Following this I surmise Professor Weiss read the Montagnier interview. In the meantime, believing that Weiss would not reply (mistakenly and I must give Professor Weiss full credit for being the only HIV protagonist who has taken the time to debate us), I emailed Rex Ranieri, a documentarian from TV Channel Nine in Australia. He emailed Weiss on 29th March:
Dear Professor,
I have been observing the HIV/AIDS debate with some interest and I have recently been contacted by Val Turner. He have sent me some questions which he has put to you recently together with the subsequent email discussion .
It appears to me that the argument for the existence of HIV is not sufficiently rigorous.
Is there a bigger story developing here?
I look forward to your reply
Kind regards,
Rex
Rex Ranieri
Channel 9
Perth, Western Australia
rranieri@perthtv9.net.au
+61 8 9449 9999
Fax +61 8 9449 9905
Mobile 0411 258344
Professor Weiss replied immediately:
If HIV does not exist, then neither did smallpox virus (variola), nor does polio virus, tobacco mosaic virus in plants, etc. etc. If you wish to deny the existence of viruses, and virus diseases, go ahead, but leave scientists like me out of the picture.
Robin A Weiss
To which Rex Ranieri replied:
Professor,
My understanding is that Dr. Turner and his colleagues have questioned not whether a number of other viruses exist. Only HIV. As far as I am aware, a scientist does not prove that a particular virus exists by pointing to the existence of others.
I am well versed with some of the argument so far (for a lay person) so naturally your response contributes little to my questions.
I appreciate your desire to be left "out of picture" however as you are a world renowned researcher who has spent some time on the question, it is difficult for me to accept that you can bow out of the discussion.
Naturally, It is your perogative not to respond, however I think that this would ultimately be damaging to both sides of the argument. We have seen many examples of media debacle which can result from lack of discussion.
I realise that your time is valuable and I urge you to respond to my questions. Please accept that my intentions are to arrive at the truth, whatever that may turn out to be.
Kind regards,
Rex
Weiss responded to this email approximately two months later (see Addendum III).
Meantime I sent reminder emails to Weiss. Eventually he replied:
March 26th
I am very tied up with work at present and will give you a considered reply in due course.
Robin A. Weiss
I responded:
Dear Professor Weiss,
Thank you for your reply. I fully understand that you have been busy moving from Fulham Road to Clevland Street and setting up your new department. I am also sure that you understand how anxious both my clinical and non clinical colleagues and I are to examine your considered reply.
Yours sincerely,
VF Turner
After more reminders Weiss emailed me:
April 15th
Dear Dr Turner
You have breached my correspondence with you as an academic colleague by forwarding it to a journalist, Rex Ranieri. In your message to him, you write that that you do not hold any great hope that I shall answer a second time, and yet to me you express your understanding that I'm busy with other things. So here is my second response. I hope it is my last response, because I find the issue of 'purification' quite sterile, and unconnected with matters of medical importance. We are simply talking at cross-purposes.
There appears to be a consortium of medical people and biophysicists in Perth who have a fixation of HIV purification. Perhaps you are influenced by this. It is also a 'cause' championed by the British based magazine 'Continuum' founded by Jodi Wells whom I knew and who sadly died of an AIDS-like disease a few years ago. He initially supported Peter Duesberg's view that HIV indeed exists and can be purified, but that it is harmless. Then he shifted into a denial that there is any such thing as HIV. With Harold Jaffe I argued against Duesberg's view in Nature nearly 9 years ago (Nature 345: 659-660, 1990). I've nothing more to say on this issue, save that with the efficacy of combination anti-retroviral drugs, Duesberg seems to have lost his constituency of support among 'lay' gay men.
Now, to turn to your points sent on March 10 regarding my first reply:-
1. and 2. You confuse isolation and purification. I see no contradiction between what I wrote - in 1986 or in 1999 - and what Barre-Sinoussi and colleagues had reported previously. One can isolate some viruses by propagating them in cells in culture. For example, HIV, smallpox virus, measles virus, polio virus. There are other viruses which no-one has yet succeeded in serially propagating in culture following isolation because they require specialized, differentiated cells; for example, hepatitis B virus, human papilloma virus types 16 and 18 (associated with cervical cancer), and so on.
In both cases, viral genomes can be isolated, indeed highly 'purified' by molecular cloning using recombinant DNA methodology. Thus it is almost routine now in our lab and may other research labs to clone the HIV genome as DNA in bacterial vectors, and then recover them again in infectious form by transferring that DNA back into human lymphocytes. It is difficult to conceive anything 'purer' than the complete cloned virus without any proteins, particles, etc.
3. I do not think Barre-Sinoussi et al misled the scientifically community by calling the 1.16 g/ml band purified virus. But if you and your colleagues prefer to call it enriched but not yet completely pure, I would happily concur with that opinion. This illustrates what I mean by a sterile argument: how pure is pure? Is distilled water 'pure'? Yes, but it will still have a few parts per million or per billion of other soluble molecules.
Are your surgical instruments sterile? Yes regarding bacteria and viruses, if they have been heat-treated or autoclaved. No, regarding the agent of Creuzfeld-Jakob disease which partially resists such treatment. Yet, every surgeon knows what others mean by sterile. Let's not get bogged down in how pure is pure.
