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
Afternoon Session
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
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
PROCEEDINGS
AFTERNOON SESSION
1:55 p.m.
Return to Table of Contents
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?
Return to Table of Contents
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.
Return to Table of Contents
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.
Return to Table of Contents
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.
Return to Table of Contents
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.
Return to Table of Contents
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?
Return to Table of Contents
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?
Return to Table of Contents
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.
Return to Table of Contents
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.
Return to Table of Contents
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.
Return to Table of Contents
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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