH: Even to children Doctor Fraumeni followed? DOCTOR LEVINE: At least out to the first three or four years, but, Jim, maybe do you know anything more about it? DOCTOR SHAH: I think they were children who got oral polio vaccine, I think, so they were probably antibody free. DOCTOR LEVINE: Well, he didn't follow the ones that were antibody free, because they didn't have antibodies, so it must have been the group that had the Salk vaccine. Other comments or questions? Doctor Strickler? DOCTOR STRICKLER: Yes, thank you. I think the big problem that we have in trying to validate any sero assays is that we don't necessarily have good exposure data on any particular individuals. We don't know exactly who is infected. We, in our one, tried to see if the virus could be detected in urines, as a manner of figuring out who is infected and non-infected, we were at least so far unsuccessful. Doctor Butel took the first step in looking at comparison groups, in which she had an understanding of the probability of exposure by looking at different birth cohorts, but that's exactly the type of thing that we need to do as we move forward with these assays. We have to be very clear about what our exposure groups are as we validate this, lab workers who were exposed to monkeys likely to be contaminated is one group, the birth cohorts that we've defined, and, obviously, the patients in whom we think that we are detecting SV40 DNA, are individuals we would like to test, but we need to compare them also to appropriate groups. For example, it would be interesting to know if Doctor Butel was able to detect antibodies with her assay in any patients with the tumors that have been found to contain SV40, but I think it's also important to look in other cancer patients, because as people become immune compromised there can be other reasons for antibodies to different viruses to increase. So, I think we need to be very careful about our comparison groups, and we need to look towards individuals who we think we understand what their exposures were, as we try to validate these assays, and not just make it based on competition assays and so on. We need to try to validate them based on the amount of exposure data that we have. DOCTOR LEVINE: Thank you. DOCTOR DORRIES: I would like to comment to the ELISA titer -- to titers in the natural monkey infection. We recently did some ELISAs on persistently infected, naturally infected monkeys, and the ELISAs were fluorescent based, and we got titers in the range of one to 5,000, and one to 10,000. So, in reviewing the new methods, one might get another possibility to check out. DOCTOR GOEDERT: T-antigen or V antigen? DOCTOR DORRIES: V-antigen, we used purified SV40 particles. DOCTOR LEVINE: Doctor Carbone? DOCTOR CARBONE: I would like to ask the panel, the members of the panel a question, that I'm not sure I understand very well, and it's the following. The oral polio vaccine were used, one of the main reasons that they were used was because they are excreted, and so you vaccinated one kid and then you vaccinated an entire family. Now, if I understand correctly, SV40 contaminated some of those vaccines, and SV40 was also excreted. So, I would assume, and I'm not sure I'm correct here, but would assume that if the other members of the family got infected and vaccinated with the polio, they also maybe got SV40. Now, I hear that there is a lot of concern whether you are checking the antibodies or the tumor induction in kids or in adults, but this may be, in fact, not completely correct, because if the kids were mostly the ones who received the vaccines, but then if the thing works the way that the polio worked, then everybody got it. Am I wrong? DOCTOR LEVINE: Doctor Shah, do you want to comment? DOCTOR SHAH: I did not exactly get the question. DOCTOR LEVINE: I think Doctor Carbone wants -- is raising the issue of whether SD40 was spread by the oral fecal route from infants who were immunized to their family members. DOCTOR SHAH: Right. In the U.S., only a few thousand infants received oral polio vaccine that was potentially contaminated, because the licensed oral polio vaccines were free of SV40. I think it is absolutely right that if there's a polio excretion in a family, every member will become infected with polio virus. I think this was done by Doctor Melnick and many other people long, long ago, and you could imagine that the similar thing could occur with the SV40, which would be in the vaccine. The studies that were done on excretion of SV40, in people who received contaminated oral polio vaccine, and they were able to do it because the stools were saved for polio studies and they went back and looked at the same stools, there were about three or four studies, maybe one or two found SV40 excretion, which was intermittent, low level for about five or six weeks, but some other studies were negative, they did not find SV40 persisting in the stools. Whether it could have infected family members, of course, I don't know. DOCTOR MORRIS: I'd like to make a comment about the individuals. You said there were very few individuals for which the exposure is known. Well, I think there were 31 volunteers participated in the studies that we carried out in the early 1960s. Those sera were stored, and they were used most recently in 1966. So, for those persons, and I still -- I believe that those sera are still stored here at NIH, if you want an individual with a known exposure, with virus recovery from throats, with antibody rises, you have subjects that are available, that is, the materials recovered from these subjects are available, and they should be stored still at NIH for use in further studies. DOCTOR LEVINE: I should point out that we have another bank of sera that may be relevant here. In the late 1950s, there was a national study, at that time sponsored by the Neurology Institute, of cerebral palsy, and to carry out that study sera were collected from 45,000 pregnant women, and those sera still exist. They date from about 1958 through about 1965, and the histories were well documented, so we know which mothers received the vaccine and which did not. The cord sera and infant sera from 20,000 progeny of those mothers exist in the bank as well, and so that's going to be a valuable resource, once we decide what assays might be applied, so that we don't squander the sera, which brings up the question, actually goes back to what we might -- DOCTOR BUTEL: Could I say something before you change the subject? DOCTOR LEVINE: Sure. DOCTOR BUTEL: To sort of address Michele's question, some of the sera, but it doesn't exactly answer the question, some of the sera that we've had access to were from daughters and mothers. The daughters were of the age to have been exposed to the contaminated vaccine, the mothers were older and would not have been. And, we looked at some of the matched sets of the mothers' and daughters' sera to see if the daughter was positive for SV40 neutralizing antibody, was there a better chance then that the mother was going to be positive, most probably because the virus had spread in the family from the vaccine. The results were that there didn't seem to be any correlation between whether the daughter was positive and the mothers were positive. DOCTOR LEVINE: Yes? AUDIENCE: I'm very encouraged this morning to hear that the government is interested in looking at the parents of the children who have SV40 associated -- DOCTOR LEVINE: Go ahead, I think the microphone is on. You were on. AUDIENCE: -- to see if they also are carrying SV40. I was exposed to the potentially contaminated vaccines in both Minnesota and Colorado, when I was a child. I think that parents or mothers in my generation would be very interested to have you do a problem study and find out how many of us are carrying it, and also to find out how many of our children are carrying it. I think it's very important for the independent researchers who came here and who have done these studies, that have pointed out this problem, be funded and be part of anything official that is done to find out if there are SV40 associated cancers that are causing health problems. I think that the government is in charge of promoting vaccination, and it took many, many years for you to admit that the oral polio vaccine can cause polio in some children and in close contacts, and the public would not have confidence, frankly, if these studies that you are talking about are only conducted by the government and do not include independent researchers in some kind of oversight. DOCTOR LEVINE: Thank you for your comment, but I would like to point out that I don't -- it's not my sense that the government has been conspiratorial or guilty of obscurantism in dealing with the issue of whether SV40 -- let me just -- hear me out for a second -- whether SV40 is in these vaccines, because as Doctor Helleman pointed out yesterday, the discovery of a new -- what was then a new virus, and elucidation of its biology, and ridding that the uncurrent vaccines of SV40 was all accomplished with a two-year period, which is quite remarkable. Secondly, I would like to point out that the data that we heard at this meeting has been entirely generated, or at least primarily generated, by non-government scientists, and, in fact, there's no reason to believe that that won't be sustained. But, finally, I do need to remind you of the data that Doctors Olin and Strickler showed yesterday, which is very powerful epidemiologic data suggesting that no harm came from the vaccines that were knowingly contaminated with SV40 with respect to cancer. Could I have the next speaker, please? Doctor Lednicky? AUDIENCE: I'd like to reply to what you said. DOCTOR LEVINE: I will let you in just a moment. DOCTOR LEDNICKY: Perhaps, the panel might address this. Now, as a virologist, I have a question about doing antibody tests when we suspect an association with tumors, because if it's not a Lytic disease can we really expect that you would have high titers of neutralizing antibody against virus? So, perhaps, as was talked about earlier, using ELISA tests directed against T-antigen might be the better way to go, because I'm not sure you can really