The important thing is serial propagation of the microbe. Koch and Petri over 700 [70] years ago 'purified' bacteria by propagating them as colonies (clones) on gelatin in Petri's dishes - nowadays we use agar-agar with nutrients in place of gelatin. Did Koch purify the microbes. Yes in his and my terms, maybe not in yours. Certainly he did purify [?not] them by biophysical methods such as sucrose gradients, but nothing else kept reproducing itself on the 'impure' nutrients. So it is the same for viruses. As intercellular [intracellular] parasites, of course, they can only be propagated in living cultured cells (or in plants, animals or humans) but one can 'plague-purify' them - a term dating from early bacteriophage studies in the 1920s. Animal viruses were similarly plague-purified: polio in 1952; vaccinia around 1955. We used a plaque 'purification' or biological cloning technique for HIV in 1989. No, these were not physically pure, but they were biologically pure, ie they were cloned. Molecular cloning, however, as I mentioned already is one step better. Both methods to my mind, are sufficiently purified to draw scientific conclusions, although one must be cautious not to draw conclusions beyond the validity of the data, including the kind of purity, biological, molecular, chemical or physical.
4. My definition of isolation of HIV by Barre-Sinoussi et al. Gallo and Levy and others in the early days of AIDS research is propagation in culture. Today, however, we more often use molecular cloning, then recover the cloned genome or partial genome and characterise its phenotype.
5. Yes, I do claim that visualization of HIV by electron microscopy was, in 1983/84, an important component of the collective data on virus isolation. Taken together with virus propagation, reverse transcriptase activity and enrichment of particles by isopycnio [density] gradients, it convinced me that HIV is a retrovirus. Even more so, it was Montagnier's electron micrographs published in April 1984 and previously shown at Cold Spring Harbor Laboratory in September 1983 that convinced me that HIV was probably a lentivrius among retroviruses, as they resembled particles of equine infections anaemia virus - a lentivirus first propagated by inoculating a filtrate too small for bacteria to pass through into horses and donkeys and causing disease. So yes, I am definitely arguing that, disregarding your meaning of 'purification', but with my meaning of 'isolation', you can make quite large amounts of HIV proteins and smaller amounts of RNA.
6. Barre-Sinoussi et al published electron micrographs of early budding forms of virus only, that were not immediately identifiable as retrovirus particles. By September 1983, Montagnier's electron micrographs looked more typical.
7. There are many different tests for HIV-specific antibodies. Today's commercial test kits are based on oligopeptides and on proteins manufactured from cloned HIV DNA. No biological test for anything is 100% specific and 100% sensitive, but today's HIV tests are as good as tests for any other human viral pathogen. Likewise, today's PCR primers are highly specific and sensitive for the major strains of HIV in developed countries. Some 'outlier' strains, especially in Gabon and Cameroon are not picked up quite as sensitively and therefore estimates of viral load with these 'outlier' infections should be interpreted cautiously.
There will always be a few people who cannot be convinced by the data before our eyes - or who emotionally wish to deny what the rest of us regard as facts. Of course, interpretation will change over time. Newetonian physios still serves pretty well for the dynamics of road accidents, but Einstein's relativity superseded it on a cosmic scale. In my view, to deny the existence of HIV is a bit like denying the Nazi holocaust.
If we are to doubt HIV as a cause of AIDS, we must cast even more doubt on variola as a cause of smallpox, and the existence of measles, mumps, influenza and respiratory syncytial virus. None of these would pass your definition of purification. None of these has been 'purified' even by culture propagation (my sense) to the extent that has been achieved for poliovirus and for HIV.
This terminates our debate.
Robin A Weiss
Although Professor Weiss terminated the debate, my colleagues and I were far from satisfied. Eleni Papadopulos and I spent many weeks preparing a response (see below). This was sent in late July with the following note:
22/7/99
Dear Professor Weiss,
I apologise for the length of the attached file [35 pages] but it is impossible to debate this topic without data and citations. I trust you appreciate this necessity.
I greatly appreciate your reply to the questions in my last email. Let me say that eighteen years ago neither I nor my colleagues in the Perth Group set out to frustrate the efforts of scientists such as yourself. Rather, we began as you and many others did in the early 1980s, to make a contribution to solving the problem of AIDS. Certainly we are poles apart from the mainstream but, as Philebus says in the Dialogues of Plato, I hope "we are not simply contending in order that my view or that of yours may prevail, but I presume we ought both of us to be fighting for the truth"..
I am disappointed that you do not intend to continue the exchange but I respect your decision. Sometime in the near future I intend to put our debate on the Perth group website. Thus, if you would like to add or alter your replies in any way I will encompass these in the posting. If you would like to contribute anything else at all, scientific or perhaps philosophical including your views on dissent and dissenters, I would be very pleased to post these as well.
I look forward to hearing from you once again.
Yours sincerely,
VF Turner
31/8/99
Dear Dr Turner
I have been in your country during August without opening my e-mails. We shall have to agree to differ on the nature and existence of HIV. I have no additional comments to make for your website.
