Re: In fact, INCREDIBLE as it may sound, he acknowledged nothing of relevance to your enduringly specious arguments...
Dear Dr. Floyd,
Permit me to introduce myself. I am a scientific journalist working
on aids research since more than 10 years, and I am particularly
interested on the controversy about the causes of AIDS. I am following
with a great interest your debate with the Perth Group in BMJ online about
the isolation of HIV. It seems in you letter from the 24 October 2003,
that you have questioned the existence of the Montagnier interview quoted
by the Perth Group and regarding the isolation of HIV. As the author of
the interview, I would like to inform you about the historic of it.
In July 1997 I did a video interview with Professor Montagnier concerning
his early work on isolation of HIV. This interview was published in
Continuum Magazine in winter 1997. Please find here the exact transcript
of the interview which was published in Continuum.
Expecting the reading of the interview will allow you (and all others
readers) to have a better appreciation of Montagnier’ words.
Producer & Scientific journalist
Interview with Professor Luc Montagnier
by Djamel TAHI - (Pasteur Institut, July 1997)
Djamel TAHI : A group of scientists from Australia argues that nobody
up till now has isolated the AIDS virus, HIV. For them the rules of
retrovirus isolation have not been carefully respected for HIV. These
rules are: culture, purification of the material by ultracentrifugation,
Electron Microscopic (EM) photographs of the material which bands at the
retrovirus density, characterisation of these particles, proof of the
infectivity of the particles.
Luc Montagnier : No, that is not isolation. We did isolation because
we "passed on" the virus, we made a culture of the virus. For example
Gallo said : "They have not isolated the virus...and we (Gallo et al.), we
have made it emerge in abundance in an immortal cell line." But before
making it emerge in immortal cell lines, we made it emerge in cultures of
normal Iymphocytes from a blood donor. That is the principle criterion.
One had something one could pass on serially, that one could maintain.
And characterised as a retrovirus not only by its visual properties, but
also biochemistry, RT [reverse transcriptase] activity which is truly
specific of retroviruses. We also had the reactions of antibodies against
some proteins, probably the internal proteins. I say probably by analogy
with knowledge of other retroviruses. One could not have isolated this
retrovirus without knowledge of other retroviruses, that's obvious. But I
believe we have answered the criteria of isolation. Totally.
Djamel TAHI : according to several published references cited by the
Australian group, RT is not specific to retroviruses and moreover your
work to detect RT was not done in the purified material?
Luc Montagnier : I believe we published in Science (May 1983) a
gradient which showed that the RT had exactly the density of 1.16. So one
had a 'peak' which was RT. So one has fulfilled this criterion for
purification. But to pass it on serially is difficult because when you put
the material in purification, into a gradient, retroviruses are very
fragile, so they break each other and greatly lose their infectivity. But
I think even so we were able to keep a little of their infectivity. But it
was not as easy as one does it today, because the quantities of virus were
nonetheless very feeble. At the beginning we stumbled on a virus which did
not kill cells. The virus came from an asymptomatic patient and so was
classified amongst the non-syncithia-forming, non-cytopathogenic viruses
using the co-receptor ccr5 . It was the first BRU virus. One had very
little of it, and one could not pass it on in an immortal cell line. We
tried for some months, we didn't succeed. We succeeded very easily with
the second strain. But there lies the quite mysterious problem of the
contamination of that second strain by the first. That was LAI.
Djamel TAHI : Why do the EM photographs published by you, come from
the culture and not from the purification?
Luc Montagnier : There was so little production of virus it was
impossible to see what might be in a concentrate of virus in the gradient.
There was not enough virus to do that. Of course one looked for it, one
looked for it in the tissues at the start, likewise in the biopsie .. We
saw some particles but they did not have the morphology typical of
retroviruses. They were very different. Relatively different. So with the
culture it took many hours to find the first pictures. It was a Roman
effort! It's easy to criticise after the event. What we did not have, and
I have always recognised it, was that it was truly the cause of aids.
Djamel TAHI : How is it possible without EM pictures from the
purification, to know whether or not these particles are viral and
appertain to a retrovirus, moreover a specific retrovirus?
Luc Montagnier : Well, there were the pictures of the budding. We
published images of budding which are characteristic of retroviruses.
Having said that, on the morphology alone one could not say it was truly a
retrovirus. For example, a French specialist of EMs of retroviruses
publicly attacked me saying: "This is not a retrovirus, it is an
arenavirus". Because there are other families of virus which bud and have
spikes on the surface, etc.
Djamel TAHI : Why this confusion? The EM pictures did not show
clearly a retrovirus?