learn anything about screening perspective -- patients with perspective SV40 tumors for antibody. The second thing is, doing neutralization tests, a lot of labs can't talk to each other because the antibody levels that are detected aren't always exactly the same, and that goes back to basic virology. One reason for this are the reagents. If you have a lot of defective interfering particles in your virus prep, for example, this can affect your result. So, I would like to suggest that we standardize that test. Doctor Butel has people doing literally hundreds of these tests a week, and it might be a good idea to standardize some of these tests by having virus come from one source. Thank you. DOCTOR LEVINE: Thank you. Would the panel like to respond? DOCTOR GOEDERT: This is, I guess, a related question. I was wondering, I was going to ask if others beside Doctor Pass and our group have tested sera from mesothelioma patients or others whom we believe would have a high likelihood of having virus detected, at least by PCR. And, you know, we did not detect neutralizing antibodies, and I think the reason may, in fact, be that they don't have anything but latent antigens expressed if they are infected, and I think there's a reasonable chance that they are, and so I'm just wondering if others have experience with populations of people who have some validation that they actually are infected with the virus. DOCTOR LEVINE: Does anybody have data? Let me finish with the panel first, Doctor Frisque? DOCTOR FRISQUE: Okay. I just wanted to make a point about the reagents and discussion of reagents. One factor we haven't really considered, I believe, is the source of the antigen used in these assays, and, clearly, there are antigenic variants in these viruses. With BK virus, for example, BK wild type versus the second strain called BKVAS, which is 95 percent sequence identity, but those viruses if you make antibody to BK wild type you will not see a cross reaction with BKVAS. On the other hand, if you make antibodies against BKVAS, you will see a good cross reactivity in the other direction with BK wild type. So, I think there is consideration in the antigen sources that we use in these tests, that that's an important factor as well. DOCTOR LEVINE: Thank you. Now, let's go back to the panel. Yes? DOCTOR KYLE: Yes, sir, Walter Kyle, I'd like to comment briefly on Doctor Carbone's question. There are a couple of things. First, I think everybody should realize, as Doctor Ratner attempted to point out yesterday but was interrupted, the inactivated polio vaccines of the '50s weren't actually inactivated. They contained live polio viruses, they caused many cases of polio. In addition, very significantly, when you mix formalin with protein you get plastic. You had plastic encapsulated polio viruses that presented themselves after 30 days of inoculation, and I assume the SV40 was live encapsulated also in a lot of those early injections. I spoke about this at the National Academy of Sciences in 1992. I've had the opportunity to gather and review a lot of the manufacturing records from that time, of the manufacturers that were making these vaccines. The other comment I have is to Doctor Shah, I keep hearing this reference to after the vaccine was licensed in 1963, there was no SV40 in it. You should all know, I know, I have the records, most, if not all of the licensing lots of the oral Sabin vaccine were SV40 contaminated. Now, if they removed it afterwards, I would hope so, the fact is that the government agencies in charge for the safety of the vaccine have been found negligent in its licensing and release. It's the only vaccine or product where the government has been sued successfully, and that's -- that case was litigated for over 20 years, I was a part of it for a while. The documents we've gathered from it may be very helpful to some of you here, and I'd be happy to share them with the independent scientists. Thank you. DOCTOR LEVINE: Well, I don't -- if I may take the Moderator's prerogative -- I don't think that there's any question that there was live SV40 in the polio vaccines. That's not been an issue. Yesterday, we heard representatives from the manufacturers describe their technology in detail, assuring us that to the best of their ability, and with all current technologies, they can't detect SV40 now, but I don't know whether any of those people are still here in the audience. Is anybody here from the -- yes, would you like to comment? May we have a response to your question? AUDIENCE: I understand what's going on now wasn't what happened in the past, and I know -- I have the records of what Doctor Kirschstein has seen in the polio vaccine over the years, and her comments and her meetings with the manufacturers. For the manufacturer to get up here and assert that there's no viable microbial agent in their vaccines belies the evidence that the NIH and the Bureau of Biologics has as to what was in there over the years, into the '70s, into the '80s, after the '80s they started cleaning the monkeys up because a big problem arose, it was AIDS. But, the retro viruses, they actually discovered in the vaccines, the oral vaccines, in the mid-'70s, permitted their release, and didn't recall them from the market in the '80s when they knew. DOCTOR LEVINE: Right. I think we are getting into a territory which is remote from what we want to discuss, but if we can have a brief response to that comment I would appreciate it. AUDIENCE: I mean, I think leaving aside the retro virus question, I'm not a manufacturer. I'm the CBER equivalent, if you like, in the United Kingdom. In 1962, there were international requirements produced, which were the consensus of national requirements, okay, which say that SV40 should be excluded. In 1965, this was made a statutory requirement, if you like, for WHO things. The requirements were updated around 1972, I think, and this remained in there, in 1989 it's still in there. So, from 1965 international requirements, which are the consensus of national requirements, require that SV40 shall be absent. At NIBSC, which is an independent testing laboratory, we have records from at least 1966 to say that we were testing every batch of vaccine that came in for SV40 and finding them negative, so we were actually checking this out. At least as far as we are concerned, from the documented point of view, from 1966 the vaccines were free. The data that Dave Sangar presented yesterday on the PCR aspects of the batches that we are looking at at the moment start around 1996, because these are the recent batches that we can do something about, we've got back as far as 1980, and they were all negative. The proposal, really, is to go back even further and show that they are negative yet. In my view, the methods which the manufacturers used to try and detect this kind of contamination were really the best available at the time, and I think that the modern methods which we are now applying prove that they actually were adequate to remove it. DOCTOR LEVINE: Thank you. Panelists, anybody? Sure, we can, but let me just finish up with the questions from the audience. Doctor Procopio? DOCTOR PROCOPIO: Yes, I just want to go back to the antibodies against T-antigen. We have screened the '60s sera from a group in Italy from different clinical groups, and some of them were from mesothelioma patients. We have seen by Western Blotting that most of them were -- that they had been reacting with antibodies against SV40 antigen specific antigen. However, we have not seen correlation with the mesothelioma or other tumors, so it seems to be that also T-antigen specific antibody, you know, you use the T-antigen as a source of specificity may over estimate the infection, potential infection. I would like a comment from the panel about whether it is possible to select specific peptides of T-antigen that may differentiate BK, JC or SV40 T-antigen. DOCTOR LEVINE: Panel? DOCTOR FRISQUE: I think it's important to point out, I guess most people understand this, but the polyclonal sera against T-antigens are highly cross reactive, so that I think it's going to be very difficult to get a specific test for T-antigen. I think that's the wrong approach to try to take. Whether you take a peptide or whatever, you may find small areas, but the homology is so high it would be very difficult to find epitopes, which probably do exist to some extent, to make a test, though, that was specific for one of these T-antigens versus another. I think it's the wrong approach. DOCTOR LEVINE: Could you leave some light on the panel, but put the questions back on the board, so we can remember exactly what it is we are addressing this morning? DOCTOR GARCEA: I just want to make a comment about the antipeptide antibodies. I mean, first of all, I'd like to say that I think that the serology problem is a big one, the technical problems are immense. The capsid cross reactivities between these viruses are immense. I don't think T-antigen is the way to go, there's even more cross reactivity. But, for a particular experiment that we've actually done, we've made antipeptide antibodies against the BC and DE hyper variable loops on SV40 in an attempt to get capsid-specific antibodies, and these antibodies generated by antipeptide antibodies cross react with polyoma, among other viruses. So, I think that there's a terrible problem here, it certainly does deserve a tremendous amount more work, but I think that, for example, even in the ELISA assays that were judged to be excellent, I think that by my criteria they are not excellent, and they are not even very good for screening. DOCTOR LEVINE: Doctor Martin? DOCTOR MARTIN: I have, one is a comment, and the second is a question, a legitimate scientific question. The first comment is, I think we are forgetting some of the historical perspective. I was in medical school between '56 and '60, and on the wards in '57 and seeing the last few -- this is in Massachusetts General, in iron lungs, and went out immediately and got inoculated with the Salk vaccine. I think it was criminal not to have given the Salk vaccine in those days, so that's the comment. The question is, in view of