Robin A Weiss
My response to this was:
1/9/99
Dear Professor Weiss,
Thank you for your email. I am truly sorry you are unwilling to continue the debate. Eleni Papapdopulos and I spent several weeks preparing what I consider a worthy reponse and believe it your responsibility to answer the points I have raised. This especially applies to the arguments related to the extant HL23V. As far as I can tell evidence for the the "isolation" and thus the existence of HL23V is better than that for HIV. Yet HL23V has disappeared from the scientific literature and no longer exists.
I am disappointed you did not look us up while you were in Australia. My colleagues and I would have been delighted to take you out for a meal in Perth's Kings Park overlooking the Swan River. We have some excellent wines which I am sure you would have appreciated. We would have enjoyed your company and the invitation is open should you ever return.
I would like to thank you once again for your time and effort on behalf of this debate. This is not a polemic about any particular person winning. Any fighting is about "fighting for the truth".
Yours sincerely,
Val Turner
REPLY TO PROFESSOR R WEISS BY DR. VF TURNER OF THE PERTH GROUP
JULY 21ST 1999
Dear Professor Weiss,
Scientists should be particularly hard on hypotheses. An hypothesis must explain and predict everything. For example, the HIV theory has to explain why, in Western countries, HIV/AIDS has remained in the original risk groups. It is claimed that HIV/AIDS is spread predominantly by sexual contact of one kind or another and "everyone is at risk" from "a virus that does not discriminate". But prostitutes who do not use drugs are not infected with HIV. In Australia for example, a decade after the beginning of the AIDS era, the number of prostitutes infected by sexual intercourse, despite their being "seriously at risk of HIV infection", was zero. In 1994, Manaloto reported on the "Natural history of HIV infection in Filipino females commercial sex workers". Over seven years 72/53,903 (0.01%) were found to be HIV positive and "Intravenous drug use was denied in all cases". We can take a step further and examine an instance where we can be sure of unprotected sexual intercourse in heterosexuals, that is, new born babies. Again in Australia, in 1989 a study testing 10, 217 blood samples of newborn babies found no babies and thus presumably none of their mothers or fathers HIV positive. These data are supported by Nancy Padian and colleagues’ ten year study of heterosexual couples (1986-1996). There were two parts to this study, one cross-sectional, the other prospective. Of the HIV negative male partners of 82 positive female cases only 2 were found HIV positive but under circumstances considered ambiguous by Padian. In the prospective study, starting in 1990, 175 HIV-discordant couples were followed for approximately 282 couple-years. At entry, one third used condoms consistently and in the six months prior their last follow up visit, 26% of couples consistently failed to use condoms. There were no seroconversions after entry including the 47 couples not using condoms consistently. Based on the 2/86 men who became HIV positive in the early study, the risk to a non-infected male from his HIV positive female partner was reported to be in the order of 1/9000 per contact. From this statistic one can calculate that on average, a male would need to have 6000 sexual contacts with an infected female to achieve a 50% chance of becoming HIV positive. If sexual intercourse were to commence at age 20 and average three times weekly, this would occupy a lifetime; If heterosexual men so rarely become infected how do heterosexual women become infected?
Despite the fact that HIV/AIDS experts continuously assert that epidemiology proves HIV causes AIDS, in my view epidemiological data are incapable of proving anything . But such data may prove scientifically useful when inconsistent with the predictions of a particular theory. As the above data amply illustrate. Thus, in my view and our Group, unless we entertain extraordinary properties for a putative infectious agent, it is obligatory for scientists to question the HIV theory of AIDS. Or, as Simon Wain-Hobson expeditiously put it, "a virus's job" is to spread. "If you don't spread, you're dead". I could not agree more. Inevitably, an exercise of this sort leads Homo sapiens to question what the antibody and genomic tests are measuring. How have their specificities for a unique, infectious agent been determined and what are they? Since these tests purport to use "HIV" proteins and RNA (cDNA) as diagnostic reagents, and since viruses are obligatorily cultured in cells where one would expect to find hundreds if not thousands of cellular proteins, RNAs and DNAs, we would expect there to be proof that HIV had been purified. Otherwise it would be impossible to claim that certain proteins, RNA or DNA are unique constituents of a retrovirus HIV. However, the data of Montagnier, Bess and Gluschankof, all HIV protagonist experts, prove there is no such thing as purified HIV and, according to you, "purification is not particularly important" and Montagnier was right "to say it was not purified virus". These being the case we are left wondering on what basis scientists such as yourself accept that there is such a retrovirus as HIV. This, of course, is what you and I are presently debating. I am sure we can at least agree that without HIV there can be no HIV theory.
Since your definition and understanding especially, but not only of purification, isolation, virus, viral cloning are different from mine, I concur we are "talking at cross-purposes". Where we do seem to agree is on the importance of medical issues, although we seem to disagree exactly what constitutes these medical issues. You state, "I find the issue of purification quite sterile, and unconnected with matters of medical purposes". However:
(a) for more than 15 years
(i) the world has been made to believe that Barré-Sinoussi, Montagnier and their colleagues claim to have proven the existence of a unique retrovirus, HIV, by purifying it using "isopycnic gradients";
(ii) in 1997 Montagnier admitted that the 1.16g/ml band in their isopycnic gradients which they claimed to have proven was "purified labelled virus" did not contain even retrovirus-like particles. Yet, this "purified" virus is accepted as being the cause of the black plague of the century;
(iii) in the name of this virus hundreds of thousands of individuals have died and the lives of millions of people have been adversely affected either directly or indirectly.
