SV40 Workshop at NIH 1997

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------------------------------------------------------------------------Simian Virus 40 (SV40:)
A Possible Human Polyomavirus Workshop
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UNITED STATES OF AMERICA
DEPARTMENT OF HEALTH AND HUMAN SERVICES
CBER-NCI-NICHD-NIP-NVPO
SIMIAN VIRUS 40 (SV40):
A POSSIBLE HUMAN POLYOMAVIRUS WORKSHOP
MONDAY, 27 JANUARY, 1997
Morning Session
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The Workshop took place in the Natcher Auditorium, National Institutes of Health, Bethesda, Maryland, at 8:30 a.m., Kathryn C. Zoon, Director, CBER, presiding.
PRESENT:
KATHRYN C. ZOON, M.D. DIRECTOR, CBER
ROB BRIEMAN CO-CHAIR
MIKE FRIED CO-CHAIR
RUTH KIRSCHSTEIN CO-CHAIR
DIXIE SNIDER CO-CHAIR
BONNIE D. BROCK, V.M.D. SPEAKER
JANET BUTEL, Ph.D. SPEAKER
MICHELE CARBONE, M.D., Ph.D. SPEAKER
KRISTINA DOERRIES SPEAKER
ELLEN FANNING SPEAKER
RICHARD FRISQUE, Ph.D. SPEAKER
ROBERT L. GARCEA, M.D. SPEAKER
ALLEN GIBBS SPEAKER
MAURICE R. HILLEMAN, Ph.D. SPEAKER
MICHAEL J. IMPERIALE SPEAKER
KAMEL KHALILI SPEAKER
ANDREW LEWIS, M.D. SPEAKER
MARIA C. MONACO SPEAKER
LUCIANO MUTTI SPEAKER
FRANK O'NEILL, Ph.D. SPEAKER
PATRICK OLIN SPEAKER
DAVID SANGAR SPEAKER
KEERTI V. SHAH SPEAKER
HOWARD STRICKLER SPEAKER
MAURO TOGNON, Ph.D. SPEAKER
JIM C. WILLIAMS, Ph.D. SPEAKER
JOHN LEDNICKY, Ph.D. PANELIST
ALSO PRESENT:
DR. GALATEAU-SALLE
HARVEY PASS
ETHEL de VILLERS
ROBIN WEISS
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CONTENTS
Introduction and Welcome by Dr. Zoon
SESSION 1 Presentations:
Dr. Fanning
Dr. Shah
Dr. Garcea
Dr. Butel
Dr. Carbone
Dr. Gibbs
Dr. Mutti
Dr. Giordano
Dr. Tognon
Dr. Shah
SESSION 2 Presentations
Dr. Dorries
Dr. Imperiale
Dr. Khalili
Dr. Frisque
Dr. Monaco
LUNCHEON RECESS Afternoon Session
Audience Participation
Presentation by Dr. Lednicky
Panel Discussion
SESSION 3 Presentations
Dr. Hilleman
Dr. O'Neill
Dr. Lewis
Dr. Brock
Dr. Williams
Dr. Sangar
Dr. Olin
Dr. Strickler
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PROCEEDINGS
8:35 a.m.
DIRECTOR ZOON: On behalf of sponsor's agencies today, which include the National Institute of Child Health and Human Development at NIH, the Division of Cancer Epidemiology and Genetics at NCINIH, the National Center for Infectious Diseases and National Immunization Programs at CDC, the National Vaccine Program Office, and the Center for Biologics, Evaluation, and Research of FDA, I'd like to welcome you to this workshop on SV40.
We, the sponsors of this workshop are pleased that so many of the national and international scientific community have come to discuss this very important topic. The workshop was prompted by recent reports demonstrating the presence of SV40 viral sequences in tissue, including certain rare, human tumors, and the fact that SV40 was an unsuspected contaminant in early polio and adenovirus vaccines.
The potential connection between SV40 and these various tumors is confounded by the finding of SV40 sequences in individuals who are too young to have received the SV40-containing vaccines. As a result, we must ask whether SV40 was present in the human population and if so, whether SV40 was present in the human population prior to polio vaccines.
Moreover, the role of the human polyomaviruses such as BK and JC, which are closely related to SV40, need to be explored. Therefore, the purpose of this workshop is twofold.
The first is to consider the possibility that SV40 is an infectious agent that is endemic in the human population; and second, is to stimulate the effort required to determine if SV40 is a causative agent in human disease.
We look forward to discussions, both formal and informal, over the next two days, that will better define these scientific issues. Furthermore, we hope that these discussions will lead to important new research collaborations.
Finally, at the outset I would like to acknowledge the enormous effort by those individuals who organized this conference, including Drs. Strickler, Levine, Egan, and particularly, Dr. Lewis.
Before I turn the meeting over to the Chair of the first session, Dr. Ruth Kirschstein, there's several housekeeping issues I'd like to go through. Lunch will be available upstairs at the cafeteria. There will be coffee breaks served in the foyer down here. Buses will return participants to the Marriott Hotel.
Transcripts and videotapes of the workshop will be available and how to obtain them is presented in your registration package. In the context of that I would like to ask those of you who come to the microphone to please identify yourselves so we will have a record of it.
The registration package also contains a comprehensive bibliography of papers relevant to the topics being discussed at this meeting. If participants know of additional materials, please send them to Dr. Lewis and we will see to it that they will be disseminated to the registrants.
Finally, we welcome the media coverage of this meeting but we ask that in the spirit of facilitating the scientific discussions that are so vital to the success of this meeting, that the media refrain from questioning the participants during the meeting.
There will be a press-availability session at the end of the meeting in conference room B, and representatives from each of the sponsoring agencies will be available. Other workshop participants are also welcome.
With that, I'd like to now turn the meeting over to Dr. Ruth Kirschstein.
CHAIRMAN KIRSCHSTEIN: Good morning. I want to add my welcome on behalf of NIH as a whole. We're pleased to be helping to sponsor this conference.
This conference is a little bit of a nostalgia trip for me since I started working in this field many, many, many years ago. And indeed, I was scheduled to Chair a different session than this one. For reasons of scheduling, I had to change that, and when I looked at the program, it is perhaps even more appropriate that I chair this session since some of the talks relate to work that I did over 30 years ago.
With that, I'd like to call on our first speaker, Ellen Fanning, who is in the Department of Molecular Biology at Vanderbilt University, who will give us a brief review of SV40 biology, and an overview of the organization and expression of the SV40 genome.
Dr. Fanning.
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DR. FANNING: Thank you very much. When I got a call from Andy Lewis asking me to review the biology at the beginning of this meeting -- of SV40 -- in 20 minutes, I felt a little bit like Sarge in the Beetle Bailey cartoon. He said, you've been working on SV40 for 20 years; you know a lot about it. I says yes, but I don't know all about it, and 20 minutes? Andy persisted, however, so it's of course a little bit daunting.
SV40 has been studied from the beginning of its first reports in 1960 by Sweet and Hilleman, by some of the brightest minds in science. This virus provided a model system to study cell transformation and growth control, eukaryotic gene expression and DNA replication, chromatin structure.
It's led to the discovery of enhancers and promoters -- many of the factors that bind to them -- to the discovery of RNA processing and eukaryotes to signals for nuclear protein transport and to the first reports of the tumor suppressor, p53. SV40 has taught us a lot about eukaryotic cell biology.
So what I'm going to try to do in the next 20 minutes is to start at the beginning, to mention a few of the milestones along the path in learning about this virus, and then over the course of the next two days, my colleagues will provide you with some data on the questions that are at hand and to be discussed in this meeting.
As was already mentioned, SV40 was a byproduct of the early polio vaccines. The virus was discovered in polio vaccines that had been produced in monkey cells in culture. The virus was named after the cytopathic effects that it produced in infected monkey cells which became highly vacuolated, hence the name. The SV40 standing for simian virus, or vacuolating virus number 40.
The virus particle is relatively simple. It's small, it's made up of 72 capsomeres that contain three different viral proteins. It's just slightly larger than ribosomal subunits. Inside the virus there's a mini-chromosome of 5,243 base pairs, which is complexed with cellular nucleosones made up of cellular histones.
The mini-chromosome as shown up here, it's a very compact structure. When it's prepared for electronmicroscopy sometimes it opens up like this so that you can see the typical beads on a string. These over here are intermediates in viral packaging.
SV40 normally infects permissive cells -- that was how it was discovered -- such as this little -- according to this little scheme which is typical of the way that it infects CV1 monkey cells in culture. The virus particle enters the cell, progresses into the nucleus. The viral mini-chromosome is set free in the nucleus, it's transcribed by cellular transcription factors and enzymes to produce the so-called early messenger RNAs.
One of the products, protein products, that's generated from this early messenger RNA is called T-antigen. This is a short abbreviation for tumor antigen. This protein was discovered by virtue of the fact that very early on, it was established that SV40 could cause tumors in rodents and that these rodents produced antibodies against a new antigen that was not found in the virus particle. It was called tumor antigen.
This protein, T-antigen, as it's now known, is found in infected cells as well as in these tumor cells. This protein migrates back into the nucleus where it carries out the next stage of the productive infection, initiating viral DNA replication and stimulating late transcription.
The late messenger RNAs encode the capsid proteins which again migrate back into the nucleus, assemble around the mini-chromosomes to produce the new virus particles.
Not all cells are permissive for SV40. Most cells in fact, are semi-permissive like this species here, or non-permissive like the rodent shown down here in the grass. The infection in non-permissive cells is depicted here in a slide borrowed from Arnie Levine's book, Viruses. It stops after the transcription of the early viral genes.
The virus particle comes into the cell, the early messenger RNAs are made, the T-antigen is synthesized, it migrates into the nucleus, and normally the infection stops there. However, infrequently, the viral genome can become integrated in the host DNA in such a way that the T-antigen continues to be expressed on the other early genes as well. This can lead to cell transformation.
All right. So we have seen that somehow the virus infection does not progress beyond the very early phase. Viral DNA replication does not take place and late gene products are not synthesized.
The year 1978 was a milestone year in SV40 research. One of the important advances that was made in this year was that the first reports of the sequence of the SV40 genome appeared by Walter Feur's lab, and independently from Sherman Weisman's lab.
You can notice several things about the SV40 genome here. There are two sets of genes. The early genes are here; the late genes are over here; one transcribed on the Watson strand; the other transcribed on the Crick strand.
You can also note that the genes are overlapping. That is, the early genes contain several protein products, there's a large T-antigen, the small T-antigen -- and not depicted on this slide is another early viral gene product called the 17KT antigen which was recently identified.
The late genes are also overlapping. The three capsid proteins: VP1, VP2, and VP3. In between these two sets of genes is a control region which is depicted in more detail on this slide down at the bottom, here.
Here we have the early messenger RNA being transcribed this way, the late messenger being transcribed this way, the alternative splice products of these two sets of transcripts lead to the different viral gene products.
If we look at these control sequences in more detail we see two prominent binding sites here for SV40 T-antigen. Now, as the concentration of T-antigen increases in the cell, T-antigen binds to these sites specifically. It down-regulates the early transcription through this binding event, and initiates viral DNA replication through this second binding event.
The sequences that control transcription are located here. There's a so-called tatabox promoter, and you'll hear more about these copies of the 72 base repeats which are called enhancers. All of these are required for transcription.
Now, as viral DNA replication takes place and T-antigen changes the activity of the cellular transcription factors, the late genes are turned by indirect mechanisms.
So if you're starting to get the idea that T-antigen is a rather complex protein, you've got the right idea. T-antigen is perhaps the most multi-functional protein that's ever been discovered. Notice all of the arms here. T-antigen is something that we still don't know everything about. Notice the mysterious spatial expression here.
We do know quite a bit about it, however. The strategies that the T-antigen follows in directing the viral infection in permissive cells is depicted here.
First of all, the T-antigen must prepare the cell to support the viral infection. It does this by kicking a quiescent, differentiated, resting cell back into the cell cycle and forcing it into the S-phase. It needs this in order to replicate its own viral DNA which is dependent on cellular replication enzymes.
Having done that, T-antigen then initiates replication of the viral DNA, recruits the cellular proteins to replicate it, and through the indirect mechanisms that I mentioned earlier, leads to stimulation of late transcription -- viral transcription.
T-antigen somehow then senses that it should not initiate DNA replication anymore, but allow the viral genomes to be packaged into new virus particles. In other words, the T-antigen function changes with time after infection.
All right. So now, how does T-antigen do all of this? Generations of graduate students and post-docs have mutagenized the T-antigen gene, and this is the simplified version of some of what they've found.
The protein encodes 708 amino acids; it's sensitive to proteases at the sites marked by the asterisks, which tells you that the protein is folded up in different folding domains. These domains tend to correlate with functional properties of the T-antigen molecule. For example, this domain is responsible for specific binding to those two sequences in the viral control region.
T-antigen has a number of other intrinsic, biochemical activities. It binds to ATP and hydrolyzes ATP in a DNA-dependant manner using sequences located in the carboxyl terminus of the protein. It uses both of these domains to encode a DNA helocase that was first recognized in 1986 in Rolf Knipper's lab.
It needs all of these activities in order to replicate viral DNA. Now in addition to these intrinsic biochemical activities, T-antigen also interacts with a large variety of cellular proteins. Some of the proteins that it interacts with are depicted here.
The DNA preliminaries alpha primase, interacts with T-antigen at two independent sites diagramed here. T-antigen interacts with nuclear location protein transport machinery located at this region here, designated NLS. T-antigen also interacts with tumor suppressor proteins such as Rb and the other members of the pocket protein family.
T-antigen also interacts with p53, and actually there are two independent regions that are involved in binding p53: one here and one here, as shown in Judy Tevethia's lab. There's also a sequence down here which was not very well understood, which helps SV40 determine what type of monkey cells it can replicate in.
Now, T-antigen's phosphoprotein, the sites have been mapped to serienes and threonines and two clusters in the amino terminus and the carboxyl terminus.
All right, so having said all that, let's try and look at how T-antigen carries out this strategy. I'm going to discuss first -- because we know most about it -- how T-antigen directs the replication of viral DNA, very briefly; and then say a few words about how T-antigen prepares the cell to support the viral infection.
All right. More than ten years ago Tom Kelly's lab developed a system which would replicate SV40 DNA in a test tube. This system was dependent only on one viral protein of course, the SV40 T-antigen, as well as ten cellular proteins that have been defined and studied in some detail in Bruce Stillman's, Jerry Hurwitz's, and Tom Kelly's lab.
T-antigen's functions in this system are threefold, basically. First of all, T-antigen binds to the viral origin of replication and assembles there as a multimer. Having done that, it proceeds to unwind the two strands of the parental DNA so that they're available to be replicated by the cellular proteins.
You can see here that a mutant T-antigen -- this is the wild type up here -- this mutant T-antigen is stuck at the origin and cannot proceed further to these bidirectional unwinding of the parental DNA.
The third function that T-antigen carries out in the viral replication phase of the infection is that it interacts with key cellular proteins involved in replication -- in getting replication started -- such as DNA preliminaries alpha primase.
Its DNA preliminaries alpha primase is the molecule which is responsible for determining the host specificity of viral replication at least, in a cell-free system, as first shown by Jerry Hurwitz's lab. And in fact, as it's turned out in studies that were carried out in my lab, it's only one of these subunits of DNA preliminaries alpha primase which is sufficient to determine whether or not SV40 DNA can be replicated by this preliminaries alpha primase.
What we did was to generate recombinant enzymes, human or mouse enzymes, or rehybrid enzymes which contain only one subunit from mouse or one subunit from human. And using this system what we were able to find was that only a single subunit, the large subunit of DNA preliminaries alpha primase must be from humans in order to allow SV40 replication. If that subunit's from mouse cells, SV40 DNA cannot replicate in the test tube.
Replication is also controlled by the phosphorylation state of T-antigen. If we look at the form of T-antigen that's most common in productively-infected cells or in transformed cells for that matter, it's a highly phosphorylated form of T-antigen; in particular at two key seriene and one threonine residues.
This form of T-antigen is not able to replicate viral DNA, although it represents the bulk of the protein in the infected cell. The form of T-antigen that's able to replicate SV40 DNA is an under-phosphorylated form which lacks phosphorylation at these two key seriene residues. This is a minor form in infected cells, but fortunately for biochemists like us who want to study it, it's the major form that's produced in recombinant baculovirus infected insect cells.
The unphosphorylated protein has made any cholase also inactive. So we have a lot of different things going on. We have a multi-functional protein whose activity is then being regulated more exactly by its phosphorylation state.
I'd like to turn then to the early stage of the infection when T-antigen is trying to prepare the cell to support the viral infection. If you look here you'll see a representation of a cell cycle in eukaryotic cells. Most cells that T-antigen would infect in an animal would be in a resting state.
And as soon as T-antigen concentration builds up, presumably in this highly phosphorylated form, it will have the effect of forcing the cell back into the cell cycle, forcing it through the early G-1 phase of the cell cycle and into the S-phase.
It does this by circumventing some of the signal transduction pathways that normally would control cell growth. It does this through its interactions with cellular growth control proteins such as the Rb tumor suppressor protein, the p53 tumor suppressor protein.
Also in 1978 it was first shown by Adolf Gressman that T-antigen was sufficient to stimulate cells to re-enter the cell cycle and progress into the S-phase. And this experiment was reproduced in my lab, shown here, either in secondary African Green Monkey kidney cells or in CV-1 cultured cells.
The cells were serum-starved and then treated with -- and were microinjected with SV40 DNA, or treated with serum and then the kinetics of re-entry into the S-phase were followed. You can see down here that T-antigen protein does this faster than SV40 DNA.
There are a number of functions of T-antigen besides binding to Rb and binding to p53 that are involved in this growth stimulation functions. Each of the mutations shown by these red bars will inactivate these growth stimulating functions but does not affect the ability of T-antigen to replicate viral DNA.
So it's possible to specifically knock out these growth stimulating functions, and this is by no means all of them. There's only four of them here. They're interacting independently with cellular growth control proteins.
To give you an idea of the importance of these interactions, in the biological activity of T-antigen in stimulating cell growth and eventually transforming the cells, bear in mind that not only SV40 T-antigen interacts with these cellular growth control proteins, but also other groups of viruses.
Adenoviruses and code early proteins that target the same cellular proteins, and the human papillomaviruses -- which we know are risk factors in human cancer -- are early proteins which target the same set of -- at least some of the same set -- of cellular growth control proteins.
So I'd like to stop there and hope that I've prepared you to fit the biology, together with some of the data that you're going to here over the course of the next two days on SV40, as a possible human pathogen. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Fanning. Indeed, you have kept the time beautifully and also prepared us for the next session of speakers.
Our next speaker will be Dr. Keerti Shah, who is in the Department of Molecular Microbiology and Immunology at Johns Hopkins, and will talk on SV40 as an infectious agent in simians and humans. Dr. Shah.
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DR. SHAH: This morning, while registering for this meeting, I met a lot of people whom I had not seen for 10 to 20 years and I said, we are all old-timers. One of them told me that I was the oldest of the old-timers.
What I want to do is, I was going to review some of the background for the infection of human beings with simian virus 40, which was a contaminant mainly in the inactivated Salk polio vaccine. And what I was going to cover was the natural infection in Rhesus monkeys in India who are the donors of the kidneys in which the vaccines were made.
I'll describe a little bit of the experimental infections in these animals because that gives us some insight into how the virus might be transmitted, and then describe briefly the circumstances of human exposure to SV40 and the early studies of finding out if this virus was pathogenic for man or not.
And I had reviewed this topic in some detail in 1976 -- and this citation is shown on the slide -- and almost everything I'm going to say is contained in that review paper.
The three viruses which are very similar is: one is the simian virus 40 of the Macacs, and the two human viruses, BK viruses and JC virus, which are very similar biology to the simian virus 40. The BCV and JCV were identified in 1971; SV40 in 1960.
They're called polyomaviruses after the polyomavirus of mice, which was the first virus of this subfamily that was characterized. And there are many polyomaviruses scattered in a large number of species -- as shown there in rabbits and mice, in parakeet, in cattle -- so that it is widely distributed. And each polyomavirus is very well-adapted to the species in which it grows. So they're highly species-specific and no polyomaviruses is shared between two different species.
The primary infections with these viruses are almost completely harmless and after the virus enters the body there is probably some multiplication at the local site. Then there's a period of viremia on the viruses in the blood and it reaches its target organs by viremia.
The target organ in most instances is the kidney. The viruses in their primary infection produce viremia that reach the kidney. There is probably some virus excretion in urine, and after that the viruses remain latent in the kidneys indefinitely, perhaps for the lifetime of the particular infected species.
They are reactivated in times of immunological impairment and transplant patients, patients with AIDS, are the ones in whom these viruses are found very frequently. Viruses are found most frequently -- BK and the SV -- in the urines of these exposed individuals.
This is the distribution of the Rhesus monkey which provided the kidneys in which the collections were made. And it's a Macacus species which lives in north India. And we have done some work on the infection in these Rhesus monkeys.
In south India there's another Macacus species, a bonnet Macacus, which is not naturally infected with SV40. But the Rhesus Macacus, whose picture you see -- here there is a female with a baby -- this is an angry male, and he looks as if he might transmit something more than SV40. Actually in India, very often these monkeys will bite individuals who are nearby.
And many of these Rhesus live in ecological contact with human beings as shown here in this temple in Nepal, where they are there in large numbers. One of the questions which I looked at at the time, was whether the virus from the Rhesus monkey was transmitted to people naturally, and if this were to happen it would occur in a country like India. And the first NIH grant that I obtained was to study if simian virus 40 was responsible for any human cancers in India.
Only some of the Macacus species are infected with SV40, and in the Rhesus Macacus only about 20 percent of juvenile monkeys, and perhaps all of the adult monkeys, have antibodies to the virus. So it is not widely prevalent.
In some situations, as in a colony that was established in the island of Cayo Santiago off Puerto Rico, where the Rhesus monkeys were brought there in 1938, they came to the island with SV40 infections, but then it was eventually lost -- the SV40 infection was lost from this Rhesus colony. So this can occur.
Although the infection is not widely prevalent in young Rhesus, when they are brought together and caged together as they were in India prior to their transport to the United States, and then in the U.S. before they were used for vaccine production, the antibody prevalence reaches practically 100 percent, because there is a great deal of transmission from infected animals to non-infected animals.
If you infect the Rhesus experimentally, it is infected extremely efficiently whether the virus is given subcutaneously or -- orally this virus was introduced into the stomach of the Rhesus monkey, or by the intranasal route. In all instances they have a period of viremia, generally in the first week that this virus is in the blood.
The period of virulia, virus in the urine which is seen two to six weeks post-inoculation, in this particular experiment the virus was not recovered from rectal swabs and throats swabs. And then all the animals, no matter how they were infected, they developed very high titers of antibodies to -- neutralizing antibodies to the viruses, and they also developed antibodies to the T-antigen that Dr. Fanning described in the earlier presentation.
There are many other studies done at the time simply to see where the virus is. And in a number of studies in the African Green Monkeys it was shown that the virus is excreted in the urine, it is latent in the kidney, and there may be a lower-level shedding of the virus during infection. In African Green Monkey the virus was recovered sometimes from throat swabs and stools.
SV40 does not produce much or less in the Rhesus Macacus. It is extremely rare that it would produce any less in the Rhesus Macacus. No tumors, benign or malignant, have been ascribed to SV40, any tumors in the Rhesus Macacus.
But just as JC virus and BK virus will produce human disease in immunosuppressed people, so does simian virus 40 produce disease in Rhesus Macacus, especially when they're immunosuppressed. When monkeys that have the immunodeficiency virus in them, the simian virus 40 will produce an illness which resembles PML, progressive multifocal leukenepalopathy, which is a degenerative disease of the nervous system, demyelinating disease.
It also sometimes produces renal pathology, renal tubular necrosis, which is very similar to that produced rarely by BK virus. So with all of these viruses, most of the illnesses occur only in immunosuppressed populations.
Now, the factors that determine how much virus will be in the vaccines are listed here. First, the source of cells that are used. Now, many of the vaccines were produced in Rhesus cells, but some were produced in cynomolgus cells, which is another Macacus species.
The Cynomolgus Monkey is not naturally infected with SV40. So if the cells were of Rhesus origin, there's a greater chance of that being contaminated than if it was cynomolgus cells.
The type of culture of the cells are grown in a monolayer culture. They expressed simian virus 40 and replicated simian virus 40 very readily, whereas in some instances the vaccines were made in what are called the Maitland cultures, where the cells are not in monolayer form, but they are in the form of minced kidney tissues. This Maitland-type of culture did not support the replication of SV40 as well as the monolayer cells.
Then in many instances the kidneys pooled. The number of studies then would show that if the vaccine was made in a single -- all the cells derived from a single animal, it has a low chance of being contaminated with SV40, but if you pooled the kidneys, then any one infected kidney would contaminate all the rest, and then you have a much higher chance of getting contaminated vaccine.
One of the big, major factor was -- especially in terms of live SV40 and only matter of importance is live SV40, not inactivate SV40 -- depended upon whether the vaccines were live vaccines or inactivated vaccines. In the live vaccines such as the oral Sabin vaccine -- which are not inactivated -- the SV40 remained in high titer; whereas in the Salk vaccine the formalin that was used to inactivate the polio virus, also inactivates SV40 to a large extent.
So people would get, in the contaminated vaccines, either live SV40 along with a good bit of inactivated SV40, and they would get smaller amounts of SV40 in the Salk vaccines than they would get in the Sabin vaccine.
And very probably, although we are not completely sure about this from the data that we had available to us in 1976, only a proportion of the Salk vaccines had contamination with SV40 because a large proportion of the vaccines were made in the Maitland-type culture which do not replicate SV40 very well.
The most important exposure is the third that I've listed here: licensed inactivated polio virus vaccine. But in 1955 and 1961 the vaccine was contaminated -- some lots were contaminated. As we said before, being an inactivated vaccine it would have low amount of live SV40, but 98 million people had received the vaccine by 1961.
The live polio vaccine which would have large amounts of SV40 in the United States, only the experimental lots contained live SV40. By the time the live polio vaccine was licensed it was required to be free of SV40. So in the U.S. people were not -- not a large number of people were exposed to SV40 in the live vaccine.
The live RS vaccine which is the first line there, it's given to very few people and it's important simply to see what happens to SV40 when it is given intranasally. The second one, the inactivated adenovirus vaccine -- I think it's an error on my part here because those were live virus vaccines that were given to military recruits.
If you give the SV40 intranasally to individuals, the virus excretion occurs in throat excretions to some extent. There's a very low level antibody response. With oral vaccine there is almost no antibody response and the virus is recovered very infrequently, suggesting that the infection is very transient. There is no information with respect to the subcutaneous vaccine, how often it is -- if the virus is disseminated from people who are infected by subcutaneous vaccine.
And I'll pass this over just to show, the people who are most likely infected with SV40 vaccine, the year of birth, 1941, '61 -- people born between 1941 and '61 -- have a high probability of being infected. Those were born up to 1963 and later, those are very small probability of being infected.
The are a number of studies -- this last slide -- the number of studies done in the United States to see if the virus had bettered in a city of poor people -- and I have just listed them. I think we may have a chance to discuss many of them in the course of the two days.
But it is summarized at the bottom that while the studies did not reveal any ill effect of SV40, they did not have enough numbers, there was not sufficient period of follow up. The most susceptible would be infants who were infected in early life; not many of them could be followed. So while the data did not show any pathogenicity for SV40, there were -- all of the studies had their limitations.
Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Shah. We will continue now with SV40-like sequences in choroid plexus tumors and ependymomas by Dr. Robert Garcea who's at Children's Hospital in Denver, Colorado. Dr. Garcea.
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DR. GARCEA: Thank you. What I would like to present today is the data from a New England Journal paper in 1992. And I'd like to present it in the context of exactly how the experiments were carried out. And so it's an historical, sociological presentation rather than a -- although I will give you all the facts, I think you'll see -- I want to give you the flavor of how we proceeded with these experiments.
My laboratory at that time was located at the Dana Farber Cancer Institute, and our laboratory is primarily interested in the structural biology of papomaviruses and really not in translational work, although I am a Pediatric Oncologist. In 1989 John Bergsagel came to the laboratory to look for a post-doctoral fellowship and wanted to pursue whether there was any link between polyomaviruses and human, in particular, pediatric malignancies.
I didn't think any existed, but I was struck -- and this paper had always stuck in my mind -- this is from 1984, Brinster and Pometer, who described -- I think it was the second transgenic animal ever made with the SV40 large T-antigen driven under its own enhancer/promoter.
These animals developed very peculiar tumors, all of them in the coriplexus of the animals. And so I thought that this was quite a striking result and therefore I told John to go learn PCR and examine all the coriplexus tumors he could get his hands on at the Children's Hospital in Boston, the presence of polyoma-like sequences.
Now, coriplexus tumors are very, very rare tumors. They're only three percent of all of childhood malignant intercranial tumors, and we estimate that there's about 30 to 60 cases per year in the United States. Very interestingly though, all of these tumors are -- the majority of them occur within the first year of life, and they are in the differential diagnosis of hydrocephalus in utero.
A related tumor that we decided to study along with coriplexus tumors, because of the animal data of tumorigenicity of SV40, and because the ependymal cells are also a lining cell of the ventricular cavity of the brain, were ependymomas. Ependymomas are a little bit more common but again, still strikingly rare.
So John went to the pathology department at Children's Hospital, to the archival specimens. And he decided to -- what I thought in my wildest hopes was that maybe he would find BK or JC in these tumors and in my less-than-wildest hopes I knew that we would probably get PCR working in the laboratory.
John was to do a computer-assisted search of the polyomaviruses and align the sequences and find out where there were very close homologies between the viral genomes to make PCR primers. And not surprisingly, what he came up with was a region just after the splice site in large T-antigen, which is the Rb binding site. This site is very highly conserved among most of the polyomaviruses. And so John made primers in this region.
And the strategy that we initially employed was to -- these regions are identical for the forward and reverse primers, for both the BK and the JC viruses. I think we were trying to economize -- at that time it was expensive to buy primers, and so I think we were trying to be a little bit economical here.
And so these primers at the end would amplify either BK or JC, and the strategy therefore, was to amplify the specimen and then probe with an internal probe that was unique for either BK or JC.
And so what we did, John went to the archives, scraped slides off -- just in passing and I think we'll get back to this -- is that at least at Boston Children's, before 1976 most of the brain samples were fixed in Bouin's solution which has I think, picric acid in it, and we found that the DNA was highly fragmented. So most of our specimens came subsequent to 1977.
So what John did was, for most of these specimens which were on slides and from paraffin-embedded material, he precipitated the DNA. He then analyzed with globin oligos to see whether the DNA was intact, then he PCR'd with the BK and JC oligos, did a southern blot, and he probed with a specific oligos for either BK or JC.
And this is one of the original blots that John got. It's what we call low stringency. Actually, the whole PCR reaction was of low stringency and that cost us some consternation. And the other problem was that John, at that point being a physician, didn't quite understand the necessity for monitoring temperature in southern blots.
But what we found in some of the first samples was that there was a -- for example, we could get amplification in many samples of BK, for BK and even of JC, but many, many times these bands were hybridizing in both situations to both the BK and JC probe. And just for interest sake, this went on for quite a number of months, about three months, before I became a little frustrated and I told John to stop this and just sequence one of them so we could figure out what was going on.
And this was when the first matrices surprise happened. And that is, when John sequenced two of these bands he found that they were neither JC or BK but SV40. And there's a very characteristic nine base pair change in this region that he sequenced between those viruses.
And when we went back and looked at the primers, indeed the primers that we had -- since this region was so conserved, would have amplified SV40 under our conditions. And therefore, we went back and we used those primers with now a probe that was more specific for an internal region of SV40, and we used higher stringency conditions in the southern blot at 52 degrees -- and John had realized the importance of temperature by that time I think.
And most of our samples then, were amplified and blotted with this SV40 specific probe, although still -- and I think this is a topic for further discussion -- some had also BK or JC sequences in them, which we could not figure out why, but we were still now stuck with the fact that we had sequenced SV40 and that many of our samples were amplified and being identified with a probe for SV40.
John then went back and made new primers. The original primers were these right here after the splice site, PY forward and PY reverse with the probe in the middle.
John made now, another set of primers that amplified across the intervening sequence, SV forward-2 and SV reverse, and also another set of primers that amplified a very small, 107 I think, base pair region that lie just outside the previous amplification.
So when John -- we were concerned here that maybe there was some CDNA contamination in the laboratory. Contamination of course, is going to be a major issue in our discussion.
And so for example, he took some of the tumor DNA -- now this is, we have found that -- we were lucky in that our first set of primers amplified about 170 to 180 base pair fragment, because from the paraffin-embedded material it was very difficult to get long amplifications, and so when we got some fresh tumor specimens it was possible to do these longer amplifications.
And this for example, is an amplification with those long primers, with those primers that amplified across the intervening sequence, and it generated a band which could be specifically cut with an enzyme that would only cut SV40 and not BK or JC. So this is, besides sequencing, another way we determine that some of our samples were probably SV40.
At that time when we got this result, I wanted to verify the data with at least some immunohistochemistry. And so I called up Janet Butel and along with Milton Finegold, the pathologist at Texas Children's Hospital, we did immunohistochemical studies of these tumors -- and this is an example of immunohistochemistry with a large T-antigen of an ependymoma, and you can see that there's some very darkly-staining nuclei in this field, whereas the others don't stain.
And this for example, is a coriplexus tumor which is hard to see, but there are very darkly-staining nuclei here. And I think we'll get to the problems with the immunohistochemical cross-reactivity of different T-antigens, but this was the data that Janet and Milton obtained.
We went back then, with the set of primers that amplified that short region, and re-analyzed all the specimens that we had in hand. And this is the table of all the tumors we had at that time. And so we had 20 coriplexus tumors, ten of which amplified with those short, 107 base pair amplifying primers, ten of 11 ependymomas amplified.
We put neuroblastomas -- we analyzed those because transgenic animals with JC or BK also give rise to neuroblastomas and so we were very curious in that, also because it was a pediatric malignancy, but we didn't find any sequences in those tumors.
These are other controls. We studied normal brains, seven normal brains. We had a problem in the beginning because it was difficult to find brains that were not pickled for a month, and seven specimens were capable of globin amplification.
One was positive for SV40, which we thought was unusual. This was a 28-week, premature infant that died shortly after birth. The neuroblastomas I talked about, and we also examined normal blood. Fifty were studied at time and they were all negative. Subsequently we've studied several hundred and they've been all negative.