Andy Lewis' comment, I wonder to what extent the adenovirus vaccines and/or isolates, random isolates of adenovirus have been looked for by PCR for SV40 sequences, because while it's true that there might be a cross reactivity with the T-antigen between BK, JC and SV40, it's also true that if SV40 is being introduced via adenovirus recombinants, naturally occurring, I'm not saying this is -- that you are never going to see them. And, if, like the LEV -- do I have the nomenclature right, it's LEV and HEV -- DOCTOR LEVINE: L-E-Y, H-E-Y. DOCTOR MARTIN: L-E-Y, thank you, it's been so long since I've been in SV40, if those things are around you are going to -- the mode of infection may be via adenovirus, but the thing that's doing the dirty work could still be SV40. DOCTOR LEVINE: Does anyone have data on the SV40 sequences by PCR in the hybrids? No, I don't think such data exists. I'd like to continue with questions that are focused on the topic at hand, which is the sensitivity and specificity of serologic assays for SV40 antigens. Yes? AUDIENCE: I think it's unfair and inappropriate for you to characterize our call for participation and funding of independent researchers in these studies that the governments are suggesting. Conspiracy was a word that you used, not me. I made it very clear yesterday that we understand that you didn't know at the time that you released those vaccines in the '50s, you didn't have the tests. However, it's very important to also emphasize that when you did know that these vaccines carried monkey viruses, that fact was not communicated to the public. And, all the epidemiological data in the world, including that that was presented by Doctor Strickler and Doctor Olin yesterday, is not going to negate the fact that there are now SV40 associated cancers that are occurring in adults and children. And, again, I think the public is interested in a full examination with the participation of independent researchers. DOCTOR LEVINE: Well, let me respond in a couple of ways. First of all, I certainly wasn't suggesting that you were accusing the government of being conspiratorial. Nonetheless, one reason for this workshop is to try to, in fact, do just what you suggest, to try to, by putting our heads and our collective data together, to arrive upon what is scientific truth. There had, however, been, before this meeting, a sense in the lay press of a conspiratorial quality in the government's actions. Once again, you needn't -- we really don't want to have a debate about this, because I'm not accusing you of having accused me of being conspiratorial. The fact remains that this is a very grey area. You heard yesterday that we are not sure, even in the best of hands, whether the SV40 footprint is or is not in tumors. Most importantly, if it is there, we don't know whether it's effect or cause. And, finally, you've heard this morning that there is much work to be done before we can, in fact, hit upon assays that tell us correctly whether this virus is or is not endemic in the human population. Doctor Weiss? DOCTOR WEISS: Robin Weiss from London. To get back to the questions on the screen, I'd like to echo what Harvey Ozer suggested, that it might be worth investing some technology in competitive assays. And, I don't mean just competitive absorption with antigen, but by taking reference sera, it could be monkey sera, it could be rabbit sera, that have a known high titer, titrating them out, labeling them, going in with your human test sera to see whether you compete out the labeled antiserum. This works superbly well with HIV, and, you know, I take in what Janet said, that SV40 is not HIV, but it enabled us to establish that slim disease in Africa actually was HIV infection, when a previous report got about 50 percent false positives. This kind of competitive assay yielded no false positives. It's enabled clinical virologists to distinguish in rapid masquerading assays between Herpes Simplex Type II and Herpes Simplex Type I, which is of the kind of order that we might need to distinguished between BK, JC and SV40. So, I think there are tricks of the trade that are reasonably well known to clinical virologists, but which most of us molecular virologists are not quite so good at, that could be applied fruitfully, or at least would be worth investigating, to try and see if we could get up a mass screening serological assay. Then those should be followed by confirmatory tests by Western Blotting other kinds of assays, assays for other SV40 proteins, and, of course, by PCR. But, I think it's worth doing, and I don't think it would be vastly expensive, and I could suggest one or two names of clever people who might be able to help. DOCTOR LEVINE: I think that would be a very good approach. If and when we get to PCR, we will, of course, have to agree on how to do it, and what it means, so are there responses from the panel, further comments from the panel on this issue? Doctor Goedert? DOCTOR GOEDERT: I would just endorse the idea of a competitive ELISA, because in addition to the examples that Robin cited, I think it worked extremely well for HTLV at a time when it was, you know, rare, and it is rare, and it has some other analogies, and I think it's the kind of thing that you ultimately do need some additional confirmatory assays, but it's the kind of thing that can be set up and executed pretty easily. DOCTOR LEVINE: I had one more question that we haven't addressed on the board, the last question, how good are assays for SV40 specific antibody in immunocomprised patients? There's no particular reason to think that they are not good, since we are finding them there, and certainly, in patients with HIV infection we find reasonable levels of antibody to at least some antigens. But, I wonder if anybody on the panel would like to address that question. Anybody in the audience? Yes, Doctor Weiss? DOCTOR WEISS: Well, in the majority of HIV infected patients, until they reach very late stage AIDS, there's actually a hyperplasia of B lymphocytes, so antibodies to all sorts of things you've had in the past go up. So, we've got to distinguish that HIV patients are selectively immunocompromised, and part of the HIV syndrome is an elevation of antibodies. So, I think if there has been past experience of SV40 infection, it's quite likely to be detectable. DOCTOR LEVINE: Thank you. DOCTOR GOEDERT: Yes, I would have to agree as well, with patients that have PML, almost invariably they have -- they are immunocompromised severely, and they still retain high levels of antibody to JC that stay fairly level. So, those antibody levels don't go down with this state. DOCTOR LEVINE: Yes? DOCTOR CHEN: Yes, this is Bob Chen from CDC. My comment is more of a second comment, just to correct the record in terms of when the vaccine associated paralytic polio cases after oral polio vaccine were more or less established and reported to the public, right after the oral polio vaccines were used in the early '60s there was a Surgeon General report that came out, and I believe it was 1962 or 1963, so I think in terms of some kind of cover up I don't think that really was the case at all. Then, the second issue is in terms of epidemiology studies, I think, from Doctor Olin's report yesterday, it sounds like in Sweden they are definitely well defined cohorts of exposure, et cetera, and I think once the serology assays are made more sensitive and specific, I think it will be very nice to go into settings like that, to kind of figure out what the exact prevalence of SV40 is, and, similarly, in the U.S. kind of every ten years the NHANES does a serosurvey, and, presumably, from those we could construct certain cohorts of seroprevalence also. Thank you. DOCTOR LEVINE: Right, and we have the child development study bank that I talked about earlier, with 45,000 sera. Just out of curiosity, does anyone know of any other serum bank that antedates the polio virus vaccine or that followed the vaccine progressively thereafter? AUDIENCE: I just wanted to comment. There's another cohort that you might want to look at, and this is people that have worked with SV40 for a lot of years, many who have kept antibody data. And, I know when I was in Paul's lab, Paul Berg at Stanford years back, that data were accumulated over years, and that some individuals had surprisingly high titers, so that might be an interesting source of information to look at. I think Chuck Cole had one to 3,000 antibody titer, for example. DOCTOR LEVINE: Right. Geissler has data, too, from the high-intensity SV40 exposure in the labs, and, again, just to remind you, the data were that 50 to 60 percent of people so exposed had titers, and they were at high levels by the plaque reduction assay. Yes? DOCTOR KLEIN: This may be quite a far-fetched question, George Klein, Stockholm, but, perhaps, it's not irrelevant with regard to the question of the SV40 tumor association in humans. In the days when Bob Shubner discovered the SV40 T-antigen by complementary -- there was then an expression called the tumor of the hamster, and the titers against the large T-antigen, these were also by immunofluorescence, were highest and as an SV40 induced tumor was growing in the hamsters. And, actually, it could be used horizontally to follow the tumor as it were. Now, with the uncertainty that now exists, as I understood it yesterday, with regard to the association between SV40 and the tumor rates detected by PCR, whether it's in all cells and all that, is there any evidence that titers are high and increase in patients with SV40 carrying tumors? And, could that be followed horizontally? DOCTOR LEVINE: That's an interesting question. Does anybody have data on the evolution of the antibody titer in people who have been bled pre-tumor, early tumor, late tumor and so forth, anybody doing that? Doctor Carbone, are you doing that, wherever you are? DOCTOR GARCEA: I think we discussed yesterday that there's a difficulty in a lot of these samples are archival, and for the IRB studies you can't go back and get blood samples. We currently have before the major pediatric oncology groups, the Children's Cancer Study Group and the Inner Group Sarcoma Study