Surely these are "matters of medical importance"?
(b) According to Montagnier, "analysis of the proteins of the virus demands mass production and purification. It is necessary to do that. And there I should say that we partially failed". In fact I venture that Montagnier totally failed since the proteins and the RNA which he and his colleagues named HIV proteins and RNA were taken from an isopycnic gradient (the 1.16g/ml band) which they called "purified labelled virus", but where, even after a "Roman effort", they could not find even particles with the "morphology typical of retroviruses", much less retrovirus particles, not to mention a specific retrovirus, HIV. The claim that some of the proteins in this "purified labelled virus" were HIV proteins defies not only scientific reasoning but commonsense. This is no different from a fisherman claiming to have processed a catch of a totally new species of fish into unique fish proteins when his net contained anything but "fish-like" objects, or me as a surgeon going into an operating room where there are many types of animals, but none which look human, removing a few hearts and kidneys, transplanting them into humans in an adjacent operating room and claiming the transplanted organs were of human origin. Yet:
(i) In the name of one of these "HIV proteins", reverse transcriptase, (based solely on the detection of which many researchers publish papers claiming "HIV isolation"), hundreds of thousands of individuals have been treated for more than 10 years with toxic drugs;
(ii) In the name of another of these "HIV proteins", protease, in the last few years even more toxic "cocktails" of drugs have been used;
(iii) Since 1984, in the name of yet another "HIV protein", gp120, hundreds of millions of dollars have been spent to develop a vaccine which it is said will be especially useful in the developing countries where the majority of AIDS cases are tuberculosis. It is the first time in medical history that a vaccine based on a protein said to be a retroviral protein, but which never has been shown to be a constituent of even a retrovirus-like particle, is going to be used to prevent a disease caused by a bacterium.
(iv) In the name of an antibody test employing these "HIV" proteins as antigens, millions of people have been told that they are infected with a lethal retrovirus;
(v) A stretch of RNA (or its cDNA) which never has been shown to be a constituent even of a retrovirus-like particle much less of a retrovirus is used as a hybridization probe and PCR primer to prove infection with a retrovirus claimed to be deadly, and in fact to quantify it.
Surely these also constitute "matters of medical importance"?
For physicians like me, stranded between laboratory scientists and patients, the meaning of the antibody test and the "viral load" are of extreme "medical importance". And whether they are aware or not, for "lay" gay men, drug users, haemophiliacs, blood transfusion recipients, pregnant women and their offspring, and all their relatives, these are also matters of extreme "medical importance".
As far as I am aware there are two groups of "anti-retroviral drugs". The reverse transcriptase inhibitors which are said to prevent the reverse transcription of "HIV RNA" into "HIV DNA", and the protease inhibitors which are said to render particles non-infectious. In other words, the effect of both groups of drugs is to decrease the synthesis of new "HIV DNA", that is, to decrease the amount of provirus. Since none of the "anti-retroviral drugs" used to date has any effect on the transcription of "HIV DNA" into "HIV RNA", a decrease in the latter, the "viral load", must always be preceded by a decrease in the former (the "viral burden"). This is not the case. While many studies report "anti-retroviral" treatment decreases the viral load from millions to undetectable levels, the "HIV DNA" remains constant. In a study published this year "of 34 patients on combination therapy who had plasma HIV-1 RNA levels of less than 200 copies/ml, replication-competent virus was isolated" from 32 (94%). In longitudinal studies it was shown that the frequency of isolation did not depend on the length of combination therapy or the time it was started. Compare this with the 37% frequency of isolation by the best HIV laboratories of non-treated patients in a 1994 WHO study. This can be explained by one or more of the following:
(a) The proviral theory is wrong, that is, there is no relationship between "HIV DNA" and "HIV RNA"
(b) Either "HIV DNA" or "HIV RNA" or both have nothing to do with a retrovirus;
(c) The drugs have no anti-retroviral effect, they only mask the measurement of "HIV RNA".
I venture to say that your present enthusiasm for "combination anti-retroviral drugs" is matched only by your former enthusiasm for AZT. However, there are at least two reasons why this drug should never have been introduced into clinical practice and while its continued use should be abandoned. First and foremost, the pharmacology of AZT proves beyond all reasonable doubt that AZT cannot act as a reverse transcriptase inhibitor, DNA chain terminator. Secondly, even before the AIDS era it was known that AZT is very toxic.
The Concorde study, the only truly clinical blind study, has vindicated both John Lauritsen's and Peter Duesberg's claims that AZT instead of saving lives may endanger them.
In another study published this year by Italian researchers, the authors followed up, for the first three years of life, HIV seropositive children born to mothers who either did or did not receive AZT treatment. The two groups of children were similar with respect to all variables (year of birth, maternal clinical condition, birth weight and treatments) apart from age at the beginning of PCP chemopropylaxis, which was undertaken earlier in these children who were born to mothers who took AZT. They found that the children born to these mothers "had a higher probability of developing severe disease" (57.3% versus 37.2%) or severe immune suppression (53.9% versus 37.5%) and a lower survival (72.2% versus 81%)".