Luc Montagnier : At this period the best known retroviruses were
those of type C, which were very typical. This retrovirus wasn't a type C
and lentiviruses were little known. I myself recognised it by looking at
pictures of Equine infectious anaemia virus at the library, and later of
the visna virus. But I repeat, it was not only the morphology and the
budding, there was RT...it was the assemblage of these properties which
made me say it was a retrovirus.
Djamel TAHI : About the RT, it is detected in the culture. Then there
is purification where one finds retroviral particles. But at this density
there are a lot of others elements, among others those which one calls
Luc Montagnier : Exactly, exactly. If you like, it is not one
property but the assemblage of the properties which made us say it was a
retrovirus of the family of lentiviruses. Taken in isolation, each of the
properties isn't truly specific. It is the assemblage of them. So we had:
the density, RT, pictures of budding and the analogy with the visna virus.
Those are the four characteristics.
Djamel TAHI : But how do all these elements allow proof that it is a
new retrovirus? Some of these elements could appertain to other things,
Luc Montagnier : Yes, and what's more we have endogenous retroviruses
which sometimes express particles - but of endogenous origin, and which
therefore don't have pathological roles, in any case not in aids.
Djamel TAHI : But then how can one make out the difference?
Luc Montagnier : Because we could "pass on" the virus. We passed on
the RT activity in new Iymphocytes. We got a "peak" of replication. We
kept track of the virus. It is the assembly of properties which made us
say it was a retrovirus. And why new? The first question put to us by
Nature was: "Is it not a laboratory contamination? Is it perhaps a mouse
retrovirus or an animal retrovirus?". To that one could say no! Because we
had shown that the patient had antibodies against a protein of his own
virus. The assemblage has a perfect logic! But it is important to take it
as an assemblage. If you take each property separately, they are not
specific. It is the assemblage which gives the specificity.
Djamel TAHI : But at the density of retroviruses, did you observe
particles which seemed to be retroviruses? A new retrovirus?
Luc Montagnier : At the density of 1.15, 1.16, we had a peak of RT
activity, which is the enzyme characteristic of retroviruses.
Djamel TAHI : But could that be something else?
Luc Montagnier : No..in my opinion it was very clear. It could not be
anything but a retrovirus in this way. Because the enzyme that F. Barre-
Sinoussi characterised biochemically needed magnesium, a little like HTLV
elsewhere. It required the matrix, the template, also primer which was
completely characteristic of an RT. That was not open for discussion. At
Cold Spring Harbour in September 1983, Gallo asked me whether I was sure
it was an RT. I knew it, F. Barre-Sinoussi had done all the controls for
that. It was not merely a cellular polymerase, it was an RT. It worked
only with RNA primers, it made DNA. That one was sure of.
Djamel TAHI : With the other retroviruses you have met in your career
did you follow the same process and did you meet the same difficulties?
Luc Montagnier : I would say that for HIV it is an easy process.
Compared with the obstacles one finds for the others...because the virus
does not emerge, or indeed because isolation is sporadic - you manage it
one time in five. I am talking about current research into others
illnesses. One can cite the virus of Multiple Sclerosis of Prof. Peron. He
showed me his work a decade ago and it took him around ten years to
finally find a gene sequence which is very close to an endogenous virus.
You see...it is very difficult. Because he could not "pass on" the virus,
he could not make it emerge in culture. Whereas HIV emerges like couch
grass. The LAI strain for example emerges like couchgrass. That's why it
contaminated the others.
Djamel TAHI : With what did you culture the lymphocytes of your
patient? With the H9 cell line?
Luc Montagnier : No, because it didn't work at all with the H9. We
used a lot of cell lines and the only one which could produce it was the
Tampon (!?) Iymphocytes.
Djamel TAHI : But using these kinds of elements it is possible to
introduce other things capable of inducing an RT and proteins, etc..
Luc Montagnier : Agreed completely. That's why finally we were not
very ardent about using immortal cell lines. To cultivate the virus en
masse - OK. But not to characterise it, because we knew we were going to
bring in other things. There are MT cell lines (MT2, MT4) which have been
found by the Japanese which replicate HIV very well and which at the same
time are transformed by HTLV. So, you have a mix of HIV and HTLV. It is a
Djamel TAHI : What's more it's not impossible that patients may be
infected by other infectious agents?
Luc Montagnier : There could be mycoplasmas...there could be a stack
of things. But fortunately we had the negative experience with viruses
associated with cancers and that helped us, because we had encountered all
these problems. For example, one day I had a very fine "peak" of RT, which
F.Barre-Sinoussi gave me, with a density a little bit higher, 1.19. And I
checked! It was a mycoplasma, not a retrovirus.
Djamel TAHI : With the material purified at the retrovirus density,
how is it possible to make out the difference between what is viral and
what is not? Because at this density there's a stack of other things,
including "virus-like" particles, cellular fragments...
Luc Montagnier : Yes, that's why it is easier with the cell culture
because one sees the phases of virus production. You have the budding.