Group, two prospective collections, where we look at all ependymoma, choroid plexus and osteosarcoma patients that will occur in the United States in the pediatric population. And, coincident with this, we'll gather the sera, and also test the tumors for the virus. But, in the retrospective archival studies, we cannot do those kind of studies, but, hopefully, we'll have the data, but I think the purpose of this discussion is actually, once you collect those sera how are you going to make sense of the titers. DOCTOR LEVINE: Yes. I would also point out that, getting back to the question of cause versus effect, that if SV40 is endemic in the human population, one could hypothesize that it finds a comfortable home in a tumor cell, as a function, perhaps, of the replicative machinery of the tumor cell. And, particularly, if its replication is episomal, as opposed to integrative, it is entirely possible that, in fact, you'd begin replicating more SV40, as well as JC and BK, as a consequence of the genome simply being in the tumor cell. So, that, in itself, is not going to separate cause from effect. Yes? DOCTOR TEVETHIA: Tevethia. I just wanted to send a word of caution about detection of T-antigen, and no two groups, as what I have read, are using the same monoclonal antibodies to T-antigen for the detection. And so, a large number of these monoclonal antibodies do react, number one, with JC, and that has to be distinguished. Number two, the number of monoclonal antibodies to SV40 T reactive with seroproteins, and they must be avoided to make sure people don't -- maybe the first thing they should know about it, second, they shouldn't use it. Third thing, they should use the monoclonal antibody as precisely mapped, you know, to about eight or nine immunoacids, and everybody should use the same monoclonal antibody that are high titers, and precisely well defined, and third thing, an important test, in my opinion, is, for example, recently our lab, well, a while back, and in Levine's lab together, and now Dick Frisque, have generated monoclonal antibodies to JC that don't react to SV40 T. So, any sample that you have reacts to SV40 but not the JC antibody, to make sure definitely you are detecting SV40 T. DOCTOR LEVINE: Thank you. We'll take a question in the back. DOCTOR IMPERIALE: Mike Imperiale, University of Michigan. One suggestion is to use RNA aptomers to look for cross reactivity, because those aptomers can be exquisitely sensitive and may be able to actually help out in distinguishing whether the antibody is reactive against one or the other of the viruses. DOCTOR LEVINE: Okay. One more question, but devoted specifically to the issue of the sensitivity and specificity of serologic assays for SV40. Do you have such a question? AUDIENCE: No. I want to ask if anybody on the panel will discuss the famous leukemia cases from Niles, Illinois, eight leukemia cases associated with one school, and it seems that nobody is discussing that. DOCTOR LEVINE: I'll be happy to discuss it. AUDIENCE: Thank you. DOCTOR LEVINE: Although I'm not an epidemiologist, but there have been many instances in the history of cancer epidemiology in which clusters of tumors have been found, the Niles leukemia epidemic, the Albany Hodgkin's epidemic and so forth. And, when the borders of these regions have been carefully drawn and redrawn in a variety of ways, the cause and effect relationships become obscure. But, I really would prefer Doctor Goedert to comment on this. AUDIENCE: Each -- DOCTOR LEVINE: Could I have Doctor Goedert respond also, and then I'll give you another chance. AUDIENCE: -- each of these leukemia cases had three or more polio shots in the '50s. DOCTOR LEVINE: Right, so did a lot of other people who didn't get leukemia. Doctor Goedert? AUDIENCE: This is a famous cluster. DOCTOR GOEDERT: Yes, I'll be happy to talk with you at length during the coffee break. Basically, cancer clusters do occur by chance alone, some of them by some kind of common exposure mechanism. It's usually an extremely difficult and inefficient way to try and sort out causation, to pursue the clusters of cancer cases, and the example that you cite, I think Doctor Levine has certainly the right answer, is that virtually every child had three polio shots, and so I think that it would not be unexpected for seven children in Niles, either randomly selected, or selected because they showed up at the hospital with a serious disease, are unlikely to be different. DOCTOR LEVINE: Okay. May I have the next slide, please? Let's go on, so that we try to finish our questions before the coffee break. Here we asked a question, should we look for SV40 specific antibody in the mothers of infants or young children with tumors that have been associated with SV40 DNA sequences. That question we have already addressed, I think everybody is in agreement that that would be an interesting study to do. Is there any other comment from the panel or the audience on that question? Doctor Morris? DOCTOR MORRIS: Yes. I'd like to make a proposal that might partially answer the last questions there, about the persistence of SV40. The experiment that you described earlier that we carried out the mid-1960s, these experiments were carried out under almost ideal conditions. The prisoner volunteers were housed in the clinical center here on this campus, very careful records were made of their experience while here, but I'd like to propose that someone be assigned the task of trying to find these people now, those who have died in the interim, what they died of, those who are still living, the health status. This could be carried out by a graduate student given a grant to do such work, but I think it's important that these patients might be followed, after all these years, more than 30 years, to find out whether or not there is a virus persistent, or whether or not there is not. Irrespective of this test, of this examination, the results would be of interest. DOCTOR LEVINE: I think that's an excellent suggestion. How many were there altogether in the original group? DOCTOR MORRIS: There were 31 prisoner volunteers, and there were 11 controls, so some of these people must still be alive, but those who have died, the cause of death would be of interest. DOCTOR LEVINE: Right. The second question on the slide, does SV40 persist in humans? Which organs does it persist in? And, is viral gene required for persistence was suggested by Doctor O'Neill, Doctor Morris has started on that question. Doctor O'Neill, would you like to dilate on the question? DOCTOR O'NEILL: Yes. DOCTOR LEVINE: I hesitate to use the word amplify, so I used dilate instead. DOCTOR O'NEILL: I think the observation that Doctor Shah made that the antibody titers to SV40 persist at a level, a steady level for several years, perhaps, 30 years, I think that suggests that the virus is persisting in those individuals. And, it would be interesting to determine which organs that virus is residing in, and how it persists, and we might try to compare that with persistence of BK and JC viruses. DOCTOR LEVINE: Does anybody have data on the issue of SV40 in human kidneys, selectively? No. Doctor Lednicky? DOCTOR LEDNICKY: Addressing the second question, we shouldn't overlook the monkey model. No one has done this recently. And, I suggest that we do that, we look at some of these monkeys and using our current methodology try to culture the virus from different organs, as well as to do PCR. And, in fact, one population of monkeys that might be interesting to look at are the ones from southern India that Doctor Shah looked at, because it would be interesting to find out if the virus was persisting in slightly different cells or organs in those particular monkeys. DOCTOR LEVINE: Thank you. Doctor Strickler? DOCTOR STRICKLER: In response to Doctor Morris, I would like to say that we had the same idea to follow up on those individuals exposed during that volunteer study, and we've not been able to find the records yet. So, if any help can be brought to be able to identify these people and so we could follow up on them, we'd be very interested. I'd like to also mention that we are also endeavoring to follow up on the patients who doctor Fraumeni identified early on in his study in Cleveland working with Doctor Mortimer there, where they were following more than 1,070 children who were exposed in the first days of their life to contaminated oral polio vaccines. DOCTOR LEVINE: Thank you. DOCTOR MORRIS: Sir, what is your name? DOCTOR STRICKLER: Strickler. DOCTOR LEVINE: Doctor Lewis? DOCTOR LEWIS: I wanted to follow up on the question that Doctor Lednicky raised about the bonnet macaques in southern India, and I was going to ask Doctor Shah if there's any overlap in the range between the bonnet macaques in southern India and the rhesus macaque in northern India, because if there is, the question is why two species which are equally susceptible to the virus, one is carrying it endemically as an infection in a fairly significant portion of the population, and the other is completely negative. And, the question comes as to whether that's sort of a model for what could be going on in humans. And, the analogy that I would raise is that it's my understanding that we really don't know how JC virus is spreading among us either. And, I'll ask Doctor Frisque to comment on that, but if we have JC viruses that is present in the population in a very large percentage of us, what, 80, 90 percent by the time we are 20 years old, and we have SV40 which is similar, both these viruses share properties in the sense that they really don't, in tissue culture, don't infect human cells very well. So, the question is whether there's some analogy here between human species and simian species in this regard. DOCTOR SHAH: I think -- should I answer? DOCTOR LEVINE: Yes. DOCTOR SHAH: I think it is quite true that some of the macaque species are free of SV40, at least as judged by serological studies of 50, 60 monkeys, that is all that this is based on, but, for example, the Swedish vaccine was made in Javanese monkeys, they were free of SV40. The study on the South Indian monkeys was based on looking at 60 or 70 specimens, and