Given these findings any disinterested scientist, clinician, layman, even politician would advise against the administration of AZT to pregnant women. But not HIV/AIDS experts. The authors of this study concluded: "Findings may suggest a need to hasten HIV-1 diagnosis in infants of ZDV-treated mothers and undertake an aggressive anti-retroviral therapy in those found to be infected", and "should not be misinterpreted as a reason not to use ZDV prophylaxis, which is effective in preventing perinatal HIV-1 infection". However, since as the authors of this study claim, "A strong association exists between high maternal viral load and an increased risk of transmission" and since none of the many studies conducted to date has shown a decrease in viral load by AZT, it follows that AZT cannot inhibit maternal transmission. In the face of such data the authors' conclusions and recommendations amply illustrate to what lengths some scientists will strive not to question the HIV theory. If you can offer any other rational explanation I would be most grateful to hear it.
According to Professor Brian Gazzard, President of the British HIV Association, treatment with AZT monotherapy is "ludicrous". Yet not only is AZT monotherapy prescribed to mothers and mothers who object to this treatment for their children are threatened with having their children taken from them. Your enthusiasms for combination therapy is shared neither by the physicians who prescribe it nor by the "lay" gay men who take it. "As physicians venture into even wilder frontiers of HIV treatment, the grand experiment with combination therapies, called Highly Active Anti-Retroviral Therapy, or HAART, is rushing forward without any data. No-one is keeping track". Apparently one of the few physicians, if not the only one who tries to make any sense of HAART is Michael Saag who supervises research and the care of more than one thousand AIDS patients in Birmingham, Alabama. "In one year, 157 of Saag's patients collectively took 189 different drug formulas, with only three patients taking the same mix of HAART drugs…despite such rigorous, individualized medical attention, Saag says, the HAART 'dam' is already leaking and there's high danger of it collapsing altogether...Failures are occurring right and left".
According to Dr Wafaa El-Sadr from Harlem Hospital "it takes a heap of denial to reach anything but sobering, even grim conclusions" regarding HAART. Let me also quote a few gay men, who, unlike the courageous Jodi Wells and the equally courageous and well informed present editors of Continuum, are not in "denial" regarding the existence of HIV. "Anything they give me, I suspect would simply create more side effects and then fail after only a few months, leaving me in even worse shape than I'm now. As my doctors see it, being of therapy is doing nothing, and doing nothing is what scares them most...So when they tell you all is well, they may be doing it less for you than for themselves" (Stephen Gendin POZ Magazine, January 1999). "None of us would say that having access to the best available scientific information every step of the way has helped us particularly...professional judgement has consistently been way out in front of evidence. We're going to have to rethink everybody". Saag agrees: "We don't know what we are doing. Hubris! Hubris! We need to be scientists every step of the way and do our darndest to seek what reality is". If the claims for actual reality of HIV are also based on professional judgement rather than evidence we are indeed guilty of arrogant presumption and must expect nemesis.
Thus, in your position, I would not uphold "anti-retroviral drugs" either in support of the HIV theory of AIDS or the existence of HIV. The only rational conclusion one can draw from the "anti-retroviral" treatment data is either there are no such drugs or there is no such thing as a retrovirus, HIV. (Additional data on PIs is at the end of the reference section*)
Bearing all this in mind, I would like to comment point by point on your answers regarding claims for the existence of HIV.
ANSWER 1 AND 2
My questions were:
1 Why, in 1986, did you and your colleagues write: "A so-called AIDS virus isolate was first reported in 1983 by Montagnier and his colleagues in France who named the material "Lymphadenopathy Associated Virus One". Did or did not Montagnier isolate, purify a retrovirus?
2. If he did not why did you say that he had? If you were aware that he did not do this, and this was the reason for you using the word "material" to describe his finding, why did you not, as a well known and respected retrovirologist, draw the attention of the rest of the scientific community to it, especially if one considers the extremely important consequences?
Your answer was:
You confuse isolation and purification. I see no contradiction between what I wrote - in 1986 or in 1999 - and what Barre-Sinoussi and colleagues had reported previously. One can isolate some viruses by propagating them in cells in culture. For example, HIV, smallpox virus, measles virus, polio virus. There are other viruses which no-one has yet succeeded in serially propagating in culture following isolation because they require specialized, differentiated cells; for example, hepatitis B virus, human papilloma virus types 16 and 18 (associated with cervical cancer), and so on. In both cases, viral genomes can be isolated, indeed highly 'purified' by molecular cloning using recombinant DNA methodology. Thus it is almost routine now in our lab and many other research labs to clone the HIV genome as DNA in bacterial vectors, and then recover them again in infectious form by transferring that DNA back into human lymphocytes. It is difficult to conceive anything 'purer' than the complete cloned virus without any proteins, particles, etc.
My response:
I do not accept isolation and purification as being different. The word "isolation" means to place apart or alone. (From Latin "insulatus", made into an island). "Purification" means to obtain something free from impurities. For a particle such as a virus, isolation and purification are the same thing. The act of placing an object apart or alone, isolation, is totally different from multiplying, transmitting, that is propagating that object. The fact that something can be multiplied or propagated through many generations of cultures neither means that it is any more or less isolated nor that you are any more aware of its identity. No object including a virus can be isolated by propagation. You, more than anybody else, know that there are some very significant differences between retroviruses and other viruses. The subject of interest for you and me is HIV. It is of no interest what methods have been used to prove the existence of other viruses and whether they are good or bad. What is sufficient for one group of viruses may not be sufficient for another. What we all want to know is whether the methods and the evidence which are claimed to prove the existence of HIV and thus of its proteins and its genome, do so. To claim propagation of a unique retrovirus, HIV, one must first prove its existence which can be done only by isolating, purifying it. To date, nobody, not even you, has achieved this.