So at that time in this paper, we concluded that a segment of DNA corresponding to SV40 T-antigen was amplified from these tumors, and we concluded that perhaps SV40 or a related virus -- at that time we didn't know whether it was intact SV40 since we'd only amplified from one part of the viral genome, and maybe some hybrid virus was involved here.
And the tumors that we were finding this in were similar to those tumors that were induced in experimental animals by SV40. And so the questions that we had at that time were: is this a hybrid virus; is there full length copies of DNA in these tumors or are these episomal or integrated; where else are these sequences found? And of course, cause and effect is always a problem here, and I think that that's an issue for discussion.
There were two very interesting patients in our first study. One was a patient with Aicardi Syndrome, and this is a very rare syndrome that has agenesis of the corpus callosum in it. There's associated other abnormalities and rarely there's coriplexus tumors in these patients.
But perhaps more interesting, one of our coriplexus patients was a member of a Li-Fraumeni family, and this of course, most are aware is, the proband is a young patient usually with a sarcoma, and there's two 1st-degree relatives with cancer -- oftentimes breast cancer or osteosarcomas.
At that time, there were two kindreds identified by Judy Garber and Fred Li, with coriplexus tumors as part of the phenotype, and subsequently several other kindreds have been found having coriplexes tumors as part of this syndrome. About half of these individuals will have some mutations in p53, and the other half I think, are still up in the air.
Because of the occurrence of one Li-Fraumeni patient in our initial series, and because David Malkin and Steve Friend were across town at Mass General, I asked David for all of the Li-Fraumeni DNA specimens he could give me in a blinded fashion. And so we analyzed 163 Li-Fraumeni-related specimens -- and I'll talk about how they were related in a minute -- because we were interested in trying to follow up this one patient.
We'd also done some other controls. We looked at Wilms tumor because it's a kidney tumor and these virus seem to be trophic for kidneys. Subsequently, we looked at lung cancer because afterwards as you'll hear, Michele Carbone had found the virus in mesotheliomas and we were interested in other lung-related tumors.
So for the Li-Fraumeni specimens we analyzed 163 of these, and only 19 were positive. Now we were amplifying for the large fragment -- that fragment that amplifies across the intervening sequence. And I think what I was struck most about this analysis -- and so my first surprise was finding the SV40 sequence, and my second surprise was in decoding the specimens and finding that five were from osteosarcomas, four were from the blood from osteosarcoma patients, and one was from a lymphocyte cell line made from an osteosarcoma patient.
Now, all of these DNAs have been prepared by David Malkin and Steve Friend. And so there was a preponderance here of, half or over half of these in a very large series were related to osteosarcomas. And at that time I think I got my first call from Michele Carbone telling me about the mesothelioma data. And I told him about the osteosarcoma data and he said, oh yes, we've seen that too.
And so at that point, that's when Michele and I started to collaborate on the bone tumors which he's going to tell you about. And we also told immediately, Janet Butel, who's also got some data on the osteosarcomas. So I'm not going to give any more of that except as an introduction to their talks.
So this is a further breakdown of the Li-Fraumeni. There were three from unknown tumor types. I would say in the 163 samples there were many, many breast cancer specimens because this was part of the Li-Fraumeni syndrome, and none of those were positive.
There were somatic sources, and this was from blood or fibroblast cell lines that had been made from these patients. But again, a number were related to the osteosarcomas.
So that in short, is the introduction to Janet's and Michele's talks, I think. And I think Dr. Butel and Dr. Carbone will give more details on the subsequent analysis of these sequences in brain and bone tumors.
And I would just like to point out my collaborators at the Dana Farber when we were there, was John Bergsagel as a post-doctoral Fellow who really, from an M.D. coming into the lab, did a spectacular job. And I have to say that we didn't publish the New England Journal paper for at least a year of repeating all of these things over and over again from John, and he was a very meticulous person.
Wendy and Kristie Johnson worked up the Li-Fraumeni specimens in my laboratory. We had a very nice collaboration with the Baylor group, with Janet, Milton, and John, and at the University of Chicago, now at Loyola with Michele Carbone.
And thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Garcea. Before we go on to the next talk, if Dr. Mutti is in the audience, I understand he wants to talk to me, and if he'll come up here we'll try to settle the problem before he's announced to speak.
Our next speaker is Dr. Janet Butel from Baylor College of Medicine. She will talk to us on evidence for the presence of authentic SV40 in human brain and bone tumors. Dr. Butel.
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DR. BUTEL: Thank you very much. I'm going to pick up the story now from where Bob Garcea left off. We thought about his finding of the SV40 DNA fragment in human brain tumors and two major questions emerged: did the DNA represent authentic SV40, or was it a new virus or some recombinant virus; and if in fact it really was SV40, were there human-specific variants that we could identify?
So in the next 15 minutes I'm going to present some evidence that suggests very strongly that authentic SV40 is present in at least a few human tumors. And since I'm going to go over several types of data rather quickly, I wanted to just summarize what the main points were going to be.
And that is, by PCR we've been able to detect SV40 fragments from four different regions of the viral genome, and sequencing showed that it's really SV40 DNA. The regulatory region structures are typical of a non-duplicated enhancer region that's found in some natural isolets.
We detected variability in the C-terminal T-antigen gene sequences. We isolated infectious SV40 from one human brain tumor, and as Bob mentioned briefly, we have some similar results with bone tumors.
So Bob sent us blinded samples of DNA from some of the brain tumors for a follow-up study. And this map shows the SV40 genome and it shows the positioning of different sets of primers that we use for PCR analysis. Here was the Rb proximal region that Bob described.
We looked at the regulatory region of SV40 down here at the end of the T-antigen gene, and then a region in VP1, the structural protein of the virus. Our idea was, that if separated regions from the viral genome could be detected, then most likely the virus that was present in the tumor was SV40.
Source controls for the regulatory region studies. We used a series of plasmas that John Lednicky had constructed previously, and these contained one, two, or three copies of the 72 base pair region; that's the enhancer region. Lab strains usually have a duplication in this region and some natural isolets from monkeys have had a single copy.
This is a gel showing the PCR results using these primers from the regulatory region. A number of these blinded brain tumor samples were positive, and the size of the fragment that was generated was the same size as the fragment containing a single 72 base pair region.
When John sequenced those PCR products, in fact the sequence was exactly SV40 from each of those, and in fact, they did contain non-duplicated enhancers.
This shows the results of PCR assays using the VP1-specific primers. The same tumors that had been positive for the regulatory region were also positive and generated a VP1 fragment. And the sequencing of those PCR fragments showed that it was an exact match with SV40.
We then examined the samples using the T-antigen primers. Now, from everything we know about SV40 we would predict that the full length T-antigen gene ought to be present if infectious virus were present or if T-antigen were involved in tumor development. And using the primers from the C-terminus of the T-antigen gene, the same tumors that had been positive for the other parts of the viral genome were positive and yielded products.
When the products were sequenced there were some nucleotide changes that were detected. This is compared to strain 776 here at the top. Each of these slides represent something slightly different about the sequence and of the five brain tumors that were sequenced, each one yielded a slightly different T-antigen sequence.
And some of those nucleotide changes would result in amino acid changes when you look at the predicted amino acid sequence for T-antigen. And that's what this slide shows. There were some substitutions, there were some deletions, and there were some insertions in what was found in the brain tumors.
We tried to isolate virus from the brain tumor samples and we did succeed in one case. The tumor DNAs were lipofected into monkey kidney cells: both TC7 and CV1. Only one of the samples, sample number 12, produced CPE. It took a pretty long time for the CPE to show up -- six weeks. The cells were extracted and John cloned the DNA and we called this virus isolet SVCPC.
The virus induces typical CPE vacuolization. We're starting to characterize the virus. Renee Steward in the lab has done growth curves comparing our Baylor wild type strain of virus with the human tumor isolate, SVCPC, in both monkey kidney cells and in human cell lines. This happens to be a renal tumor cell line that we got from the American Type Culture collection.
And the point is that both the wild type virus and the human isolet grow very well in both monkey and human cells. So it's clear that SV40 can grow well in human cells.
The next question is, were there changes in other parts of the T-antigen gene among different virus isolets? So Renee sequenced the entire early region, not only of SVCPC but of several other isolets including two old human isolets, SVMEN isolated by Dr. Kreig in 1984 from a meningioma, and SV40PML isolated in '72 by Drs. Weiner and Shah.
There were a few nucleotide changes that were found but no huge differences, and bear in mind we're comparing here both monkey and human isolets and viruses that were recovered over a span of about 35 years.
Now, when the nucleic acid sequence was converted to the amino acid sequence, it was really remarkable to discover how conserved the T-antigen protein is. There were no changes in the protein at all among any of these isolets until we got down to about the last 90 amino acids, and then we saw a cluster of changes. So on this basis we refer to this as the variable domain of T-antigen and this is the region where we're seeing some variation in the tumor associated sequences.
Renee has just about completed sequencing the late region of SVCPC. This slide summarizes the results from the coding regions. If there's a vertical line below this horizontal bar that means there's an amino acid change, and we didn't find any amino acid changes in the agnoprotein, in VP2 or VP3, and a single change in VP1. So it's clear that these human isolets are typical SV40.
Now, when I heard from Bob Garcea and Michele Carbone that they were finding SV40 DNA in osteosarcomas, we wondered first of all, could we confirm that on an independent set of samples, and then secondly, perhaps it would be possible to identify a distinct bone tumor associated virus variant.
So my pathologist colleague, Milton Finegold, obtained for us ten osteosarcoma samples from St. Jude's Hospital, and this slide just summarizes some of the patient information. I want to point out that all of these patients were born 1965 or later, after the use of the contaminated vaccine was discontinued.
The samples were provided to us blinded, they were extracted -- John Lednicky will discuss some of the important steps in processing these samples and analyzing these samples by PCR during the panel discussion. But we used the same four sets of primers to analyze the osteosarcomas that we had used in the brain tumor study.
This shows the PCR results using the primers for the regulatory region of SV40. Five of the ten osteosarcoma samples were positive and that they yielded a product. And when John sequenced those, each contained a single 72 base pair region -- that is, a non-duplicated enhancer -- and the sequence was typical SV40. We also used primers specific for JC and BK regulatory region and didn't pick up anything in these osteosarcomas.
This just summarizes the VP1 results from the osteos. The same five samples were positive and when the products were sequenced, again, there was an exact match with SV40, VP1.
The T-antigen gene was also present in the same five samples. We got positive signals with both sets of primers: those from the Rb proximal region and then those from the C-terminus of the T-antigen gene. I'm just showing the amino acid calculated sequence.
The take-home lesson is that again, that each of the osteosarcomas contained a slightly different T-antigen sequence when we looked at the variable domain of T-antigen. Three of the tumors yielded new sequences that we hadn't seen before, and in two cases, it was a known sequence.
So finally, I want to end by telling how we addressed the question of whether this variable domain at the end of the T-antigen gene is highly mutable -- meaning it changes rapidly over time -- or in fact, is it stable and the variation that we were observing would reflect stable strain differences.
So to do that we analyzed high and low passages of two different strains of SV40 for which we had a known history. And this study was possible because I had frozen away in my freezer, some low passage stocks that had been frozen down for more than 25 years. And I'm just going to show you the story with VA4554.
This virus was received at Baylor in 1967. It was passed a couple of times and a stock frozen down in 1971. We reconstructed the history. We know that Dr. Hilleman sent the virus to the Enders laboratory in the early-60s.
Peter Tegtmeyher used this virus, manipulated it in all of his genetic studies. Then in the mid-70s he sent the virus to Judy Tevethia's lab where she used it in her genetic studies. And then in the early-80s Judy cloned the virus and sequenced it.
So John Lednicky went to this very low passage stock of VA4554 that we had, he cloned out isolets from that stock, and then compared the sequence of these isolets with this sequence that Judy had determined on this lineage of virus that had a very different history.
John cloned out two types of viruses. One had an archetypal regulatory region, one had a duplicated enhancer from the low passage stocks. The sequence of each was exactly like the sequence that Judy had determined for her virus.
And then finally, when we examined the sequence at the C-terminus of the T-antigen gene, the sequence was exactly what Judy had determined for her virus. So our conclusion is that the sequence is stable and we think that these variations reflect viral strain differences.
So I've run out of time and in summary, this just reiterates what I had said earlier was going to be the take-home lesson. And these are the people who have been involved in the work.
CHAIRMAN KIRSCHSTEIN: Thank you very much, Dr. Butel. I want to repeat, if Dr. Mutti is here I need to speak to him.
The next presentation will be by Dr. Michele Carbone, who is at the Loyola Medical Center in Illinois. Evidence for SV40-like DNA sequences in human mesotheliomas and osteosarcomas. Dr. Carbone.
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DR. CARBONE: Thanks. And first of all, I would like to thank Dr. Lewis and Dr. Levine for having invited me here. I did my post-doctoral training with Dr. Lewis and I was hired by Dr. Levine as a visiting associate in his lab, and he gave me the possibility to start my career, so I'm particularly grateful to the organizer of this meeting for what they did for me.
Today I am going to talk about the evidence that we have for the presence of SV40 sequences in human mesotheliomas and in human osteosarcomas. Tomorrow I will present our new data that suggests that not only these sequences are present in the tumors, but they may also in some instances, contribute to the transformed phenotype. And tomorrow we'll also talk about, as before, oncogenicity in hamsters.
In order to start my -- this is SV40 viruses, small DNA tumor viruses. In our studies in humans we are prompted by our findings in hamsters. What we found when we injected SV40 intracardially into animals, was that only particular tumor types developed, specifically, mesotheliomas, osteosarcomas, lymphomas, and on the -- sarcomas.
The reason that we injected the virus in the heart was to expose more cell types to the virus and to see whether every cell could be transformed by SV40 or only specific cells were transformed. Now, from our tissue cultures -- not ours, but from tissue culture experiments that other people did -- we know that SV40 will infect more or less, every cell. Even in humans it can easily infect karyotinocytes.
You can clearly see that the most common cancer, at least those that developed in humans, never developed in a hamster when you inject SV40. We have never seen a carcinoma. I was particularly struck by the fact that the mesotheliomas developed and so we repeated the experiment injecting as before, into the pleural space. And in that case, 100 percent of the animals came down in tumors in three to six months.
Other investigators had found that when you inject SV40 intracranially in animals, only ependymomas or ancyroid plexus tumors develop. So the conclusion of this experiment is obviously, SV40 is a virus that for some reason, even if it can enter different cell types, will transform only particular cell types. They must be more susceptible to transformation by this virus.
Again, I was particularly struck by this tumor. This is how it looks histologically. This is a mesothelioma. Mesotheliomas are a tumor with incidences increasing inordinately. Today we have 2,000 or 3,000 cases in the United States. Actually, I just heard from the -- that 4,000 are projected for '97.
And this is a high number if you consider that until 1950 or so most books of pathology denied the existence of mesothelioma between they were so rare in that many people so that they didn't exist at all. So it's like going from zero to 4,000.
The reason for that is the use of asbestos. At the beginning of the century asbestos was used largely in all the western world, and it's obvious that exposure to asbestos induced mesotheliomas after approximately 20 to 50 years.
However, 20 to 50 percent of mesotheliomas -- and that depends what study you look at -- are not associated with asbestos exposure. Now, that's a big number because obviously there is a large number of mesotheliomas that are increasing from 1960 for which we cannot account. So we ask it, could SV40 or a related virus be related to the development of mesotheliomas?
And this slide summarizes whether that time was known as SV40 human pathogen. SV40 can transform human cells in tissue culture. SV40 human transformed cells induce tumors when injected into human volunteers. Millions of people were injected with SV40 contaminated adeno and polio vaccine and after 1963 vaccine should be SV43.
While I was trying to convince my -- at that time I was supervising Dr. Levine's lab -- but I couldn't convince them to look at these SV40 tumors because obviously it was a very risky project. So while I was struggling to convince somebody to work on this with me, Dr. Garcea published in New England Journal of Medicine that 60 percent of human ependymomas contained SV40-like sequences.
It's a study that was later confirmed by other investigators, including a recent paper by Martini, et al, in cancer research a couple of months ago, and from the laboratory of Dr. Butel where Dr. Lednicky isolated infectious SV40 from one of these tumors indicating that at the least, in that case, the SV40 life sequence was infected as we thought.
So eventually I was able to convince Dr. Procopio that had done his post-doctoral training at the NIH and was a tenured professor in Italy, to come to the NIH and do this work together to see whether there was any SV40-like in these mesotheliomas.
We used the same technical approach that had been used by Dr. Bergsagel in his studies and the reason is, as Dr. Garcea indicated before, that the Rb binding domain should be there if the T-antigen is doing something, because the Rb pocket binding domain is that region of the antigen that binds Rb, p107, Rb2 or p130. So the cell are proteins that must be inactivated by the antigen in order to receive transformation, therefore, that would be the region that you most likely would expect to find.
We started a great collaboration with Dr. Harvey Pass, that at that time was the Chief of Thoracic Oncology here at the National Cancer Institute. Harvey has an incredible collection of mesotheliomas and he candidly gave access to us to his collection and also he worked very closely with us during these experiments.
And this is a summary what we found; that is, 29 of these 48 mesotheliomas that Harvey gave us tested positive with primers that amplified the Rb pocket binding domain of T-antigen. Sequence analysis confirmed that those sequences, the type that amplified with the specific proper SV40, were in fact SV40, and the arrow points to that unique region of SV40 that Dr. Garcea already talked about so I'm not going to repeat it.
Immunoperoxidase staining indicated that some mesotheliomas cells contained a nuclear antigen that strongly reacted with the monoclonal antibody against, as before, the T-antigen. So we were seeing either as before, the T-antigen or something very close to it.
And actually, if you look at that again you can see that only the tumor cells stain and in fact, that the reactive fibroblasts that are around the tumor cells do not stain. And immuno-precipitation studies from frozen tissue precipitate a 90 kilodalton protein with the monoclonal antibody against the antigen that reacted in western blot with the monoclonal antibody against the antigen.
And this was the conclusion of that paper, we found SV40-like DNA sequences in 29 of 48 mesotheliomas stasis and demonstrated the antigen expression in 11 of 14 specimens. The associated lung did not contain SV40 sequences although they contained asbestos.
We suggested that an SV40-like virus may act independently or as a co-carcinogen with asbestos. Moreover, the selective T-antigen expression by mesotheliomas and not the surrounding pulmonary barenchyma, may have diagnostic and therapeutic implications.
At that time I gave the code to Bob Garcea and from what we knew in animals, there was another possibility for finding SV40 among the many human cancers was obviously osteosarcoma. And the other question was, are there other tumors in humans that may contain SV40 sequences?
So we started a collaboration -- me, Bob, the laboratory of Dr. Pass, and the laboratory of Dr. Procopio in Italy -- and we studied 345 different human samples including 159 bone tumors and sarcomas for SV40-like sequences by PCR.
Only after all four laboratories completed the analysis was the code identifying the origin of the bone tumor specimen broken and the results compared. And these are the results that we published in Oncogene last summer.
Fifty-three of 159 bone tumors and sarcomas -- that is exactly one-third -- tested positive. Of the 186 known bone tumor sarcoma samples only one out of one neurofibromatosis type, one was positive. And the other 185 were all negative.
Ten samples, all from bone tumors, gave conflicting results, meaning that at least one reaction in one of the four laboratories was positive. These samples were considered negative.
And these are some of the results that we obtained. In this particular study we used a different set of primers that are the long primers that Bob referred before. And they are indicated up there -- I don't have a stick to indicate them -- but you can see a hoop at the top line. They are indicated by SV42 and SV-rev.
They amplify the same RV pocket binding domain that we used to amplify before that is indicated by the primers before SV-rev. But they also amplify the intraregion of the antigen. And the reason to include the intraregion was that while you want to find SV40 T-antigen there so that's why you put the Rb pocket binding domain there, it would have been nice to find some mutations, because there is always the question when you do PCR, people will ask, are you sure that there's no contamination?
Well, this is what we found. This is the 574 page paper that you expect to amplify with this parameters. And the last time in the right under the H letter that indicates hamster, is a hamster osteosarcoma. So you can see that in addition to the 574 base pair band there are other bands with different molecular weights.
Particularly prominent is the band around 300 or so base pair. When Dr. Rizzo, that is my research associate, sequenced those bands that were hybridizing with the probe specific for SV40, the sequence indicated that that was SV40 T-antigen, but that there were deletions within the intraregion. And we never found deletion outside there; we never found deletion in the Rb pocket binding domain.
For some samples we also tested the other region of SV40 genome to see whether they were there, and for example for those two samples indicated with the numbers 103 and 105 that were positive for SV40-like sequences, in panel B we are amplifying the capsid throat in PV1 and in panel C we're using two sets of parameters that amplify the carboxyl terminal domain of the antigen.
So for at least these samples a lot of the SV40 genome was there. We also sequenced the regulatory region of SV40 and we found 272 base pair.
This slide points to an intriguing coincide that comes out from these studies. And these are the tumors associated with SV40 in humans. Ependymomas are induced by SV40 hamsters and in humans contain SV40-like sequences. The same is true for choroid plexus tumors, the same is true for mesotheliomas, the same is true for bone tumors, the same is true for sarcomas.
The last one, true histiocytical lymphomas that are induced by SV40 hamsters, are so rare in man that many people doubt that they exist at all.
And this is the conclusion for, this talk at least. The significance of SV40 and SV40-like sequences in human tumors is presently unclear. Specifically, it is unknown from what source these sequences originate; whether these sequences contribute to tumor development. And again, I am going to present some data suggesting that in some cases they can, and Dr. Weiss has been very kind to offer me to present this data at the beginning of this round table tree tomorrow afternoon.
And whether these sequences can be used as targets for designing new immunotherapeutic and molecular approaches for the management of malignancies expressed in T-antigen. And while that can seem futuristic or too much optimistic, I think that in fact, it's a very exciting area.
This is one of the best immunoperoxidase that I have, of course, but this is a human mesothelioma and you can see the staining of these mesothelioma cells that clearly distinguish them from the surrounding non-malignant cell. Now obviously the presence of a unique antigen on a tumor cell gives you, at least in theory, the possibility to attack those cells, and we currently are working on this hypothesis.
Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Carbone. Our next speaker is Dr. Allen Gibbs who will present SV40 DNA sequences in mesotheliomas. Dr. Gibbs is from Llandough Hospital the United Kingdom.
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DR. GIBBS: First of all, thank you. I want to thank the sponsors for inviting me to present the work that has been conducted in Cardiff in this. I'd just like to point out that the pronunciation of the hospital is "thrandock" and not "thrandough", but I will excuse the American pronunciation.
I'd also like to acknowledge Dr. Bharat Jasani -- he was also at this meeting -- who really is responsible for the actual methodological approach of the study.
Why did we conduct this study? Well first of all, we had seen the paper by Dr. Carbone on the finding of SV40-like sequences in mesothelioma; that my particular hospital has had a long association with examining mesotheliomas and I have an archival store of several thousand mesotheliomas. And these go back to the days of Chris Wagner who worked in my hospital and in fact, was the person who put the association with asbestos and mesothelioma on the map in 1960.
And we were particular interested, really, in looking at our archival material because a considerable proportion of that has been well worked up, both diagnostically and from the point of view of exposure data, and that includes a very detailed mineral analysis on the lung tissues.
So we were interested in trying to exploit this material, looking at the possible role of SV40 and asbestos in causation of mesothelioma. So this was a, basically a pilot study to examine frozen and archival, surgical and post-mortem mesothelioma tissues for SV40 DNA-like sequences, and we were using the method of PCR.
I'm not going to talk about the Tab expression using immunohistochemistry now, but it may possibly come up in one of the later sessions.
The materials and methods that we started with are cases. There were nine pleural mesotheliomas. I also took nine adenocarcinomas that were metastasized to the pleura, and nine reactive pleura. These varied from reactive eosinophilic pleuritis to non-specific chronic inflammation of the pleura.
All the cases had formalin fixed and paraffin embedded material, but in addition I mentioned to, as we went along, obtained four mesotheliomas which we had frozen down. These were complementary to the four other surgicals -- three surgicals and one post-mortem.
There were three post-mortem cases and six surgical biopsy cases when it came to the mesotheliomas. The breakdown was eight males and one female. The age range varied from 38 to 73 with a mean of 58.8, and there was a mixture of histologies here. There were both the epithelial, biphasic and the sarcomatous types. All of these had a history of exposure to asbestos.
We basically used the same technology that Michele Carbone used in his paper, and we employed the Bergsagel primers, the direct known as the SV40, specific DNA sequences which was 107 base pair, and then one -- sorry, 105 -- and then the 172 base pair sequence that recognized the papomaviruses BK, JC, etc.
And for controls we had some SV40 transfected human thyroid cells, one positive control and one negative, and the positive contained one copy per cell. And employed the controls both on frozen material and on formalin fixed, paraffin embedded material.
This shows the gel electrophoresis on the -- here's the mark here which tells you the size of the protein that's found. This was one of the mesotheliomas which was negative. This was the positive thyroid transfected cells, and then these other four were the four positive mesothelioma cases.
This shows the controls of three positive controls; again the marker here. This was the frozen, positive control and these two were both fixed in different ways, and they were positive, too.
So to summarize the results of our PCR analysis on these cases, we found the SV40 specific segment detection in four out of the nine mesotheliomas: one out of three post-mortem cases, three out of six surgical biopsy cases. We didn't find any present in the reactive pleurae and we didn't find any in the adenocarcinomas.
Just to go into some more detail on the actual mesotheliomas showing the concordance, or discordance between the SV40 and the papomavirus sequences, that the SV40 here was consistently present, both in the frozen and the paraffin material for each case that was positive, but that we got some cases which were negative to the SV40 which had the other sequence.
And again, these show the same thing for the extra cases. These were all just the paraffin embedded surgical cases.
This is just to compare our study with the published literature on studies of mesothelioma that -- Michele Carbone has already gone into this; that there's a somewhat similar rate in the two studies. A recent study by Strickler in fact, failed to find any SV40-like sequences in 50 cases, and then there's another study by Cristaudo that found positivity in 72 percent; again on archival material.
So the conclusion to the study was a simple one. That we found SV40 DNA-like sequences in some British mesothelioma cases. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Gibbs. The next talk will be shared by two speakers. First, Dr. Luciano Mutti who will speak on SV40 on mesotheliomas. Dr. Mutti is at the Fondazione San Maugeri in Italy. And the second part of the talk will be presented by Dr. Antonio Giordano from Jefferson Medical College, who will talk on a retinoblastoma family in mesothelioma. Dr. Mutti.
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DR. MUTTI: First of all, let me thank the organizer of this fine workshop for my invitation. My work has been -- I share my work with Dr. Giordano from Philadelphia. And very briefly, as you can see, SV40 has been able as stated, to induce some tumors on hamsters, and as far as mesothelioma is concerned, injection of SV40 in pleural space, able to induce in 100 percent, mesothelioma, and while injecting in peritoneal space, is able to induce it near 50 percent of the animal.
In humans, raw data showing that SV40-like sequences are detectable in mesothelioma cells in about 60 percent of cases, and the T-antigen is detectable in a good number of cases. And SV40 has been so considered as a powerful cofactor in addition to asbestos fiber, for example, in inducing this tumor.
We studied this population of ten patients with this type of size of neoplasperitoneoma and nine of pleural mesothelioma, and with this type of histology. Some of these patients had not been exposed to occupational asbestos fibers, but had been exposed to an environmental exposure of a -- they lived in an area wherein a plant working on asbestos items had been working for at least 20 years.
So pollution of at least free fibers for meter cubed has been, nowadays, even now, detectable in this area. And these patients had been an occupational exposure for this period.
And there is another of these patients that had been exposed both to occupational and environmental exposure, because they lived in this plant -- they worked in this plant and they lived in this area.
Very briefly, we found SV40-like sequences in three out of ten patients we studied. And as you can see, one patient had both occupational and environmental exposure and the other two, only occupational exposure.
So we can see that there are some implications of these conclusions because SV40 can be considered actually as a tumorigenic factor, but it's important for us to assess -- to state that SV40 could be considered as an inducer, as a tumor, as an antigen, for mesothelioma.
So far it's not been possible to induce an effective neuro-response in mesothelioma cells, but there are a lot of studies that can demonstrate that a self antigen, a tumor or self antigen of mesothelioma cells do exist.
And one of the most important strategies to increase the new response is to increase the self-antigen expression, and with perspective in treatment of the -- of mesothelioma cells or mesothelioma is, without any doubt, to find out tumors that are dangerous, such as the antigen against it's possible to induce an immune response and a T-mediated response.
Just very brief, so I'll leave my talk to Dr. Giordano because he carried out another study about these patients, studying especially every family protein.
CHAIRMAN KIRSCHSTEIN: Dr. Giordano.
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DR. GIORDANO: Okay, so thank you, Dr. Mutti, actually for allowing me to present some common data that we carry out in collaboration. In the first light --
CHAIRMAN KIRSCHSTEIN: Before you start, I need to make an announcement. Dr. Chanock has an urgent message at the message center.
DR. GIORDANO: Okay. So very quickly, the retinoblastoma families is formed by three members, Rb 107, and the Rb2 p130. The Rb2 p130 is the family member cloned a few years ago in my laboratory -- we code Rb2 and then code for under 30 kilo often. The 03 family member, okay, they share a pocket region, so-called pocket region because the functional domain that is a point of the structure is tackled by different genus tumor viruses like the virus C1 as is before T-antigen. Is seven of papomavirus and do it as a function of domain because this is how this family or protein go through the cell cycle machinery.
So once we cloned Rb 2 we went on in the collaboration with Dr. Knudsen at Fox Chase Cancer Center. And with Dr. Knudsen we marked the Rb 2 on a chromosome 16, 12.2. And that start actually, our interest in the mesothelioma, because 16.2 by Dr. Testa's group at Fox Chase has been found also being the region found deleted in 16 out of 25 mesothelioma tested by Dr. Testa.
So we went on and carried on a series of experiments. And as a first characterization that we did, we found that Rb 2 is important to growth suppressor gene. When you applied to different tumor cell line the gene, basically you express in appropriate way, you achieve a growth suppressive property that is pretty dramatic in several tumor lines: like osteosarcoma, like glioblastoma, like nasopharyngeal cancer.
So by immunochemistry, okay, immunochemistry we went home and first test a -- cell line cancer tissue. And what do we found? We found that in line cancer actually, there is -- we divide in different histological groups. We found very interesting, retinoblastoma family in line cancer, especially the Rb 2 P 130, as in vast relationship, okay, with the aggressiveness of the disease. More aggressive is the tumor, undetectable is the Rb 2 gene.
So again, we went on and start the collaboration with Dr. Mutti and we start to test the mesothelioma lines in the tumors, actually, from tumors that also we obtain from different sources.
If you see here, we just use the primers that amplify the regional T-antigen that is required to tackle the retinoblastoma family. And we found the sequences of T-antigen in four of the patients of the nine patients that we tested.
But more interestingly, when we performed immunohistochemical analysis, when we perform immunohistochemical analysis on these mesothelioma, alike up in the line cancer, we find 11 of the Rb family do not change. So there are high, medium high level of the Rb family in all the mesothelioma tissue that we tested.
So T-antigen, sequence of T-antigen we found in these patients, and when we extract actually, the protein from this fresh tissue using antibody -- monoclonal antibody, this T-antigen -- we find the protein the right size, okay, in which T-antigen migrate. This one is a cos cell line that -- this place, SV40 T-antigen, and this one a cell line HLA 60. This does not see any DNA tumor virus. So there is no expression of T-antigen.
So Rb family highly expressed in all mesothelioma lines. So a mechanism that the work probably we could predict, I mean, just considering the data by several laboratories, that T-antigens and other genus tumor virus binds in a physical association with the original blastoma family, lead us to an experiment in which we took the source of T-antigen in these patients having mesothelioma, and we ask if there is any physical association with the original blastoma family.
As you can see here, okay, the Rb, the p107, and the pRb 2 physically interact; they interact in a physical pro-interaction with those three family members. So no wild conclusion from this of course, but clearly there is no blastoma family, okay, member in -- that in line cancer for instance, and don't see intermediate cancer in the screening of studies we did, correlates, especially the Rb 2, P 130, correlates with the aggressive disease in mesothelioma do not change.
And one of the mechanisms that probably we suggest is that known mechanism suggested by several laboratories that T-antigen by binding these original blastoma family does not allow them to perform the suppressive property. Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, DRs. Mutti and Giordano. Our next speaker is Dr. Mauro Tognon from the University of Ferrara, Italy, who will speak on SV40 and mesotheliomas.
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DR. TOGNON: Thank you very much, the organizer, for inviting me to this interesting meeting. And congratulation also, to the Chairperson for the very nice pronunciation of my name.
Probably the people are already tired to listen to the PCR amplification of the Rb pocket domains of the T-antigen, but in our investigation we did exactly the same thing. And in these experimental control we have in the first lane, the BK, the second, JC, and the SV40. One other, 72 base pair were amplified by the primer that is being named P4 and P5.
And as you can see ethidium bromide staining, we have the amplification of this region, and because this region is common to the three different polyomaviruses, we set up an experiment with the internal liogprobe that recognized specifically the three different polyomavirus.
We used a very high stringency condition just to be sure that this time we did the experiment with these two primers that amplify the Rb pocket domain of the large T-antigen which is common to the three polyomaviruses. All the time we have clear results by hybridization. In this case, we used the recombinant plasmid DNA that contains the entire genome of the three viruses.
In these experiments we analyze different human brain tumors and in particular I would like to say about the experiment, because we always start with 500 nanograms of human genomic DNA, and all the time we extracted DNA in -- what can I say -- in old fashioned way, we tried to avoid all the time to centrifuge or to precipitate the DNA with ethanol.
And the reason is that in our experience, if you precipitate the DNA with the ethanol, or you extracted DNA with the commercial kit, you lose the signal after the hybridization with the internal olioprobe.
So probably this makes sometimes the difference, is a possibility, between the high level internal percentage that we found in our human brain tumor and we can see a little bit later, also in the normal brain tissue, of the positive sample that we found.
In this case the control was always present. This is the 172 base pair. This is the only possibility because it's the control to see the amplified bands in the ethidium bromide staining, but in the other samples that are under analysis, the amplified bands never appear. In this case we amplify the DNA for 35 cycles.
The results come out only with the internal olioprobe that is specific in this case for SV40. As you can see, there are in these typical experiments, some primary tumors that come from ependymoma, across the plastispapilloma, plasticytoma, and neoblastoma. The neoblastoma was negative and also the sponsoblastoma was negative.