they were free of SV40 antibodies. Even in the rhesus macaque, the infection can be lost, and there was one documentation of it, because a group of rhesus monkeys were moved to Cayo Santiago, which is off Puerto Rico, for a natural history study by some ecologists, primotologists, in 1938, and no new monkeys were added. And, in serological studies we were able to show that one of the -- some of the monkeys that were born on the island of Puerto Rico had antibodies, so the infection was brought to the island, but all the animals which were under a certain age were completely free of antibodies, so as if the infection was brought, it persisted for a while, and then it was lost. If you look at human populations, I think you can see similar things, for example, measles can be lost if the number of people in a population -- it will not survive, they cannot sustain it, and I think even in Doctor Brown's study and some of these isolated populations, I think they had some instances where the antibody prevalence was quite low. So, with the current density of human populations, we can maintain these viruses, but in small populations I think they could be lost. DOCTOR LEVINE: We do have access, of course, to the regional primate centers in this country, which would be an excellent resource for looking at the natural history of SV40 in various macaque and other old world/new world species. So, that's another resource that's available. DOCTOR BUTEL: Can I? DOCTOR LEVINE: Yes, Doctor Butel. DOCTOR BUTEL: I'd like to ask Doctor Dorries or Doctor Frisque to just bring us up to date on the most current thinking about the transmission of JC and BK. DOCTOR DORRIES: I think at least in part urinary excretion and transmission is correlated very closely, and several Japanese studies from families who really very nicely show that it is transmitted in the families, and the genetic footprints really show that some of the families got the virus by the parents and the virus types were, similar virus types, were transmitted to the children. And, in other families, different virus types came in the family after the kids came to school. So, I think that the transmission, at least in part, is going within the families, and as we have heard yesterday, probably respiratory tract infection also might be involved in JC virus infection, as it is shown for some years in BK virus infection. So, both sides of infection might be responsible for the transmission. DOCTOR LEVINE: Doctor Frisque? DOCTOR FRISQUE: I would have given the same answer, I believe. The only thing I would add is, again, to remind you that this is infection that occurs in children primarily, and an excretion occurs in adults at a high level, perhaps, over 40 percent of us will excrete JC in our adult life at various times, and so that, it is probably through the urine that this virus leads, as I said yesterday, probably as archetype, and that's what enters our body usually at a young age. DOCTOR LEVINE: Yes, Doctor Dorries. DOCTOR DORRIES: I have another comment to the Puerto Rico monkeys. They were negative for SV40 up to the last year that we got monkeys from Puerto Rico, to getting in Germany. And, shortly after the monkeys seroconverted to SV40 positivity, and we have seropositive monkeys in Göetting, so I think transmission also was going on by urinary excretion in these monkeys, because they are caged in different cages. DOCTOR LEVINE: Okay. I'd like to get to the last slide, the last two questions, because we've already touched on them and we're -- our time is growing short. The questions are as follows: Could recombinant viruses, which include SV40 sequences, affect our interpretation of the endemicity of SV40 in humans? And, could SV40 strains isolated from human tumors differ from the archetypical SV40, and if so, could this affect our interpretation of the endemicity of SV40 in the human population? These are both questions that we've already touched on, but, perhaps, Doctor Frisque, you can continue with either the first or the second question. DOCTOR FRISQUE: Yes. I might take your second question first, and that is, in terms of the archetypical type of SV40, that is found certainly in animals, and I would say that if you look at the sequence, small amount of sequencing that we've done, that the sequence is essential identical that we've looked at with wild type forms, forms that have been rearranged. So, in terms of changing antigenicity, I think most of the changes that occur in archetype versus rearranged forms of these viruses occur within the transcripts control region, not within coding ranges where you might see antigenic changes. That's not to say that antigenic changes, antigenic variants do not occur, but if you are comparing archetype with rearranged forms, I don't think that's the point. In terms of the other question, whether there's recombination with human viruses, again, we talked about that possibility yesterday, and I think it's possible. It certainly could complicate things, depending on how much rearrangement occurred and how many sequences were rearranged. Small parts of SV40 put into JC might make JC a bit more active, maybe more tumorigenic, but it might be difficult also to define those SV40 sequences in those JCs unless you do a lot of sequencing. DOCTOR LEVINE: Right. Other comments from the panel? Doctor O'Neill? DOCTOR O'NEILL: Is there any indication that there are individual cells that contain both JC and BK virus? DOCTOR LEVINE: Anybody have data? Doctor Dorries? DOCTOR DORRIES: Actually, I would expect that similar cells might be co-infected, but I think nobody has really shown it up to now. DOCTOR FRISQUE: I certainly haven't seen it. DOCTOR LEVINE: Doctor Carbone? DOCTOR CARBONE: Just a comment on the last question. From the data that we heard yesterday, it's obvious that today fortunately all the vaccine that we have are SV40 free. However, from what is written there exactly, and from the data of Doctor Butel, it is also clear that there are some differences with all the strains, that differ from the 776 that we dealt in the past, that is the virus that, the Lyse quickly CV1 cells. And so, it's at least conceivable to think that a virus that is only 172 base pair, or something like that, may grow less efficiently in CV1 cell, and that if that happens, and we are just testing the CV1 cells for lysis, if one is particularly unlucky could miss it. So, what I was just suggesting is that, given the fact that -- is the PCR, whether one could just simply add what you think of that, simply add to those tests and just look at lysis of disease in molecular tests that is very sensitive, to exclude the viruses that are not SV40, as we call this before today, but are similar to it, may eventually one day be there. DOCTOR LEVINE: You mean PCR with sequencing the product? DOCTOR O'NEILL: Yes, any PCR amplification of that would completely rule out that something that is close to SV40, but is not the SV40 776 that we have been talking about that is a virus that will grow and lyse the cell, and so it will be obvious that it's there, could be missed. DOCTOR LEVINE: Right. Doctor Dorries? DOCTOR DORRIES: I have a question. Has anybody really sequenced another wild type virus completely? DOCTOR BUTEL: Yes. Judy Tevethia had sequenced VA 4554, and we've sequenced the Baylor strain of SV40, Renee Stewart, in the lab, has done that, and we've sequenced the SVCPC isolate, and SVPML-1 and SVMEN. DOCTOR LEVINE: You've sequenced the entire genome? DOCTOR DORRIES: And, they are all conserved? DOCTOR BUTEL: Yes. DOCTOR LEVINE: Doctor Lednicky? DOCTOR LEDNICKY: Something else to consider related to question one. We should also think about DI particles. Now, recall that when Krieg cloned what he called SV -- what we call SVMEN, there was another virus that was cloned, and in particular it was a truncated version that had two ecolar one cites, it wasn't a complete virus. I think it was something like 3.5 kb. So, the point is, it could also be that in some of these infections that we clear the wild type virus but some DIs linger, and, you know, we might be detecting DI particles in some of these cases. DOCTOR LEVINE: That's an interesting point. Are there any other comments from the panel or from the audience? Doctor O'Neill? DOCTOR O'NEILL: Well, one of the problems with the defectives is that if you clear the infectious virus, the defectives probably won't hang around very long, because they need the wild type helper, and once that helper is gone, a particle that had a genome that's only 3.5 kb is not likely to be infectious on its own, so it probably will be lost. DOCTOR LEVINE: Doctor Dorries? DOCTOR DORRIES: We -- in JC work, we never have seen really defective particles, in terms of rearrangement, major rearrangements, whether the TCR archetypes might be defective in a certain sort of way we don't know yet. DOCTOR LEVINE: Doctor Oxman? DOCTOR OXMAN: Art, can we have a word from Andy Lewis on what we know and how much follow-up of E46 with respect to the first question, the military who received E46 adeno SV40 hybrid vaccine? DOCTOR LEVINE: Doctor Lewis? DOCTOR LEWIS: Mike, I'm not aware of any data that was done on an analysis of military recruits. I don't think it's been done. DOCTOR LEVINE: Did Steve Baum have any data on that? DOCTOR LEWIS: No. DOCTOR LEVINE: No? Okay. Any other questions or comments? Well, if not, I think our time is virtually up. I did want to make one summary comment, though, before everybody gets up, because I think it's important to try to put the things that we hear at this meeting in perspective. I did want to point out that I think on the basis of our discussion it's fair to say that despite the question of specificity of the earlier assays that have been used to determine whether or not neutralizing antibody to SV40 is present in the population, the fact is that virtually all studies have shown that while there is some cross reactivity, the homologous reactivity is at least 100 fold greater than the heterologous reactivity. Therefore, putting all the data together that I've heard, I think it's safe to say that SV40 is endemic, at least at a very low level, in the human