Charles Dauget (an EM specialist) looked rather at the cells. Of course he
looked at the plasma, the concentrate, etc...he saw nothing major.
Because if you make a concentrate it's necessary to make a thin section ,
and to make a thin section it's necessary to have a concentrate at least
the size of the head of a pin. So enormous amounts of virus are necessary.
By contrast, you make a thin section of cells very easily and it's in
these thin sections that Charles Dauget found the retrovirus, with
different phases of budding.
Djamel TAHI : When one looks at the published electron microscope
photographs, for you as a retrovirologist it is clear it's a retrovirus,
a new retrovirus?
Luc Montagnier : No, at that point one cannot say. With the first
budding pictures it could be a type C virus. One cannot distinguish.
Djamel TAHI : Could it be anything else than a retrovirus?
Luc Montagnier : No .. well, after all, yes .. it could be another
budding virus. But we have an atlas. One knows a little bit from
familiarity, what is a retrovirus and what is not. With the morphology one
can distinguish but it takes a certain familiarity.
Djamel TAHI : Why no purification?
Luc Montagnier : I repeat we did not purify. We purified to
characterise the density of the RT, which was soundly that of a
retrovirus. But we didn't take the "peak"...or it didn't work...because if
you purify, you damage. So for infectious particles it is better to not
touch them too much. So you take simply the supernatant from the culture
of lymphocytes which have produced the virus and you put it in a small
quantity on some new cultures of lymphocytes. And it follows, you pass on
the retrovirus serially and you always get the same characteristics and
you increase the production each time you pass it on.
Djamel TAHI : So the stage of purification is not necessary?
Luc Montagnier : No, no, it's not necessary. What is essential is to
pass on the virus. The problem Pr. Peron had with the multiple sclerosis
virus was that he could not pass on the virus from one culture to another.
That is the problem. He managed it a very little, not enough to
characterise it. And these days to characterise means above all at the
molecular standard. If you will, the procedure is done more quickly. So to
do it : a DNA, clone this DNA, amplify it, sequence it, etc..So you have
the DNA, the sequence of the DNA which tells you if it is truly a
retrovirus. One knows the familiar structure of retroviruses, all the
retroviruses have a familiar genomic structure with such and such a gene
which is characteristic.
Djamel TAHI : So, for isolation of retroviruses the stage of
purification is not obligatory? One can isolate retroviruses without
Luc Montagnier : Yes .. one is not obliged to transmit pure material.
It would be better, but there is the problem that one damages it and
diminishes the infectivity of the retrovirus.
Djamel TAHI : Without going through this stage of purification, isn't
there a risk of confusion over the proteins that one identifies and also
over the RT which could come from something else?
Luc Montagnier : I repeat if we have a peak of RT at the density of
1.15, 1.16, there are 999 chances out of 1,000 that it is a retrovirus.
But it could be a retrovirus of different origin. I repeat, there are some
endogenous retroviruses, pseudo-particles which can be emitted by cells,
but even so from the part of the genome that provides retroviruses. And
which one acquires through heredity, in the cells for a very long time.
But finally I think for the proof, because things evolve like molecular
biology permitting even easier characterisation these days, it's necessary
to move on very quickly to cloning. And that was done very quickly, as
well by Gallo as by ourselves. Cloning and sequencing, and there one has
the complete characterisation. But I repeat, the first characterisation
is the belonging to the lentivirus family, the density, the budding, etc..
the biological properties, the association with the T4 cells. All these
things are part of the characterisation, and it was us who did it.
Djamel TAHI : But there comes a point when one must do the
characterisation of the virus. This means: what are the proteins of which
Luc Montagnier : That's it. So then, analysis of the proteins of the
virus demands mass production and purification. It is necessary to do
that. And there I should say that that partially failed. J.C. Chermann was
in charge of that, at least for the internal proteins. And he had
difficulties producing the virus and it didn't work. But this was one
possible way, the other way was to have the nucleic acid, cloning, etc.
It's this way which worked very quickly. The other way didn't work
because we had at that time a system of production which wasn't robust
enough. One had not enough particles produced to purify and characterise
the viral proteins. It couldn't be done. One couldn't produce a lot of
virus at that time because this virus didn't emerge in the immortal cell
line. We could do it with the LAI virus, but at that time we did not know
Djamel TAHI : Gallo did it?
Luc Montagnier : Gallo? .. I don't know if he really purified. I
don't believe so. I believe he launched very quickly into the molecular
part, that's to say cloning . What he did do is the Western Blot. We used
the RIPA technique, so what they did that was new was they showed some
proteins which one had not seen well with the other technique. Here is
another aspect of characterising the virus. You cannot purify it but if
you know somebody who has antibodies against the proteins of the virus,
you can purify the antibody / antigen complex. That's what one did. And
thus one had a visible band, radioactively labelled, which one called
protein 25, p25. And Gallo saw others. There was the p25 which he called
p24, there was p41 which we saw ...