We analyzed also the normal brain tissue from people that died by car accident and we extracted the DNA in the same fashion, and the bands that amplify the control is always present. You can see the hybridization band that comes out after three or four days of exposure, our radiography, but none of the six samples that were analyzed in the experiment were positive for the SV40 sequences.
Okay, this is another experiment that we set up to investigate the presence of SV40-like sequences in the peripheral blood cells. As you can see, after the amplification and hybridization two of these samples are positive and indeed, we have the results of peripheral blood cells that sometimes are positive for these sequences, even if the percentage of positive sample in peripheral cell of normal individual.
This is not blood that comes from the patients but we took 70 different blood samples from the blood bank of the general hospital at the University of Ferrara.
Because we found the sequences specific for SV40, both in the tumor sample and in the peripheral blood cells of normal individuals, we think that probably there are sometimes contamination of the blood of our biopsies that come from the neurosurgeon.
And so we investigate the same kind of tumors, starting this time from the tumor cell line. All these samples were from glioblastoma cell lines. We analyzed, if I remember correctly, 18 glioblastoma cell lines. Nine were set in the lab of the Department of Pathology at the University of Verona, and they never worked with SV40, and the other nine were purchased from European culture collection.
And surprisingly, when we found that the results of our data after the hybridization with the internal olioprobes, comes out that practically all the Italian tissue cultures cell lines from neoblastoma were positive and none were positive from the tissue cultures that we purchased in England.
This is another data that comes from the experiments we did in the same fashion with other tumor -- primary tumors from the -- primary brain tumors. And in this instance we isolated the RNA from the tumor cell line, the glioblastoma cell lines, and among four different glioblastoma cell lines, three were positive for the messenger RNA. Even in this case, the experiments were set up with the specific primers from the Rb pocket domain.
This RT-PCR has been recently conduced, not only with the glioblastoma cell line but also with the osteosarcoma cell line. It's a new experiment that we set up recently. And some new data is now out from the normal tissue from people that are not affected by any kind of disease. These are three samples that come from buffy coats.
These are three different cell lines that were obtained from the Institute of Medical Genetics. Also these people -- of the University of Ferrara -- also these people never work with SV40. And these are three samples that come from the sperm fluid in another University, University of Modena.
As you can see, these experiments is only representative of the sample that I analyzed. Some of them are positive, some other are negative, and at the end I'll show you the table that summarizes all this data.
When we try to specify if the Rb pocket domains that the large T-antigen sequences are specific of SV40, we clone in sequence at the beginning from a glioblastoma primary brain tumor, a glioblastoma, the SV40 sequences, and they came out -- it was practically the same sequences of the SV40 wild type.
The difference that was also pointed out during the talk of Dr. Bob Garcea, is that the nine base pair insert that are present in BK and JC are always absent in the SV40 sequences, and also there are some other differences among the other bases that we sequence. So by comparison and by checking in the databank the sequence, we know there is a real SV40 -- I'm sorry -- okay, got it.
These are different sequences that were obtained from other primary brain tumors. This is an ependymoma and the sequence is the wild type SV40. These two come from two different glioblastoma cell lines, and we found a mutation in this position. The sequence is A, C, G, T.
And this is another primary brain tumor, a neoblastoma, that has two different mutations. Both are transitions in the first base of the triplets and at the end when we checked the amino acid we found that there are a substitution of the amino acid in this position.
Okay, after checking the specificity of the sequences for SV40, we checked also in two of the blastoma cell lines, the presence of the large T-antigen. And in this case we used a specific monoclonal antibody that react specifically with the large T-antigen SV40, and at the same time we compared these data with a polyclonal antibody that recognize SV40 and the BK virus T-antigen.
As you can see the results are similar in terms of positive data, but if you check here -- this is the control of BK transformed cell that is called T-53 -- this light doesn't react with the monoclonal antibody, specifically SV40 and the polyclonal antibody reactor, and we can see in the nuclei the positive fluorescence.
On the top we have the cos cell that are transformed with the large T-antigen, and expressed the large T-antigen in the nucleus, and as you can see even here, the monoclonal antibody, specifically SV40 react, and the polyclonal antibody that recognize both the large T-antigen, react with the same cells.
In a previous area of experiments we did more or less the same experiment in searching for the homology, not only for the SV40 but also for the BK. Actually, this was a first series of experiments and also in this case we used the internal olioprobe specific for BK to discriminate along the other polyomavirus sequences that eventually are present.
In this case, the same sample that I showed you before, ependymomas and plastispapilloma, so now to be positive for the BK, the same for the three or four blood cells, and the same data are for the RT-PCR in the glioblastoma cell line.
When we sequence the BK positive for the large T-antigen Rb pocket domains, it turns out in 12 different samples, ten from the tumor samples and two for normal tissue, that all the sequences were the same compared to the BK virus Dunlop strain. We couldn't find any mutation in the 12 different sequences that we performed in 12 different samples.
This tells me, I have to show you the table that summarizes -- wait a second, I go back for a second. No, it's just I want to show you this transparency. Okay. This is the table that summarizes the data for the detection of the specific BK virus DNA, and detection of SV40 DNA. And as you can see, all the primary brain tumor that was positive for the BK DNA were also positive for SV40 DNA in terms of much more positiveness for the BK with respect to the SV40 DNA.
Similar data were obtained also for the cell lines, and these tell us that the positive doesn't depend on the blood -- contaminated the samples, and we have some new data compared to the previous publication directed to the primary bone tumors, and the data are very similar to those that we had before from Dr. Carbone, Dr. Garcea, and Dr. Butel.
Approximately the five percent of the osteosarcoma are positive. We analyzed also some huge tumors, and it turns out that 33 percent are positive. And once again we analyzed those of the cell line and the osteosarcoma are 43 positive for SV40, but all of them are positive for the BK. And interesting, these giant cell tumors, these rare tumors, 80 percent are positive for SV40. And the small osteosarcoma is a particular kind of osteosarcoma -- 36 percent are positive for SV40.
Also some other tumors came out to be positive, but they are less of an extent for SV40 and positive also in the 42 percent for BK. But interesting, the normal brain tissue is quite different in terms of original data compared to the results that we heard before by other speakers.
And we have for example, the normal brain tissue, only one out of 13 is positive for SV40; the bone, none are positive; but the peripheral blood cell in general, that we obtained from density gradient centrifugation, 23 percent are positive for SV40.
But if you take the B and TD 4 side that are cell lines, in this case transformed by the ABV, you see that the SV40 is approximately 11 out of 15 samples were positive; 73 percent is quite high. Even the TD4 side contains the SV40 and as we published already, the nine out of 20 sperm fluids were positive for SV40.
The new and the last -- when you work with the PCR of course, you think sometimes you contaminate your sample. So this is a new analysis what we did very recently by PCR in human tumor and tumor cell lines from DNA that was extracted ten years ago, and in the laboratory that never worked with any kind of virus.
And as you can see from the results, the percentage of positive samples for SV40 are more or less the same that was entered in the previous table, both for the primary tumor and for the cell line.
So in conclusion, it seems to me that there are some different regional distributions of the SV40 sequences. And compared, as I say, to this country or in England, we have much more positive samples from the normal tissue compared to other data.
I have also a couple of more results I would like to tell to the people, that are obtained recently in the Institute of Microbiology by Professor Barbanti-Brodano, that unfortunately today is not here because he got a recent operation. And we tried to rescue the viruses of the SV40 by transfecting with lipofecting or lipofit -- I mean DNA from brain tumors, brain tumor cell lines, and from preferable cell experiments.
The only two isolates that we rescued so far were from preferable cell sample from the dysplasia of a vulva sample that was even in this case, obtained from a collection of a large DNA sample that was at minus 80 since 1985.
Now we are characterizing it to isolate it. The first analysis -- I mean the first sequence analysis seems to indicate that to isolate it at the two SV40, and in particular, the difference that we see as a variant are in the origin of replication. This data are just preliminary so we have to control a little bit in detail, these results.
Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Tognon. Our final presentation today is by Keerti Shah: A Search for SV40 in Human Mesotheliomas.
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DR. SHAH: We were also very intrigued by the finding of Dr. Carbone about the presence of SV40 in mesothelioma tissue, and we attempted to reproduce that. And this has been written up and published in this journal in 1996. We were not able to detect simian virus 40 sequences in pleural mesothelioma.
We also looked at serum specimens from mesothelioma patients and osteosarcoma patients for antibodies to simian virus 40. And we were not able to detect any evidence of SV40 neutralizing antibodies, the results documented in this paper.
And more recently -- something which is not in this paper -- we tried to look for simian virus 40 in urine specimens collected from immuno-compromised and non-immuno-compromised patients. We looked for BK virus, JC virus, and also simian virus 40, and we were not able to detect any simian virus 40 sequences in these urines.
So this is the mesothelioma study. There were 50 pleura-mesothelioma samples collected at the Armed Forces Institute of Pathology over a number of years -- this was from 1987 to 1992 -- and obtained from many different hospitals and clinics in the United States.
The patient's diagnosis was between -- age year diagnosis was between 43 and 88, and median age was 68 years. These were archive samples so we tested the paraffin sections with proteinase K and we amplified them with two sets of primers for SV40, and another set of primers for beta globin to see if the DNA was suitable for PCR.
The two primers for SV40 we used were described by Dr. Garcea. They amplified 103 and 205 base pair regions of the T-antigen for SV40. And they are shown here in illustration.
This is what we found. These are three identical filters. The top one is with beta globin probe, and these are amplified with the beta globin primers. And most of the specimens we were able to amplify beta globin easily. There were some failures -- one here, one here -- so in 48 out of the 50 mesothelioma tissues we were able to amplify beta globin so that the DNAs were thought to be suitable for PCR studies.
These are the same identical filters here and here they are tested with an SV1 probe, a probe that was described by Dr. Carbone. These are all mesothelioma samples. None of them show up SV40 sequence. And we have done a titration of SV40 cos-1 cells.
Cos-1 cells have one copy of the SV40 genome per cell, and these are 300 cells, 30 cells, three cells, and actually less than one cell. So with this particular primer path we were able to amplify one copy of this SV40 genome just by this particular data with the second set of primers; again 300, 30, and three SV40 copies.
So that you -- we thought that our technique was quite sensitive but we were not able to detect any SV40 genomes. So we also looked at 105 serum specimens: 35 were derived from mesothelioma, 35 from sarcomas, and 35 controls, matched to the mesothelioma patients. Then 97 -- and these were tested in SV40 plaque neutralization assay in BSC-1 cells -- 97 were completely negative and five were partially protective.
They did not completely neutralize the simian virus 40, but reduced the number of plaques. And they were scattered in the three groups. There were three out of 34 mesothelioma, one out of 33 osteosarcoma, and one out of 35 controls.
Then we looked at the urines and we collected -- these are 165 urines provided to us by Dr. Strickler and Dr. Gater -- and they came from a study of homosexual men, some of whom were HIV positive -- seven urines which were HIV-positive men and 78 from HIV-negative men. The median age was 38/39; ethnicity, they were 91 percent white.
And this time we thought that if we do not have good positive controls for SV40 no one is going to believe our data. So we took SV40 cos cells and spiked normal urine with the cells, and these tubes we sent back to the NCI where they were processed with the other urines.
And then when we received these 165 urine specimens they also contained other tubes marked similarly, which could not be distinguished but which contained 17 specimens which came from the spiked urine which were the SV40 positive urines, and contained approximately 300 cos-1 cells or 300 copies of the SV40 genomes.
These are the SV40 data. There are -- 17 urine specimens that we got were positive for SV40 and there were -- each of the 17 of the positive controls, the urine specimens that were spiked with SV40. The other 165 urine specimens, none of them contained SV40, but the prevalence of BKV and JCV was quite high. About 50 percent of the urines contained either BKV or JCV and some contained both.
This is the data from the -- for BKV and JCV from the urine specimen. In the HIV negative urines, or urines from HIV negative people -- only 2.3 percent were positive with HIV negative, and it increased with SV40 positivity and the degree of immunosuppression, something that has been reported before.
The SV40 prevalence was very high in both HIV negative urines as well as HIV positive urines, and we did not see any much greater increase in prevalence from what was already a very high prevalence in the HIV negative individuals.
So we could not detect SV40 sequences in mesothelioma tissues although we did 50 tissues. From the results of previous speakers we should have picked up at least 20, 25 positive specimens. We did not find serological evidence of SV40. One would think that if these people were developing tumors because of an SV40 infection, one would expect some evidence of SV40 infection, and the antibodies to SV40 would be a very good sensitive measure. Plaque neutralization test is very specific; we did not find this.
Now, this is just a beginning study. At least in this group of patients, immuno-competent patients and immuno-compromised patients, we did not find any virus in the urine.
One of the puzzles in all of these studies is that all these patients in whom SV40 has been found, many of them have been born after the vaccines were cleared of SV40. So this suggests that SV40 must be circulating in human communities.
And to get some evidence for that, whether SV40 is circulating in human communities, we looked at these urines from groups with generally shared lots of polyomaviruses like BKV and JCV. And so we did not find evidence of that also. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Shah. This concludes the first session, a very interesting session. There will now be a coffee break until 11:15.
(Whereupon, the foregoing matter went off
the record at 11:03 a.m. and went back on
the record at 11:30 a.m.)
CHAIRMAN BRIEMAN: I'm Rob Brieman. I'm the Acting Director of the National Vaccine Program Office, and I'd like to welcome you back for the next session.
I would like to say that the NVPO is very happy to sponsor this meeting and I think it is providing the opportunity to look at very interesting data and to examine the implications of the information presented from a public health standpoint.
The first speaker for this session is Dr. Kristina Dorries from the University of Wurzburg in Germany.
And I'm sorry. Before we begin, let me just say that if anyone has additional data that they would like to present in a short presentation -- either positive or negative information -- in time for the next panel, or the panel discussion which will be this afternoon and led by Dr. Fried who's standing here --please see him either during this session or immediately following to arrange that presentation.
So again, our first speaker will be Dr. Dorries.
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DR. DORRIES: Thank you very much for the invitation, to the organizers, and we will proceed now to the human polyomaviruses, BK and JC virus, and I will try to summarize some of the essential molecular, biological, and pathogenic features of both viruses.
A common infection of BK virus is early in childhood with zero conversion rates of more than 90 percent in the young. In contrast, JC virus is coming a little bit later up and there we have zero conversion rate of 70 to 90 percent in the adult population.
According to PCR analysis, a virus persists in nearly 100 percent of young adults and both viruses persist in kidney epithelium. They are transmitted, at least in part, by urinary excretion. Both viruses are discussed to be involved in tumor induction in men.
These have the viruses -- and both viruses in common with SV40, but additional DNA homologies and protein homologies are followed by similar morphology of virus particles and by the same replication strategies of the three primate polyomaviruses.
This is a genome of JCV and you'll see small T-antigen and large T-antigen coding in the early part of the genome, VP1, 2, and 3 in the late part, and there is an open reading frame for the adenoprotein.
These DNA sequences are very homologous between the two viruses, however the TCRs, the transcription control region is different.
This is the non-coding control region with the origin of DNA replication. That is conserved with T-antigen mining sites 1 and 2, in the whole family of the primate polyomaviruses and the transcription control region is shown from SV40. And we have here heterogeneity of single and double promoter elements that are present in different -- that might be present in different organs.
The conserved first in hearts or promoter element contains binding sites for cellular factors that are gliocell specific or associated to the basic activation -- cellular activation -- and are associated with signal transcription elements.
The cause of human polyomavirus is not quite clear. We don't know the entry site, but primary infection always is followed by live, long persistence of the viruses. We don't know whether the infection is latent or low copy infectioned, but we can find viral DNA, episomal DNA in the affected organs.
Under limited changes in the immune state, as induced by pregnancy or older age, we find a temporary activated infection with short episodes of virus production and messenger RNA viral proteins that can be detected. This situation is asymptomatic.
In contrast, in the clinical overt state of infection under severe immune deficiency as induced by lymphoproliferative diseases, immunosuppressive therapy, or AIDS, we find an unrestricted virus grows with efficient virus production and the lysis of viral target cells.
Polyomavirus associated human disease are only described in the central nervous system for JC virus. It's progressive multifocal leukoencephalopathy, and for BK virus there is strong association with the urogenital system. However, recently there is a systemic disease described that involved tubular lynenphorytis and disthicia pneumonitis and the subacute meningeal encephalitis for BK virus.
This is summarized here on this slide but I would like to concentrate on the cell type specificity. Cells of the connective tissue are clearly associated with BK virus infection at epithelial cells as fibrocytes of the three organs, and specifically in the meningeal encephalitis we find epidermal cells associated with BK virus infection and interestingly, astrocytes.
The astrocytes are in contrast to JC virus infection from the glio type of cells -- oligodendrocytes predominantly are infected.
This is only shortly in in situ hybridization of the epidermal layer on the ventricular system. This is -- the brown color is BK virus DNA specific hybridization, and this is a double immuno staining of GFAP astrocyte specific protein, and in the nuclei polyomavirus specific antigen is found.
This BK virus associated disease is a rare disease. It was described once in Germany, and we might now have a second case. However, this is completely different to JC virus associated PML as found now in steadily increasing numbers.
The characteristics of PML is disseminated focus cytolytic infection of oligodendrocytes in cortex and white matter of the brain, and this is followed by the loss of myelin sheath.
This is a cartoon of a typical PML lesion with the oligodendrocytes filled with virus particles and the rim of the lesion, and at the center you see reactive astrocytes and giant cells in mitosis. This is believed to be a semi-permissive JC virus infections and there are cases where virus particles are found in these cells.
Additionally, it might be associated with transforming infection. Limited infiltration of lymphoid cells is described recently in predominantly in AIDS associated cases.
Here you'll see in situ hybridization of typical PML lesion. You'll see the highly concentrated virus DNA by the black grains. It's radioactive hybridization. This is at rim of such a lesion with less concentrated DNA in freshly-infected cells.
In contrast, in the periphery we have only an attenuated infection. These are epithelial cells surrounding the renal tubules, and although this cell contains not only DNA but also a viral antigen and an electromicroscopy virus particles where detected, you see that this is persistent, infected in the activated state -- a more or less attenuated infection.
A molecular characterization of the virus DNA population in these different sites of infection in the brain revealed presence of heterogenous TCR, transcription and control elements, that are repeated at the GSP prototype from Wurzburg, and in the kidneys you find the single, so-called archetype elements.
In the time before AIDS, lymphoproliferative diseases were closely related. About 62 percent of all cases in '84 were the basic diseases for PML, but now in the AIDS era we discuss between five and ten percent of AIDS patients with neurological symptoms might eventually die by PML. And therefore, several laboratories are now interested in factors or situations that might lead to the induction of disease.
And I summarized several factors that are discussed in the moment that as, first, virus-dependent factors and host-dependent factors. In case of virus-dependent factors, conditional viral dissemination is discussed that probably primary -- accidental primary infection of the effector organs, the central nervous system under immunosuppression as leading to disease.
The second possibility is that the genetic heterogeneity of the promoter/enhancer elements is involved in pathogenic -- in the induction of the disease, and this might be associated with the neurotropic selection of highly active, cell specific transcription elements.
From the host-dependent factors, genetic predisposition can be assumed, but what is obvious is the control of virus host interaction by the immune system really plays an essential role in the activation processes.
The first possibility was that primary infection might only happen under immunosuppression. Under this condition, no involvement of the central nervous system in persistent infection should be expected and therefore we analyzed cellular DNA from non-PML patients. And altogether I think we analyzed 70 patients by PCR analysis. And here, from several regions of the genome these were hybridized with JC virus-specific probes and the products were sequenced.
What came out is summarized here. This is a T-antigen amplification product that hybridized with JC virus DNA. And interestingly, from the same -- hybridization from the same material revealed that not only JC virus is present, but also BK virus is present. And these probes are species-specific for both viruses.
From this data we can say now that probably JC virus is persisting in the central nervous system and reaches the central nervous system long time before the induction of disease.
And that is a requirement for the selection of neurotropic lysal-specific TCR variants that might be selected by cytolytic -- might be selected by activation processes in the persistent state.
And to analyze this we characterize the variants that were present in the different ethnification products by cloning PCR amplification products and sequencing them afterwards.
And altogether we found seven different TCR subtypes. From the TCR type 1, three subtypes in TCR type 2, two subtypes type 1. Repeating the tatabox elements type 2 leaves tatabox element out and repeats only conserved sequences that are following.
What was interesting is that we found single elements -- these are so-called archetypes -- we found double elements here and here. And here, those are other duplicated elements. And additionally, we found triplicate from TCR type 1 DNA.
This was a finding that was astonishing but what really was new is that W1 and W6 were the dominant subtypes that we found in persistently infected brain tissue. W1 is similar to the mat 1 isolet from medicine, the American isolet, and the TCR type 2 is similar to the GSB isolets. The predominant type altogether was med types -- American subtypes.
Then we thought that possibly the situation might -- that these rearrangements were -- came up under persistent infection and we studied then a kidney tissue. And interestingly we only could find W1, double enhancer promoter elements in kidney tissue of eight different patients. Only the single W4, single archetypes are found in only low percentages.
From these data we only can assume that the different rearranged elements are present in the human population and are not rearranged anew in each patient.
This brings us back to the third possibility that changes in the virus host interactions due to severe breakdown of the immunological surveillance are the real requirement for the induction of clinical, overt CNS disease.
However, we have not mentioned under these conditions recent results that were found in lymphocytes. First it was described that V cells in PML tissue were actively infected by JC virus and in addition, later on, peripheral blood leukocytes were analyzed for the presence of JC virus DNA.
And as you can see here again, it has T-antigen PCR hybridization -- with T-antigen specific probe revealed that almost all healthy individuals were carriers of JC virus DNA. To a certain extent we also detected BK virus DNA. However, the concentration is lower than the JC virus concentration, although the primer is similarly sensitive for both viruses.
This opens up now, a complete new field of possibilities, how the virus could interact with host-related mechanisms and will bring us further I think, in the next time.
To summarize, the conditions that are associated with the pathogenesis of PML under immunocompetence, we find an asymptomatic JC virus infection of the central nervous system, life-long persisting viable JC genomes with highly active TCR types are present in the central nervous systems, a growing virus load in advanced stages of life was detected, and also in individuals undergoing changes of the immune state as induced by systemic tumors carry an enhanced virus load.
A virus infection is probably controlled by tight immunological surveillance, and the severe changes of immunological virus/host interaction and probably comes to a 2-step situation. First, we find an activation of viral expression in peripheral organs, we find a growing virus load by the activation event and peripheral tissue and in the cells, and this really probably is followed by an enhanced seeding of virus to the central nervous system.
And a second all simultaneous step we find an unlimited activation of viral expression in the central nervous system, and then CNS tissue is destructed by cytolytic multiplication, and additional enhancement of the virus load by infected lymphocytes may happen also.
And then possible interaction with the viral transactivators should be discussed. In vitro experiments with cytomegalovirus and HIV early antigens have shown that the early antigens are interactive with JC virus, and it could be that under these conditions we find an enhancement of virus replication.
Thank you.
CHAIRMAN BRIEMAN: Thank you very much, Dr. Dorries for that elegant presentation that was, I might also note, precisely timed -- perfectly. I need to announce again that Dr. Chanock needs to check at the desk because he has an urgent message.
The next speaker will address the question, "is BK virus a co-factor in human cancer?", and it's Dr. Michael Imperiale of the University of Michigan.
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DR. IMPERIALE: I'd like to thank the organizers for inviting me to talk. What I'd like to do during my talk is sort of move into the realm of molecular biology a little bit.
Okay, so you've already heard a little bit about BK virus but I just want to repeat a few salient points here. The first is that there's a subclinical, persistent infection in about 80 percent of the population. BK virus encodes a T-antigen which is about 75 homologous to SV40 T-antigen which you've heard a lot about this morning. However, that homology is much higher in the transforming domains that Ellen Fanning referred to in her talk.
It's been shown that over-expression of T-antigen or a co-expression of T-antigen with an activated ras gene can transform cells -- this is both in vitro and also in transgenic animals.
And finally, as you heard before, BK virus has been associated with various types of tumors and also with hemorrhagic cystitis in amino-compromised patients.
So about two years ago what we wanted to set out to do in my lab was to ask the question regarding these tumors in an indirect sense and that is, does BK virus have the tools that it would need to be involved in human cancer?
This is just a table from a paper that was published in Virology in 1995 from the group in Ferrara, where they showed that BK viruses associated with various urinary tract tumors -- and indeed, in some of these tumors they found that they could detect viral sequences by southern blotting rather than by PCR, which implicated that there might be at least a higher amount of viral DNA in those tumors.
Okay, so the first thing that we wanted to look at was the interaction of viral T-antigen with the Rb family of proteins. This is just to remind you that these proteins inhibit cell cycle progression. They do this by binding to family members of the E2F transcription factors and block E2F function.
The next slide just talks a little bit about E2F. It's a cellular transcription factor. It's actually a family of heterodimeric proteins, and the function of these transcription factors is to regulate genes that involved in S-phase progression.
And the next slide is just a model of what's going on here. So this is a normal cell. Up here at the top, Rb and p107, p130 are bound to E2F. When the cell receives a signal to divide the proteins become phosphorylated; this results in release of E2F which is now able to activate transcription of its target genes which then drive the cell into S-phase.
In the model that's been worked out for SV40 T-antigen is shown down here at the bottom, which is that T-antigen binds to the retinoblastoma protein and its other related family members. This has the same end effect as a mitogenic signal up here, which is that the E2F is released and the cell can now enter the cell cycle.
So we wanted to ask whether BK virus T-antigen was functioning in a similar manner. The first thing that we did was to just look and see whether T-antigen could complex with the Rb family members in cells -- and these are a series of immuno-precipitation assays where we immuno-precipitate with antibodies against T-antigen, run the proteins on a gel and blot and probe for the three different Rb family members. And you can see that we find complexes of T-antigen with Rb, p107, p130 in the cells that are expressing T-antigen.
Now, what I need to point out here is that, in order to see this we have to use 100 times as much protein from these BK virus T-antigen expressing cells, as from cos-1 cells which express SV40 T-antigen. And the reason for that is that there's a hundred-fold less T-antigen in those cells.
So this is a T-antigen blot. We have to load 100 times as much protein to see the T-antigen as we do here. So any effects that BK virus T-antigen is having here as compared to SV40 T-antigen, these effects are occurring in spite of the fact that there's a hundred-fold less protein there. And that's something I'd like you to keep in mind as I go through this.
The next slide on the right is just a whole cell lysate blotted with antibodies against these proteins. And if you look at the normal cells compared to the cells expressing BK virus T-antigen, you can see that there's less Rb, p107, p130. What's left is mostly in the hypo- or under-phosphorylated form. And I think Jim DeCaprio is going to talk more about this tomorrow, so I'm not going to address that issue.
But what I want to point out here is that what this is saying is that most of what's left in the cell here is in this form, which should be the growth inhibitory form, and also most of what's there is not bound to T-antigen because there's so little T-antigen present in the cell. So the question is, are we getting an induction of E2F activity?
So to look at this, what we did was to take a gene that expresses a CAT reporter, under the control of E2F DNA binding site, and transfect that into cells, and the results have shown -- the next slide on the right -- so these are just CAT activities of these different cell lines.
You can see when we transfect this reporter gena to normal kidney cells we don't get any activity. If we put it into cells expressing BK virus T-antigen we get an induction of activity. SV40 T-antigen we also get an induction.
Notice that the induction only differs by about 4- or 5-fold even though again, there's 100-fold difference in the amount of T-antigen in these cells. And these are just transient transfections where we've done the same sort of assay showing the induction by BK virus T-antigen compared to another protein that interacts in the system, which is the adenovirus C1A printing.
So in spite of the fact that there's very little T-antigen binding to these proteins that we can detect and that most of the protein that's there in the cell is in this form here which should be bound to E2F, it looks like we're getting induction of E2F activity.
So the question we wanted to ask is, what does the E2F look like in these cells? What we've done then is to look at E2F binding activity by doing DNA band shift assays. The left side here are just straight DNA band shift assays, making extracts either from normal monkey kidney cells, cells that are expressing BK virus T-antigen, or cells expressing SV40 T-antigen.
What I want to show you on this side here is that, this is the free E2F, so this is the theoretically, transcriptionally active form of E2F. And although this is a little bit overexposed so that you can see some of these other bands, there is an induction of the amount of free E2F in the cells expressing the viral T-antigens as compared to the normal cells.
And I should point out, this is not due to differences in the number of cells that are in different portions of the cell cycle.
Now, the right side of the -- I'm sorry, the left slide over here shows that even though T-antigen is present in these cells, there's a significant amount of E2F that's still bound to Rb. And that's shown here. You can see that there's a little bit of E2F Rb complex that's left here, and this side of the slide just shows how we identify the complex, as this complex goes away if we add antibodies against Rb; this complex goes away if we add antibodies against p107.
This is to show that there are Rb E2F complexes still present in the cell. What we've done here is to use antibodies against the Rb protein to immunoprecipitate and then release any E2F that was present, bound to that Rb, and put that into the band shift assay.
And you can see that there's still E2F activity that's associated with Rb, both in the BK virus T-antigen expressing cells and in the SV40. And again, this is about equivalent, despite the huge difference in the levels of T-antigen in these two different cell lines.
Finally, the last slide on the left. So the question is, if we're getting induction of E2F activity, are we getting induction of cell growth? So these are some growth assays of these cells. If we take these cells and grow them in ten percent serum you can see that all of these different cell lines grow well.
However, if we put these cells into medium-containing load serums -- so this would be an assay for serum independent growth which is a hallmark of transformed cells -- we can see here that two different parental monkey kidney lines do not grow well in low serum, whereas the cells expressing BK virus T-antigen or SV40 T-antigen both grow in low serum.
So what this tell us then, is that BK virus has the ability to induce cells to grow. So this would be one of the things that one might expect for something that might be involved in tumorigenesis.
The next thing that we wanted to look at then, is the interaction of T-antigen with p53, and this is just a little cartoon here to show you how p53 works. Normally, if a cell is getting ready to divide and that cell receives DNA damage, there's an induction of p53 levels, and that results in arrest of the cell before it enters S-phase. That allows the cell to either repair the damage or if the damage cannot be repaired then that cell will be induced to undergo programmed cell death or apoptosis.
Now, in the presence of SV40 T-antigen what happens is, T-antigen binds to p53, the cell receives DNA damage, there's no induction of p53, no G1 arrest, and the cell can go on to divide with damage, or if there's enough damage there will be mitotic failure and cell death.
So we wanted to ask what was the interaction between BK virus T-antigen and p53. The next slide on the right is just an immunoprecipitation similar to what I showed you before. For the Rb proteins, if we immunoprecipitate with antibodies against T-antigen, we bring down all of the p53, or over 95 percent of the p53 protein that's present in the cell.
So this says that even though there's very low levels of T-antigen present in the cell, it is able to bind most, if not all, of the p53 in those cells. So the question is then, is it having any effect on p53 function?
So what we did then, was to take normal cells or cells that are expressing T-antigen and irradiate them and look at the response to DNA damage. The next slide on the right is just an amino blot -- oops, on the left, sorry -- it's just amino blot where we've looked at either the induction of p53 or one of its important target genes, p21, upon irradiation.
So the left side of this slide shows the normal cells; increasing amounts of irradiation we get an induction of p53 levels; and we get a concomitant induction of p21 levels.
In the cells that are expressing BK virus T-antigen, first you can see that there are higher levels of p53 to start with. This is because T-antigen binding stabilizes p53. However, there's no further induction of p53 upon irradiation and there's also no induction of p21 upon irradiation.
The next two slides just show some cell cycle analysis of this cells, the normal cells on the left, the transformed cells on the right, and you can see that when these cells are irradiated, the normal cells, one gets mainly arrest here in the G1 phase of the cell cycle. If you look at the cells expressing SV40 T-antigen however, one does not get arrest in G1; instead, one mostly gets arrest in G2 M-phase of the cell cycle.
So what this says then is that BK virus T-antigen is interfering with p53 function and removing its ability to block cells in the G1 phase of the cell cycle upon DNA damage.
Okay, finally then, we did an experiment based on some old experiments that were published for SV40 many years ago, where people showed that SV40 infection can induce chromosomal damage. And so we wanted to ask whether BK virus can also induce the same sort of chromosomal damage.
So what we did then, was to take some primary human fibroblasts and infect them with either BK virus or also JC virus, and look at their chromosomes. And the next slide on the left shows the result.
This is a karyotype from normal cells here. These are the karyotypes from either BK virus-infected cells or JC-infected cells. And in both cases you can see that we're getting chromosome fragmentation, there are translocations. And moreover, in the presence of BK virus, we're also seeing this thickening of the chromosomes which is indicative of endoreduplication.
And this actually makes sense when one looks at the cell cycle analysis I just showed you; that the cells may be getting hung up in M-phase. So BK virus is able to induce DNA damage in these cells.
And what I want to point out here is that we did this at a rather high multiplicity of infection so that we could get enough karyotypes -- enough infected cells to actually see a karyotype. And we assume that BK virus may be inducing a lot of undetectable chromosomal damage, even at lower multiplicities of infection that could potentially be occurring during a persistent infection.
So the next slide on the left then, I'd like to just sum up by asking a question -- and let me state from the outset, I don't know what the answer to this question is -- but the question is, is BKV a co-factor in human cancer?
As I showed you, it can induce DNA damage, it inhibits the p53 response to this damage so if cells receive damage then they're unable to arrest in G1 before that damage can be repaired, and moreover, it's inducing cellular DNA synthesis so it's inducting E2F activity. And the question is, can this lead to increased mutation rates which then potentially could lead to carcinogenesis.
And so I think I'll stop there, and just on the next slide acknowledge the people who have done this work. In my lab, Kimya Harris, Joan Christianson, and Eugenia Chang. And our collaborator in the Department of Human Genetics, who's done the karyotype analysis, Tom Glover. Thank you.
CHAIRMAN BRIEMAN: Thank you, Dr. Imperiale. That was very impressive. The next speaker from Allegheny University for the Health Sciences, is Dr. Kamel Khalili who will speak about JC and BK sequences in human tumors.
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DR. KHALILI: I guess what I'm going to do today is focus my talk on JC virus. Can I have first slide, please?
This slide just demonstrates that the JC virus is not another SV40 or humana SV40, and it has its own personality and character. Kristina Dorries very elegantly laid out some of those characteristics, and I'm going to bypass some of these slides which I have initially, to describe a JC viral control region or lytic cycle and then demonstrate that.