population, and that shouldn't surprise us since we know that the infection is transmissible at some level from monkeys to people, and we also know that lab workers who have been exposed to high levels of SV40 for long periods of time have high titers of antibodies, at least some of which appear to be specific. Nonetheless, there's little question that we need to improve and to standardize our assays. A suggestion of using, of course, a blocking antigen is a very salient suggestion. Whether or not we go to molecular techniques to determine endemicity will depend upon our agreement that we are using the right assay with the right conditions, the right standardization, the right sensitivity, and the right specificity. It's not an easy question in molecular biology. And finally, once we've done all that, we must beg the question of transmission, and there the concept of looking at mothers and infants, families of people known to be infected, workers with monkeys and so forth, all become germane. So, on that note, I thank you. Enjoy the coffee break. (Whereupon, at 10:28 a.m., a recess until 11:06 a.m.) DOCTOR MAHY: It's time to start this session, so please take your seats. My name is Brian Mahy, I'm Director of the Division of Foreign Rickettsial Diseases at the Centers for Disease Control, and we're moving on to a subject now which, if you like, is a little less debatable, the question of SV40 and its oncogenicity. The papers we are going to have this morning are going to be primarily concerned with oncogenicity in rodents and cells in vitro. We do have the opportunity, if people speak for less than the appropriate time, to have some questions, and I'd quite like that. But, the first speaker has just ten minutes, Michele Carbone, from Loyola Medical Center in Maywood, Illinois. Are you here, Doctor Carbone? God. DOCTOR CARBONE: Thank you. In this talk, I was asked to review the data about SV40 oncogenicity in hamsters. Can I have the first slide? Great, okay. So, I'm going to review the data about SV40 oncogenicity in hamsters. In 1961, Doctor Eddy reported that tumors were induced in hamsters by monkey kidney cell -- and a year later, in 1962, she identified the substance present in these kidney cell -- responsible for the oncogenicity of SV40. These were subcutaneous tumors that from an histologic point of view would be called sarcomas. But, at the same time, Doctor Kirschstein reported that if you injected the virus in the brain of hamsters, they would develop ependymomas, and ependymomas also develop in mastomys, which I understand is a kind of rat, when they were injected with SV40 into the brain. Some years later, Doctor Diamandopoulous started what would happen if you injected the virus systemically, and he injected SV40 to the femoral vein. And, when he did that, with his great surprise, he found that only specific cell type would develop, and they are indicated here. One hundred and fifty animals were injected, 125 of them developed tumors, the tumors, as listed below, obviously, more than one tumor developed in some animals, and you have the tumors that developed were abdominal and mediastinal lymphomas, also sarcomas, and one single lymphocytic lymphoma. Now, the oncogenicity of SV40 is related to the large T-antigen. The role of the small T-antigen, if any, in the oncogenic process was unknown. And, Doctor Lewis and Doctor Martin studied this problem and addressed it. And, they published that if you injected hamsters with small T -- they published a study in 1979 in PNAS -- that if you inject hamsters with SV40 multi mutants these mutants are still able to induce tumors. However, they found it prolonged the latency. A few years later, Kathleen Dixon reported that if you injected these multi mutant subcu, in addition to these tumors with the prolonged latency that were reported before by Doctor Lewis, some animals developed abdominal metastases. Now, these metastases never developed in animals injected with SV40 wild type. But, at that time, actually, two years later, I began a post doc in the lab of Doctor Lewis, and we studied those tumors, and to our surprise we found out that those so-called metastases were not metastases, those were primary lymphomas that would occur in these animals. So, for some reason, when you inject the se multimutants subcutaneously a portion of animals would develop primary abdominal lymphomas, it's an ugly terminology, but these are macrophage lymphomas, or to use a human term, true histiocytic lymphomas. We were particularly intrigued by this finding, why that would happen, and why that would happen only with multimutants, and so we decided to inject a variety of multimutants systemically through the heart into hamsters to see what was the oncogenicity of these multimutants when different organs were exposed to the virus, and, of course, we had the control group animals that was injected with wild type SV40, and in the control group of animals we found the most unexpected results, but let's go in order. These were the tumors that developed in animals injected with multimutants, these are lymphomas, and all of them were -- actually, not all of them, most of them were true histiocytic lymphomas, few of them were bilymphomas. So, for some reason when you inject the virus systemically, that's basically the only tumor that you see when you use this multimutant. There must be a different reason, and I don't have time to go through all of them. Rarely, a multimutant injected animal will develop an osteosarcoma that is shown here, also sarcomas did develop in the control group of animals that was injected iwth SV40 wild type. The most surprise thing was that in the control group of animals injected iwth wild type, we saw these tumors, and these tumors are mesotheliomas and you can see in A and in C the epithelial -- the basic pattern that is kind of characteristic of this malignancy. Now, we were very surprised by that, because, as I mentioned yesterday, mesotheliomas have never been associated with anything else than asbestos exposure, at least in mammals. In D, you can see a cell line that -- cell culture derived from one of those tumor, and this is one of the tests that we did to characterize these tumors in these cells. On the last day of the original tumor, -- of the cells derived from it, indicating that they are representative of the original tumor. They really look like a -- and there are some of the characteristic electron microscope in these mesotheliomas, including those branching microvilli that some of you can appreciate. Obviously, you would like to see that the virus is, in fact, integrated into these tumors, and that is responsible for these tumors, and that is, in fact, the case. In panel A, you have the line alternate, tumor cell line, tumor cell line, each line is derived from the tumor, and they are cut with a single cut of Bam HI or EcoRI. It's obvious a number of things. First of all, that the pattern of integration in the tumor and in the cell line is quite different, and so probably rearrange them and -- at least in cell culture, maybe in the tumor, too. The second thing that is obvious is that there is -- in A but there is also about 5.1, 5.2 kb band, which could represent episomal DNA. So, in B, we got to that DNA with a non-cutter, and when we use a non-cutter for SV40, only high molecular weight is there, indicating that the -- tumor at least, all or most of it, is SV40 integrated and not episomal, and finally in panel C Hind III showing the characteristic pattern of the early region, that is, the region that calls for large T and small T, and that you would expect to find there if the tumor if the virus is playing at all in tumor -- in oncogenesis. The lines that are white in panel C, the panel cut with Hind III, are from the heart of these animals, indicating that at least by Southern Blot you cannot detect SV40 in the organs of these animals that do not contain tumor, but only the tumor. These cells express large T-antigen, 90 kilodalton, and if they derived from the wild type also there's multi-antigen, 17 kilodalton, 19 kilodalton. They were strongly positive by immunoperoxidase experiments, and these experiments were done by Doctor Harvey Pass at NCI a few years later than those I showed before. When you take this, these are hamster mesothelioma, SV40 induced mesotheliomas and cell lines derived from these tumors, when you take these cells and you inject them into hamsters they are highly oncogenic, they are oncogenic up to 102 cells, when you inject 102 cells they will develop tumors in about eight weeks, when you inject 105, 106 cells they will develop -- the animal will develop tumor in about four weeks. And, it doesn't matter where you inject it, you can inject subcu and you get the same thing, the tumors grow very quickly. So, these are the conclusions from the hamster studies, at least these are our studies. Wild type SV40 injected intracardially, 60 percent of animals develop mesotheliomas, 40 percent true histiocytic lymphomas, five percent osteosarcomas, five percent sarcomas, when we inject the wild type SV40 into the pleural space, 100 percent of animals develop pleural mesotheliomas in three to five months. When wild type SV40 is injected intra peritoneally, mesotheliomas and true histiocytic lymphomas can both develop. When wild type SV40 was injected intracranially by other investigators, ependymomas and choroid plexus tumors developed, and if you delete this multi-antigen and you inject the virus systemically only true histiocytic lymphomas develop, true histiocytic lymphoma will also develop if you inject the virus subcu in a minority of animals, in addition to the local sarcomas. And so, these are the conclusions of this study. In hamsters SV40 preferential induce mesotheliomas, osteosarcomas, sarcomas, specific types of lymphomas and ependymomas. These same tumor types have been shown to contain SV40-like sequences in humans. Deletions of this multi-antigen out of the oncogenicity of SV40. Thanks. (Applause.) DOCTOR MAHY: Thank you very much, indeed. There's time for one quick question, if anybody has one. We are short of time, but Doctor Shah? DOCTOR SHAH: In the