Djamel TAHI : About the antibodies, numerous studies have shown that
these antibodies react with other proteins or elements which are not part
of HIV. And that they can not be sufficient to characterise the proteins
Luc Montagnier : No! Because we had controls. We had people who
didn't have AIDS and had no antibodies against these proteins. And the
techniques we used were techniques I had refined myself some years
previously, to detect the src gene. You see the src gene was detected by
immunoprecipitation too. It was the p60 [protein 60]. I was very
dexterous, and my technician also, with the RIPA technique. If one gets a
specific reaction, it's specific.
Djamel TAHI : But we know AIDS patients are infected with a multitude
of other infectious agents which are susceptible to induce crossreactions.
Luc Montagnier : Yes, but antibodies are very specific. They know how
to distinguish one molecule in one million. There is a very great
affinity. When antibodies have sufficient affinity, you fish out something
really very specific. With monoclonal antibodies you fish out really ONE
protein. All of that is used for diagnostic antigen detection.
Djamel TAHI : For you the p41 was not of viral origin and so didn't
belong to HIV. For Gallo it was the most specific protein of the HIV. Why
Luc Montagnier : We were both reasonably right. That's to say that I
in my RIPA technique...in effect there are cellular proteins that one
meets everywhere - there's a non-specific "background noise", and amongst
these proteins one is very abundant in cells, which is actin. And this
protein has a molecular weight 43000kd. So, it was there. So I was
reasonably right, but what Gallo saw on the other hand was the gp41 of
HIV, because he was using the Western Blot. And that I have recognised.
Djamel TAHI : For you p24 was the most specific protein of HIV, for
Gallo not at all. One recognises thanks to other studies that antibodies
directed against p24 were often found in patients who were not infected
with HIV, and even certain animals. In fact today, an antibody reaction
with p24 is considered non specific.
Luc Montagnier : It is not sufficient for diagnosing HIV infection.
Djamel TAHI : No protein is sufficient.
Luc Montagnier : No protein is sufficient anyway. But at the time the
problem didn't reveal itself like that. The problem was to know whether it
was an HTLV or not. The only human retrovirus known was HTLV. And we
showed clearly that it was not an HTLV, that Gallo's monoclonal antibodies
against the p24 of HTLV did no recognise the p25 of HIV.
Djamel TAHI : At the density of retroviruses, 1.16, there are a lot
of particles, but only 20% of them appertain to HIV. Why are 80% of the
proteins not viral and the others are? How can one make out the
Luc Montagnier : There are two explanations. For the one part, at
this density you have what one calls microvesicles of cellular origin,
which have approximately the same size as the virus, and then the virus
itself, in budding, brings cellular proteins. So effectively these
proteins are not viral, they are cellular in origin. So, how to make out
the difference?! Frankly with this technique one can't do it precisely .
What we can do is to purify the virus to the maximum with successive
gradients, and you always stumble on the same proteins.
Djamel TAHI : The others disappear?
Luc Montagnier : Let's say the others reduce a little bit. You take
off the microvesicles, but each time you lose a lot of virus, so it's
necessary to have a lot of virus to start off in order to keep a little
bit when you arrive at the end. And then again it's the molecular
analysis, it's the sequence of these proteins which is going allow one to
say whether they are of viral origin or not. That's what we began for p25,
that failed ...and the other technique is to do the cloning, and so then
you have the DNA and from the DNA you get the proteins. You deduce the
sequence of the proteins and their size and, you stumble again on what
you've already observed with immunoprecipitation or with gel
electrophoresis. And one knows by analogy with the sizes of the proteins
of other retroviruses, one can deduce quite closely these proteins. So you
have the p25 which was close to the p24 of HTLV, you have the p18.. in the
end you have the others. On the other hand the one which was very
different was the very large protein, p120.
Djamel TAHI : Today, are the problems about mass production of the
virus, purification, EM pictures at 1.16, resolved?
Luc Montagnier : Yes, of course.
Djamel TAHI : Do EM pictures of HIV from the purification exist?
Luc Montagnier : Yes. of course.
Djamel TAHI : Have they been published?
Luc Montagnier : I couldn't tell you...we have some somewhere .. but
it is not of interest, not of any interest.
Djamel TAHI : Today, with mass production of the virus, is it
possible to see an EM, after purification, of a large number of viruses?
Luc Montagnier : Yes, yes. Absolutely. One can see them, one even
sees visible bands.
Djamel TAHI : So for you HIV exists?
Luc Montagnier : Yes it's clear. I have seen it and I have
Competing interests: No competing interests