By molecular cloning it is meant the production of identical copies of, for example, a DNA molecule, any DNA molecule, from an ancestral molecule by splicing it into a suitable cloning vehicle, for example a bacteriophage. The DNA molecules can be either of viral or cellular origin. Or they could be artificial. The molecules within the bacteriophages can, in their turn, be introduced in any cells. If you want to call these molecules an "infectious form" (I will not) then you must accept that any piece of DNA, no matter what its origin, is an "infectious form".
Unquestionably, by molecular cloning one can obtain large amounts of "purified" DNA, but equally unquestionable is the fact that molecular cloning tells you nothing about the origin of a particular DNA molecule. To clone "the HIV genome" one must know beforehand that a particular DNA is the HIV genome. In other words, one must start with "HIV RNA" which can be obtained only by prior purification of particles proven to be retroviral particles. Since this has not been achieved, then what is the origin of the DNA fragment which is known as the "HIV genome" which you routinely clone in your lab? If you are using "material" similar to that used by Montagnier in 1983, in which there is no proof there were even retrovirus-like particles, then surely nobody, not even you, can claim this RNA (cDNA) is the genome of a retrovirus, not to mention a unique retrovirus, HIV.
As an undergraduate medical student I was taught that viruses are infectious particles of particular morphologies containing nucleic acids, proteins and lipids. According to all other retrovirologists including Montagnier, Gallo and Gelderblom, retroviruses are particles with a diameter of 100-120nM containing "condensed inner bodies (cores)" and surfaces "studded with projections (spikes, knobs)". If, unlike other viruses, HIV consists of nothing else but an "infectious form" of DNA molecules (as you claim: "It is difficult to conceive anything ‘purer’ than the complete cloned virus without any proteins, particles, etc"), then why do all other retrovirologists, without exception, claim that the gp120 protein is absolutely necessary for HIV infection? If HIV is nothing else but a pure nucleic acid molecule (RNA) "without any proteins", then why is there so much effort, not to mention expense, to develop a vaccine based on the "HIV" proteins?
ANSWER 3
My question was:
In 1983, when B-S et al published their paper entitled, "Isolation of a T-lymphotrophic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)", and called the 1.16g/ml band "pure labelled virus", did they mislead the scientific community?
Your answer was:
I do not think Barre-Sinoussi et al misled the scientific community by calling the 1.16 g/ml band purified virus. But if you and your colleagues prefer to call it enriched but not yet completely pure, I would happily concur with that opinion. This illustrates what I mean by a sterile argument: how pure is pure? Is distilled water 'pure'? Yes, but it will still have a few parts per million or per billion of other soluble molecules. Are your surgical instruments sterile? Yes regarding bacteria and viruses, if they have been heat-treated or autoclaved. No, regarding the agent of Creuzfeld-Jakob disease which partially resists such treatment. Yet, every surgeon knows what others mean by sterile. Let's not get bogged down in how pure is pure. The important thing is serial propagation of the microbe. Koch and Petri over 70 years ago 'purified' bacteria by propagating them as colonies (clones) on gelatin in Petri's dishes - nowadays we use agar-agar with nutrients in place of gelatin. Did Koch purify the microbes. Yes in his and my terms, maybe not in yours. Certainly he did purify them by biophysical methods such as sucrose gradients, but nothing else kept reproducing itself on the 'impure' nutrients. So it is the same for viruses. As intracellular parasites, of course, they can only be propagated in living cultured cells (or in plants, animals or humans) but one can 'plaque-purify' them - a term dating from early bacteriophage studies in the 1920s. Animal viruses were similarly plaque-purified: polio in 1952; vaccinia around 1955. We used a plaque 'purification' or biological cloning technique for HIV in 1989. No, these were not physically pure, but they were biologically pure, ie they were cloned. Molecular cloning, however, as I mentioned already is one step better. Both methods to my mind, are sufficiently purified to draw scientific conclusions, although one must be cautious not to draw conclusions beyond the validity of the data, including the kind of purity, biological, molecular, chemical or physical.
My response:
We cannot call Barré-Sinoussi's 1.16g/ml band pure or even "enriched" since the scientists who prepared the material conceded it contained no retrovirus-like particles, much less the particles of a unique retrovirus. Perhaps it is inconceivable to you that Montagnier made this admission. In that case let me present you with the actual transcript from the videotape given to me by Djamel Tahi, the French investigative journalist who conducted the interview at the Pasteur Institute in July 1997. Montagnier was asked, "Pourquois les photographies du EM publiées par vous, proviennent de la culture et non de la purifcation?" Montagnier's reply was: "Il y avait tellement peu de production que c’était impossible de voir soit dans un culot de virus à partir d'un gradient. Il n'y avait pas assez de virus pour faire ça. Bien sûr on l'a cherché, on l'a cherché aussi dans les tissus de départ, de la biopsie également. On a vu des particules, mais elles n'avaient pas la morphologie typique des retrovirus. C’était trés différent... relativement différent. Donc avec la culture il a fallu beaucoup d'heures pour trouver les première images. C’tait un travail de romain".