In the next slide -- this is a virus which infects greater than 92 percent of human populations and as you heard from Kristina's talk, perhaps 100 percent PCR. And it's a virus which has no animal reservoir, and perhaps more importantly, it's a virus that we can talk about. When you talk about an AIDS epidemic you cannot ignore JC virus.
It's a virus which used to be rare and the disease of the virus was rare but not any more. And perhaps I should mention that greater than ten percent of AIDS patients which no logical disorders, which by itself is greater than 70 percent, come down with PML.
So suggesting that the virus has a chance to become reactive replicates in cells. The cells of the virus is replicating oligodendrocytes.
This is oligodendrocyte which is responsible for formation of myelin sheaths around the axon. In fact, oligodendrocytes sends a processes that -- this is an electromicrograph of normal brain -- shows that the oligodendrocyte sends a process, circulating processes, and these processes form the layers, very compacted layers, around the axon, an important event for the insulting axon.
Now, cytolytic destruction of these oligodendrocytes upon reactivation of endogenous JC virus, all the JC virus which gets to the brain by B cell or any other vehicle, results in the demyelinating disease of PML.
As Kristina mentioned, the JC control region sits between the viral early and late genome, and it has a unique characteristic. It has a 298 base pair repeat sequence and old genome viral DNA replication. It's a bidirection promoter of single to SV40. In one hand it shoots for the early gene expression; on the other hand, the other sides controls late gene expression.
Now, if you really go through the sequence you do see that there is not much release sequence homology or similarity between the JC control region and SV40 control region. That's one characteristic of the virus counts.
We spent about eight years -- over the last eight years -- trying to understand what confers the tissue specificity to the viral gene expression. We knew that early gene expression is make or break for the virus. So if you reduce immunosystem, virus gets opportunity to replicate, but it does not replicate in every cell. It replicates in all oligodendrocytes, the cell which I showed previously.
So there is two barriers in the viral gene expression: first is immune system and the second is its cell-type specific barrier. The latter was easier to address. We have the similar cell lines and we went on through the standard biochemistry to understand that one of the sequences within the viral control region, which, upon interaction with cellular factor, turn on early gene transcription.
Again, Kristina demonstrates a slide summarizes all the transcription factors which could bind to a JC control region. And through those studies, we and others learn that perhaps interaction of the protein some were inducible, some were tissue specific factors.
So the control region, per se, is not -- it's essential but still does not put the virus through the lytic cycle. So it seems that the interplay between protein upon the interaction plays more important role in this event.
So this slide is about a 3-year old slide. It just shows some of the proteins that we and others purified by standard cold room chemistry and cloned the genes, and demonstrated that association of the protein, for example in this case, YB-1 with the JC control region, is important to induce viral gene expression couple-folds, but sufficient -- it wasn't sufficient to put the virus through the lytic cycle.
Then once other proteins were purified, we realized that the communication of this protein with each other perhaps plays a more important role.
Now, another feature of the virus which I'm going to little bit talk about that, is the TAR region. TAR is a sequence which first identified in HIV genome, and this region, located downstream from the HIV transcription initiation site, and it's responsive to tat protein, a protein from HIV which is important for the viral replication.
Now, a higher incidence of PML among AIDS patients suggests that perhaps in addition to immune system, there is some direct communication between HIV-1 and JC Virus in the brain parenchyma.
So we asked the question of whether or not HIV regulator protein included in tat could activate JC promoter in the choleal cells. The answer to that question was yes, and we demonstrated that this activation of the viral promoter, JC promoter for HIV tat was mediated through the sequence, TAR log sequence which is located in the leader of the late gene transcription -- very reminiscent to the structure described in HIV promoter.
In addition, it was demonstrated that tat could bind to another cellular protein; a protein which initially identified based on this ability to transcribe, stimulate JC promoter, and as a result of this interaction, augment expiration of the viral JC virus.
So you see that we learn much more by studying JC virus in terms of the mechanism of viral-viral interaction and the cell type of specific transcription.
Now, let me move on, on the next slide. So the question was JC virus, under proper conditions, proper environment, produce a T-antigen, and once the T-antigen is produced such as other papomavirus, induce viral DNA replication, late gene transcription, and certainly the destruction, the virus causes PML.
Now, what happens if you block replication event so you still produce T-antigen under certain circumstances, and what happened to that T-antigen? Does T-antigen, the absence of replication that's supposed to be crossed, could induce the tumor?
So the first clue for that came from the animal studies, hamster model, where the JC virus cannot efficiently reproduce. In the replication does not happen because of the primary unit of DNA prolimerase is species-specific, cannot turn on JC viral DNA replication in hamster.
Therefore, if you take a JC virus, inject in the brain of a newborn hamster, what happens after two months, you get tumors. Eighty percent of the injected animals come down with a tumor. This was demonstrated by a number of people, and also later on, Sid Hoff and others demonstrated that intercerebral inoculation of the JC virus in our monkey, the squirrel monkey, induces glioblastoma multiformity. A very devastating brain tumor which we still don't know about the pathogenesis of the tumor and also we don't basically have a cure for this tumor.
So this slide illustrates the gross section of the brain of the newborn hamster. This is a tumor for an open injection of JC virus and this is a normal area. Now, if you take the tumor results here and stain it for T-antigen, you basically get 100 percent staining of the T-antigen in almost every cell.
There's a series of slides which -- studies that we did, and I'm not going to go through that -- and we will learn that these cells do have their own characteristic in terms of the cell growth and other factors; factors which are important for control of the cell proliferation.
And even the extent of the T-antigen in a variety of the cells were different. We cloned these cells; we are in process of analyzing it -- how low, high, and medium dose of the T-antigen in the various cells could utilize the different pathways which leads to the formation of the tumor.
And at the same time, we are utilizing the hamster model as a measure for studying formation of the tumor. This is a very unique model. You can inject JC virus in the hamster brain with notion that 80 percent of the injected animals come down with the tumor. So what you could do is, you could monitor development of the tumor; things that you cannot do in any other animal species, and clearly we cannot do in human.
So how you can do it in vivo, we have taken this hamster model and in during MRI on the living animal, we are monitoring formation of the tumor from the time that injected to the time that becomes significantly big and close to kill the animal.
So in parallel we are devising histopathological studies to understand the interplay, because some of those regulatory factors that Mike talked about, that E2F for example, cyclins during the formation of the tumor, and at the same time we are trying to see that whether or not the ring value stage of the tumor, the partner of the T-antigen changes.
We know that the T-antigen binds to p53 and Rb. These experiments are done in cell line, but we can utilize this study, animal model, to do experiments in whole animal model.
Now, another model is the transgenic mice, that expression of the T-antigen induced tumor in the transgenic mice. And analysis of the tumor showed that a tumor is formed in the abdominal area. It was NSC-positive, normal crest origin tumor.
And when you do a histopathological analysis of this tumor formed upon expression of JC in trans-genic mice, you see that they're highly differentiated cells that -- tumor cells, basically infiltrates between the wall of the colon and the muscle cells here. These are all the tumor cells and these are all T-antigen-positive cells.
Now, what we are trying to do, we are trying to use these transgenic mice to study the importance of some of the cell cycle regulators in whole animal model information of the tumor. First we demonstrated that T-antigen was expressed in RT-PCR, in almost in every tissue of this experimental animal.
But when you look at the Weston blot you see that the detectable level of the T-antigen was obtained only in the tumor but not in any other tissue derived from this animal.
So in the next slide here, we looked at the interaction of T-antigen with p53 tumor suppressor, our favorite experiment as Mark demonstrated, by coming and precipitating with one antibody, probe the Western blot with another antibody. The take-home message is yes, p53 associates with the tumor and the T-antigen associates with p53 in the old 2-reciprocal experiment.
And the working model was is that if the JC virus T-antigen associated with p53 takes away p53 from the loop as a result on same target to p53, p21 WAF, a protein which could suppress function of cyclin's associated kinases and the complex which eventually could phosphorylate Rb and liberate E12, could be other function in this case.
And what we know is, each WAF could regulate number of other cell cycle genes and put the cells into the rapid proliferating stage, and at the same time, T-antigen could associate with Rb, another way that liberates E12.
Now, we really went on through every step of this and examined every step in this tumor. For example we asked, what happened to WAF gene in this tumor tissue? We realized that there is no WAF gene, basically WAF protein in the tumor cells. And then we asked, if you don't have WAF, what happens to all the cyclin and CDKs?
What I'm going to do in the next slide just showed two examples of that. This is cyclin E and this shows the level of cyclin E, and this shows that it's a kinase activity.
And the partner to cyclin E is CDK2 -- the level of cyclin is CDK2 and its kinase activity. So you see that it is highly active in these tumor cells. What happens if the cyclin E, CDK2 is very active? After the previous pathway -- in the next slide -- we examined the Rb phosphorylation and you see Rb, unphosphorylated Rb which is supposed to be the single band in the tumor, becomes two bands. The top band is phosphorylated.
If the Rb phosphorylated dose E12 liberates from the Rb -- next slide -- it does, and it all regulates itself and you see massive amounts of E12. And if E12 is functional or not -- yes it is, because it activates PCNA -- it's on the same target and expressed in the tumor.
So you see that we can utilize this model to really go through all those cascade of events that has been described in the cell cycle, and identify the regulatory factors which really is important for the pathogenesis of the JC-induced tumor.
Now, what I'm going to do is, we switched to the human samples, and we all learned that JC virus could be associated with a number of tumors in humans, we learned this morning, and these are studies done previously by many other people.
Recently, maybe two years ago, a 61-year-old, immunocompetent, HIV-negative -- this is very important clinically -- individual came to neurology service and he had a multiple grand mal seizure.
This individual, after MRI, showed that the hypointension area in the left frontal lobe of the brain -- after about a year, gallilinium staining shows a new enhancement which sits right in the center of the previous enhancement, suggesting that the tumor is formed here.
Now, if you see that the tumor mass was so big that it pushed the lower, the left anterior -- the formal -- this structure, and you see that this structure, which is ventricular, should be like this. But the tumor is pushing this toward the right lobe.
Now anyway, so we got the samples from these patient, and the RNA protein DNA was extracted and we did RT-PCR for the presence of T-antigen DNA which was there, and the RNA was utilized for the primary extension for expression of JC virus RNA, and the results is here. Basically says yes, early RNA was expressed.
And then when we look at the protein by Western blot, here is a T-antigen of the JC in the tumor specimen. And if you do immunohistochemistry you see that the T-antigen is present in 50 percent of the tumor cells.
This slide shows the staining of cells with Chi-67. It's a self-proliferating marker showing that the cells are highly proliferating and this is Luxor blue staining showing that the level of the myelination -- which shows they're heavily myelinated.
Now, if that T-antigen doesn't belong to a JC virus, we amplify the control region of this virus, and after sequencing they turn out to be member of a JC virus -- it's a mat-4 strain of the virus which has a characteristic of 98 base pair and 79 base pair sequence.
Now, if the virus is there, why does it cause PML? Why cannot replicate and destroy the oligodendrocyte? We thought initially that it might be a mutation, replication which does not respond to T-antigen. As a result, you create a situation like hamster; that you produce T-antigen in the brain but DNA replication does not take place. T-antigen triggers a cascade of events, and lead to the formation of the tumor in this patients.
Clearly the T-antigen was intact. That was a wrong assumption. Then we learned from SV40 there's another protein in the leader of the late gene -- adenoprotein.
We learned that if you introduce mutations in the AUG of the adenoprotein -- this is worked on by Tom Shank many years ago and also some of the mutants that Jim Pepper has created, the mutation which has a T-antigen deletion of the carboxy terminal of a SV40 T-antigen -- what happens is you do not get a viral protein, late protein, be transported to the nuclei and form the virions.
And when we look at the adenoprotein of the JC virus we found that there's a mutation -- three nucleotides right at the initiation site which put the adenoprotein on a frame. At present we don't know that's the cause, that's the important event that put the virus through the other pathway, the tumor pathway or not. So that's a question that we're asking.
So I'm going to stop here, but before ending I would like to -- well, Dr. Shah mentioned something very interesting this morning. He mentioned that this meeting reminds him about 10, 15, 20 years ago when the people, the polyomaviruses were gathering and talking about this system. And indeed it does. It resembles like at those meetings that we used to go to, Cold Spring Harbor and talking about polyomavirus, mostly on SV40.
But there's one person that's missing in this today, and that's a person who did a lot of work, a lot of contribution on SV40; things that we learned on enhancer regulation of SV40, and the T-antigen. This person is George Khoury who left us about ten years ago. In 1987 he died of lymphoma, and I think it's very appropriate that at this meeting we just remember him and his contribution.
Thank you.
CHAIRMAN BRIEMAN: Thank you very much, Dr. Khalili. Our next speaker is Dr. Richard Frisque of Penn State University, and the title of his presentation if, "Rearranged and chimeric primate polyomavirus genomes".
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DR. FRISQUE: Well, I'd like to offer my thanks also, to the organizers for the opportunity to speak. The focus of my laboratory is on the pathogenic and oncogenic potential of JC virus. Now, much of our work has relied upon the biological and molecular comparisons with related viruses BK and SV40.
This slide shows the comparison of the JC and SV40 genomes, and these are very similar. They share 69 percent sequence identity. JC and BK share even higher: 75 percent sequence identity. The organizations as you can see, are essentially identical.
There are three regions as already have been pointed out. I'll just quickly do that again. The early region which includes this T-antigen that we've all been talking about. In the case of JC there are five different proteins produced, now we know, in the early region, based on alternative splicing mechanisms.
The late region includes the capsid proteins and then this control region -- or the regulatory region as I call it -- is also present; the third region. It includes the replication origin as well as the signals for transcription.
Now although many biological similarities occur between these three viruses, JC is distinct. It has a prolonged lytic cycle; it is very inefficient at transforming cells in culture; and its expression is restricted to only a few cell types.
Over ten years ago we asked the question, what makes JC biologically distinct? Which regions of the genome, which sequences are important? And we began making in our first approach, chimeric virus between JC, BK, and SV40.
Now, our prediction was that if we replaced some of the JC sequences with those of SV40 or BK, we might make a more active virus and identify some of those sequences that contribute to JC's unique biology.
Now, in terms of relevance this workshop, I think that if co-infections to occur in the human host with one or more of these viruses, that in fact, if a combination can occur between these three viruses then perhaps we might generate more viable, perhaps more active, recombinance.
Alternatively, or maybe in addition, we also may see complementation occurring, where one virus could complement the growth of the second co-infecting virus. So that's the relevance then, to this workshop.
This slide shows you the first of the chimeric genomes that we've produced. These are what I call regulatory region chimeras. What we do is, we take the coding region of one virus -- in this case, JC -- replace its regulatory sequences with those of BK or SV40. And similar things were done with the other two viruses.
Now, if you can see it with this color -- I'm not sure how it looks back there -- but what we first did with these chimeras was to look at their transforming behavior in rodent cells. And what we found was that when we had the same coding sequences linked to the various regulatory regions, in all cases SV40 was always more potent, JC was always by far, the weakest at transforming cells.
Similarly, if we took viruses that had the same regulatory region and different coding regions, that again SV40 was always the most potent, JC was always the weakest. And in fact, the coding sequences seemed to have more of an influence upon this restricted behavior of JC than even the regulatory sequences.
Now, we've also done some tumorigenicity studies. This has been done with transgenic mice, and as others have shown as well, the regulatory sequences of JC, SV40 types of chimeras, the regulatory sequences are influencing the location of where the tumor occurs, whereas the coding region -- again the T-antigen, primarily -- is involved in the tumor induction itself.
Well again, these same chimeras are shown in this slide, but in this case now, we're looking at lytic behavior in terms of DNA replication and the ability to produce viruses themselves, and also looking at host range effects.
And what we found that, somewhat to our surprise, that those constructs that had JC regulatory sequences in either SV40 or BK coding sequences, that in fact, these were viable viruses -- viruses produced. And in fact, they were more active than wild type JC itself.
In addition, when we took one of these chimeras, JC SV40, we found that its host range was expanded over that of wild type JC. In fact, these not only grew in human cells but they also grew in monkey cells as well.
Finally, the last surprise from looking at lytic behavior of these chimeras is shown over here on the right, when we had constructs that had JC coding sequences linked to BK or SV40 regulatory sequences, which are more potent, that in fact these were dead viruses.
So if I could have the next slide, I would like to then look at that last point in more detail. Here what we've done is make another kind of chimera. In this case we've made chimeric replication origins. We've made chimeric regulatory regions to see if we could find out why the JC coding sequences in the particular T-antigen was unable to interact productively with the origin of SV40 and BK.
Now what we've done is, we've put the regulatory sequences of the various viruses onto a plasmid that does not contain any other sequence information for viruses, for the viral proteins. So just the regulatory sequences are on this plasmid.
And as we've shown already with the JCT protein, when these constructs were put into cell lines expressing JCT protein, both BK and SV40 were unable to -- their origins not replicate in the presence of JC T-antigen.
What we found from the chimeras was, when we had a JC on the early side of the replication and origin, core origin, that SV40 sequences on the late side, that in fact again, JC's T-antigen was not able to productively interact with those sequences. In other words, sequences on the late side of the core origin were responsible for JC's T-protein's ability to distinguish between the two origins.
And in fact, we've gone through the site- specific mutational analysis now, where there's three nucleotide differences within that small core region within the AT-rich region, that allows JC to distinguish between the two origins.
On the other hand, all the replication of origins, chimeric or wild type, were able to interact with the SV40 T-antigen as shown on the right.
On this slide I'm showing you a new set of chimeras. In this case we're producing chimeras that had T-antigens that were exchanged for sequences in three locations: either at the amino terminus, in the central region of the T-protein, or the carboxy terminus.
And in this slide I'm showing you a regulatory region -- and in this case, SV40's regulatory region, although we've also made chimeras with the JC's regulatory region as well. I'll just show you the data with the SV40 origin and transcriptional control signals.
And what we've found is, when we look at transformation in rodent cells, that as expected, JC's T-antigen if it was wild type, transformed very poorly. By replacing the amino terminus with SV40 sequences, we did not see much of an increase in transformed behavior. However, as we started to replace carboxy and central regions of the T-antigen with SV40 sequences in place of the JC, transforming behavior went up until we used the wild type SV40 T-antigen which is very potent.
Now in addition, in studies done in collaboration with Dr. Frank O'Neill, we did some immortalization studies with these chimeras, in human cells. And what we found was that most of these chimeras could not immortalize human cells. SV40 does immortalize human cells, but ten percent of transformed human cells become immortal.
What we were surprised to find was that constructs that had JC or BK at the carboxy terminus, these were also able to immortalize human cells and in fact, it's a much higher level about 50 percent of the lines that were looked at.
Well, in following with the way I've been going here, we've also taken these same kind of T-antigens and looked at their ability to stimulate DNA replication. Again, those are the same constructs as I showed you on the last slide using the SV40 replication, origin, and transcription signals.
And what we found was that the constructs that failed to interact with the SV40 origin for replication -- for DNA replication -- were those constructs that had JC within the central region of the T-antigen. So those, in human cells, were unable to replicate, thus identifying the part of T-antigen which was able to distinguish between the two replication origins: JC or SV40.
We also found that two of the constructs, those that had SV40 sequences at the carboxy end, again were able to replicate in monkey cells as well as human cells. So expanded host range involved as expected, the carboxy terminus of T-antigen.
Well, I just wanted to summarize the chimeric data at this point and again try to show you some relevance to this workshop. If in fact, co-infections do occur, we have the question of whether or not recombination can occur between these viruses. And if so, if a recombinant does occur, what would we predict, based on some of this data and some others, is the phenotypes would be relative to wild type SV40.
And I would like to suggest as possibilities, that the recombinant would probably show reduced transforming behavior, but perhaps higher or lower mortalizing potential. We'd expect probably, DNA replication activity would be reduced as well as virus yields, we'd expect that the host range would perhaps be more restrictive than SV40, and we would expect that the recombinant itself might actually be more active than wild type JC.
Alternatively, we can look at co-infections as a possibility of complementation occurring. Again, based on the data that I've shown you, we would predict that JC probably would not enhance SV40 growth if they co-infected the same cell. On the other hand, we might predict that SV40 would enhance JC growth.
I'd like to switch gears at this point and tell you about some experiments that we've been doing looking for JC's presence in human tissues, again as you've all heard, by PCR analysis and then followed by sequence analysis.
We have not found any evidence yet for recombination occurring between these three viruses. What we do see, considerable rearrangement occurring within the promoter/enhancer elements for transcription. And that's what I want to tell you about next.
What I'm showing you here is JC, and we're looking at the regulatory region, in particular, the transcriptional control region of the promoter/enhancer signals. In humans there are two types of JCs that can be found: this archetype that you've heard about, and what I call the rearranged form of JC.
Now archetype is shown at the top in the schematic, and what's unique about the archetype genome is that it has a single copy of the promoter/
enhancer region; there are no large tandem duplications. On the other hand, I show you four examples of rearranged forms of JC's transcription control region. These all came from different PML patient's brains.
And what you can see is that, unlike the archetype, there are differences in rearrange between each other as well as with archetype. And they involve deletion events usually involving the 66 base per block of information in archetype shown here, and sometimes its 23 base pair block of information is missing as well.
Following these deletion events, then duplication and sometimes even triplications occur, such that these sequences here are duplicated relative to archetype. What we believe is happening is that archetype, which is found in the kidney and in urine of healthy people, circulates in the population. We become infected by that.
Within our body we believe that these rearrangements may occur involving deletion events and duplication events. And these rearranged forms have been primarily found in brain, sometimes in PML kidney, and in lymphocytes.
In our first PCR experiments that we conducted about seven years ago, we first started by looking at PML tissues. Obviously, we were expecting to find JC in those tissues. And what I show you here are the tissues of brain and kidney from five different PML patients.
Over here, the brain specimens. Lane 7s in each case are the positive controls; lane 1 are the negative controls. These are the five brain specimens; these are the five kidney specimens; and as expected high levels of JC were found in these tissues.
When we went ahead and sequenced these isolets -- these PCR products, what we found was that these in fact, were rearranged forms of JC. As well in the kidney, we found primarily rearranged forms as well, although we have found archetype in some PML kidney.
More surprising at the time when we published this was that in fact, JC was also present in normal brain tissue. In other words, we've had 18 different specimens shown on this slide in which people had died of things unrelated to neurological complications. And in fact, these are the positive controls; negative controls are shown in the first part of each panel. And these are the positive samples that we've seen when we were doing PCR analysis and then blotting the PCR products with the JC-specific probe.
So JC we found, was present in greater than 50 percent of normal brain specimens that we looked at. When we sequenced these, as I said -- I believe I said -- these are rearranged forms. When we looked at normal kidneys, on the other hand, normal kidney was always archetype JC.
Well, at the same time when these initial studies were being done we did have access to five tumor specimens. These were five different patients with medulloblastoma. So we also looked for the presence of JC in those tumor samples and in fact, we found high levels of JC in each one of those five.
This here in lane 8 represents a normal brain -- one of our best normal brains in terms of the amount of JC that was present. You can see the difference in the amounts that were present. Again, this is a positive control and a negative control.
Now, more recently our PCR analysis has been conducted on a single PML patient. What makes this patient interesting we believe, it is unusual in terms of the way PML occurs. This occurred in a 5-year-old child. PML usually occurs later in life.
This child had severe combined immune deficiency. And so in addition to being young, we believe also that the PML arose following a primary infection rather than in most cases of PML where it occurs following the reactivation of an earlier persistent infection.
In addition, most cases of PML you can find JC in the brain, in the lymphocytes, and in kidney. When my grad student, Jason Newman, began looking at this patient, we had specimens from eight different tissues. And he found JC present in each one of these: brain, coeliac plexus, spleen, kidney, lymph node, liver, cardiac muscle, and lung. So JC was present in all of these, and this is just a southern blot to confirm the identity as JC.
Now, Jason was using primers that would lie outside of the regulatory region of JC in a highly conserved region for all JC isolets looked at so far. And so the PCR primers he was using would allow to amplify either archetype or rearranged forms of JC if they were present.
What Jason found -- this is again, the comparison, we're all going to compare with this archetype strain called CY. CY is a strain that was isolated by Dr. Yogo in Japan a number of years ago and I'm using that as a reference.
What Jason found was that in all the kidney clones that he looked at in sequence, they were identical to CY except for a single change at a hotspot of rearrangement for archetype at position 217. He also found, in cardiac muscle, archetype-like JC. That is, there are small deletions or intermediate size deletion without duplication. He also found in lung, another archetype-like strain.
This slide again -- with comparison to CY shown at the top -- this is the other tissues that he looked at. And what was he found was that there were multiple rearrangements occurring within these JC isolets. In fact, this is again unusual relative to most PML patients that are looked at.
Usually when you look at a rearranged form in a PML patient there's only one or maybe two types of rearranged forms. Here, we found multiple kinds of rearranged JC, and this is actually only a subset of what he found.
Now you also notice that in some cases the same clone was found in multiple tissues. In other cases you found that in the same tissue -- brain, brain, brain -- you found multiple clones. So there's a lot of rearrangement going on here.
Now as I said, the primers that Jason was using were laying right outside the regulatory region so they would amplify whatever was predominantly there. We wanted to see if archetype might be present in tissues other than kidney, since that really hadn't been looked at too closely.
This slide was constructed following JC's work with archetype-specific primers. In other words, he had two pairs of primers that he used, whereas one member of each pair lay within the 66 base pair sequence which would be archetype sequence that was present. Rearranged forms essentially in this patient, were always losing this region. So it would be specific for the presence of archetype JC.
And what Jason found, was in the brain and the lymph node that he could find the archetype JC or archetype-like variance within brain and lymph node, and this is the first time this has been shown.
So if I could have the last slide -- just some conclusions from this work with the pediatric patient that I've just been talking about. We believe that the immune status at the time of exposure -- this child has severe combined immune deficiency -- probably contributed to this widespread distribution of JC that we see in her body, and to the extent of rearrangement of this transcriptional control region.
The data I believe, shows us that rearrangements can occur early, perhaps after primary infection. This is in contrast to the past where we've been thinking that it usually occurred following the reactivation of a persistent infection. So at least sometimes, rearrangement occurs quite soon.
We believe lymphocytes are probably involved; that whether they're involved directly in rearrangement process we're not sure yet -- we may hear some more information about that next; and we believe that they certainly are involved in seeding the virus to secondary sites of infection.
As already mentioned, we do not know where the primary site of infection is, but lymphocytes may take it, wherever that is, to these other tissues that I've been showing you.
Finally, we would suggest that because we see different predominance of archetype and rearranged forms in different tissues, that in fact, this might indicate that the replication potential of these two forms differs in the differing tissues.
Now, given everything I've shown you so far, as I said, we have not seen anything to resemble recombination between JC, BK, and SV40 from these kinds of studies. I should add quickly, that we really haven't really looked that hard until recently. So in terms of whether or not rearrangement occur, that question is open.
Well, and partly for what I've shown, I think rearrangements certainly do occur; they occur in JC as well as in BK and SV40, leading to these archetype to rearrange the types of transitions that we see, so that this a highly plastic region of the genome that might be interesting to look at further.
Thank you very much.
CHAIRMAN BRIEMAN: Well, thank you, Dr. Frisque. You covered a lot of material within the time limit and I think that was very nicely done.
The next speaker is Dr. Maria Chiard Monaco of NIH, and she's been given a very limited time period to address the question of JCV infection of peripheral lymphoid tissue and the implications for viral latency.
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DR. MONACO: Good afternoon. First I want to thank Dr. Lewis to give me the possibility to present my data today. As we heard from the previous speakers, JC is a DNA virus of the papomavirus family and as Dr. Khalili mentioned before, more than 80 percent of the human population has been infected by JC virus and few conversion occurs during childhood.
JC is thought as a neurotropic virus but as we can see from these slides, JC can also infect cells -- non-neural cells as well. Back in 1988, Dr. Sid Hoff and Dr. Major found that JC virus DNA associated to capitalize a lymphocyte from two PML patients in the bone marrow and spleens. And we expounded a host range of JC virus, focused our attention particularly on cells of lymphoid tissue.
In this slide we have a nested PCR amplification from two AIDS patients: patient 1 is not PML, and patient 2 was at the onset with PML. And this line marker, there's normal B cells contains DNA from a B cell isolated from peripheral blood mononuclear cells of JC virus-negative individuals.
Patient 1 and patient 2 -- in the lane of patient 1 and patient 2 we have DNA extracted from unfractionated peripheral mononuclear cells, or B and T subpopulation isolated by cell sorting technique.
And we found no evidence of JC virus DNA in the T cells from the PML patients and in the unfractionated peripheral mononuclear cells, or B and T cells from the non-PML patients. So this data shows us that PML can infect the peripheral lymphocytes in vivo, in particular, this population.
So next question we asked was, whether progenitor cells could be susceptible to JC viral infection. And to answer this question we looked at two progenitor cell lines, KG1 and KG1-A. These lines are derived from a patient who presented with leukemia but eventually developed acute myelogenous leukemia. But these two lines, CD34 antigen, that is a marker from stem cells.
As we can see in these slides we have in A, T-antigen-positive cells, and in B, T-antigen-positive cells detected with two different monoclonal antibodies, and in C we have some hybridization with the KG1-A positive cells using a biotinylated JC virus probe.
And the interesting feature of these cell lines is that when they are treated with formalizers they can differentiate -- KG1 can differentiate into a macrophage -- a mature macrophage. Instead, KG1-A cells are not affected by this treatment. So for this reason, we infected both KG1 and KG1-A untreated, and we saw susceptibility of these cells by JC virus.
When we infected KG1 treated, PML-treated cells, differentiating cells with microphage-like characteristics, these cells were no longer susceptible to JC virus infection. So this data clearly demonstrated that cells with monocytic lineage are not susceptible to JC virus infection.
And we confirmed the susceptibility of some cells, of precursor cells, with the primary CD 34, human parameter CD34 cells as we can see in these slides.
Previously, we described some of the interaction between tonsillar thermo cells, in particular B lymphocytes. Thermo cells are an important constituent of lymphoid organs, so we want to evaluate their involvement in the polyomavirus infection.
In this slide we have a representative field of tonsillar thermo cells infected by JC virus. The percentage of thermo cells infected by positive for JC virus T-antigen and viral DNA, was only 2.5 times lower than the percentage of human fetal iligos cells that are recognized as the most susceptible cells to JC viral infection.
So if JC virus occurs by respiratory route, tonsillar thermo cells, because they have relatively high susceptibility to JV viral infection, and for the natural interaction with the progenitor cells and lymphocytes are ideally positioning to be a site for initial infection and possibility to disseminate virus.
We are testing this working hypothesis by examining tonsillar tissue from children and other donors for the presence of JC virus DNA, and so far we've found 20 percent of these tonsils positive for JC virus DNA. We don't know yet in which cell type the virus, the DNA was sequestered, but this data are additional evidence that tonsils or other lymphoid organs could be a reservoir for the virus, or also initial site for primary infection.
This work has been done in Gene Major's lab and I want to thank him for his work. I also want to thank Blanche Korfman, Peter Jensen, Dala Galanti, Cathy Connor, for their help. Thank you very much.
CHAIRMAN BRIEMAN: Thank you, Dr. Monaco. Now, before going off to lunch in what, for those of you who haven't been here before, I think you'll find to be a very atypically excellent cafeteria for, you know, government cafeterias, let me remind you that we will return here at 1:40 for a panel-audience discussion that will be moderated by Dr. Mike Fried. And the topic is, "Issues related to the detection of SV40 DNA in human tissues". And again, if you have additional data, either positive or negative, regarding detection that you'd like to have presented, please contact him. Thank you.
(Whereupon, a luncheon recess was taken at 1:00 p.m.)
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Last Updated: 2/12/2001

Afternoon Session
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The Workshop took place in the Natcher Auditorium, National Institutes of Health, Bethesda, Maryland, at 8:30 a.m., Kathryn C. Zoon, Director, CBER, presiding.
PRESENT:
KATHRYN C. ZOON, M.D. DIRECTOR, CBER
ROB BRIEMAN CO-CHAIR
MIKE FRIED CO-CHAIR
RUTH KIRSCHSTEIN CO-CHAIR
DIXIE SNIDER CO-CHAIR
BONNIE D. BROCK, V.M.D. SPEAKER
JANET BUTEL, Ph.D. SPEAKER
MICHELE CARBONE, M.D., Ph.D. SPEAKER
KRISTINA DOERRIES SPEAKER
ELLEN FANNING SPEAKER
RICHARD FRISQUE, Ph.D. SPEAKER
ROBERT L. GARCEA, M.D. SPEAKER
ALLEN GIBBS SPEAKER
MAURICE R. HILLEMAN, Ph.D. SPEAKER
MICHAEL J. IMPERIALE SPEAKER
KAMEL KHALILI SPEAKER
ANDREW LEWIS, M.D. SPEAKER
MARIA C. MONACO SPEAKER
LUCIANO MUTTI SPEAKER
FRANK O'NEILL, Ph.D. SPEAKER
PATRICK OLIN SPEAKER
DAVID SANGAR SPEAKER
KEERTI V. SHAH SPEAKER
HOWARD STRICKLER SPEAKER
MAURO TOGNON, Ph.D. SPEAKER
JIM C. WILLIAMS, Ph.D. SPEAKER
JOHN LEDNICKY, Ph.D. PANELIST
ALSO PRESENT:
DR. GALATEAU-SALLE
HARVEY PASS
ETHEL de VILLERS
ROBIN WEISS
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CONTENTS
Morning Session
Introduction and Welcome by Dr. Zoon
SESSION 1 Presentations:
Dr. Fanning
Dr. Shah
Dr. Garcea
Dr. Butel
Dr. Carbone
Dr. Gibbs
Dr. Mutti
Dr. Giordano
Dr. Tognon
Dr. Shah
SESSION 2 Presentations
Dr. Dorries
Dr. Imperiale
Dr. Khalili
Dr. Frisque
Dr. Monaco
LUNCHEON RECESS
Audience Participation
Presentation by Dr. Lednicky
Panel Discussion
SESSION 3 Presentations
Dr. Hilleman
Dr. O'Neill
Dr. Lewis
Dr. Brock
Dr. Williams
Dr. Sangar
Dr. Olin
Dr. Strickler
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PROCEEDINGS
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AFTERNOON SESSION
1:55 p.m.
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MODERATOR FRIED: We have a couple of more presentations of people who will continue from Session 1 this morning, who have positive and negative data on different detection of sequences in different types of tumors.
In addition, we want other audience participation because you didn't really get a chance to ask any of the speakers questions this morning. So that we will go through a discussion and then you can ask the speakers and the panel will discuss various points on detection of sequences.