earlier studies in the '60s, the lots of SV40 innoculated hamsters, did they also detect mesotheliomas, or this is a new finding? DOCTOR CARBONE: There is one single report of a cell line that was derived from mesothelioma. I am very sorry I can't remember the first author of that paper, however, it went this way. There was the cell line, it's called TU something, and I called him, and he explained to me that at that time, in the '60s or so, they were injected 100s of hamsters between the scapula, and that then they would send these tumors to pathology, and that one of these tumors came back saying that this was a mesothelioma, and that everybody was intrigued with that and he'd send the cell line around to many places. And, his own opinion was that since these were small animals, he thought that probably the technician who did the injection in that case went deep enough to reach the pleura and that's why the mesothelioma came out. So, there is one only report and the cell line is called TU-800. DOCTOR SHAH: Thank you very much. DOCTOR MAHY: Okay. We're going to move on now to a paper by Priscilla Furth, who is from the new Institute of Human Virology in Baltimore, Maryland, on SV40 rodent tumor models as paradigms of human disease transfusing mouse models. DOCTOR FURTH: Thank you, and Doctor Lewis invited me here to discuss some of the transgenic mouse data using T-antigen. First slide. And, as a way of introduction, of course, the vast majority of transgenic mouse studies do not look at viral infection, rather, they focus on a specific viral protein, and I'm going to tell you this will be the SV40 large T-antigen. In many of the constructs, the small T-antigen is there as well, but it may or may not be expressed. So, these studies differ considerably than from what you've heard before, because we are looking at the action only of the transforming region from the SV40 virus. Most of the studies have focused on viral oncogenesis at the process of viral oncogenesis, and they have looked in a number of different tissues. I would venture to say that the large T-antigen coding sequence may be the most frequently injected DNA sequence in transgenic animals. It's been expressed in a wide variety of tissues, and it's also been used as a tool, basically, to look at some new technologies as well. So, there's a number of studies out there, and what I will do is present data from my own laboratory which illustrates some of the common themes that have been seen. I would mention that there is a transgenic mouse model which does use the promoter from SV40, and it does target to the choroid plexus, and Terry van Dyke's laboratory has done a lot of work on that particular model. But, if you look around and do a MEDLINE search you can come up with models, liver, pancreases, Doug Hanahan has done a lot of work there, intestine, lung, kidney, the lens of the eye, bone and cartilage. In my own laboratory, we focus on the mammary gland and the salivary gland. The large T-antigen is interesting for those of us who are interested in viral oncogenesis, because, of course, it binds to and inactivates two very important tumor suppressor genes, the retinoblastoma protein and P53. So, now I would like to discuss our mammary model, and similar to all of the other animal models, if you express SV40 large T-antigen in mammary epithelial cells you will, in fact, produce tumors, and this is a transgenic mouse which has a mammary tumor. Mice, of course, have ten mammary glands, five on each side. These tumors come up in these mice after about three to four pregnancies, or about four to five months of age. So, there's a considerable latency there. What's interesting to us about this particular model is that we can look at early steps which follow expression of large T-antigen. The promoter, which we use to drive T-antigen expression to the mammary epithelial cells, is under a strict developmental control. We use the whey acidic protein promoter. What's important for you to remember is that this promoter turns on around day 13 of pregnancy in these cells, and what we've done is then look at the glands at day 18 pregnancy. This would be five days after initial T-antigen expression, to see what, in fact, has happened to the cells. On the top panel, this is alveolus in the mammary gland here, and this is another alveolus that's here. These alveoli are embedded within a fat pad in the mammary gland. This is T-antigen immunohistochemistry, and so you can see the typical staining of the nuclei in these cells. In this particular transgenic line, T-antigen expression is virtually homogeneous in these cells, in other words, all the cells express T-antigen. One of the interesting findings that we saw was that expression of T-antigen in these cells actually induced programmed cell death or apoptosis, and you can recognize the cells which are undergoing programmed cell death because they appear brown here in this in situ stain, which can identify cells undergoing this process. So, again, we have the alveolar structures here, and you can see one to two cells in each of these alveolus is actually undergoing programmed cell death. This was somewhat interesting to us, because, of course, the P53 and retinoblastoma protein are bound by T-antigen, P53 has been implicated in a number of processes involved in programmed cell death. In these animals, P53 is, in fact, bound up and inactive, yet we see induction of apoptosis, and the studies that we followed up with, in fact, demonstrated that this is P53 independent apoptosis. So, we see that very early after expression of this oncoprotein, the cells are fighting back and trying to eliminate probably those cells which may, in fact, have DNA damage. One thing I should mention is that when we express T-antigen in these cells, virtually, all the cells are polyploid, so if we do a fogan stain, which can recognize DNA and stains do DNA, all these cells will light up with multiple copies of their cellular DNA, so we've introduced into these cells an abnormal cell cycle, and it may well be that these cells are undergoing programmed cell death because they are recognized as being defective. What happens during the process of oncogenesis, however, is that the cells develop a resistance to P53 independent apoptosis, and this we can see in the mammary gland by looking at the involution. Involution is the process which follows lactation. It is the time when all the mammary epithelial cells die, through the process of programmed cell death, and what you can see in this normal gland, which is now 13 days after lactation has ceased, you can still see all of the residual ductal structures, but you do not see the epithelial cells here. In contrast, this is a T-antigen animal, which has undergone three pregnancies, three lactations, and what you'll see now is that this gland no longer regresses, and this is evidence that the gland has developed resistance to P53 independent apoptotis, so this would be one of the themes that you see during the process of T-antigen oncogenesis. We've also looked at these cells at the day 18 pregnancy for other evidence of abnormalities. This is an H&E stain, again, the alveoli. This is the fat pad within here. You can, in fact, pick up those cells which are undergoing apoptosis, they stain darkly within these alveoli. What I want you to note on this particular slide is that the cells do not appear dramatically transformed. They are single layer cells, and they are resting upon a basement membrane. Nevertheless, the cells have considerable functional abnormalities. One of the things that happens in the mammary epithelial cells is they are no longer able to process and secrete milk proteins, and this is immunohistochemistry in the T-antigen animals here, and controls, we look for milk proteins, one of them the whey acidic protein, and this is an antibody that recognizes total milk. You can see that the T-antigen animals do not secrete any of these milk proteins, and this can be demonstrated again on a Western Blot, three, four and five are transgenic samples, one and two are wild type animals. Initially, we thought this was because the cells was dedifferentiated, T-antigen has been "associated" with the dedifferentiated phenotype, but we found, in fact, that these cells were largely differentiated from mammary epithelial cells. In this Northern Blot, we are looking at the expression of milk protein genes in mammary epithelial cells. These can be used as a measure of differentiation, and what we saw, that this is control animals here, beta casein, wap, alpha lactalbumin, we can see that in these T-antigen animals we saw RNA expression of all these genes, this means that the deficit that we are seeing in these animals is very specific and related to protein synthesis. And, here's your T-antigen expression. While these genes are expressed normally, there are examples of other genes which have their transcription patterns interrupted. One of the interesting ones is something called WDNM 1. It's interesting because this gene was originally identified in mammary cell lines as a gene which is down regulated in metastasis. Metastasis, of course, is a very late phenomenon in oncogenesis, but we see down regulation of this gene only five days after T-antigen expression, so it probably indicates that this gene is not, in fact, a metastasis factor, but something related to early changes within the cell. And now, to look at some of the later steps in oncogenesis, I'm going to turn to a different transgenic model, and this model we used a binary system, and the reason that we have chosen to do this is we would like to temporally control the expression of T-antigen, in other words, we want to use an animal, but we want to be able to turn T-antigen expression on at a specific time, and then later we want to be able to turn it off at a specific time. This enables us to perform experiments in which we can look at the time dependency of viral oncogenesis. The system that we use is a tetracycline responsive gene expression system. It consists of two genes here, and what we do is drive T-antigen expression off a promoter, which I've termed here the tet-op promoter, this