Consider your analogy of pure water. Suppose you are doing a vital experiment which requires pure water. Your colleagues on the floor above provide a bottle labelled "pure water" which forms a critical part of your experiment and ultimately the results of this experiment are used in a way which affects the lives of millions of people. Fifteen years later you are informed that the bottle your colleagues gave you, far from being pure water, contained not even one molecule of water. Would you not think that your colleagues were wrong to label it "pure water" and that they had misled you? Would you then be happy to call it water enriched?
It is not valid to argue that the technique of Koch and Petri justifies the method used to prove the existence of HIV. They did not define purification or, if you like, isolation as "propagation in culture", as you do. They did not "purify them" by biophysical methods such as sucrose gradients because this method is a much later invention and is not even necessary. In their cultures Koch and Petri had gelatin plus particles which could be unambiguously identified as bacteria using the light microscope. Any proteins (other than gelatin) RNA or DNA found in the culture could only be of bacterial origin. Any biological effect, including a plaque produced by an aliquot from this culture which is not produced by gelatin alone must be induced by the bacteria. But it is not the "same for virus", especially retroviruses. Unlike the bacteria, dishes which in addition to bacteria contain only one known protein, gelatin, the "HIV" dishes contain cells, cellular fragments and perhaps bacterial fragments as well as mycoplasmas and viruses. As far as the presence of HIV is concerned, it is sufficient to mention that some electron micrographs of culture supernatants have shown some particles with some of the morphological characteristics, but no particles with all of the morphological characteristics of mature retroviruses.
Even assuming that the particles were those of a unique retrovirus, the proteins and RNA present in the culture or even the culture supernatant cannot be assumed to belong to the virus. Induction of a biological effect by the culture supernatant including "plaque" need not be due to the virus, but to any of the many other factors present. I am not aware of any publications describing "plaque 'purification' or biological cloning technique of HIV in 1989". I would be interested to know how one can know that a "plaque" or any other biological effect is induced by a specific object without prior proof for its existence. "Plaque" and other biological effects are induced by addition and interaction while isolation (purification) is a process of subtraction and separation. Biological effects including plaque can be used only to detect a retrovirus, and then, if and only if, there is a prior knowledge they are specific to the retrovirus, which can be done only by first isolating the virus.
I agree with you that one must be cautious not to draw conclusions "beyond the validity of the data". However, unlike you, I believe that the claim for the existence of HIV is based on interpretations which go far "beyond the validity of the data".
ANSWER 4
My question was:
Since it is generally accepted, and makes common sense, that the existence of a new retrovirus can be proven only by isolating it (both B-S and Gallo claimed to have proven the existence of HIV by isolating it) what is the scientific basis for your claims that B-S et al discovered a new retrovirus?
Your answer was:
My definition of isolation of HIV by Barre-Sinoussi et al. Gallo and Levy and others in the early days of AIDS research is propagation in culture. Today, however, we more often use molecular cloning, then recover the cloned genome or partial genome and characterise its phenotype.
My response:
In 1983 Barré-Sinoussi et al, in the T cell culture derived from the lymph nodes from a patient at risk of AIDS, detected reverse transcriptase activity. The cells from this culture were cultured with T cells from a healthy donor whereupon the detection of reverse transcriptase activity in this second culture was considered proof for isolation of a retrovirus which originated from the patient. Detection of reverse transcription, even if detected in thousands of consecutive cultures is not proof for the isolation of the enzyme reverse transcriptase, much less the isolation of a unique retrovirus, HIV. The measurement of cardiac or liver enzymes in cases of myocardial infarction or hepatitis respectively cannot be construed as "isolation" of the heart or liver. In fact, detection of reverse transcription is not even proof of reverse transcriptase as other enzymes ably perform the same task.
Reverse transcription is non-specific. Indeed, in 1996 you wrote: "Now we know that a broader group of genetic elements than retrovirus utilise reverse transcription at some stage of replication; these include hepadnoviruses (including hepatitis B virus), cauliflower mosaic virus and retrotransposons of eukaryotes and prokaryotes". If this is the case, given the right conditions, one would expect to detect reverse transcription in all cultures, irrespective of the presence or absence of a retrovirus. Then, surely you would agree that the finding of reverse transcription is not even proof for the detection of a retrovirus, much less its isolation, even if one uses your definition. In 1984, Levy et al cultured cells from patients with Kaposi's sarcoma a disease which everybody agrees is not caused by HIV. The supernatants were tested for RT activity, the cells for reactions with serum from Barré-Sinoussi's patient BRU. Some cultures were examined by electron microscopy. The finding of a positive result with "any of those tests" was considered proof for HIV isolation.
In 1984 Gallo said that Montagnier's group data did not constitute proof for isolation, and defined isolation as the detection of more than one of the following:
"reverse transcriptase activity in supernatant fluids;virus observed by electron microscopy [retroviral particles in the cultures]; intracellular expression of virus-related antigens detected with antibodies from seropositive donors or with rabbit antiserum to HTLV-III; or transmission of particles". By transmission of particles was meant detection of reverse transcriptase or particles in cultures of "human cord blood, bone marrow, or peripheral blood T lymphocytes", cultured with concentrated fluids from the cell cultures from tissues obtained from AIDS patients.