So to start off, we'll have Robin Weiss give his presentation. Robin's from the Chester Beady Institute in London.
DR. WEISS: Well, thank you, Mike. Mike Fried's asked me to speak first because I'm using the overhead and then we can rid of that as well as me. I'll just put up the overheads because we got involved, like so many others, into just looking to see whether there were SV40-like sequences in human tumors.
Worked on by a student in our lab, Dave Griffiths -- it's all his work -- because we had DNA samples already and that the Institute and Cancer Research were adjacent to the Royal Marsden Hospital, which is a cancer hospital, and the Royal Brompton Hospital is the chest hospital. So there's quite an archival store of mesotheliomas.
So that's what we starting looking at. And we've done nothing original. We adopted, in the first place, the primers within the T-antigen region that were described in the paper, New England Journal, by John Bergsagel and others that we've already heard about from Bob Garcea this morning, and one that is supposed to work for SV40 as well as BKV and JCV.
And like Keerti Shah showed just before the coffee break, David Griffiths thought he'd better calibrate the primers first, and there came the first surprise. That if you take some plasmid or spike it with DNA, we find very different sensitivities.
The primer 2 and the generic primer, they're primer pairs that are very sensitive -- we can detect between one and ten molecules of DNA -- but if we go to the shortest fragment, the 105 base pair fragment that is supposed to be specific for SV40, it's at least 100 times less sensitive in amplification.
Paradoxically, the primer pair that's least sensitive gave us 100 percent positivity with mesothelioma, but we had to run to 40-plus cycles. So I think perhaps we need more discussion about the efficiency of the primers, and Keerti's talk was the only one that I really heard this morning that titrated that out against cos cells, which Dave Griffiths in our lab has also done.
We also looked at semen samples -- this is whole, unseparated semen -- against samples we had prepared for a study of HHV8 in AIDS. These are all HIV-positive patients. And contrary to the situation from Ferrara, at least the Po Valley, we got zero. And these tissues were from three or four patients who died from non-malignant causes where we happened to pick up one sample.
If we look at the mesotheliomas, these are mesotheliomas from patients who presented in London, a rather slightly different set from those in South Wales, then with this least sensitive primer pair we're getting some positive signal, if you go on cycling enough by PCR.
And curiously, with the generic sequences, it ought to pick up all primate papomaviruses, polyomaviruses, we get many fewer. And with the SV40-specific sequence here we're getting only four. And so you use different sets of primers, you get different results.
You go on far beyond the number of cycles you would need if this genome was in every tumor cell, and then you begin to get positive results. Our conclusions would say it's not clonal, we've not done immunochemical staining yet, but I'd be very surprised if they came out like Michele Carbone's, because there's simply not enough DNA there to get T-antigen expression in every tumor cell. But that's still to be done; we'll have to cut more sections.
And we checked on the sequence for the four that were clearly positive, with the second set of generic primers. This is 105 base pairs and here's a prototype SV40. I think we amplified this out of cos cells. Here's the four mesotheliomas we tested; here's BKV, JCV.
And whatever we have amplified is clearly SV40 over this small region, and in fact, there's only one nucleotide that's different from the prototype and they're missing this 9 base pair region that's in the two well-known human viruses.
So there we are; that's our little bit of extra information which we can add to this analysis. I don't think it clarifies the subject at all; I think it further confuses it. But that's my feeling at this stage of the meeting, is that we don't know too much about what's there and there's a lot of variation between different labs.
And I think the sooner we start exchanging blinded sets of samples so that the different labs can look at the same set and one central lab should then decode it, the more we might get to grips with whether these are technical difficulties, whether our positivities are false positives or whether there's a very low grade real presence there, and whether there are genuine geographic differences, or differences in collections.
Thank you.
MODERATOR FRIED: Thank you, Robin. So basically, when you do about 30 cycles you don't see it, and when you keep going you find it, is that the take home message?
Have we lost Ellen Fanning from the panel? Okay, we also have some comments from Ethel de Villers from Heidelberg, who's been doing something and she will just tell us about it. She has no overheads.
DR. de VILLERS: Thank you, Mike. Well actually, we came in from the cold because we've been working on papilloma viruses for many years now, and my main aim was to characterize and identify new papilloma viruses, and then we decided to broaden this to polyomaviruses as well.
So we actually started off applying the methodology in a broad sense, to the polyomaviruses that we've been doing with the papilloma viruses. During the last three years we've been able to identify and partially characterize, 43 new papilloma virus types. So we were very optimistic about the polyomavirus types, but to tell you the truth, we haven't found anything.
And I just want to give you a few details. I haven't got any overheads or anything; it was a quick decision to be here. But I think the experimental part is a very important one, and I think we heard very little about that this morning, and hopefully we'll have more discussion this afternoon.
First of all, we started off using the VP conserved, amino acid conserved region, and we chose four different primers which we split up in degenerative primer pairs in order to identify all known polyomavirus types, including the mouse types: the kilham, the hamster, bovine, as well as the parakeet.
By doing so we do 12 different primer combinations on one biopsy and we actually -- well, we think there should be more than two human polyomavirus types. I think many people have the same idea. We then looked at many different types of tumors -- the numbers are still small -- but I'll just mention what they were.
We looked at normal lymphocytes of 12 samples, glioblastomas, five astrocytomas, ten cell lines of astrocytomas, five meningiomas, six lymphomas -- Hodgkin lymphomas, actually -- and ten lymphoma cell lines.
Then we didn't only stick to the VP1 area; we constructed primers in the T-antigen region too, in the conserved region. We didn't find anything with that, either. At a later stage we included the Kaposi sarcomas -- we looked at 14 of them -- we did 20 bladder carcinomas. And by not getting any positive results we decided what we'll do is maybe go into the literature and try some of the primers that have been published.
With great difficulties, with some groups we got hold of primers -- which was not the ones that they described in the papers -- but nevertheless, they gave us some primers and in the end I think the majority of people used the Bergsagel primers.
We applied these primers in exactly the same way as been published, and I do not think one can consider the conditions of the PCR as stringent conditions. In other words, you do pick up other sequences, we do get a smear of cellular sequences in the background using those same conditions.
If you use TEC gold you get a lot of bands, not only the smear. If we hybridize we get the smear hybridizing as well under those conditions. In some instances we got a little bit of a stronger band in the area where you would expect -- on this size that you would expect.
What we usually do in the papilloma viruses is we clone and sequence. We are absolutely convinced there is no way you can get around that in any positive signal. So we cut out that area and we clone it and we sequence at least ten clones.
We haven't found any polyoma viruses in any of these tumors or cell lines that we've looked at. What we did find is that we found, for example, many cellular sequences. One cellular sequence, for example, had 78 percent homologies to the Rb gene.
We had another clone which had more than 70 percent homology to sequence in the fetal brain. So if you look in the databank you can find many sequences in varying homogies to these cellular clones.
So that's the situation we are at now. We're progressing in this. What I would just like to mention is that what I miss in the data presented and as well as published, is the sensitivity which Robin talked about now, and on the other hand, our experience is that if you do not test your sensitivity by mixing your positive control to, say placenta background, then you get a different degree of amplification than you would use only the plasmid.
On the other hand, all the negative controls for example, placenta DNA, water, and so on, are very often missing. The other thing is, we find that if we use more than 50 to 100 nanograms of cellular DNA input, we get a reduction in the efficiency of the PCR reaction. So those are just things that I would like to mention.
MODERATOR FRIED: Thank you. I'm sure we'll cover some of these points more in the general discussion. Another presentation we have is by Harvey Pass from Wayne State.
DR. PASS: Thank you, Dr. Fried. As you know, Michele Carbone is my collaborator and he starting working in my lab, which was SV40 negative before I left the NCI. And when Michele left to go to Chicago I was excited but also skeptical about these findings.
In my unique position as a surgeon who takes care of patients with mesothelioma, I was able to recruit the first 48 for the first set of patients, but felt it would be necessary to re-establish this in a completely separate series of patients that were operated on by me at the NCI. That was done after Michele left and that would be done by people in my laboratory who essentially were learning the techniques.
Could I have the first slide please? And I'd like the lights down please. Maybe I don't do the ethidium bromides as well as everybody. But we therefore took a series of 42 patients that were operated on since the first set, and not only looked at the amino terminus region, the Rb binding pocket for T-antigen as Michele has done, but concentrated on the larger fragment -- the 500 or so base pair fragment.
But also with the help of Janet Butel, looked at the enhancer/promoter region for T-antigen and then also used primers to amplify the carboxy terminus in these patients, essentially. So we're concentrating on this primer here, primer pair, which amplifies a 574 base pair region of the Rb binding pocket which Michele touched on.
Primers 7 and 8 -- that's my connotation of primers from the literatures that were described to amplify a 281 base pair of the carboxy terminus. And then finally from Janet's work, we use her RA1, RA2 to amplify 310 base pair region that was the regulatory region.
Essentially, this just shows the primers. Essentially, this is the 7/8 primer which is the carboxy terminus, and then the RA1, RA2 here, so there's just the preliminary data.
And again to refresh your memory about SV42, it essentially amplifies a larger fragment of the Rb binding region that when you look at your southern hybridization you may get two bands, one of which will reflect the presence of the centron and the other reflects that it is not there -- about 300 base pairs.
Well, when we then did the ethidium bromides -- these are the positive controls which is a hamster mesothelioma tumor -- we weren't very impressed with SV42 on the ethidium bromides, but using the SV probe -- next slide -- here is the original.
In the 42 or so new specimens you can see that we have some positivity, and in fact, in its 13 out of 42, which is 25 percent had -- we were able to amplify this region. And in some patients both species are present, but in most it's a single species.
In the carboxy terminus region for amplification we found that 38 percent were essentially amplified using that primer, but again, we didn't have a probe for this so using BsaB1 digestion we took our positives, and this is the positive control, the hamster tumor that shows that it cuts, and then this is a positive that cuts, and this unfortunately is light, but another one that cuts. So it seemed like we had the same sort of amplification that we did in the control.
So to reiterate, again, we found 38 percent positivity but again, with restriction enzyme digested, reflected what the controls were. No, we have not sequenced that.
With regard to the regulatory region, using the primers described by Janet, we found very close to her data, about 50 percent seemed to be positive. And in fact, we had a unique restriction site here which we used Fok1 -- next slide. The positive control is here with an uncut, cut, uncut, cut, uncut, cut. Very similar in all these patients to the positive control, but we did sequence four of these patients that were positive, cloned out the product. Next slide.
These four patients -- here is the original sequencing gel. To sum this up it was exactly homologous to what we found with H9A.
But that wasn't enough. We wanted to go back and take another vial of tumor and then re-extract the DNA from another vial of tumor from these patients, and then do the digestion again to see if it corroborated our previous work.
And indeed, when we re-amplified and then extracted a new specimen from those patients that were positive -- here's the positive control: cut, uncut, cut, uncut -- we found the same sort of digestion pattern.
If you summarize all the data, then with these three areas this reflects the ethidium bromide data for the smaller fragment, 24 percent of the patients -- at least in the new series, the 42 patients apart from the original series -- have amplification of these three regions.
I absolutely agree with the comments that have been made by the previous two speakers. I absolutely agree with the exchanging of specimens and standardization of this. Because the data that I'll talk about tomorrow which has to do with therapy, is going to be useless unless we find that this actually a true phenomenon.
And I thank you for this time.
MODERATOR FRIED: Thank you, and we have one more relevant to the last talk, by Dr. Galateau-Salle from France who will use one of the microphones.
DR. GALATEAU-SALLE: Sorry for my transparencies and thank you to let me just give our result. We have looked for SV40-like DNA sequences in pleural mesothelioma, bronchia pulmonary carcinoma, and non-malignant pulmonary diseases that the study has been performed in Caen, France.
We have studied 147 frozen sections including 15 mesotheliomas, 63 bronchia-pulmonary carcinoma, eight other tumors, and among them, one parietal osteosarcoma and metastasis, 71 non-malignant samples, and six mesothelioma cell lines.
The DNA extraction was from fresh frozen biopsy and they were cut on ice under sterile condition, then was extracted by phenol chloroform method. Then amplification was performed with the primer designed by Bergsagel, amplified the conserved sequenced of large type and polyomaviruses, SV40 173 base pair, JC virus 129 base pair, and BK virus, 182 base pair. And to avoid false positive we considered OD index separated to 1.5.
All samples were tested twice or three times. So we find positivity in 30 percent of bronchial carcinoma, 50 percent of mesothelioma, and 60 percent of non-menign pulmonary disease, and we find also the parietal osteosarcoma was positive.
The DNA sequences were not related to BK virus sequences but three of our samples were also positive for JC virus sequences. The mean age of patient was 63 years old: the youngest was 41 and the oldest was 74. And the male/female ratio, we find 35 positive male patients out of 105, and six females out of 20.
And if we consider persons of our sample exhibiting DNA-like sequences, a value of index according to disease, we find that in our adenocarcinoma, the OD index was higher than in mesothelioma, and we find that's all the peripheral adenocarcinoma, papillary carcinoma, or mesothelioma -- just adenocarcinoma was positive, and it was the same in non-malignant pulmonary disease.
We find positivity in the peripheral line. And if we compare that mesothelioma to organizing priorities, we don't find any difference between the positivity in mesothelioma and organizing priorities.
Now we have also studied the relation between asbestos exposure and SV40 DNA-like second positivities. We have studied on all the higher mesothelioma except one, where exposed to asbestos. And only 40 bronchia pulmonary carcinoma were exposed to asbestos and we haven't found any correlation between positivity and asbestos exposures.
Now, regarding vaccination, it was very difficult because all the people have remembrance of the way that have been vaccinated and what type of vaccine. But all the people who were positive were old enough to have been vaccinated and born before 1963, and we haven't found any positivity in people born after 1963.
The last result is, we looked for SV40 TAC expression by immunohistopathology and we haven't found any nuclear staining. Thank you.
MODERATOR FRIED: And finally, before we start the panel we have -- Keerti Shah from Johns Hopkins will talk about BK in some brain tumors.
DR. SHAH: May I have the first slide please? In this study we had looked for BKV-specific sequences in brain tumors.
There have been a number of reports, most clearly from the group of Dr. Barbanti-Brodani from Ferrara, Italy, that they found BKV-specific sequences in human brain tumors, especially in glioblastomas. So we had done this study a couple of years ago and it has been published in Journal of Neural Oncology.
We looked at malignant gliomas in 31 instances. We had purified DNA from frozen tumors, and these were obtained from Dr. Bert Vogelstein's lab. He had already processed them and we got the purified DNA. And we also got 47 paraffin sections from Johns Hopkins Hospital, and they were largely glioblastoma multiforming, but most all of them were malignant gliomas.
We looked at them with two primer sets. This PEP-1 and PEP-2 are the ones which were developed in our lab by reactor, and those have been published. And then amplify 173, 176 base pair regions of T-antigen, and there are identical sequences here for both BKV and JCV.
So we would amplify with a single primer pad, this one, and then hybridize with different probes; one for BKV and one for JCV. And this is the other primer, which is for the regulatory region of BKV which was used by the Italian group to detect these BKV sequences. So we obtained those primers from them.
And these are the results. We were able to, by globin amplification, all of the 31 purified DNAs gave very good globin bands, and 44 of the 47 paraffin sections gave good globin bands.
The sensitivity we thought was 100 to 100,000 copies of BKV or JCV, we would have picked up 100,000 copies total. And all tumors specimens were negative for both BKV and JCV DNA.
From the tumors we had gotten from Dr. Volgelstein from which we had purified DNA, we could estimate the cell equivalent of tumor DNA, and we thought that we had at least 40,000 cell equivalents of the human DNA.
And we thought that we would have picked up the viral DNA if only one of 40 of the tumor cells had a single copy of the viral DNA. And so the sensitivity was quite good. We still failed to detect the viral DNA in the human tumors. Thank you.
MODERATOR FRIED: Okay. I would like to stop these more formal part of the discussion and the way I'd like to do it is shown on the first slide that I have.
So we should be talking about: PCR conditions -- the sensitivity, the specificity -- since we have positive and negative; the methods of identification of the PCR products; the possibility of contamination where the people have SV40 or SV40 constructs in their labs, and what it means, the detection in normal and neoplastic tissues; what are the differences; why are we seeing this; and if there's any recommendations for the future.
So before we go through and discuss with the different panel members the differences that they find about -- and the different PCR conditions and whether we would hope to, out of this meeting, get some sort of standardization -- John Lednicky will be giving a presentation from the panel about different technical details. John?
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DR. LEDNICKY: Well, as we all know, those of us who are looking for SV40, the samples are using PCR -- which is a powerful technique -- but our findings are often discordant. And knowing that many factors affect PCR reactions, it's likely that our results are affected, not only by sample choice, but also by specimen quality and PCR methodology.
So I'd like to identify some problem areas with a goal of having this audience suggest ways to improve these tests.
So I guess the central question to be addressed is: What is it about the PCR methodology that could be affecting the reproducibility of results among different labs? If I can have the first two slides, please.
So particular questions to consider are shown in slide 1, and the first question I'm going to pose is: What's the most effective method to extract DNA from paraffin-embedded tissues?
And we should consider: what PCR conditions should be used; are the primers really SV40-specific; how should we go about using and setting up positive and negative controls; and how should PCR products be verified?
Now, a lot of people think they're PCR experts and they really don't have a feeling for what samples extracted from paraffin slides often look like. So in the upper panel here I've shown total tumor DNA extracted using -- chopping up tissue, doing a proteinase K digest, and precipitating all the DNA out looks like.
And what you see in reality, although it hasn't stained, a lot of the high molecular weight DNA hasn't gone into this one percent agarose gel, and you see prominent mitochondrial bands here.
In contrast -- and this comes as a very big surprise to people who have never looked at these, and the majority of labs never do this stuff, they just set up PCR conditions, assuming that they've recovered a certain amount of DNA -- and that is, oftentimes with DNA extracted from paraffin slides, you get very fragmented and degraded DNA.
The reason we should of course, decide what might be the best way to extract these DNAs is, when you have fragmented and degraded DNA, this is really going to affect the PCR sensitivity; the efficiency of the PCR reactions are diminished.
In these next two slides I'm showing some very basis problems. Now, we also need to be aware of problems arising from DNA preparation methods, and in this slide I show some purified DNAs that we received from other labs. In fact, this particular sample was hand-delivered to me by the person who supervises the PCR work in that lab.
By the way, none of these DNAs were from Bob Garcea's lab; I just wanted to make that clear.
So seeing such sloppily prepared DNA, how can you assume the DNA is not contaminated with other DNAs? And for people who are setting up positive controls based on amplification of alpha and beta globulin genes, how do you know what you're amplifying from samples like this, don't derive from the skin flakes of the technician working up these samples? It's a basic question, but it's something we really need to think more about.
Now, with samples like this we really need to consider whether reliability is a problem if we subcontract PCR work. Like, what assurance is needed that the DNA is being handled properly? And here in the U.S. this is a very contemporary concern these days because, as my colleagues say, there are a lot of rent-a-techs in core facilities and maybe some sort of oversight is needed.
Another type of DNA preparation problem is shown in this slide. And in this demonstration slide what I'm showing is DNA that was spooled from SV40-infected tissue. And what I've done is amplify the regulatory region: these two are control lanes; this is a negative control lane; this is a lane that has total DNA, just precipitated from a sample.
Here I've resuspended the spool DNA and as you see, I don't get any signal, whereas what's left in the tube does give me a signal. Now, spooling is still used by many labs working with eukaryotic DNA and I'd like to note also that working with coded samples and not knowing beforehand how a sample was prepared, in our lab we have not just detected SV40, JC virus, or BK in a single spool DNA sample we've looked at.
So I'd like to discuss PCR conditions and to demystify some of the methods our own lab has developed. Now, a primary question when PCR signals are seen is: Is it really SV40? Now, our lab's approach is first of all, look at more than one site of the SV40 genome.
So we look at some of the sites. We typically look at the regulatory region, Rb proximal binding site, carboxy terminus of T-antigen. And in particular, the carboxy terminus of T-antigen shows variability between SV40 strains and sequence data from the site may be useful for taxonomic and epidemiological studies.
Now, other sites such as the regulatory region, are useful when the target DNA is episomal, and that the regulatory regions of SV40, JC, and BK are distinct. In this slide which is pretty busy, I'm just showing primers and PCR, annealing temperatures we use.
Now, one of the commonest questions we're asked is, why do you use so many cycles? And when you're working with paraffin samples, why do you use so many cycles?
Well, when you have a lot of fragmented DNA, I guarantee you, you need to use more than the standard 30 cycles that a lot of people normally use. And here what I've listed is two temperatures. The temperature in parenthesis which is lower than the one to its immediate left is the temperature we use when we're working with samples extracted from paraffin.
So notice, these are what I refer to as lower stringency conditions. We have found that it's not possible to use more stringent conditions, and a lot of labs seem to do this.
Now, very importantly, since more than 40 cycles are needed, we should discuss detection sensitivity as some people have said earlier, because a lot of labs overestimate their detection sensitivity. And the biggest problem is they use plasmids without spiking them with additional DNA, and that really decreases the sensitivity.
Can I go back to the other slides? Now, I'd like to give a warning -- and it's not a good idea at all to use lower stringency conditions when you have highly intact DNA -- and this is something else a lot of labs do. And the reason for that is numerous, non-SV40 PCR products are formed. And we have also actually sequenced some of those bands and confirmed they're not papomavirus bands.
So the problem is setting the appropriate PCR conditions for these samples derived from paraffin samples is really more of an art than a science now, and we really need to put our heads together to try to come up with, you know, realistic protocols.
Additionally, the conditions cannot be universally applied, and in particular -- for example, when we use these two primers we have to use a lower, what I call high stringency condition.
And the reason is, with these particular primers which amplify the carboxy terminus of T-antigen, if we go much higher than 60 degrees, we get truncated T-antigen products in addition to the full length product. So you have to be careful about some of these conditions.
The next two slides, please. Now, another reason for using different sets of primers is that it's possible that DNA sequence changes might occur in different strains of viruses, and in particular, the regulatory region of these viruses might be somewhat different.
The primers we use seem to work for different strains of SV40 even those with rearrangements like SUPML-1, but there is a danger, and I'd like to bring to everyone's attention that the primers being used might not be specific for SV40, even though very sophisticated computer programs tell you that they would, under the conditions you want to use them.
And so for any set of primers you have, you really have to test them. It's very hard to use computer programs to really predict whether they're going to work.
So for example here, using RA3 and RA4 primers which have quite a few mismatches with JC virus, even under relatively stringent conditions, we're actually able to amplify the JC regulatory region.
Here I've amplified the med1 regulatory region and sequenced it in both directions. So we find that we can actually amplify JC and BK virus. The point is, these findings highlight the need to verify the identity of PCR products. You can't go just by seeing a band on the gel.
Another important question is, how do you distinguish between true positives and false positives? Now false positives can usually be traced to contamination by controlled DNAs, and our lab solution is to substitute SV40 templates for natural templates.
And in this slide, what I've done is create some SV40 templates which have unique XHO, or SAL 1 sites. So when you amplify them, the product is about the size of what you'd get off a natural template, but then now you can do an XHO 1 digest and only the artificial template gets caught by XHO 1.
And we're developing similar constructs for other regions that we analyze and we think this is a really good idea for people to use for positive controls.
Now, another approach we're trying to perfect is that of using long PCR to amplify the whole SV40 genome. And this slide shows some of our findings. Using a commercial kit we're able to now amplify an entire SV40 genome from plasmids environ cell lysates.
And the procedure, the way we use it, works fast. If you remove some of the high molecular rate DNA first and then do your amplification -- I won't go into any more details -- but I think this approach eventually may be useful in that it will be possible to not only answer whether episomal DNA is present, but also because it will be possible to amplify the entire genome for cloning and additional analysis.
So I'd like to discuss the merits of DNA sequencing. So in this slide the sequence DNA band -- I'm sorry, the sequence PCR DNA band is clearly different from that of the control template. There are two changes done here, but if you scan up here you'll see that there are indeed, changes.
Now, the question is, how do we know these changes aren't artificial? And if you look at this slide, the answer is evident in that you see repeating patterns of 9 base pair deletions or insertions that aren't seen in our template for control positive DNA.
Also, the sequences we've come up with are different from the standard SV40 strain that's present in our laboratory, which is the Baylor strain of SV40. We have found that just merely doing southern blots may be a tricky thing, because as one of the speakers said earlier, if you play around with the hybridization conditions, you actually non-specifically light up unrelated DNA.
And I hope this presentation put some of these problems in perspective, and thank you for your attention.
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MODERATOR FRIED: Thank you, John. You've opened up a lot of different aspects of the PCR technique that I think we should discuss. Could I have my next slide?
So what we have discussed, we have some positive results, we have some negative results, but what's quite clear is that if there was one copy of SV40 per cell, per tumor cell or per normal cell, it would be very easy to detect. And it's not very easy to detect, so there's probably a very low level or the primers people are using are not very specific.
So the question is, what are the limitations, how can we increase the sensitivity of the PCR and the relevance of the copy number? Could I ask the different members of the panel how much they think they're detecting in copies per cell? Does anybody want to volunteer? Michele?
DR. CARBONE: We have done the original experiment that's indicated. We are able to detect one genome in our PCR reactions. I would like to comment just briefly on what you just said --
MODERATOR FRIED: Sorry -- one genome per what?
DR. CARBONE: One SV40 genome.
MODERATOR FRIED: If you have in your whole PCR reaction, one genome, you could --
DR. CARBONE: One SV40 we've detected. Actually, ten genomes. At one genome -- between one and ten genomes we are able to detect. Now, the real problem -- not the real problem, but one problem that should be considered in what you said about one copy of the cell is that here we're not talking about cell cultures, we're talking about tumors.
And obviously, in a tumor we have -- the majority of the cells often are not cells that are tumor cells. Many of them are reactive cells that are not malignant cells and other cells are necrotic cells. So that should be considered when we talk about the level of sensitivity.
MODERATOR FRIED: Fine. I would agree with that, but still, I mean, I'm sure we're going to get to the cancer whether this is related to cancer in other sessions, but it's clear that from all the other papomavirus or the polyomaviruses, we know they don't get lost -- I mean, they stay there.
And the only reason that possibly an episomal copy would be doing something if it got into a cell and excited that cell, stimulated that cell to secrete factors which would make other cells divide, so then you could have low copy number. But certainly, you know, if it was cancer cells we would have plenty of copies, I think.
DR. CARBONE: We have at least, however, one bovine papilloma virus, type was 4, that get lost.
MODERATOR FRIED: So Dr. Shah, how many copies do you think you could detect, or not detect?
DR. SHAH: I think we detect perhaps, ten to 100 copies, genome copies, of the virus in our PCR reaction. Not per cell, but of the virus. Just as --
MODERATOR FRIED: The whole PCR realm --
DR. SHAH: -- Michele say is one to ten, I would say ten to 100. We believe that even with paraffin sections we are processing at least 1,000 or 5,000 cells. So we would detect, if there was this one copy in ten cells or 50 cells -- if there was one copy of the viral genome in ten cells, I think we would detect it. This would take care of the problem that Michele described, that not everything in the paraffin section is tumor cells.
MODERATOR FRIED: But sometime you have fresh tissue also. I mean, have you not --
DR. SHAH: We had fresh tissues -- we did not have fresh tissues for the mesotheliomas, but we had fresh tissues for the brain tumors. For that we were -- because we knew how much DNA we were processing, we thought that that was DNA which would come out of something like 40,000 cells, but that was with the BKV system.
So if we can detect 100 copies and we are 40,000 cells, then we can detect -- one complete genome in 40 cells was our estimate for the BKV study.
MODERATOR FRIED: But you're not detected what other people are. I mean --
DR. SHAH: That is true.
MODERATOR FRIED: And you're also not using radioactivity hybridization, you're using biotin. Do you think that's less sensitive?
DR. SHAH: I don't think so. We used to do radioactive probes until about three or four years ago. When we changed to the biotin label probes we examined it very thoroughly and also solves the experience of many people in the papilloma virus field. We do this routinely, hundreds of times, for studies on human papilloma virus in cervical cancer, and there is no loss of sensitivity by moving to the non-radioactive detection system, that we have observed.
MODERATOR FRIED: John Lednicky suggests that most of the DNA they're detecting is episomal -- I mean, not integrated. I mean, is there anything that possibly --
DR. SHAH: No, we do not precipitate the DNA. We proteinase, extract it, and test it in the same tube. So we do not have this problem of spoiling and losing some portion of the DNA.
MODERATOR FRIED: Could anybody else suggest why they think there's differences between positive and negative?
DR. TOGNON: Yes, I would like to comment on the sensitivity. Just to have a rough idea how many molecules in terms of genomes we have in our detection experiments, we did a sort of reconstruction experiment. Since we start always with 500 nanograms of human DNA we mixed in dilutions different amounts of SV40 DNA. And at the end, it turns out that we have the sensitivity of around ten molecules in our assay.
I would like to say something else about the negative results that we heard before. I wish to point out once more, the problem that's related to the extraction of DNA is not enough to digest with proteinase K and SDS. We usually extract several times with phenol and chloroform the DNA, and at the end instead to precipitate the DNA, or instead to directly amplify the DNA, we dialyze for two or three days, the DNA.
This is very, very important because usually the BK DNA or the SV40 DNA, or JC DNA usually in episomal state. And the amount of the DNA of the viral origin is always very, very low. We estimate in our assay is approximately .1 fentagram. That means practically nothing.
MODERATOR FRIED: But it's been pointed out by Barielle and discussions, one of the classic ways people purify plasmids or SV40 or circular molecules is by precipitating the DNA, so why do you think you'd be losing the episomal, especially when you have so much carrier DNA to bring it down?
DR. TOGNON: The difference is the amount, because if you precipitate your DNA, you precipitate the high molecular weight DNA. If you have enough episomal DNA, episomal DNA can't precipitate with the human DNA, but if the amount of the episomal DNA is very reduced, you've lost the DNA and you've lost the signal during the PCR.
This is a very, very simple experiment, because you may reconstruct in the laboratory, okay. You can make ten or 20 different Eppendorf tubes with different amounts of your episomal DNA together with the 500 nanograms of human DNA. And at the end you eventually can repeat the extraction and you see the difference.
MODERATOR FRIED: Dr. Gibson?
DR. GIBSON: This is something to consider, and we learned this by trying to purify DNA using a number of commercial kits. But a lot of people lose even mitochondrial DNA when they're precipitating or somehow collecting their high molecular weight DNA. So as a rule of thumb, I think it's a good idea to look for your -- to see if your methods are bringing down the mitochondrial DNA. So mitochondrial DNA is circular, it's about 16 kB, and any purification methods that will work for SV40 will also work for mitochondrial DNA.
But we have actually gotten samples from other labs that have a lot of high molecular DNA and you just don't see any mitochondrial DNA. And I'm sure if the SV40 or whatever out there -- papomavirus you're looking for is in there in an episomal form, you'll never see it.
MODERATOR FRIED: What about increasing the sensitivity? Ellen Fanning, did you have some points to make?
DR. FANNING: Well, I was wondering, maybe some of the people who were doing this routinely could respond. Whether it's not possible to construct a competitor molecule that uses the same primers and thereby eliminate the variable activity of different primers -- the efficiency with which different primer pairs will amplify a target sequence.
You could construct a competitor. There's some effort I guess, already in that direction, which has a different size or which has a restriction side or something like that, so that you could distinguish it from the products that you're trying to look for.
MODERATOR FRIED: That that would help avoid contamination of people who are putting in SV40 to use as the primers. So you would put a primer, some junk DNA, whatever, and the other primer. And this could be any size. And you could spike this in to just check, you know, and it wouldn't be falling off people's hair because it could be something else.
What about other -- you're also suggesting maybe, other types of PCRs in situ?
DR. FANNING: One other thing that one wonders, particularly with tumor cells that appear to be staining for T-antigen, is whether those cells couldn't be used. Certainly, the tumor cells could be distinguished as tumor cells when you look at them.
For example, do in situ PCR on tumor cells and ask whether those cells, rather than the contaminating, normal tissue around them, may be the cells that are specifically containing the viral sequences. Is this feasible?
MODERATOR FRIED: Anybody?
DR. CARBONE: I show tomorrow, some RNA hybridization. We did not do in situ PCR on the cells. We hope to do it soon, but there comes one problem, if I can address that. I mean, I agree 100 percent with John Lednicky, what he said. He presented an excellent presentation of what should be done, and I'm sure that if we do what he says it's always going to work. It fact, it works in our lab and in fact, that's the way that we work and that you have seen the presentation of Dr. Pass, that's the way he's working.
But then comes in terms of practical problems and that is, that sure, you want to use many primers, you are using in situ hybridization, you want to use as much as you can. But all this takes time, it take money. And that has to be taken into consideration because it's a factor that has affected this research considerably.
In other words, if you want to test 100 samples and you want to test 100 samples with primers for many different regions, you need to have the resources to do that, otherwise, it would be impossible.
A more practical approach that also has been taken is the one of sequencing the DNA. I think, in my opinion, that that's probably the best approach if you don't want to do extensive studies, meaning using a lot of primers, a lot of hybridization, and a lot of work. Not because you don't want to do it, but because you don't have the resources to do it.
And for example, if I can answer to what, the excellent presentation that Robin Weiss gave before in which he showed something that I think is in fact, the point of the discussion today. The point is, he said I use different primers for this before, and here I'm getting different positivities. I go from 100 percent when I use SV3 primers, to -- I don't remember how much -- when he used the beef primers, to something less when he used longer primers.
That seems to be the argument, not that much why some lab is not finding it, because it seems to me that overwhelming we are finding it. But why is that using different primers we get different percentage, and what primers, what set of primers should be used?
My experience has been that initially we used that set of primers that gave the 100 percent positivity that was reported before, or the other one that is called beef set of primers. These are shorter primers.
The problem that you can have then, is that you have to rely to hybridization, and also the problem of the temperature was brought up. When you rely on hybridization there can be cross-reaction with BK or JC, and you hope that in fact, what you're seeing is true, but you cannot be absolutely certain.
And that's why in our last paper, together with Bob Garcea, we went to the longer primers, that they are the SV two sets of primers -- because in our experience, at least using that set of primers, is big enough that you can be more assured that what you see in there in hybridization is in fact, true SV40 DNA and not something else.
If in fact, you're using the shorter primers, the one that used beef primers, for example, in my experience you need to use those primers when you use for example, for my fixed tissue, because you can't amplify 574 base pair many times, so you need to go to a shorter primer.