is a minimal promoter, it contains 50 base pairs around the CMV tata box, and it is linked to seven tet-operator sequences from the E. Coli repressor transposon. Those DNA sites which are linked the CMV minimal site, contain no eukaryotic transcription factor binding sites. What that means is, this promoter is, essentially, silent in a eukaryotic cell. So, it's off. We turn this promoter on by expressing a specific transactivator, which can bind to the promoter and increase gene transcription. This transactivator is contained on a second transgene here. It consists of the protein domains which can recognize and bind to these DNA domains, and also comes from the repressor, from the E. coli transposon. This is linked to an activator domain from herpes simplex virus. And, what essentially that does, this hybrid transactivator is a eukaryotic transcriptional activator and has turned what was a repressor protein in a bacterial system into an activator. We control the binding of this transactivator by the administration and withdrawal of tetracycline. The DNA sequences from the repressor contain a binding site for tetracycline. When tetracycline binds to this particular protein, it changes its confirmation, and it can no longer recognize and bind to DNA. However, in the absence of tetracycline it can bind to this promoter, and you see gene transcription. So, this is a system then that we can specifically turn gene expression on and off at time points. The cells that we used the system to look in are the striated ductal cells of the submandibular salivary gland, and we chose to do the experiment in those cells for a very good reason. We had, in this particular transgenic model, very homogeneous expression of T-antigen in those cells, and this would allow us to read out, if we turned off T-antigen expression in a large population, the phenomenon that we wished to look at. Striated ductal cells are characterized by these pink striations in here. They are absorptive cells. These are their nuclei, rather round here, and placed eccentrenically. This slide, we sometimes will put a reporter gene here besides T-antigen. This is a nuclear localized betagalactacydase gene, and what it demonstrates is we target betagal expression to the striated ductal cells, similar to what we did with T-antigen, so these are these cells with T-antigen expressed in them for approximately four months, and what you can see now is the cells have, in fact, become transformed, the nuclei are eccentric, and you can see a hyperplasia is developing off here. This is a Western Blot demonstrating expression of T-antigen in the presence of the transactivator here, and you can see we see excellent levels of expression. This is actually the earlier model, you looked at the mammary gland model, you can see we express actually significantly greater amounts of T-antigen protein using the binary system here. This is a single transgenic animal. It contains only this construct, and you can see that there's no T-antigen expression in that animal. If we look over time in these animals, the first time point that we've been able to examine is two weeks of age, and at two weeks of age you can find foci within the submandibular salivary gland which are hyperplastic, but if you look in a larger field you'll see that these are rather limited, and there are many cells which do not exhibit any hyperplastic changes. If you follow that mice up to about four months of age, however, you'll see that the vast majority of the striated ductal cells are now hyperplastic. We chose this time point then to turn off gene expression and ask whether or not the hyperplasia that we were observing was dependent on continued T-antigen expression. In other words, if we turned off T-antigen expression, would the hyperplasia reverse or would it be maintained. So, to turn it off then, we placed these animals on tetracycline, and what we saw, in fact, was a very dramatic reversal of the hyperplastic changes. Coming across here, this is the nuclear localized lacZ that you saw before, but you are seeing it at a lower power. It outlines the structure of the striated ducts. This is a non-transgenic animal, you can see that there's pink staining within here. Those are all striated ductal cells. This is a single transgenic animal, it does not express T-antigen, and it looks indistinguishable from wild type. This is a double transgenic animal, in which T-antigen has been expressed for four months, we see dramatic hyperplasia here. We placed this animal on tetracycline for three weeks, and what you see is extensive reversal of this hyperplastic phenomena, and this would suggest then the hyperplasias at four months of age are, in fact, dependent on continued T-antigen expression. One of the interesting controls about this is that you can see that the density of the striated ductal structures is actually increased over this animal, and that let's you know that, in fact, hyperplasia was there. If we look at a higher power, we can see an animal that is not on tetracycline, so T-antigen expression, hyperplasia, we see the immunohistochemistry for T-antigen. This animal has been on tetracycline for three weeks. The histology of the cells is reversed, and there is no T-antigen on immunohistochemistry. We then performed the same experiment at seven months of age and asked the same question. And, in contrast to the results at four months, by seven months of age we find that reversal of the phenotype is very limited, and this is illustrated here. This is a seven month sample. You see hyperplasia in the absence of tetracycline. We placed that animal on tetracycline for three weeks and the hyperplastic changes remain. This would suggest then that this hyperplasia no longer needs T-antigen expression, and in a brief look at mechanism we'll return to our four month sample, one of the things that you do find as T-antigen expression is correlated with the appearance of polyploidian cells. And, this is a fogan stain here, the DNA which is stained in multiple copies, appears dark pink here, these are cells which have T-antigen in them, and these cells are polyploid. When we turn off T-antigen expression at four months, there are a few foci which remain transformed. They do not express T-antigen, however, they maintain their polyploidy state, and, therefore, we would suggest that there may be genes, cellular genes, which have become mutated in this process, which are now sufficient to keep the cell in a transformed state. Thanks. And, I'd like to acknowledge Ming Lan Lee, is a Post-Doc in my lab that worked on both of the projects, and Dagmar Avol, a Post-Doc in -- lab that did the second project. (Applause.) DOCTOR MAHY: Thank you very much, Doctor Furth. That was a very interesting presentation. We have time for a couple of questions. AUDIENCE: Have you looked for P53 localization at any time during this? For example, is P53 localized to the T-antigen expressing cells early on, and does it stay in the nucleus, for example? DOCTOR FURST: Yes. Most of the P53 studies I've done are on the mammary gland, and when you express T-antigen P53 is localized to the nucleus in those cells. In mammary epithelial cells, there's actually very low levels of P53, although, it's somewhat strain dependent. So, it's difficult to pick up, but it's there, it's in the nucleus. AUDIENCE: And, that reverses upon tetracycline? DOCTOR FURST: I did those studies in the mammary gland, not in the salivary gland. Probably P53 plays a different role in the salivary gland, and it may well -- that will be something interesting that we could look at. If you take an oncogeny, and you express it equally in mammary tissue and salivary tissue, you breed that animal into a P53 null. You will find that you don't change the mammary tumor incidence, but you do change the incidence of salivary tumors. Bottom line, P53 may play a more prominent role in salivary gland tumorigenesis than it does in mammary gland tumorigenesis. DOCTOR MAHY: Question in the back? AUDIENCE: Yes. A very nice study. The question is on the mice that you did show that the reduction hyperplasia, when you didn't have complete removal of hyperplasia, did you look at the promoter to see if it was mutated or recombined, or was it just the distribution in tetracycline? Can you explain why you didn't get complete reversal? DOCTOR FURTH: In the four month, why that one foci remains there? AUDIENCE: Yes. DOCTOR FURTH: I suspect it's because those -- you put T-antigen in, you cycle these cells, and you start to accumulate DNA damage. A lot of it may be silent. Some it eventually statistically will hit on a critical gene, maybe rats or something, mutates it. So, my working hypothesis is that particular clone of cells contains a mutation in another oncogeny, and we need to demonstrate that. DOCTOR MAHY: Last question, Doctor Ozer? DOCTOR OZER: Harvey Ozer. The question I have is, I'm familiar with the tet system, and we've used it, and some people do find a trace of leakiness in some of the systems. So, I was just going to ask you, how close to zero is the T-antigen gene in these cells, and have you done RT-PCR or something? DOCTOR FURTH: I have not done RT-PCR. There may be a threshold. All I can say is that we are below the threshold of detection by immunohistochemistry, because we're not so much interested in RT-PCR as we are interested in protein. There are studies, of course, that have looked at levels of T-antigen and oncogenesis, and there may be some correlation there. So, I know that we are down below the level that we can detect on a protein level. One of the other provisos with the tet system is that, yes, in tissue culture cells I've got my own data where it can be leaky. We have, reassuringly, found that in transgenic animals it has acted fairly tight, but, again, I can't tell you if we went in with RT-PCR if we might pick up a few transcripts. But, I'm looking at a particular threshold. DOCTOR MAHY: Thank you very much, indeed. This is a very nice study. Now, we're going to move on to transformation of cells in vitro, and Kathy Rundell from Northwestern Medical School is going to talk to us about transformation of rodent cells. |