Well, in that case, rather than relying on an hybridization where you always can question, was 58 degrees enough, should we go to 60 degrees, you can just do direct PCR sequence. You have cloned your PCR products and checked that.
So shortly, what I was suggesting that the approach that John suggested is the ideal approach if one has the resources to use that approach. If one does not have the resources to do that, the alternative is to use the set of primers that gave the lower number of positive results but still gave positive results that are the SV2, SV ref set of primers that we use for example, in the Oncogene paper.
Or if you're dealing with the formalin fixed DNA, then use the beef set of primers that will probably take also BK or JC, but if you sequence it you should be able to distinguish among them.
Then the question, why you get more positive when you use the beef set of primers or you use the longer set of primers? I don't know, but obvious we have an explanation that seems plausible and that is, that those primers may well cross-react with BK and JC.
And so it is possible that when you're using that set of primers you have seen, not only SV40, but you are seeing BK or you are seeing JC. And the only way to know that would be to sequence the DNA.
And I took too much time, I think.
MODERATOR FRIED: People from the audience? Ethel?
DR. de VILLERS: I would just like to make a comment again regarding the papilloma viruses, and I think we're not so far away --
MODERATOR FRIED: Is that microphone on?
DR. de VILLERS: I hope so. Is it on yet? Now? Can you hear me? In the papilloma virus work we've done, we do not find any difference whether you spool the DNA or where you precipitate it. We do precipitate the 8 kilo base pair plasmid with the DNA, and we do spool it out if we spool out the DNA. I don't think there's that much difference between the five and the 8th Kb fragment, or the episome.
And the other question is, or the other thing is that I wanted to mention, was that if you -- well actually, I want to be mean because what I wanted to do is make a comment, what I read last week in the "PCR Protocols", in the small book where they quoted Cary Mullis.
And he said, if you need more than 40 cycles to amplify a single copy gene, then you have serious problems with your PCR. And I just want to mention that our PCR that we're using, we go down through one genome copy per cell in our detection method, and we still do not find any polyomavirus in these tumors.
MODERATOR FRIED: Thank you. Is there anybody else from the audience who would like to make a comment?
DR. VILLARREAL: I wanted to comment. I'm Luis Villarreal. I've been studying episomal states of polyoma, mass polyoma for about ten years now, looking at low-level episomal persistence, about one copy per cell. And this problem that you've encountered of physical conditions for the purification and precipitation of the DNA strikes me as odd. I've never seen that as a phenomenon.
And I suspect the situation may not simply be the size of the DNA but the way it's being handled: the precipitation g forces involved, the salt conditions, etc. There are a lot of other variables that affect the yield. So that's one thing to consider.
I guess I'll let the other speaker for now.
MODERATOR FRIED: Do you want to go up to the microphone? Could you identify yourself?
DR. OXMAN: Mike Oxman, San Diego, pre-historic SV40. I have two questions. One is, if you're talking about polyomavirus tumors, one wouldn't expect episomal DNA; one would also expect integrated DNA. So that may not be such a big problem.
The other question is, I would love to hear the people who are using more than -- who are showing fluorescence or immunoperoxidase stains in which a number of cells shows T-antigen, and are still using more than 40 cycles. And I wonder what the explanation is for the need for that many cycles?
MODERATOR FRIED: You'd like to answer? You had 50 percent? I mean, you showed one where there was a lot of T-antigen --
DR. TOGNON: Sure, sure. I'll answer, first of all, to the problem related to the papilloma virus. We have similar experience. We don't have any problem with the papilloma virus. Indeed, the number of genomes of the papilloma virus in the tumor samples is always higher compared to the -- in my experience, in my experience -- is always higher compared to the polyomavirus.
And for the presence of integrated state of the different polyomavirus, we found that usually the percentage of integrated polyomavirus in the tumor DNA is always very low; let's say approximately 20 percent of all the sample. In that case of course, it doesn't make any difference because the DNA is integrated in the human genome.
And for the presence of the antigen, the various breaks in the cell lines, the polyomavirus -- SV40, JC, and BK -- usually infect the cell in foci. If you have for example, 110 cell -- 106 cells, only let's say, 100 on 1,000 are infected and expressed the last T-antigen. So you have always the foci of discreet presence of the last T-antigen, but not all the cells express the last T-antigen.
DR. BUTEL: Mike has asked a very important question relating to the integrated state of the DNA in these samples. And the fundamental answer is that we don't know whether it's integrated or not. We have not had enough sample size to do the right experiments to tell whether there's any DNA integrated.
I mean, it's conceivable that we're detecting episomal DNA, it's conceivable there would also be integrated DNA, but we haven't been able to do those experiments to answer that question.
DR. SHAH: I think our real problem is not so much to increase the sensitivity of the assay, because everyone seems to think that they're detecting one copy in ten cells, whatever. Our real problem is to make sure that our specificity is good. And I don't know of any DNA tumor virus where you could not detect the genome by non-amplification-based assays.
What is desperately needed is to take some of these positive samples and see in a simple certain hybridization without amplification, whether you can detect the correct bands or not. I think that is really needed.
DR. BUTEL: There's not enough DNA when you're just dealing with one tiny little amount.
DR. SHAH: Yes, but --
DR. BUTEL: If we had larger pieces of DNA than those experiments --
DR. SHAH: How many micrograms of DNA is obtained from these tumors?
DR. BUTEL: We've only been dealing with, you know, a paraffin slice.
MODERATOR FRIED: But now you know what you're looking for. You don't have to go back to archival material. I mean, there should be more material that should come up right away.
DR. CARBONE: May I intrude into this discussion? Could it be possible that now that we have a new technology -- that I agree with you, that before it was not possible to -- the DNA tumor virus was detected by southern blot, but that was also true because there was not PCR.
Today we have a new technique, and so given the fact that we have a new technique that is much more specific, it is very possible that today we are able to detect things that in the past we simply were not able to detect.
And actually, if you look at some old papers -- there is one in PNIS; I think it's Krieg, the first author; I'm not 100 percent sure -- he shows southern blot showing that brain tumors contain SV40. But the bins are dirty. I have done the same southern blots and I could show those southern blots and I believe that depending whether the reviewer is a friend or not, he could believe it or not.
In other words, the signal is not that strong that you can sell it for sure that the signal is specific. But certainly, you'll see something there. And what I'm suggesting is that today we have a new technique -- the polymerase chain reaction was not available ten years ago -- and we may be seeing things that before was not possible to see.
MODERATOR FRIED: Yes, I think PCR is obviously more sensitive than southern blotting; I think there's no doubt. And I mean, the question is, because there's positive and negative, can we get to some consensus where maybe an agency would send out different cells blind to the different labs and, you know, standard sets of conditions that people could look at them and come back, or people can contribute to --
DR. CARBONE: But this would meet -- Bob Garcea, Dr. Pass, and Dr. Procopio just did and published in Oncogene.
MODERATOR FRIED: That's right.
DR. SHAH: May I suggest? There's a strategy which has been proposed by Howard Strickler from the NCI, which I think really will address some of these problems, which will examine the different labs and the ability of the labs to reproduce their results. I think that would clarify much, and I wonder if Howard would comment on it?
DR. STRICKLER: My suggestion was, in the face of the uncertainty of the data, that what we really need is an exquisitely controlled third-party study. The Oncogene study was a very nice project involving four different laboratories, but it's somewhat difficult to follow exactly where DNA was extracted, who handled the samples, which laboratories worked with them.
It wasn't -- considering how important this issue is and how easy it should be to clarify these questions, it seems that really, we should just move forward and do a study in which multiple laboratories, using their own methods, test specimens, and we can directly measure the intra and interlaboratory reproducibility of the results, and we can talk about the results afterwards.
As long as I'm up at the podium though, I'd like to address a question which is, in those laboratories in which positive findings are being obtained, doesn't the extreme sensitivity of your own assays concern you? There's only one study so far which presented data suggesting an approach where they examined whether or not the virus was actually in the tumor cells.
And it's amazing to me -- unless I'm missing a point, which could be -- that in situ hybridization data isn't available yet, the PCRs are picking up what seems to be low copy numbers. Maybe SV40 is there. Is there additional data that someone can point to suggest that these viruses are actually in the tumor cells?
MODERATOR FRIED: Michele?
DR. CARBONE: I'll answer your question. I'll show some in situ hybridizations tomorrow. I wouldn't say that it's so amazing that no more data are being presented because again, I don't want to act like I am baby. But we have been working with no money, and when you work with no money you can't expect too much. And actually I think that working with a little amount of money that we're working, we have produced a lot of results.
The other point is that here, everybody seems very concerned about these 40 cycles. Now, I have to admit my guilt here that I've never tried 20 cycles. And the first thing that I'm going to do when I go back to the lab is to check what's going to happen if I do 20 cycles or 30 cycles. I didn't think that this was such a big issue.
The point was, if there is not there. Once something is -- if something is not there, I mean, if I take a negative sample I can amplify 100 times; still he would remain negative. So 40 cycles, I'm not sure that that's certainly the limit.
And for the question that the Doctor asked before, saying, you see that in immunohistochemistry, why you need to do 40 cycles? Probably for those samples I don't need to do 40 cycles; it's just the standardized thing. You have a number of samples, many of them will not look that good.
Of course, one shows slide that is the best slide is not going to come here and show that slide. So why show a sample that shows a lot of positive cells? And I'm sure -- not I'm sure -- I think it's a plausible question, it's possibility that if I go 20 cycles with that sample I'll get it.
MODERATOR FRIED: Okay, why don't you do 20 cycles and come back?
DR. CARBONE: I'll do.
MODERATOR FRIED: They're lining up on the microphone there first. Go ahead.
AUDIENCE PARTICIPANT: The primer pairs that have been used so far are very interesting and they are pairs for regions that are control regions of T-antigen and of the enhancer/promoter region, which interact with cellular components and are likely to have cellular analogs.
It would be interesting and I think increase my confidence in the data, if you use sequences to viral structural proteins like VP1, which would not have cellular homologs and which would still be very good at detecting BK, JC, and SV40.
DR. BUTEL: We did VP1.
MODERATOR FRIED: Yes, there may not be some conserved, so you don't really know --
DR. GARCEA: I would love to find the cellular homolog to the Rb binding pocket of SV40. I'd switch my projects over.
DR. LEDNICKY: I think he raises a very important point. And actually, we would also like to do more of those studies but there is a problem; there's only one strain of SV40 that's been fully sequenced, and we need to increase the database -- our lab's beginning to do this.
We don't know, for example, that there aren't other serotypes of DNA, and for people who are looking at antibody reactions there might be something we're missing, for example. But that's a very intriguing point.
MODERATOR FRIED: Hopefully, with John's long PCR, then you'll come around and go through both the control region and the viral protein, so satisfy everybody. Bob?
AUDIENCE PARTICIPANT: Two trivial questions --
DR. GARCEA: One thing I want to point out about that. I mean, I found that one of the most striking results -- I mean, I'm a complete skeptic, and it's just surprises that make me less skeptical. But one of the biggest surprises was finding 172 base pair repeat. That is simply diagnostic of a virus that's come very soon out of an animal. I mean, Ron deRogers has shown that. So I think that that is a very --
MODERATOR FRIED: But on the other hand, Michele found two 72 base pair repeats.
DR. BUTEL: I wanted to respond to Howard's comment though, that there is very little new information here. I would disagree with that. We took a different approach in our study instead of just continuing to look at more and more samples.
And that was to try to look very carefully at the sequences that were being detected, because we too, were very concerned about whether there was some odd, contaminant that was being picked up that was slipping in from somewhere -- even though we're very careful to always do negative controls and we set up the experiments in different room and do all those kinds of things.
But I think the bottom line is, when you sequence and you find one 72 base pair repeat, and we don't have anything in the lab like that, and the variability that has been discovered at the end of the T-antigen gene which doesn't correspond to any laboratory viruses or to the sequences that we had found in the brain tumors -- we found different sequences in the few osteosarcomas that we looked at.
And so I think that is new information and it says that there are -- in my opinion it suggests that there are different strains out there that are somehow or another, present in the tumors that are being examined.
MODERATOR FRIED: But we're limited by our primers of what we're going to detect. I mean, if we don't have the right primers we're not going to see --
DR. BUTEL: There are going to be other things that are not being detected --
DR. GARCEA: One more quick thing before -- I'm sorry -- because Janet failed to mention it in her talk. When we gave her these samples to transfect into cells, they were all blinded, and only sample number 12 gave a virus out. When we decoded those samples, samples one through 11 were from paraffin block specimens. Sample number 12 was the only fresh tumor specimen. I just want to point that out.
MODERATOR FRIED: Okay. Bob?
AUDIENCE PARTICIPANT: Two trivial questions. One is, why don't you throw in a set of primers totally unrelated -- say, hemoglobin primers -- and see whether or not in the same reaction -- in the same reaction, so you always have within the same reaction, you know your PCR reactions work. Number 40 doesn't bother me in the slightest. I've seen coli, repeatedly giving a negative result when the sequence is there.
So that way you'd have an internal control. In every single one you get another -- you get a -- some other size.
MODERATOR FRIED: That was suggested. I mean, the people --
AUDIENCE PARTICIPANT: Yes, just throw them in the same --
MODERATOR FRIED: But I mean, there's always a chance that it comes from the operator, if you're looking for human. I think maybe what Ellen was suggesting, putting primers on blind pieces of coli --
AUDIENCE PARTICIPANT: Yes, but then you get the same problem with the contamination from the blind primers. You can do it either way. That's fine; yes, I agree.
The other thing is, in terms of knowing whether or not it's free DNA or not, why don't you use the complements of the same primers you've used and run them in the opposite direction? You'll get multiples of unit length SV40 if there's full length SV40, and you'll know.
MODERATOR FRIED: That's what John was saying.
DR. LEDNICKY: This is one reason we're trying long PCR. But keep in mind that when you're working with archival samples, there's a limit to the size of the DNA that you cam amplify, and standard textbooks will say, 500 base pairs. So we found this is true and in fact, our signals in general, decrease with respect to the size of the amplified product -- archival samples.
AUDIENCE PARTICIPANT: I would like to make a question to Dr. Shah about the extraction of the samples from mesothelioma. When we made the first experiments with Michele that had been published in Oncogene, we were using only fresh tumors.
Instead, when I went back in Italy and I start to look at the statistic that is in paraffin-embedded tissue, we had a lot of difficulties and it took a while to sort out why the first screen we had positivity and the second screen we had negativity of the same samples.
And it came out that it was crucial for us, how long you take your sample after extraction. This maybe, was just a problem in our lab, maybe. The DNA was not completely destroyed so we were getting results after extraction but not later on.
But I would ask you if you are sure that this could not affect your results?
DR. SHAH: We tested the specimens soon, very soon after the proteinase K selection; within one or two days.
AUDIENCE PARTICIPANT: Within one or two days? This is the problem. Within one -- two days we were not able to get the same quality of results.
DR. SHAH: I think it is quite true that if we have fresh tumor DNA we would have a better chance of finding something which is already there. We have the controls for the globin amplification, and we used this thing very extensively in many other studies. So this is not the first time that we were doing this.
AUDIENCE PARTICIPANT: Sure. However, I would like to point out that the control -- also we are running the same controls, but these controls are not the best we can get because you know, you are comparing genomic with maybe episomic material.
MODERATOR FRIED: I don't know about these things, but do you need to use archival DNA? I mean, if you know what tumors you really want to look at, are they not able to that fresh anymore?
DR. SHAH: I think it would be wonderful to take fresh tissues; there's no question.
DR. GOEDERT: Jim Goedert from NCI. I'll comment on that. The tumors you're talking about are extraordinarily rare. I mean, they're going to be hard to find except at very major places where the lab is close to the clinic.
I actually wanted to raise a question about specificity. I was very impressed by the sequence data that Dr. Butel had presented and others, and I think that's usually considered the gold standard.
But I wanted to ask the panel members or others in the audience, whether they thought there was a possibility that artifacts, either in the amplification process or actually in the sequencing, could draw some question as to the specificity of those results?
DR. BUTEL: Let me answer. One reason, I don't think that there's a big problem with PCR artifact. If you consider the brain tumor sample 12 where we had the information, say the T-antigen sequence based on what was in the tumor, and then after the virus was rescued and it was sequenced, the sequence for that part of the gene was exactly what had previously been determined in the tumor specimen.
So certainly there is an example of where there's no PCR artifact involved. And it would seem that there would be artifacts popping up in the other gene regions as well, and there's not been any variation found in the fragment of VP1 that's been amplified, for example, or any changes in the Rb domain.
MODERATOR FRIED: So there's no PCR sequence -- I mean, could you say all your sequence differences are due to --
DR. LEDNICKY: That's a question we get asked a lot. And this concern -- people shouldn't be overly concerned with this in that, yes it's true. If you clone the DNA that you PCR amplify and then sequence that, certainly you'll see spots where you'll have possibly artifactually-induced changes.
But if you do a direct sequencing reaction on the primary PCR band and run that out in a gel, you'll pretty much be able to tell what the sequences -- you might see small bands showing up occasionally where probably there was exactly that -- the change at certain sites.
DR. WEBER: Thomas Weber, Hamburg. Maybe I may lead you back to the question of quality control. Under the auspices of the European Union, we have done a quality control study on the amplification of JC virus from one biological fluid, which is CSF which may be different.
These samples were sent out to nine laboratories throughout Europe and it didn't matter what kind of extraction method the laboratories used, it didn't matter what time it took from the central laboratory in England to come to the receiving laboratory, whether or not the colleagues detected the DNA or not.
What came out basically is, like in your report, that using primers centering around the T region, you are about by a factor of ten to 100 more sensitive than taking from the late gene region. That was the down to earth message.
So I can strongly encourage and urge you to develop a quality control panel for paraffin-embedded sections, and I think once you have established that, you should go out and do sequencing, not jump ahead or do sequencing first before you haven't done the quality control studies.
MODERATOR FRIED: You said you send them out to nine different labs?
DR. WEBER: Nine different laboratories --
MODERATOR FRIED: Was it consistent -- the results?
DR. WEBER: The results were consistent except for one laboratory that detected JC viral DNA in every sample and the dilutions were like, from one million copies per hundred microliters, to .001 copies. And they detected it everywhere. So that one laboratory had a contamination problem. The other eight laboratories detected between one and hundred copies per hundred microliter of the sample, or ten microliters of their reaction.
MODERATOR FRIED: Somebody in the back?
DR. LOWE-FISHER: My name is Barbara Lowe- Fisher and I'm cofounder and president of the National Vaccine Information Center, which for the past 15 years has been representing consumers who are concerned about vaccine safety.
Before I ask a question of Dr. Garcea I'd like to commend the organizers of this conference for bringing together independent researchers to talk about their meticulous research into the possible role of a monkey virus in human cancer. This is the kind of quality research that deserves recognition and priority funding because it could someday lead to more effective cancer therapies.
I'd also like to say that parents across America are contacting our organization and they are not as concerned about whether or not you've proven, beyond a shadow of a doubt, that monkey viruses do cause cancer or other problems in humans.
What they're concerned about is that monkey viruses were present in polio vaccines in the past and that no one knew, and that today monkeys are still being used to produce vaccines and it's still not known whether or not there are monkey viruses in them that you have not yet -- don't have the technology to detect. So parents are most interested in using vaccines that do not use monkeys for production.
And this bring me to the question for
Dr. Garcea. How many parents of young children with cancerous tumors that have SV40 in them, how many of these parents have been tested for the presence of SV40 in their bodies?
DR. GARCEA: I don't quite know what you're asking. I mean, when we did the original study, we did not -- because of IRB regulations, decode and go back to the parents of these families and talk to them. So what you're asking is, subsequent to the study, have we analyzed other tumors that we've received because of this, and talked to the parents? Is that what you're saying?
DR. LOWE-FISHER: No. Wouldn't it be interesting to know if, in these children -- these very young children who would not have received the vaccines that contained SV40 -- wouldn't it be interesting to know whether or not their parents are carrying SV40 --
DR. GARCEA: I think it would be a very interesting study, and as a part of a prospective study in looking at this, I think that that would be part of a sero-epidemiological study that you could do prospectively. But retrospectively, we can't do that, unfortunately, right now.
DR. LOWE-FISHER: It would also be interesting to go back to the contaminated vaccines and PCR off the virus that's in them, and then compare the sequence that the sequence people are finding.
DR. GARCEA: But let me just comment on your -- we can't do that because -- I would just mention, for the past seven years we have not had any money to do any of these experiments.
DR. LOWE-FISHER: Well, we are hoping that this kind of research by independent scientists will get the funding it deserves because the public is most interested. And this is fine science and we're very interested in supporting that research.
MODERATOR FRIED: Thank you.
DR. LEDNICKY: Can I make a comment on that? This might be a little speculative but, the problem is it could also be that SV40 was always a human virus. It may have been in the human population a long time. And if you speculate and say, maybe SV40 was around much longer than JC or BK virus -- because lower primates predate humans -- maybe it's had a long time to adopt to humans.
So it could be that it's in humans and just because some of us detect it in tumors, we need to prove that it's causing the tumors -- actually, one possible interpretation is, if someone has a tumor they might have an immune problem -- maybe immunosurveillance isn't cutting down an SV40 and other papomaviruses, and then so what we're detecting is circulating SV40.
MR. KYLE: Could I comment, please? My name is Walter Kyle. I'm an attorney from Hingham, Massachusetts. I've been doing polio vaccine litigation for 25 years -- or 20 years, I should say -- primarily on behalf of plaintiffs.
I know very little about genes; it's hard for me to distinguish them from a pair of levi's, but I do know that I have records going back into the late '50s, testimony before Congress, when Dr. Roderick Murray headed the Division of Biological Standards, in which he testified that no SV40 was ever found in inactivated polio vaccines.
However, at the same time, in the same period of time at NIH in transcripts, Dr. Murray commented that it was entirely acceptable for SV40 to be present in SIV. I have both transcripts. Dr. Murray continued with this type of regulation of the polio vaccines.
In 1968 after the discovery of the Marburg virus in the oral vaccine, he met with Lederle Labs and the same discussion was held. Yes, it's okay if you have these viruses in oral preparations because there's no evidence that it causes any harm.
I think we've now come across the evidence that these viruses have caused harm. I think it was disingenuous for Dr. Murray to testify under oath in a prepared statement before Congress, something contrary to what he told his colleagues at NIH.
I also would have a fundamental objection to the premise at this conference, that vaccines were clear of SV40 after 1963. You all know and I know, that every seed lot of Sabin vaccine is contaminated with SV40. What has occurred in the first production step is that you neutralize it with an anti-serum.
Which leads you to the question of, how much is neutralized and how many people have gone back and looked at the harvest fluids in that first manufacturing step, to determine -- maybe if you had something creep through, something like JVC. The initial reports of progressive multifocal leukoencephalopathy found that two people that had it had only been exposed to oral polio vaccine.
So to this day we have seed strains of the oral vaccine contaminated with SV40, and I don't think they changed their production methods in the early '60s for the IPV. There was no cutoff date, you didn't hear anybody at NIH come forward and say, we recalled that SV40 vaccine. It was not recalled.
And I don't think anything was done. Murray testified before Congress that nothing needed to be done, because by the inactivation methods in effect at the time, that there was no SV40 out in the vaccine. And I don't think that's true, and the people that are familiar with this issue know that it's not true also. Dr. Shah pointed that out this morning.
MODERATOR FRIED: Thank you for your thoughts. Ethel?
DR. FANNING: I apologize for coming back to the papilloma viruses every time but I think maybe if some of you can learn a few things of all our traumatic experiences over the years, because we've gone through many of these discussions many years ago as well.
We have looked at many archival specimens and what we see is that even -- it doesn't matter how these tumors were fixed; we have even degraded DNA going down to 100 base pairs -- but we can still amplify viral sequences up to 600/700 base pairs. Which means that these viruses are very, very resistant, and apparently polyoma is not much different.
So that the method of fixation does not influence the stability of the virus. You still have viral particles in these tumors from which you can extract the DNA later on. So I tend to disagree with that point for the polyomaviruses.
The other thing is about integration or episomal. In the cervical carcinomas that have been looked at, the majority of tumors have been looked at in the L1 region, also viral capsid protein, and in the majority of those tumors these viruses are integrated. And with the L1 primers the majority of the cervical carcinomas do contain papilloma virus DNA.
So I think that should not make too much of a difference even if we don't know whether it's integrated in these tumors or not at this stage.
And the third point that I just want to make is, if you're having a quality control, what you should maybe look at is where these tumors are coming from. If you're doing it from archival smears, how are you making those sections? Are you cleaning every little brush and are you using new blades between cutting every sample?
It's not enough to just have an empty, sort of an odd slice in between. You have to clean everything from the beginning; the whole machine between tumors. The other thing is that we've had the experience that, in three cases we've received from three different clinics, batches of tumors which contained the same, sort of in the -- one batch would contain the same HPV type throughout the tumors, although they were completely different types of tumors.
So in other words, in handling these tumors in the clinic, dividing it or sending it or packing it or whatever, it was contaminated during this process, and it was not contamination in the laboratory. So these are maybe things that one should keep in mind.
MODERATOR FRIED: Thank you. John?
DR. BERGSAGEL: John Bergsagel from Atlanta. I would agree with several of the panel members who have said that -- PCR in my opinion, doesn't prove anything. It's a screening method and you have to do something else to prove that what you found is what you think you found.
But inasmuch as PCR is a useful technique for screening these extremely rare tumors, wouldn't it be useful to look at the animal models for these tumors if the real question is, whether SV40 causes these tumors, such as choroid plexus papillomas and carcinomas from hamsters and mice?
DR. CARBONE: SV40 does cause exactly these tumors in hamsters.
DR. BERGSAGEL: Yes, but if you use the exact same techniques, PCR amplification of fresh -- and even more importantly, formalin fixed and paraffin-embedded tumors from hamsters -- would you get the exact same results that we get from humans?
DR. CARBONE: We used the hamsters as causative controls in our experiments, so the answer is yes.
DR. BERGSAGEL: And the materials were handled in exactly the same way?
DR. CARBONE: No.
DR. BERGSAGEL: In other words, paraffin-embedded --
DR. CARBONE: No -- well, depends from what point. Obviously, the humans come from a surgery room, and the animals come from another route.
DR. BERGSAGEL: Right, but if you took the animal's tumor and formalin fixed it and paraffin-embedded it to prepare your DNA as a control, and from multiple animals instead of just one which is known to be positive?
DR. CARBONE: That's what we do. That's what we use. Of course, multiple animals -- a number of animals -- it depends what "multiple" means. But that's what we do. We use, for our experiment, hamster mesothelioma, SV40-induced hamster mesothelioma, and for our bone tumor experiment, SV40-induced hamster bone tumors.
And we are very aware of the risk that there is when you use a microtome, when you're cutting this paraffin-embedded section and we certainly change blades, we change gloves, we clean everything, and then we start over again. That means, that takes a long time.
MODERATOR FRIED: Okay. Go ahead.
AUDIENCE PARTICIPANT: I think it's terribly, terribly important to stress for the two speakers before -- the lawyer and the representative of the public-at-large -- that not one of the speakers here today -- every one of them has been exquisitely careful not to claim causality. So please do not extract from what has been said, that it has been proven that SV40 is a cause of these tumors.
MODERATOR FRIED: I think we all would agree with that.
AUDIENCE PARTICIPANT: I'd just like to say something here. I was trying to be very careful to also say that the public is not as concerned about the fact whether or not you have proven beyond a shadow-of-a-doubt that there is causation. What they're concerned about is the fact that monkey viruses were in polio vaccine in the past; that we still perhaps, do not have the technology to totally guarantee they are not currently in the vaccines, and that they are concerned about the continued use of monkeys in the production of vaccines. So I just want to make clear that I wasn't implying that you had already come to the conclusion that there was causation.
MODERATOR FRIED: Okay. Thank you very much.
AUDIENCE PARTICIPANT: I'd also like to echo the comments of the former speaker here, that we haven't talked about causality. But I'd also like to emphasize what John Lednicky said, and as a person who deals with mesothelioma his points about basic immunosuppression are incredibly important.
I mean, we know that these patients who are exposed to asbestos have T-cell subsets that just don't work well. We know that asbestos causes certain changes in their basic immune system that's going to make them functionally immunosuppressed.
So I don't think we can say, wherever the T-antigen is from that that is it, that is complete. And I personally feel in dealing with these patients, that it is a complex intermix of whatever's going on, independent of the T-antigen situation, that the patients to begin with have some functional deficit.
MODERATOR FRIED: Robin?
DR. WEISS: We'll get on to causality tomorrow, but I have a question for Allen Gibbs. He told us this morning that he has more than a thousand --
MODERATOR FRIED: Could you speak into the microphone?
DR. WEISS: Allen Gibbs mentioned this morning that he has more than a thousand mesotheliomas, this bountiful collection that Chris Wagner started. Do any of them go back earlier than 1955?
DR. GIBBS: No, the earliest are 1960's, I think -- 1961/62, that sort of period. But I think there is a location where there may be a few that are pre-1960, and possibly back to 1955.
MODERATOR FRIED: Because I think that's an important point, to look at things before the vaccines came about.
DR. GIBBS: I'd just like to emphasize that I, being a pathologist, actually think that the archival material has a lot to tell us, and that's why we need to employ these techniques only on the archival material. And I understand what the concerns are and why there's an enthusiasm for using fresh material.
But I think that if we all agree after a certain point in time, that the techniques are working and we are actually detecting SV40 virus, then it is important to exploit that archival material for the purposes of looking over different periods of time, and also looking at that proportion of mesotheliomas that we believe are reasonable evidence, and not asbestos-related.
AUDIENCE PARTICIPANT: That brings me to my second question -- just one comment. A little concerned that we should not be matching historic and archival specimens with the controls that have been drawn from somewhat similar groups: age, sex, occupation, and perhaps most importantly, immunization history. That's the comment, and whether that's right you'll tell me.
I'm glad that Allen raised the issue of non-asbestos versus asbestos. As far as I can tell, the mesos that have been discussed here have been entirely asbestos-related, or thought to be, is that correct? Anyone want to amplify that?
DR. GIBBS: Certainly in my group that is the situation, but this was very much a pilot study and like Michele, we did this without any money, basically. And in terms of the controls, we did use pleural-based adenocarcinomas and non-malignant pleurae.
The age ranges were similar but of course the mesotheliomas were dominated by asbestos exposure. But I think that's a study further down the line.
DR. GOEDERT: Jim Goedert from NCI. Howard Strickler and I have discussed a number of different epidemiologic studies and to answer Robin's question, there are in fact, resources of specimens from pre-1955 from the U.S. Armed Forces Institute of Pathology that we can delve into.
But you know, our priority was to try and come up with an adequately-sensitive and specific and reproducible assay before trying to delve into those specific epidemiologic questions. But I think the materials ought to be available and the controls, obviously, are critical in terms of how they would be matched.
MODERATOR FRIED: So they could be sent out to people here on the panel? Yes?
DR. RATNER: I'm Herbert Ratner, the former Health Officer of Oak Park, Illinois, and the announcement was made April the 12, 1955, Tommy Vance has reported that the vaccine was safe and effective. And within a few days the National Foundation had this vaccine -- I won't go into the past history of that vaccine -- but it was delivered throughout the United States so that every 1st and 2nd grader, as a free gift of that vaccine -- every 1st and 2nd grader -- and in the next week or two, that vaccine was given to every 1st and 2nd grader.
I think Oak Park was probably the only one who decided to sit down that free gift, vaccination gift, just to see how things were going along. There were other reasons, too. But I decided that before parents signed an authorization slip, which makes it possible to get the vaccine, that I should make available to them -- which I did in 11 talks that week -- be willing to answer questions that they had in terms of the risk of polio that summer, etc.
By just taking a neutral position at that time, you had all the pressure from the Foundation to get that vaccine going because of an impending summer polio epidemic -- the usual summer epidemic -- and that was the only thought in people's minds: how fast, how well do mothers love their children? They didn't rush to get the vaccine, and things like that.
And in the midst of my talks -- I had two days of my talks -- my community got very upset that where everybody else was giving the vaccine, we were holding out. And it caused quite a consternation in the Chicago area. It got to the science -- Art Snider who was the Science writer for one of the major newspapers -- he said Herb, what's going on there? I said, well come out and listen to my talk, etc.
I have the talk on Tuesday and Wednesday he called me up and said, you're more right than you know. Because they just got the first report of the Cutter vaccine situation where six cases in San Francisco and one in Chicago area, both from the same manufacturer, both from the same lot number, and we were in consternation three.
I had to postpone -- actually, I was about the only one in the country that was in a position of not having anybody in my community immunized, and so I could sit it out. And I made one appointment to use the vaccine, to give that, give to their parents -- one week later or two weeks later, whatever it was -- and after that, the Cutter situation got worse.
And the local paper, as a result, had a story, checked around, in which they thought I had a very unique opinion that I hadn't given the vaccine.
MODERATOR FRIED: I think we're going to discuss the vaccines more tomorrow. I mean, this is mainly for the techniques, so --
DR. RATNER: Can I have about a minute more?
MODERATOR FRIED: One minute.
DR. RATNER: Yes. Keep up my same thought. The day that the local paper came out with the backing of all of the -- everybody in the community, kind of -- Seeley, the Surgeon General, called up the program because he wanted to make a safe vaccine safer was his exact terms.
They had to stop that thing because of the difficulty of the vaccine. And if all of you knew the difficulties they had with the Salk vaccine, whose position on inactivation turned out to be false -- universally accepted as false -- and how they kept packing it up and packing it up and packing it up, and they had to keep the program going and going.
But I'm telling you that every 1st and 2nd grade child in the United States, which represented about 85 or 90 percent, got a vaccine which had live polio viruses in it, definitely established, and at that time they found out that the SV40 was --
MODERATOR FRIED: I --
DR. RATNER: Just one sentence, please. That the SV40 was not activated, and so that meant that there was SV40 in all of the vaccines around the country, and that was confirmed by -- this is my last sentence -- that was confirmed by anybody who focused epidemiologically. There were cases popping up all over the States -- and this was confirmed by the German Health Ministry who were doing the same thing in Germany -- that polio virus was being distributed. And if you people could see --
MODERATOR FRIED: I think I have to stop you, because we --
DR. RATNER: Could I just have a half-a-sentence?
MODERATOR FRIED: You've had a half-a-sentence.
DR. RATNER: If you people sit here and say that the vaccine didn't pass on polio or SV40, you don't know what happened in those times. And I'm talking about 1955, for the next ten years or more. It's strange to me, as an epidemiologist working right on the field, to hear people somehow deny the vaccine -- one more sentence, please.
Harry Francis was attacked right after his report --
MODERATOR FRIED: I think -- why don't you save this for tomorrow?
DR. RATNER: Okay.
DR. URNOVITZ: Hi, I'm Howard Urnovitz and I'm from Berkeley. That's the other coast. First let me thank the -- I want to say thank you to the FDA, NIH. I think it's a brave move to have us all come together; I think it's very productive.
I think everybody's going to work out false positive problems. You just send samples to each other and I think that's not going to be a problem. And Dr. Carbone shouldn't get rattled. There's a lot of us who believe that what you've done is a breakthrough, and most of you here, we're very excited about it.
I want to make a comment that the chimera thing is of interest to me; that Dr. Frisque had said with the SV40 and JC virus. Is that there were dozens of viruses in those preparations. I think it's -- this is an important first step to talk about SV40 because we know a lot about them and we could start this as a springboard.
I don't think anybody here would walk away saying there's a cause of cancer. It's probably multifactorial and we're looking at the components.
The question to the panel is, as you go forward building your primers and as you see there are certain primers well lighted up, is to be mindful of the fact that some of those might be other types of hybrids. Certainly we know about SV40 adenovirus, but there were also coxacky and other adenoviruses in there, there were herpes viruses in those preparations.
There may have been chimeras and those in themselves might be important too. So as you do your primer sets, has anybody looked at doing multiplex as the screen and then sequencing as the verification?
MODERATOR FRIED: Anybody want to take that? Janet?
DR. BUTEL: We haven't done that.
MODERATOR FRIED: Okay. I think we've run out of time. We've had a very fruitful and interesting discussion. I think people would agree that the techniques are getting down to detecting things now and maybe we can get a coded test panel of cells to go to the different people interested.
We obviously need the finances for this, and maybe people should be doing PCR of the vaccines to see exactly what strains were in that and how they match up to what people are finding; whether there's really an endogenous virus or it came from somewhere else.
Okay, thank you very much to all the panel members.
(Whereupon, the foregoing matter went off
the record at 3:55 p.m. and went back on
the record at 4:20 p.m.)
CHAIRMAN SNIDER: We're ready to start this last session. We're at perhaps, the most difficult part of the day, but I think one of the most important parts of the day.
This session is on human exposure to SV40. My name is Dixie Snider; I'm the Associate Director for Science at The Centers for Disease Control and Prevention in Atlanta. We too, are happy to be co-sponsoring this meeting and look forward to the rest of the meeting and to deliberating on the significance of the outcomes.
Our first speaker for this session is well-known to everyone in the vaccine field. It's Dr. Maurice Hilleman who is at the Merck Institute for Therapeutic Research. Dr. Hilleman.
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DR. HILLEMAN: Well thank you, Dr. Snider. Having been there, perhaps I can recite the history. The development of both killed and live poliomyelitis virus vaccines was at the pioneering forefront of what was to become a new golden age of vaccinology.
For polio virus vaccines, new technologies needed to be conceived and developed, and as might be expected, there were significant challenges which related mainly to whether the polio virus in killed vaccines was completely inactivated by formaldehyde and whether the virus in live virus vaccines was underattenuated and caused poliomyelitis in human beings.
Adding to these complexities, both kinds of vaccines depended upon polio virus propagation and Maitland-type minced renal tissues of monkeys, or in cell cultures of monkey kidney. Both cultures, it was later to be determined, were commonly infected with any of more than 40 different indigenous viruses of monkeys.
The most commonly used monkeys were the Macacus rhesus and the Macacus cynomolgus species. Now, as part of the requirements for killed polio virus vaccines promulgated by the NIH's Division of Biologic Standards, later named the Bureau of Biologics, it was necessary to demonstrate the inactivation of all detectable viruses.
Live polio virus vaccine, by contrast, followed different rules that required the cell cultures to be free of known viruses from the start. All manufacturers who distributed polio vaccine in the United States were required to meet the U.S. standards.
Well, the prevalence of contagious viral infections in Macacus monkeys was vastly amplified by the shipping and caging conditions which were standard at the time, including necessary contact between animals which occurred during transport, on holding at airports, or on housing at the final destination.
The modes of viral transmission between monkeys were possibly by the respiratory route or by ingestion of monkey urine or maybe even feces containing the agent.
Well, the discovery of SV40 virus was born of change and serendipity. An urgent need for monkeys for research and development of other live virus vaccines led to a search for monkeys with as few wild virus infections as possible. This caused the speaker to consult Dr. William Mann who was then Director of the National Zoological Park in Washington, D.C., for advice on how to capture and transport monkeys with the least chance for virus exposure.
Well, Dr. Mann advised that African Green monkeys, that is, cercopithecus aethiops, could be caught in West Africa, transloaded at Madrid where there was no traffic and non-human primates, and then transported to New York and on to our laboratories. Heeding Dr. Mann's advice, these monkeys were obtained and they provided a source for kidneys.
Well, most surprising, the cercopithecus cultures showed remarkable capability for propagation with cytopathic change of a little of a hitherto unknown, indigenous, Macacus virus that was otherwise undetectable at that particular time.
Now, this virus was noted to produce vacuolar, cytopathic changes in the cytoplasm of cercopithecus renal cultures in culture. It was called a vacuolating agent and it was later renamed simian virus 40, or SV40.
Preliminary findings were presented at the June 1960 meeting of the Second International Live Poliomyelitis Vaccine Conference, which was held under the sponsorship of the Sister Elizabeth Kennedy Foundation, at the Pan American Health Organization headquartered in Washington, D.C. Thereafter, studies of the SV40 virus were continued, both in our laboratories and elsewhere.
The SV40 virus was reported at the meeting to be a hitherto, unknown agent whose small size -- and was cytopathic for cercopithecus kidney cells. By contrast, it caused only an inapparent, non-cytopathic infection in primary Macacus kidney, and in primary or continuous passage human cells. All isolates that were examined were antigenically homogeneous as determined in serum-neutralization tests.
It was reported at the June 1960, meeting, that cercopithecus kidney cells in culture were nearly always free of SV40 virus, but cultures of Macacus monkey kidneys, Sabin live polio viruses, and seed stocks of viruses used to prepare experimental killed adenovirus vaccine were found to contain the virus.
At the same meeting it was reported that cercopithecus monkeys were free of SV40 antibody, but that sera from most Macacus monkeys were positive. More than half of all the sera from the human recipients included in our study who had received killed Salk or adenovirus vaccines that had been prepared using virus grown in Macacus cell cultures, were positive.
The antiviral antibodies that were demonstrated in the sera of recipients of the killed Salk and adenovirus vaccines were appropriately interpreted as having been induced by the inactivated SV40 virus that was present in the preparations.
Recipients of Sabin vaccine -- that's the live vaccine -- were devoid of antibody even though it was shown later by others that the SV40 virus infects the human gut and is excreted in the feces, with probably lack however, of significant, systemic viral infection.
Well, at the time of that June meeting the vacuolating virus appeared to be of essentially universal presence in Macacus Rhesus monkey kidney cell cultures, frequently present in Macacus cynomolgus kidney cultures, and relatively rare in African Green monkey kidney cultures.
The new virus appeared different from other known monkey viruses such as those described by Hull, because of the distinctive vacuolating type of cytopathic change seen in infected cercopithecus kidney cell cultures. Failure of the vacuolating virus to cause cytopathic changes in Rhesus or cynomolgus monkey kidney cell cultures was a hallmark for the vacuolating agent.
Resistance of the virus to ether and failure of hemagglutination and hemabsorption such as shown by the mix of viruses were also distinguishing characteristics. The vacuolating virus appeared to be just one more of the troublesome simian agents to be screened for and eliminated from, virus seed stocks and from live virus vaccines.
Lack of antibody response in human subjects who were fed live polio vaccines containing the vacuolating agent, suggested the lack of substantive proliferations of this semi-permissive virus in the human being under the conditions employed.
Well, discovery of the SV40 virus was possible only after a cell culture system was available that would detect its presence. And that's important. The detection in Green Monkey kidney culture of this inapparent virus infection of Rhesus and cynomolgus monkey kidneys represented the first instance of demonstration of a non-detectible, indigenous monkey virus using a monkey renal cell culture.
Then in September of 1960 the inactivation kinetics of the vacuolating virus using one to 4,000 formalin at 37 degrees -- the conditions used to inactivate polio virus vaccine -- were described.
Inactivation of SV40 virus having a rate constant similar to that of poliomyelitis virus, was observed. Under the conditions used in this study, our testing indicated that the vacuolating virus was destroyed during the polio virus inactivation process.
The optimal solution to the live virus vaccine problem however, appeared to lie in total elimination of the virus from the production system as soon as possible.
In 1961, when SV40 virus of higher infectivity titer was available, and when more sensitive tests for its detection were developed, a new and unique pattern for its formaldehyde inactivation kinets were found, as shown here in red. These studies disclosed an asymptotic relationship in the inactivation curve after about 99.99/100th's percent -- that would be 4 logs to the base of 10 -- of the virus had been killed.
Virus that was subcultured from the plateau portion of the curve showed the same inactivation pattern as the original. Just why approximately one in 10,000 SV40 virus particles are refractory to inactivation by formaldehyde has been an enigma for more than three decades.
It is now known, however, that the closed, double-stranded circle of the SV40 viral DNA genome is super coiled, but that a single break or a nick in one strand of the double strand gives a relaxed ring. A double break gives a linear double strand.
Well, completely double-stranded DNA provides no exposure of immuno or amino groups with which formaldehyde can react. This might give an explanation for the means by which the chance presence of a single, resistant virus particle in every 10,000 SV40 virus particles can escape inactivation.
Reports to the Division of Biologic Standards of survival of this very small fraction of SV40 virus, led the Division to require demonstration of freedom from detectable, live virus -- SV40 live virus -- in the final product when a volume of 500 doses of finished vaccine per lot was tested in cercopithecus renal cell cultures.
As part of our studies to characterize SV40 virus, newborn hamsters had been inoculated subcutaneously and intracerebrally with live SV40 virus to test for possible oncogenicity such as had been shown for SE polyomavirus of mice.
Hamsters that are less than 24 hours of age have a relatively deficient immune system and provide an in vivo animal model to study viral oncogenesis; albeit, without it having any known or established relevance to the human species. And I would emphasize that.
In the test, mildly invasive fibromatous tumors appeared after five to ten months in nearly all hamsters given massive doses of SV40 virus. Now, this was 320,000 50 percent tissue culture, infectious doses per hamster -- a huge dose.
Tumors did not appear in appropriate placebo controls. The tumors were transplantable to new animals and markers for SV40 virus were shown present in the tumors by specific virus recovery and by immunofluorescent identification of the T-antigen.
Well, it's notable that SV40 virus tumorigenicity in hamsters is highly dose-dependent, and that no tumors appeared following injection of less than 1,000 tissue culture doses of the virus. It was shown also that SV40 virus tumor appearance was highly diminished when non-replicable, whole, cobalt-irradiated SV40 tumor cells were given prior to or as late as, 76 days following injection of the homologous virus into newborn hamsters.
This anti-cancer vaccine proved to be both prophylactic and therapeutic. It was a new principle. The appearance of tumors in hamsters inoculated with SV40 virus gave an explanation for the findings by Eddy that injection of extracts of ground, primary cell cultures of Macacus monkey kidney-induced tumors in newborn hamsters.
For want of detection of any oncogenic stimulator, Eddy referred to the tumor-inducing entity as an oncogenic substance. In a later publication, after SV40 had been discovered, Eddy reported isolation of SV40 virus in cercopithecus cells from the same monkey kidney preparations used in our earlier study.
Well, while the studies at Merck were in progress, the early results of the neonatal hamster tumorigenicity tests were reported by us to the division of biologic standards and in turn, to the technical committee on poliomyelitis vaccine.
This committee was a group of leading scientists who served as polio virus vaccine advisors to the U.S. Public Health Service. The division and the committee had previously received reports of possible, live, SV40 virus in commercial, killed, polio virus vaccine.
The view of both the division and the technical committee was that no untoward effects in human subjects could be attributed to the agent. They also concluded that there was no evidence that the small amount -- very small amount -- of live, SV40 virus which also was subsequently determined to be only semi-permissive for man, was capable of producing disease in human beings when introduced subcutaneously or intramuscularly in a formalinized vaccine.
Further, the committee stated that although the presence of the vacuolating virus in the killed vaccine does not prevent the development of immunity against polio in vaccinated persons. The elimination during the process of manufacturing polio vaccine would constitute another step in the continued improvement in the potency and the purity of the product.
Well, by late summer of 1962, the Division of Biologic Standards recommended that all pools of polio virus and adenovirus be shown free of SV40 prior to the addition of formaldehyde. SV40 virus-free pools were made a requirement early in 1963, but by that time, you know, all three serotypes of Sabin live polio vaccine had been licensed by the Division for use in the United States.
The Sabin live virus vaccine was readily accepted by the physicians and public health practitioners because of the simplicity by which it could be administered orally. Salk vaccine use was diminished and it almost disappeared.
Well, now in closing, I think it's worthy to note that within a relatively short period of time following the discovery of SV40 virus, the agent had been found present in poliomyelitis vaccines, it had been shown to be incompletely inactivated by formaldehyde, and had been shown to be oncogenic when tested in newborn hamsters.
In another short time period, the methodologies for excluding SV40 virus were developed, validated, and ultimately utilized. And it was of importance that during the time period prior to licensure of the live Sabin vaccine, the Division of Biologic Standards had been able to clear sufficient Salk vaccine for distribution to allow the large poliomyelitis immunization campaign in the U.S.A. to continue without interruption.
Because of this, thousands of cases of poliomyelitis that would otherwise have occurred, were averted. Thank you.
CHAIRMAN SNIDER: Thank you very much, Dr. Hilleman for that excellent background and historical perspective. Our next speaker is Dr. Frank O'Neill from the VA Medical Center, Salt Lake City, Utah, who will speak on the host range analysis of SV40 and SV40/BK hybrid genomes and virus latency. Dr. O'Neill.
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DR. O'NEILL: First I'd like to thank Dr. Lewis and all the meeting organizers for inviting me to this meeting. One of the projects in my laboratory over the last several years has been an analysis of SV40 growth in a variety of human cell types. And we've tried to determine which cell types SV40 grows well in, and which cell types it does not. And in those cell types where SV40 grows poorly or slowly, what about the virus is causing this slow growth?
And I'd like to summarize our findings in the following points. One is that SV40 grows well in some human cells types. In cell types which it does not grow well in, like fibroblast and human embryonic kidney cells, this slow growth appears to be caused by some function of the SV40 late region, because when we replace the SV40 late region with the late region from BK virus or RF virus -- a variant of BK -- we now get rapid growth in human embryonic kidney cells and in fibroblast.
Finally, in fibroblasts then, in human embryonic kidney cells, wild type SV40 produces very small amounts of T-antigen but it produces very large amounts of the capsid protein, VP1. And in fact, there may be 150 times more VP1 than there is T-antigen. And this overexpression of the VP1 gene, or the late region, appears to inhibit T-antigen production.
So this is an outline of the talk. There are three kinds of theoretical growth patterns of SV40 in cells: semi-permissive cells where there's very slow growth and not much virus produced -- and very little cell killing also; fully permissive cells like simian cells, CV1 monkey cells which virus grows rapidly and it kills almost all the cells; and there may be totally non-permissive cells. There may be some human cell types that are totally non-permissible.
There may be very little T-antigen expression that has been reported previously, but I'd like to qualify that and say that, in some of these studies that showed no T-antigen production in human embryonic kidney cells and in fibroblast, a lot of those plasmids had the viral DNA still covalently linked to the plasmid. And we've shown recently that plasma DNA strongly interferes with the expression of the T-antigen gene in human cells.
And as I mentioned earlier, point 3 here, the mechanisms of growth for slow growth in human embryonic kidney cells, appears to be the SV40 late region. And I also have some experiments about viral latency but it's highly unlikely I'll have time to get into that.
So these are some of the features that are the growth of SV40 in human cells: human embryonic kidney cells and fibroblasts. Only about 20 percent of the cells initially appear to be infected. And as I mentioned, little T-antigen is produced and ultimately the cells become morphologically transformed.
So we went on and started to analyze a variety of human cells types to see if SV40 would grow in other cell types besides fibroblasts and human embryonic kidney cells. And you can see on the first line we have monkey kidney cells, BSE1, TC7, and CV1s, and growth is optimal in those cell types.
But as I mentioned, HFF fibroblasts and HEK cells, the virus grows poorly or slowly. But then we looked at some neural cells, neuroblastomas; the virus seemed to grow fairly well. But after a couple of rounds of the replication cycle of the virus, the cells become resistant.
In two glioblastomas, A172 and A182, SV40 seems to grow quite well. In a lung cancer cell line, AT357, SV40 grows very well. It grows as well in those cells as it does in simian cells. And in two rhabdomyosarcoma cell lines, again, SV40 grows very well. And one renal carcinoma cell line, SV40 grows quite well also. So there's a variety of human tumors that support lytic infection by SV40.
And on the second line you'll see fetal brain cells. Fetal brain cells that are rich in spongioblasts support rapid growth by SV40. SV40 grows as well in those cells as it does in Green Monkey kidney cells.
Now, one of the things that has been indicated previously, that in human embryonic kidney cells and fibroblast there is very poor growth of SV40. And we agree with that unless you let the cells -- unless you maintain the cell cultures for long periods of time. If you harvest the cells to extract viral DNA within a week to three weeks after infection, you find very little DNA, and those experiments appear in lanes 3, 4, and 5.
Lane 1 is the amount of DNA that you would see classically, after extraction of infected monkey cells. But if you let the human cells that have been infected, you maintain them in culture for at least six weeks, you then see a lot of viral DNA. And that's Lane 6. There's as much viral DNA from those cells as you would get in a lytic infection of monkey cells.
In lanes 7, 8, and 9 are BK virus infection of human embryonic kidney cells or fibroblasts, and BK virus of course, grows well in both of those cell types -- those cell types that allow SV40 to grow only slowly.
So SV40 will grow in these so-called semi-permissive cells. If you wait long enough you can observe that good growth.
Now, one of the things that I want to mention about SV40 growth and neural cells, like spongioblast and some glioblastomas, is that the virus makes a lot of mistakes. When you isolate the DNA, even after you've started an infection with plat purified virus or molecularly cloned viral DNA, you see a lot of defective, interfering viral DNA particles.
And when you analyze even those particles that don't seem to be defective, you can see a lot of mutations. You see rearrangements in the regulatory region, in the 72 base pair repeats, and in sequences between the 72 base pair repeats and the beginning of the VP2 gene.
We also see mutations, deletions, base substitutions and insertions at the 3-prime end of the T-antigen gene, and the 3-prime end of the VP1 gene.
Another thing that we see in neural cells is that the viral DNA seems to split. Instead of having all the viral sequences necessary for an infection in one molecule, we see the viral DNA sequences split into two, complementing, defective molecules -- like on this slide.
And the circle on the left, that's a genome that contains just a T-antigen gene; the late region has been deleted. On the genome circle on the right we see the late region that has all the capsid genes, but the T-antigen gene has been deleted from that.
Both of these molecules, when introduced together, will produced a lytic infection, and they have the same host range as wild type SV40. Now, there are similar viruses that have been described for JC and for BK. Two BK variants called RF and MG, have the same genome organization and they show this genome organization isolated directly from the patients.
So one of the things that we wanted to do was determine what causes slow growth of SV40 in fibroblast and human embryonic kidney cells? And we know that in those cell types, BK and the RF variant of BK grow quite well. So what we did was, make reassortments of viral genomes using early SV40 and late RF, or early RF and late SV40; a variety of combinations between SV40 and BK or SV40 and JC.
And what we found initially was that every time we had the SV40 late region complementing BK or JC, virus growth was very poor, very slow. But in cases where we had late BK complementing early SV40, virus growth was rapid. Those hybrid viruses appeared to grow almost as well as BK or RF did in human fibroblast and kidney cells.
So that suggested that there was something in the SV40 late region which was restricting growth. What we found when we do this experiment -- if you will assume that that left circle is early SV40 and the right circle is late RF -- what we find is that there's always recombination between SV40 and RF such that the RF genome acquires an SV40 regulatory region, and that always happens every time we do the experiment.
One of these variants of late RF that has an SV40 regulatory region is called clone-H. and we decided to determine if clone-H could stimulate the growth of wild type SV40 in human fibroblast. So we introduced both clone-H and wild type SV40 -- and that's a map for wild type SV40 -- and to human fibroblast.
So what we did is, we introduced both viral genomes into human fibroblasts and the virus growth was very slow. But when we analyzed the viral DNA after the first passage, we could see very little wild type SV40 DNA. When we took that lysate and passed it several more times, the wild type SV40 DNA had totally disappeared and it was replaced by a variant of SV40 that had only the early region in it; the late region was deleted.
So late RF could not help -- late RF clone-H could not help wild type SV40 grow in human cells. The SV that did grow had lost the late region, suggesting that there was something in the late region that had some cisinhibitory effect. So further evidence that something in the late region was inhibitory to growth.
The next thing we did -- so in lane 1 you can see the bottom band is late RF clone-H as to one passage. And lane 2 is after three passages, and that bright band is early SV40 that has lost the SV40 late region and it's now complemented by late RF clone-H.
So then we decided to ligate the late RF sequence to the early SV40 sequence to make a hybrid genome or a chimeric genome that had both DNAs in one circle. And that's shown in the bottom circle in this slide. And that virus, or that viral DNA, has the same phenotype as the other hybrids I've described.
This virus now grows in human cells and it also grows in monkey cells. So this virus with a chimeric genome grows as well in monkey kidney cells as it does in human embryonic kidney cells. So again, it looks like there's something in the SV40 late region which restricts growth in fibroblasts and in kidney cells.
So the next thing I'd like to address is, what do the proteins look like after you transfect or infect human cells with wild type SV40, early SV40 which has a deleted late region, or the chimeric genome?
And if you look at lanes 1 and 2, that's early SV40 DNA minus the late region in human cells for day 3 or day 6 in lane 2, and you see there's a fair amount of T-antigen. In lanes 3 and 4 is wild type SV40 and you see there's very little T-antigen at day 3, and at day 6 it's almost undetectable.
But if you look at the bottom of those lanes you'll see plenty of VP1. A lot more VP1 than T-antigen. In human embryonic kidney cells you get a similar result. You get plenty of T-antigen with just the early regions but very little T-antigen when you use wild type, but also a lot of VP1. So VP1, the late region appears to be overexpressed compared to T-antigen, and you can show that in northern blots.
When you use the chimeric genome, T-antigen is poorly expressed early, but after a few days you see plenty of T-antigen. Again, the late region is overexpressed.
And the same results appear on this slide, but in addition we show what kind of amounts of T-antigen are produced in monkey cells with early SV40 and with wild type SV40. Again, you can see that when the late region is present you get lots of VP1 and you inhibit expression of the T-antigen gene, so you get less T-antigen.
In the human cells, at days 3 and 6 and 10, you can see that with wild type, T-antigen starts to fall off, as it does also with early SV40. With wild type, after day 10 you start to see the reappearance of T-antigen and also more VP1.
So that by about six weeks after infection when the maximum amounts of viral DNA are present and almost all the cells are T-antigen positive, you see huge amounts of VP1 but still very small amounts of T-antigen. Much less T-antigen; there's about 150 times more VP1 than there is T-antigen.
In monkey cells as the infection progresses, you see more and more T-antigen and about ten times more VP1. So in human cells, VP1 is overexpressed about 150-fold and in monkey cells, VP1 is overexpressed about 10-fold. And that could have something to do with the slow growth of SV40 in human fibroblasts.
Now, this shows just a replication assay for wild type SV40 in human cells. What we've done here, in the odd numbered lanes we've -- after transfection for two or three days we isolate the DNA, cut it with an enzyme that linearizes the wild type DNA molecule. In the even-numbered lanes, after digestion with the enzyme that linearizes the molecule, we've digested also with MBO1 which cuts only the DNA which has become unmethylated because it's replicated.
And you can see if you look at all the even numbered lanes, that all of the DNA is digestible by MBO1 so the DNA has replicated. So even though very small amounts of T-antigen appear in human cells, enough T-antigen is present to allow the viral genomes to replicate.
So SV40 produces very small amounts of T-antigen in fibroblasts and in kidney cells, but it's enough T-antigen to replicate the viral genome efficiently, and it's enough T-antigen to cause the production of the VP1 and other late proteins.
So in summary, the poor growth, SV40 grows well in a variety of cells types and a variety of human tumor cells lines. In neural cells it makes a lot of mistakes; there's a lot of mutations in the viral genome, and fibroblasts and in kidney cells, the slow growth appears to be caused by the presence of the late region. You can aggregate that inhibition of cell growth by replacing the SV40 late region with that from BK virus or RF virus.
The actual sequences involved in the BK late region are being investigated. We'd like to see if it's actually the BK VP1 gene that's responsible for more rapid growth of the chimeric genomes in human cells.
Thank you very much.
CHAIRMAN SNIDER: Thank you, Dr. O'Neill, for helping us understand how growth is regulated. Our next presenter is one of the main organizers of this meeting, Dr. Andrew Lewis, from the Food and Drug Administration, who is going to speak on SV40 and adenovirus vaccines and adeno-SV40 recombinants. Dr. Lewis.
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DR. LEWIS: Thank you, Dr. Snider. Dr. Frisque and O'Neill raised the issue about recombinants and their possible role in SV40 as it might spread in the environment and in human population.
I'm going to talk about the possible role that adeno-SV40 hybrids might have in suggesting other but similar mechanisms, that SV40 could in fact, be an environment contaminant.
I thought I'd just begin my talk by describing what an adeno-SV40 hybrid, or recombinant is. And I think as you can see illustrated very simply in this figure, adeno-SV40 hybrid is formed when portions of the circular SV40 chromosome of about 5,000 base pairs are recombined with the adenochromosome which is about 35,000 base pairs. To accommodate packaging in an adenovirus capsid, recombinants between these chromosomes result in the deletions of segments of the adeno-DNA at the point where the SV40 DNA is inserted.
Adenoviruses cause colds, pneumonia, conjunctivitis, and acute respiratory disease at military installations. The discovery of adenoviruses by Rowe and Hubner and Dr. Hilleman in the early 1950s created an interest in the development of adenovirus vaccines. However, human adenoviruses only grow efficiently in human cells and the only human cells that were available in the mid-1950s for large-scale tissue culture, were derived from human tumors.
When confronted with the possibility that adenovirus vaccines would be prepared in human tumor cells, the decision was made that only normal cells could be used for vaccine development.
At this time, the polio vaccine were prepared in Rhesus monkey cells, and these vaccines had been developed and were being used. Given the use of normal Rhesus monkey kidney cells to produce polio vaccines, it seemed reasonable to try to adopt adenoviruses to grow in Rhesus cells for vaccine production as well.
The first seven adenovirus serotypes were adopted by Hartley and Hilleman to grow in Rhesus monkey cells. When these monkey-adapted vaccine strains formed, an inactivated adenovirus types 3, 4, and 7 vaccine were prepared and studied in the military recruits between 1957 and 1960.
Following the discovery of SV40 in these vaccines in 1960 as described by Dr. Hilleman, the SV40 contaminant was removed from the adeno-3 and the adeno-7 vaccines by antibody treatment. However, SV40 could not be eliminated from the adenovirus 4 vaccine stock.
The discovery of the adeno-7 SV40 hybrids in the adeno-7 vaccine strain by Hubner and others in 1963, prompted us to look for adeno-SV40 hybrids in the other adeno-7 on the other adeno strains that had been adapted to grow in Rhesus monkey kidney cells.
And the outcome of this study are presented in the next two slides. Could I have the slide on the right, first, and on the left as well? The second slide on the right, please.
After multiple patches of these monkeys -- I'll refer you to Table 1 -- after multiple patches of these monkey kidney-adapted adenoviruses with SV40-neutralizing antibody, the viruses were then patched without antibody and tested for the presence of infectious SV40 virions.
As you can see from the Table 1, the adeno-1 and adeno-3 were free of SV40 in this assays, while the adeno-2, adeno-4, adeno-5 serotypes contained infectious SV40 -- in spite of treatment with concentrations of SV40 antibody that were adequate to remove SV40 from the monkey adapter strains of adeno-1 and adeno-3.
As you can see in Table 2 on the left, whether they contained SV40 virions or not, each of these monkey-adapted adenoviruses induced SV40 T-antigen during infection in human kidney cells. The ability of the virus to induce T-antigen was blocked by treating them with an adeno-specific antibody but it was not blocked by treating it with SV40--specific antibody.
This information suggested that the virions that were inducing the SV40 T-antigen were in fact, neutralized by adenospecific antisera and not by SV40-specific antisera, indicating that the viruses were inducting the SV40 T-antigen possessed adenovirus capsids and were most likely adeno-SV40 hybrids.
After the discovery of the adeno-SV40 recombinants in the monkey-adapted adeno strains, the adeno serotypes that were used for vaccine production were re-derived in human cells and shown to be free of SV40 and adeno-SV40 recombinants.
Adeno vaccines were redeveloped beginning in 1964 and 1965 in human cells using these fresh isolates. And the adeno-7 and adeno-4 vaccines that are in use today, are made from these re-derived SV40-free adenovirus isolates.
Now, a variety of recombinants have been recovered from the monkey-adapted adenovirus strains, and a list of these recombinants is shown in the next slide -- on the right, please. These recombinants fall into two categories: those hybrids which are defective and those hybrids which are non-defective.
Adeno-SV40 hybrids that are defective contains large deletions of adeno-DNA that's essential for viral replication. Thus, the defective hybrids are incapable of producing hybrid virus progeny unless the cells they infect are co-infected with non-hybrid adeno-virions.
The defectiveness of these hybrid particles shows that this type of adeno-SV40 hybrid could not be maintained as an infectious agent outside of the laboratory. The defective hybrids can be further subdivided into those that produce SV40 progeny like the adeno-2, and 4, and 5 hybrid particles, and those that, due to the deletions of SV40 DNA, do not produce SV40 like the adeno-3 and 7 hybrids.
Non-defective hybrids are non-defective because they contained lesions of the E3 region of the adeno genome that's not necessary for viral replication. Due to the nature of the deleted adeno DNA, the non-defective hybrids are capable of independent replication without the assistance or help of virus.
Now, if SV40 chromosomal information is spreading in the population as some of the data that have been presented at this meeting suggest, then studies of the adeno-SV40 hybrids suggest there are at least two ways that SV40 recombinant viruses could be involved.
The first possibility is existence of a non-defective hybrid which resembles the non-defective adeno-2 SV40 hybrids. Examples of the genomic structure of the non-defective adeno-2 SV40 hybrids are presented in the next slide. The slide on the right, please.
I need to point out that the representations of the genomic structures in this slide are not to scale. When you compare the genomic configuration of the ND4 hybrid -- this one here -- with the genome of the parental SV40 at the top and of the adenovirus 2 at the bottom, what you can see is that portions of the E3 region of ND4 between map position 80 and 85 -- in this little divot here -- represents the deletion of the adeno genome.
So this region between 80 and 85 has been deleted, and in its place has been inserted a segment of the early region of SV40 between map position .11 and map position .63.
The ND3 hybrid at the top contains the smallest segment of SV40, a DNA of any of the non-defective hybrids. Now, in addition to the ND3 and ND4 hybrids, three other non-defective hybrids were recovered from the same non-defective hybrid stock. They were the ND1, the ND2, and the ND5 hybrids.
Each of these hybrids contains a segment of the SV40 T-protein encoding region that's larger than the segment in ND3, but smaller than the segment in ND4. Pictures of heteroduplexes of the adeno-2 non-defective hybrid is shown in the next slide on the left, please.
Now, when you denature and reanneal hybrid and non-hybrid DNA in the same reaction mixture, heteroduplexes form in which the deleted segment of the adeno-2 genome containing the SV40 DNA insert fails to reanneal with the adeno-2 DNA sequences present in the parental adeno-2 DNA forming the loops that you can see in these pictures.
These types of experiments reveal the true structure of adeno-SV40 of the non-defective adeno-SV40 hybrids. These pictures were taken by Dr. Kelly here at the NIH in 1972.
Now, it's theoretically possible that non-defective hybrids resembling the adeno-2 SV40 hybrid could be spreading in the population. However, it's unlikely that a non-defective adeno-SV40 hybrid could have established itself in humans for the following reasons.
First, human adenoviruses do not actually replicate in monkey cells. When the monkey cells are infected simultaneous with adeno and SV40 however, adeno replication is greatly enhanced by the SV40 T-protein function.
Due to the SV40 enhancing function, adenovirus produced progeny in monkey cells almost as efficiently as they do when they infect human cells, thus there's a strong survival advantage in monkey cells for adenovirus recombinants containing SV40 DNA -- the codes for the enhancing function.
As human cells are natural hosts for adenoviruses, no survival advantage for an adeno-SV40 recombinant containing the SV40 DNA to grow in human cells or to infect humans.
The other ways that SV40 recombinants could contribute the spread of SV40 in the population is by the existence of a hypothetical, non-defective SV40 recombinant that contains the entire SV40 genome.
For reasons that I've already given, it's unlikely that the defective adeno-SV40 hybrids that contain infectious SV40 could be sustained outside the laboratory.
However, it is conceivable that SV40 DNA could recombine with a DNA virus with a very large genome and create a non-defective hybrid that contains infectious SV40 DNA.
Now, I think I need to emphasize that this is really pure speculation, because I'm not aware of any survival advantage that such recombinants would have as infectious agents either in tissue culture or in the environment. But one of the purpose, I think, of this workshop is to consider the possibilities.
So it's in the context of the possibilities that the adeno-2 LEY and adeno-2 HEY hybrids which produce SV40 progeny, suggest the types of SV40 producing recombinants that could form. The organization of the LEY genome is shown on this slide.
Now again, I need to point that this slide is not to scale because the SV40 DNA sequences in the LEY hybrid are at least twice the size of the ones in the ND4 hybrid.
And what you have here in this particular construct is a deletion of the adeno sequences between 80 and 93 with an insertion of 1.03 units of SV40 DNA into this region. This is more than one complete SV40 genome. Now LEY stands for Low Efficiency Yielder, and this means that only one in every 10,000 hybrid virions produce SV40 progeny in these populations.
And in contrast the LEY hybrid, the configuration of the HEY hybrid is shown on the next slide on the right, please. I think you can see from the slide of the HEY hybrid, it's a mixture of particles containing either 40.4 percent, 1.4 percent, or 2.4 percent of SV40 DNA units. One unit being a complete SV40 DNA genome.
The large size of the SV40 segments in the HEY2 and HEY3 hybrids permit the induction of infectious SV40 with an efficiency of about one for every ten hybrid particles, hence the name HEY or High Efficiency Yielder.
Now, if non-defective HEY/LEY type recombinants were present in the environment, they could be sources of infectious SV40. A summary of my thoughts on the implications of these hybrids for the polyomavirus workshop are on the next slide, please, on the right.
SV40 has the capacity to combine with unrelated viruses to produce new viruses with different biologic properties. It's theoretically impossible that SV40 could recombine with other viruses and be carried in humans as a recombinant.
Due to defectiveness of most the adeno-SV40 hybrids however, that have been isolated from monkey kidney-adapted adenoviruses, they lack growth advantages in human cells and it's unlikely that they are environmental contaminants. The current adenovirus vaccines are methodically tested and shown to be free of SV40. Thank you.
CHAIRMAN SNIDER: Thank you very much, Dr. Lewis. Could I ask if Dr. Brock from Praxis-Lederle is here?
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DR. BROCK: Good afternoon. I'm Bonnie Brock from Wyeth Lederle. I've been asked to provide a brief overview regarding the quality control testing of the oral polio vaccine. I'd like to start by providing you with some product background on OPV.
The oral polio vaccine is a trivalent preparation of attenuated Sabin strains of polio virus types 1, 2, and 3 in an oral dosage form. The vaccine induces an immune response comparable to the natural disease. The vaccine is credited with the eradication and control of wild type polio in the United States.
Lederle Laboratories has distributed over 650 million doses since the licensure of Orimune in 1963. The viral content of the vaccine is specified by FDA regulations. The individual three polio virus types are combined in specific ratios to assure that all three stains immunize effectively.
The manufacture and testing of Orimune is a multi-stage process that's closely monitored by the FDA following explicit protocols and requires extensive quality control testing.
I'd like to describe cell culture preparation. Preparation of the cell substrate is in primary monkey kidney cells obtained from Green Monkeys that do not harbor the SV40 virus. The monkeys used as a source of kidney tissue are purpose-bred in isolated breeding colonies. They're tested for tuberculosis and viral antibodies. They're held in isolation quarantine under strict veterinary supervision.
A kidney perfusion process is performed under aseptic conditions which liberates kidney cells in preparation for cell culturating. Perfused kidneys are then delivered to the cell culture laboratory.
The cells are disbursed into monocellular suspensions under aseptic conditions. The cells are diluted into a growth media containing the nutrients necessary for growth and replication. Cells are planted into roller bottles and incubated to form a cell monolayer.
Cells are grown and observed for at least 11 days in the cell culture laboratory. After cell growth is completed, 75 percent of the roller bottles are sent to the virus production laboratory for polio virus inoculation. The remaining 25 percent of the roller bottles are sent to quality control for testing.
Fluids from all the roller bottles are tested to detect the presence of any transmissible, microbial agent by inoculation into four cells lines -- Cercopithecus monkey kidney cells, CMK cells -- for an initial 14 days, followed by a 14-day subculture, again in CMK; Rhesus monkey kidney cells for at least 14 days; rabbit kidney cells for at least 14 days; and BSC-1 cells for at least 14 days.
The 25 percent of all the cell culture bottles that are sent to quality control are then observed in their original control bottles for at least 14 more days, followed by a test to detect hemabsorptive viruses.
At day-4 of the quality control observation period, fluids are removed from the original bottles and again tested in the same cell systems I previously described. Again, to detect the presence of any transmissible microbial agent. We always include that additional 14-day subculture on CMK.
Again, at day-14 of the quality control observation period, fluids are again removed from the original bottles and again tested in those same cell systems, including a 14-day subculture in CMK. Therefore, every individual cell batch is observed for a total of more than 50 days in culture. The appearance of any sign of contamination at any stage of testing results in rejection of the cell batch.
I'd like to move on to virus production. One of the Sabin attenuated strains is prepared to inoculate production bottles. Master polio virus seed stocks are maintained in a viable state in liquid nitrogen storage.
Master viral strains have been prepared in the presence of SV40 virus neutralizing antiserum. All subsequent working seed strains have been prepared in CMK tissue and screened to assure they're free of SV40 virus.
The same level of virus is used for each group of bottles inoculated. Production bottles are examined and records checked. Only one polio virus type is processed at a time and incubated. At the appropriate time, post-polio virus infection, fluids from infected tissues which contain polio virus are harvested.
I'd like to describe viral harvest testing now. Viral harvest samples are sent to the quality control laboratory for evaluation and the rest of the harvested fluids are stored frozen until testing is completed. Fluids from these bottles are again tested to detect the presence of any transmissible microbial agent in CMK for 14 days, followed by a subculture in CMK for another 14 days.
Viral harvest fluids are also tested again in Rhesus monkey kidney cells, rabbit kidney cells, and BSC-1 cells, all for 14 days. Samples are also tested to demonstrate the absence of microplasma.
Quality assurance releases a virus harvest for further processing when all testing has been completed with satisfactory results -- for the original cell culture, the cell culture fluid testing and subcultures, and the viral harvest samples.
In summary, over 4,000 individual cell culture observations are made during the quality control testing of a single trivalent bulk lot. Any product contamination observed at any point, results in rejection.
When the appropriate number of harvests for a single polio virus type are released by quality assurance, they are thawed and combined to form a monopool. Samples from an unfiltered, prorata monopool are tested to ensure freedom from adventitious agents in rabbits, guinea pigs, adult mice, and newborn mice.
The production monopool is then passed through a .22 micron filter. Samples are taken for monopool testing by quality control to include testing for potency, testing for polio neurovirulence, testing for markers of attenuation. The appearance of any adventitious agent at any stage of testing results in rejection of the monopool. This process is repeated for each monopool virus type.
A document is then prepared containing the production history and test results on the monopool by quality assurance. This document is submitted to FDA Center for Biologics, Evaluation, and Research, along with monopool samples for testing. The FDA reviews the manufacture's test results, performs tests as appropriate, and provides notification of the release of the monopool for further manufacture.
Released monopools, one for each type, are combined with diluent to make a trivalent vaccine bulk preparation. Samples are tested by quality control for potency and sterility. The vaccine is aseptically filled into a single dose final containers. Samples are tested for quality control, for potency, identity, and safety. Final container samples are also sent to the FDA with a final protocol for the release of the final filled container vaccine for distribution.
And that completes my talk. Thank you for your attention.
CHAIRMAN SNIDER: Thank you, Dr. Brock, for that information. And now, Dr. Jim Williams from Pasteur-Merieux Connaught will talk about testing for SV40 and their viral vaccines. I believe we're going to use the overhead?
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DR. WILLIAMS: Right. Thank you, Dr. Snider. We've heard a very detailed description from the previous speaker and since this is a presentation that we're concerned with SV40 infection, that's all we're going to talk about. We go through the similiar controls that was just described for our inactivated killed product that I'll be describing.
It's important to note that the seed stocks that are used are prepared in primary Macaque kidney cells for the products that I'm going to be talking about, but the production is done in master cell banks that are qualified for production of polio virus vaccine.
I would just like to note the participation of my colleagues that are here with me: Dr. Bernard Montagnon, Jean-Claude Flaquet, Ms. Irene Clement, Paul Austin, and Howard Six.
We have two licensed inactivated polio vaccines in the U.S. Both of these are free of SV40, as I'll show, through extensive testing. The vaccines are poliovax and Ipol, and type 1 mahoney, type 2 MEF1, and type 3 socket strains.
Poliovax is produced in human diploid MRC 5 cells; Ipol is produced in viral cells obtained from ATCC. Currently, Ipol is the only IPV distributed in the United States by our company.
I'm going to cover a period of time and really focus on SV40 testing, so this period, the Canadian product, Poliovax, covers the period from 1963 to 1987. Cercopithecus aethiops primary kidney cell substrates were used to produce the seed. SV40 testing was according to the U.S. requirements, as you've heard extensive discussions about.
Working seeds produced in the primary kidney cells and tested for SV40. All individual lots were tested for SV40. This particular vaccine was licensed in the U.S. on January 24th, 1963.
For the period 1988 to 1997, used the human diploid cell substrate MRC 5. All working cell banks were tested for SV40. Master seed produced in primary Macaque kidney cells were also tested for SV40.
Working seeds were produced in the MRC 5 cells and all working seeds were tested for SV40. The U.S. license was obtained on November 20th, 1987. Distribution was switched to Ipol in 1991 due to the licensure of Ipol.
The next vaccine I'm going to be discussing is Ipol and the period of time I'm concerned with is '83 to '97. IPV has been produced in viral cells as purified inactivated vaccine and SV40 tested according to U.S. requirements. The viral master seed is produced in PMKC cells and also tested for SV40.
The process contains testing at critical points in which are the viral master cell bank, viral working cell banks, viral cell production lots, vaccine concentrated monovalent lots, and vaccine concentrated trivalent lots. So the whole process and the manufacturing at critical points are tested for SV40 as well as other adventitious agents and various other bacterial and mycoplasma testing.
Approximately 100 million doses have been distributed as vaccine worldwide, and this is approximately equal to 450 monovalent lots that are all negative for SV40.
The important point is that the qualified viral cell line was used to produce the IPV, and this is free of SV40. And the licensure of this product was December 21st, 1990.
To sort of recap, the process steps in which SV40 is tested and various other testing occurs, the viral cell controls, the virus harvest, concentrated monovalent pools, concentrated inactivated monovalent pool, and the concentrated 5X trivalent bulk before final vial is filled.
That's all I have. Thank you.
CHAIRMAN SNIDER: Thank you very much, Dr. Williams. And now we're going to move to the U.K. Dr. David Sangar will talk about testing of the polio vaccine. He is from the National Institute for Biological Standards and Control in the U.K.
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DR. SANGAR: Okay, I'm going to give some preliminary results on some SV40 we've been doing at the National Institute of Biological Standards and Control on some vaccines that have been in the freezers there for up to 30 years.
The results are preliminary for three reasons: one, on the number of samples we've examined so far; two, on the fact we haven't got any real accurate quantitation; and three, on the fact we haven't got any false negative controls in any of the samples so far.
The first slide please -- should give the method we're using to test for these samples. So 500 microliter of the tissue culture medium is extracted with proteinase K, SDS, and phenol chloroform, ethanol precipitated, pellets dissolved in 10 microliters, and one microliter of that used in the PCR reaction.
The PCR reaction is hotstart, 40 cycles using those primers from the VP1 region of SV40. And then the product is separated on 2 percent Separide gels.
Now, it's obviously a legitimate question to ask why we're using those primers and not the normal primers from the large T-antigen, and I would like to not answer that question but to be honest, I will. The reason is, I have found it so far, impossible to obtain reagent-negative control using those reagents from the SV40.
So we've obviously got a contamination problem here, but I would say that we've done all the obvious things. New primers have been made, not only in-house but from outside companies. All reagents, including water, is brought in from commercial companies.
The positive control we use is cos cells 50 microliters in the bottom of an Eppendorf tube which was a gift, and is added after all the other reagents are added in one lab, in a laboratory several buildings away from where the PCR is done.
Nevertheless -- if we look at the next one -- this is an agarose gel with the first two lanes on your left are the positives. The next lanes are supposed to be negative reagent blanks. That 100 base pairs has been sequenced and it is from the large T-antigen.
So that's why we moved on to the VP1 primers, and fortunately when we did that, this contamination problem went away. Although we're still examining where that problem is.
So the first thing we did with our new primers was to take some vaccines which were an experimental oral vaccine produced before the SV40 problem was known about, but never used in the clinic because SV40 appeared before it was used. We had five vials of these covering all three types of polios. So two vials with type 1, two vials type 2, one vial of type 3.
And I'm just going to show you the results from one of the type 1s. The lane on the right is one microliter of the water sample from one of the previous slides which I told you, diluted in one mil of water and then one microliter of that taken. And then going towards your left, that ten times dilution of that. So this particular vaccine developed before 1960 contains something like 106 PCR genome equivalence per mil.
The sequence of that sample and all the other five has also been found. They're all identical and they are all identical to the SV40 sequence for the VP1 region published in the 1982 Cold Spring Harbor book on SV40.
So after we did that we then looked at several vaccines made after 1970, after the SV40 problem was known and should have been cleared up. This is a breakdown. They came from 1971 to 1996. There were 32 type 1s, 12 type 2s, 33 type 3s -- all orals.
And just to give you a flavor of what they looked like, this is just an agarose gel. Most of them are vaccines intermixed with negative controls. The lane 1 from the right is obviously the marker lane, and the lane right on the right is the positive cos-1 cells.
So in summary, we have looked at a large number of vaccines now. We're continuing to looking at them. We found that the early vaccines before the SV40 were indeed, by PCR, heavily contaminated it. But the vaccines made from 1971 to the present day, we have not been able to find any evidence of any SV40 contamination.
Thank you.
CHAIRMAN SNIDER: Thank you very much. And now we're going to discuss the epidemiology. Our first presenter on that topic is Dr. Howard Strickler from the National Cancer Institute.
Oh, okay. Dr. Patrick Olin will be going first. He's from the Swedish Institute for Infectious Disease Control.
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DR. OLIN: Thank you very much for inviting me to this conference and to relate some of the experience from a small country in Europe. First slide, please.
This is actually a slide relating the vaccination program in Sweden when we started to battle the polio epidemics in the 50s, and it was done by my father in 1960. We started to use polio vaccines on the national scale in 1957, and it was directed mainly to school grade children and children in pre-school ages, so it was well-defined to the age cohorts born in 1946 to '49, and 1950 to '53.
At that time, the Swedish production hadn't got started to the full extent, and only Salk vaccine was available. And about 700 individuals in these age groups received American vaccine. Very few outside those age groups got that vaccine. There's some conscripts of that year and from private physicians, a few thousands.
We knew also how large proportion of the population in that age group that received those vaccines. From 1958, only Swedish vaccine was used. This was produced by a variant method developed by Svangard, and this was made on Japanese macaque, which were incidently, free of SV40.
And by intimate contact in those small groups of virologist worldwide, working during the '50s, the Swedish investigators were informed already in '59 about the problems of SV40 in the U.S., and the quality control the Swedish vaccines started already there.
And from 1961 and onwards, both prospective and retrospective tests, all lots where shown to be free of SV40. So we can essentially, that in Sweden we had a brief exposure during 1957, of potentially SV40 contaminated inactivated vaccines.
You were shown some fancy pictures from virology, and I thought I should show fancy picture from epidemiological studies. And just to try to sort out how to look at these exposed cohorts and to relate that to cancer epidemiology.
We have in Sweden, the National Cancer Registry which started to collect data in 1960 through 1993, and we get that in age bands of -- five age groups from zero to four, five to nine, etc. And here is just shown in this slide, how large percentage of each age group in different specific years that actually were exposed to the SV40 -- potentially SV40-contaminated vaccines.
And you can see that there are three distinct years, peak years, between 70 and 64 percent, which brooks its way through the different age groups or age bands that we're studying. And we can contrast those with the closest years with no exposure to see what relative risk increases or decreases there are between these two points.
And I then talk about the specific tumors that have been discussed over this conference. The overall incidence, age standardized of brain cancer or malignant brain tumors in Sweden from 1960 to 1990, is shown in this slide, indicating that you have an increase in brain cancer incidents in both sexes, around eight to ten in the hundreds, up to 13/14 of the hundred-thousands.
And you can see that there are a sizable amount of cases each year, rising from 300 to 600 in each sex. Translating that into the age groups that we are talking about, here is, in the upper rows, the same incidence rates as shown in the figure, and here is for females and males, the three exposed years I was talking about and the unexposed two years closest to those, and the relative risk for females and males.
And what can be shown here is that in essence, these numbers -- the relative risks are around one. There are some exceptions, but here this two -- relative risk increase to two, stands for three or four cases in females, and it's not substantiated by any of the adjacent years. So in essence, the overall incidents rates of brain tumors is not affected by the exposure.
Looking at brain ependymomas in Sweden, of course the numbers here are much lower. We have only between a few to ten, maximum 15, 16 cases a year in Sweden, so the incidence rates are jumping from year to year.
Here you can see there is the relative risk is -- there is no difference between the exposed and the unexposed groups, so we can definitely say that we have no indication that the exposure during these years had any influence of the development of ependymomas in these age groups.
Ovarian cancer in Sweden is then a more common affection, with around 700 to 900 cases a year. There is no distinct trend to increase over this years. I have no explanation for the increase around 1975. Again, looking at the females then, the relative risk between exposed cohorts and unexposed in the different age groups, are none.
Likewise, with osteosarcoma which is a rare disease with very few cases, a few cases each year. The relative risk in both sexes is not to disfavor of the exposed cohorts.
More interestingly, the pleural mesothelioma in Sweden, as in the U.S., increased drastically from 1960 through 1990. It's a 10-fold increase in the age standardized incidents rate over these decades. And as mentioned, the interpretation of this has been that this is related to asbestosis exposure, which is also clear by the predominance of males and this increase.
Looking at the exposure figures, again we can say we don't see mesotheliomas in the age groups which so far these kids that were born in 1946 to '53, have reached. And in essence, there is no indication whatsoever that the exposed groups have had any increase in mesothelioma.
On the other hand, one should remember that mesothelioma is a disease which start to show as expected, some years -- 20 to 30 years after exposure to asbestosis, and what you see here is that the increase from 1960 to 1990 is explained by an increase in the age group which is older than the ones exposed in Sweden.
And I think that it's important to realize that this figure here, 15 per 100,000 in the eldest age group, it's actually higher than those reported from the U.S.
I would like to show just a few comments on the overhead, if I could get it. Could I have the overhead machine, please?
This is just the same figure with brain cancer as with mesothelioma, that the increase that we have seen in Sweden, between '60 and 1990 is explained by an increase in the age groups about 50 years of age, indicating that also this increase is independent of exposure to the SV40.
So in conclusion I can say that, in 1957 inactivated polio vaccines, potentially contaminated by SV40, were used in Sweden for approximately 700,000 individuals born between 1946 and 1953. There is no indication for increased specific cancer incidence rates in those exposed cohorts. The increased rates of brain cancer and pure mesothelioma from 1960 to 1993, are independent of the SV40 exposure in Sweden.
Of course, these data are reassuring from the Swedish Public Health perspective, but one should remember that in Sweden, mainly four to 11-year-olds were exposed, whereas infants below one year of age at exposure, may be at great risk of latent cancer development, and also that the exposed cohorts have not yet reached the age where the increased risk of mesothelioma and other tumors have been observed. So continued surveillance, during at least the next decade, is warranted.
Thank you.
CHAIRMAN SNIDER: Thank you very much, Dr. Olin. Indeed, it sure is reassuring to Swedes. And now I'm sure we're all anxious to know about the U.S., and Dr. Strickler will get the last word of the day to speak on the epidemiology of cancers reported to contain SV40 DNA in the U.S.A.
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DR. STRICKLER: Good evening. You should all be congratulated on your stamina. Could I have the first slide, please?
We studied U.S. cancer incidents and mortality data in order to address the question: Has the risk of cancer been greater in people possibly exposed to SV40 contaminated polio virus vaccines? Obviously the question on everyone's mind.
First I'd like to thank my collaborators at the National Cancer Institute, Division of Cancer Epidemiology and Genetics: Dr. Philip Rosenberg, Dr. James Goedert, Dr. Susan Devesa, and at Information Management Services, Joan Hertel.
In way of background, I'd like to just give a brief overview of some of the earlier epidemiologic studies. In general, epidemiologic studies of exposure of SV40-contaminated polio virus vaccines and cancer have been limited by the unavailability of specific, individual exposure data.
We only know the probability that certain individuals became exposed. And with few exceptions, they have tended to be small studies with few cancers of any particular type.
Two exceptions were Fraumeni, '63, and Geissler in 1990. Fraumeni in '63 looked at the 10 million children, six to eight years old, given the IPV -- that's important -- the inactivated polio vaccine in 1955, and compared them according to whether they received high SV40 titers, low SV40 titers, or no SV40 titers in the vaccines, and found no differences.
This is one of the few studies in which they had an opportunity to test the lots and compare the groups according to their level of exposure. The grave limitation on that study was that they were only able to have four years of follow-up.
The Geissler Study was set in Germany and they looked at the 900,000 children who received oral polio vaccine as infants, and compared them -- who received the contaminated SV40 oral polio vaccine -- to individuals who came along just a couple of years later and received SV40-free vaccine, and they also found no differences after 22 years of follow-up.
Notably, with that level of follow-up, they should have been able to observe any changes in ependymoma or osteosarcoma incidence rates. Obviously, mesotheliomas after 22 years of follow-up, they may not have detected.
There were two positive studies I'd like to point out: Heinonen in '73 and Farwell, '79/'84. These two groups investigated in utero exposure, by which I mean maternal vaccination. They had increased risk of neural tumors in both the studies, notably. However, they both had small numbers of cases to observe. In fact, Heinonen only had seven neural tumors; they were mixed types, and only three of them were of the central system.
Farwell saw increased gliomas and medulloblastomas, but again it was a small number of cases and they only had 40 to 60 percent response rate. Almost all the other studies found negative results. The one other study with slightly positive results were the Innis in 1968, where they found that childhood cancer cases had an 88 percent exposure rate to IPV as compared to an 81 percent rate in matched controls.
In summary, the early investigations had sometimes, conflicting results. However, the largest studies, particularly the Geissler Study with 22 years of follow-up, showed no significant effects. And you just saw the data from Sweden where only a very small segment of the population, a single group of children, were exposed and there was no effect.
This is the first data slide. These are age-adjusted incidents rates of selected tumors. Here you see several different common cancers: prostate, breast, lung, colon; an uncommon cancer for way of comparison: kidney cancer. And here are the cancers we've been talking about all day long, those that might contain SV40 DNA: ependymomas, osteosarcomas, and mesotheliomas.
And you can see they're quite rare tumors in the United States -- less than one case per 100,000 individuals. And I include here brain cancers because you've also heard in today's earlier presentations that perhaps additional brain tumors may also contain SV40.
But these are the ones we're really going to give a lot of attention to. I'll talk about brain cancers as well, though.
The implications to the low incidence here is, first, that it gives you an upper bound on the number of people likely to have been affected, and at this point in time the number seems to be small. The number would become bigger if additional cancers were found to possibly be SV40-connected.
The second thing is, just like with Karposi sarcoma which was a rare tumor that suddenly increased after the AIDS epidemic, if a sudden increase in these tumors started to occur, it should be a detectable to us. It should not be a mystery to us; we should be able to see it.
The next thing is however, the corollary to that point I just made is, if SV40 exposure only resulted in a small increase in risk, that would be difficult to detect because it would mean just a small number of cases would have occurred.
In any case, the tumors we're talking about are debilitating and often deadly, and if additional cancers were to turn out to be SV40-connected, the number of individuals possibly affected would increase.
This slide shows a brief timeline which you've already heard about, which I'll go through in two seconds. The mass immunization program began in 1955; the vaccine was contaminated at that point. The SV40 virus was detected in '60.
In '61 the virus was found to be tumorigenic enhancers. That same year the government blocked release to further SV40-contaminated vaccines; however, because the already distributed vaccines may have also contained SV40, a diminishing number of the inoculations may have, up until 1963, also contained SV40. In 1963 also, the licensed OPV was released and it was SV40-free.
The next slide please. This is an important slide. This slide shows our exposure groups that are our comparison groups. This is the risk of exposure to SV40-contaminated vaccines by birth cohort. Here's the essential group, 1955 through 1961. High level of probability of exposure in infancy. Which according to the rodent studies, we at least hypothesized this is our particular period of susceptibility to exposure.
In 1964 and later, no risk of exposure. Individuals born '40 to '54, moderate level of possibility of exposure as children. In 1921 through 1939, moderate level of exposure, but as teens and adults when we think they may be less susceptible. And before 1921, low to very low risk of exposure.
I'm going to start by discussing brain cancers because it includes ependymomas and because of the great interest in this topic. And what you can see here is age -- this is brain cancer incidents, data coming from the SEER program which only goes back to 1973 but contains very high quality data on a histologic-specific basis.
And what you can see is that brain cancers are primarily a cancer affected the oldest individuals. The biggest peak is here. There's a small, initial peak in the youngest age groups, too. The other thing to notice is that brain cancer incidence is increasing.
Years 1973 to '79 is shown in blue; '80 to '86 is shown in red; and in white, is '87 to '93. Now, people have been pointing to this issue for a number of years and have focused on occupational exposures, exposures to nitroso compounds and radiation and other environmental effects to explain this.
And it's important to remember that these are the oldest individuals -- they only had a low to moderate risk of exposure to SV40, what we consider a less vulnerable period -- and the same effect was seen in Sweden where the vaccine received by adults was free of SV40.
But what about this increase down here and in the individuals exposed as infants? Well, this is mortality data now, rather than incidence data. Mortality data goes all the way back to 1950, the period before exposure. It tends to be a little bit less detailed and we don't have the exact histological type, and possibly more prone to misclassification. So keep that in mind.
But the data are quire clear. Here we see in red, indicated the unexposed groups; individuals born after 1964. Here you see the individuals in blue -- set of different birth cohorts -- all of whom were high probability of exposure to the contaminated vaccine as infants. And in black, we are indicating those individuals at high probability of exposure as children.
You can see that for most of the ages -- this is age down here -- for most ages which goes up to 29 because we only have overlap between our exposure groups up until age 29 -- that you see that the mortality rates are about the same.
The one point of difference is in the youngest age groups, and what's noticeable here is the group, 1947 to '49 seem to have the highest rates. And notice, these individuals did not receive the vaccine until they were six to eight years of age. At this point in time they are unexposed.
In addition, most of the cohorts which were exposed as infants, have the same exact mortality rate essentially, as individuals who were later unexposed.
This is another way of looking at this issue. This is brain cancer incidence by birth cohort. Now we're back to our incidence data. The unexposed group is shown here in red; the exposed group shown in blue. The group exposed as children -- I'm sorry, in blue is exposed as infants; black is exposed as children.
And as you can see, for most ages -- this goes from age ten, overlapping at age, about 11, up until it began about age 29 -- and you can see that years in which the age groups in which there is overlap, that the lines are essentially entirely overlapping so that we see no difference.
Now, the youngest age groups particularly included ependymomas, but only about five to ten percent of childhood brain cancers are ependymomas. We looked at ependymomas separately, and what you can see is, as I suggested, ependymomas are primarily a tumor of the youngest age groups. The incidence is about -- is essentially flat thereafter.
There is some suggestion in the most recent year, of an increase -- again, this is 1987/1993; the two previous periods though, are essentially overlapping -- and because this is such a rare tumor, this is really just a few cases different. This is 72 cases for example, versus 50 cases.
Again, in later age groups there seems to be a slight difference with incidents maybe a little bit higher in the most recent period, but again, the previous periods are entirely overlapping and this is just a few cases.
Here again you see brain ependymoma incidence according to age, in the unexposed, in the individuals here in blue exposed as infants, and in black, exposed as children. You can see for most of the ages in which the three cohorts overlapped, their rates are very similar.
Here you see a slight peak in those individuals exposed as infants. Again, just a few cases made this difference -- five cases. And here it's just one case; the reason being, this is probably an edge effect. I mean, very few people actually made it out in this group to this age and contributed data, and so just even one case is able to make the difference. This is just a variable point.
The limitation of the previous data was however, we only looked at, starting at age 11. What we wanted to do, really, is also be able to look at individuals going all back to infancy. And what we see here -- this is child cancer incidence in Connecticut -- the one registry in the United States which goes back to the 1930s.
And here you see the age group zero to four, the group that we're most interested in. Here is the period 1950 to '54. Cancer incidence is about 0.4 per 100,000. And you can see from that time -- which is before the vaccine is distributed -- to the time, 1955 to '59, when the vaccine that is contaminated is first distributed.
There's a slight increase, but notice that in '60/'64 when in fact, we would expect to see the greatest effect because we were getting the cases of individuals exposed in '55/'59, plus the new cases that were occurring in '60/'64 -- if anything, the incidence rate is a little bit lower than in the period prior to exposure before the contaminated vaccines were distributed.
Overall, we are unable to detect an effect on cancer incidence in Connecticut, in childhood age cohorts, related to the period during which the vaccine was contaminated.
To summarize this lengthier part, the brain cancers and ependymomas, you can see that brain cancer mortality rates show no differences between exposure and unexposed groups, particularly in the youngest age categories. The brain cancer incidence rates also were not different, though data only covered young teens to late 20s.
When we looked at ependymomas specifically, it showed no relation to exposure. Ependymoma incidence was not different between the exposed groups -- again, because the incidence data only goes back to '73; this was limited to teens and late 20s -- but when we look back to the Connecticut data which goes back to the 1930s, we still saw no association with the period of vaccine contamination.
Another tumor which has been suggested may contain SV40, is osteosarcoma. Here again you see the age-specific incidents of the cancer -- age along the bottom -- and you see that there are two peaks: one in the teenage years dropping just before age 20, and again later in life.
Note here that individuals born -- excuse me, who develop osteosarcomas during the period 1973 to '79, were those individuals who received contaminated vaccines during the 50s and 60s, and their cancer incidence during their teenage years is if anything, a little bit lower than those who became teenagers in the periods that later -- who received vaccines that were free of SV40, roughly suggesting no effect.
But to look at this in detail, again you see our cohorts -- age is shown along the bottom -- and this is the incidence in red of individuals who were unexposed, in blue individuals who were exposed as infants, and in black, individuals who were exposed as children.
And you can see for almost all age groups starting from age 13 on, up till age 29, the lines are essentially overlapping. Again, we have a single point which seems a little bit high, but again this is probably an edge effect, and in any case for almost all the critical teenage years, the lines are essentially overlapping.
We also looked at bone cancer mortality rates in children, to examine this issue from yet another perspective. And now it's important to mention that because we do not have specific, histologic diagnosis when we look at mortality data, this is all bone cancers which include several other different types of sarcomas, which is of interest but in any case, predominantly reflects osteosarcomas which are the major form of bone cancer in these age groups.
And here's the critical age group -- 15 to 19 years of age -- and you can see that there has been a regular decline in the United States in bone cancer mortality from the period 1950 through 1990; that this decrease has been absolutely regular; and that's there's no change in that pattern in and about the time during which the vaccine was thought to be contaminated.
In summary regarding osteosarcomas, we saw no differences in osteosarcoma cancer incidence rates between individuals exposed to SV40-contaminated polio vaccine as infants, as children, or unexposed. The decreasing bone cancer mortality rates over time showed no apparent change in pattern from before, during, or after the vaccine contamination.
Now we're looking at mesotheliomas which is difficult for a couple of reasons. This is again, cancer incidence rates by age, and you can see that mesotheliomas are cancers of the oldest age groups. This is a problem because again, as Dr. Olin pointed out, the individuals who exposed to contaminated vaccines as infants and children, have not yet reached the age at which we expect them to begin to develop mesotheliomas.
It's also difficult because you see the increases in incidence in the United States during the different periods, but we have a well-known exposure -- asbestos -- which peaked in its use in the 1970s, so that we expect to see large numbers of cases going into the next millennium by that exposure alone.
However, despite these limitations, there are a number of things that we can look at to examine this issue. These are mesothelioma cancer incidence rates in the United States and again, by age. Our unexposed group here in red; our exposed as infants group here in blue; and our exposed as children group in black.
And then you can see that the lines are essentially overlapping up until age 29, and we can say that at least for these younger ages where a virus may have begun to have an effect, we do not see any relationship between exposure to contaminated vaccines and the development of cancer.
And what's very important to note that in Sweden, where only a small number had received the contaminated vaccine and the rest of the population received vaccine entirely free of SV40 for all times, that they, as Dr. Olin pointed out, experienced similar increases -- in fact, probably greater increases -- in mesothelioma incidence over those periods of time, suggesting that the known exposures are probably adequate to explain the increase in mesotheliomas.
Mesothelioma cancer incidence has increased by only in older individuals. These were individuals at low, maybe moderate risk of having been vaccinated, and only as adult -- a period that we consider possibly at low risk. Incidence rates in exposed and unexposed show no differences up until age 29, and in Sweden where the polio vaccine was free of contamination -- which can act as our unexposed group to compare to in this case -- they experienced even greater increases in mesothelioma cases than in the United States.
Next slide, please. I'm not going to go through this data, but we also studied incidence in mortality rates in the United States according to all cancers combined. We looked at non-Hodgkin's lymphoma and leukemias since the virus was detected in some studies in the peripheral blood cells.
We looked at ovarian cancers because these tumors, histopathologically, looked very similar to mesotheliomas and are often confused as mesotheliomas and metastasized to many of the same sites.
In all of these cases we saw no increases in cancer rates attributable to SV40-contaminated polio vaccines, that we could detect.
I want to point out some of the limitations to the analysis that we did. We did not examine the role of SV40 in cancers except as a contaminant of the polio vaccine, thus we did not address the issue: is SV40 a natural, human pathogen in any specific way.
Our analysis was probably insensitive to small increases in risk because these are rare tumors. In addition, exposures were often misclassified since actual SV40 titers each individual received was not known.
We did not specifically examine in utero exposures which is an issue, since at least two earlier studies had weak suggestions that that might be a particular concern -- although a third study failed to show that effect; I'll mention as an aside.
And our analyses could have been affected by changes in diagnosis, treatment, and nomenclature over time, although we worked very hard to keep our comparison groups close in time in terms of their birth cohort -- the years in which they were born -- in order to minimize that effect.
And 30 to 40 years of follow-up may not be sufficient for certain tumors like mesotheliomas.
We studied brain cancers, ependymomas, osteosarcomas, mesotheliomas, non-Hodgkin lymphomas, leukemias, ovarian cancers, and all cancers combined. No epidemic or increases in cancer rates attributable to possible exposure to SV40-contaminated polio virus vaccines could be discerned.
Cancers reported to contain SV40 DNA were rare, and are rare. Ependymomas and osteosarcomas are remaining rare. Mesotheliomas and brain cancers are increasing but mainly in the oldest, unlikely to be related to vaccine exposure.
There is one more slide, if you would please. Just to -- I think it's important to remind all of us what happened to the number of polio cases in the United States after the introduction of the vaccines. Thank you very much.
CHAIRMAN SNIDER: Thank you very much, Dr. Strickler. And I thank all the speakers for their excellent and useful presentations. I would like to thank the staff, particularly the audio/visual staff who helped us today. And thank all of you for sitting through all of this. Look forward to seeing you in the morning at 8:30.
(Whereupon, the Workshop of Simian Virus 40 was concluded at 6:19 p.m.)
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Last Updated: 9/24/1999