However:
1 As can be seen for Gallo et al, like for Barré-Sinoussi et al, the detection of reverse transcription in two consecutive cultures is proof for isolation, although a decade before the AIDS era Gallo himself reported such activity in normal cell cultures. In the AIDS era such activity was reported in the non "HIV" infected H9 cell line from which Gallo "isolated" HIV as well as many other cell lines used to "isolate HIV" and normal cell cultures.
2. Alternatively, according to Gallo's criteria, HIV can be "isolated" in the absence of reverse transcriptase activity, considered by Gallo to be the "sine qua non" of a retrovirus.
3. It is impossible for "rabbit antiserum to HTLV-III [HIV]" to predate proof for the existence of, and pure HIV. It makes no sense at all for Gallo to claim use of an antiserum to prove the existence of a virus when the production of that antiserum is totally dependent on use of the same, purified virus.
4. From the interaction of a protein with an antibody it is impossible to determine the origin of the protein or the antibody. From such a reaction one can determine the origin of only one of the reactants, and then if, and only if, the origin of one of the reactants is known and if there is proof that the interaction is specific. The specificity of such reaction between the "HIV" proteins and antibodies present in patient sera can be proven only by using HIV isolation, purification as a gold standard. Gallo et al claim that the interaction between proteins present in the cells and antibodies in the patient sera proves not only that the proteins and antibodies are HIV protein and antibodies, but the detection of such reaction together, for example with reverse transcription proves HIV isolation. In fact, Gallo has conceded that for him, an antibody test is the quintessence of "HIV isolation". Interviewed in camera by Hugh Christie at the 1998 Geneva AIDS conference he answered, "Sometimes we had Western blot positive but we couldn't isolate the virus. So we got worried and felt we were getting false positives sometimes so we added the Western blot [sic]. That's all I can tell you. It was an experimental tool when we added it and for us it worked well, 'cos we could isolate the virus when we did it". In other words, Gallo knew full well that to prove the specificity of the antibody test it was essential to isolate the virus. When he saw no correlation between antibodies and virus "isolation" Gallo resolved the dilemma by defining virus "isolation" to include the Western blot antibody test.
5. Both groups, Gallo’s and Montagnier’s, found proteins in non-infected cells which reacted with sera from the AIDS, patients.
6. Gallo’s claim for proof of isolation that the findings of particles in the culture supernatants of two consecutive cultures, or in one culture together with reverse transcription or antigen/antibody reactions is puzzling. A decade earlier, Gallo warned that "Release of virus-like particles morphologically and biochemically resembling type-C virus but apparently lacking the ability to replicate have been frequently observed from leukaemic tissue", that is, these properties are not proof for the existence of such viruses.
7. In the 1970s, such particles were frequently observed in human leukemic tissues, cultures of embryonic tissues, and in the majority, if not all, of human placentas. In the AIDS era retrovirus-like particles have been revealed in non "HIV" infected H9 (the leukemic cell line which Gallo used to "isolate" HIV), as well as other cell used for "HIV isolation", CEM, C8166, EBV transformed B-cells and umbilical cord blood lymphocytes.
8. HIV experts are unanimous that for infectivity, gp120 is essential. Indeed, in 1988 your colleague JN Weber and you wrote, "The first step in any viral infection is the binding of the virus particle to a component of the host cell's membrane...For some time it has been known that the binding takes place when CD4 interacts with an "envelope" protein of the virus called gp120". However, Hans Gelderblom and his colleagues at the Koch-Institute in Berlin, who have conducted the most detailed electron-microscopy studies of "HIV particles" have shown that the knobs on the surface of the particles, where the gp120 is found, are only present in immature (budding) particles, which are "very rarely observed". "Mature", cell-free particles do not have knobs, that is, gp120.{Hausmann, 1987 #600 Thus it would impossible for Gallo to produce "transmission by particles" by cell-free culture supernatants.
Surely, one would have to conclude, given the non specificity of particles, reverse transcription (assumed to be due to reverse transcriptase and no other enzyme activity) and antigen/antibody reactions, Barré-Sinoussi's et al, Gallo's et al and Levy's et al data do not prove the isolation of a retrovirus, even allowing your definition.
When asked in 1997 by Djamel Tahi if Gallo purified HIV, Montagnier replied "I don't believe so". He also added that when one uses leukemic cell lives derived from patients with adult T-cell leukemia (as is the case with the H9 cell line) which Gallo claims is caused by the retrovirus HTLV-I and thus must be present in the culture, "You have a mix of HIV and HTLV, it is a real soup". In other words, even if one has proof for the isolation and the propagation of a retrovirus, it is not proof that the retrovirus is HIV. It could very well be HTLV-I. Montagnier forgot to mention that in the soup one could find at least one more retrovirus, an endogenous retrovirus. The difference between retroviruses and all other viruses is that while the finding of a virus in a culture is proof that the virus has been introduced from outside (the virus is exogenous), the finding of a retrovirus is not. You know better than me that Varmus pointed out "one of the most striking features that distinguish retroviruses from all other animal viruses is the presence in the chromosomes of normal uninfected cells, of genomes closely related to, or identical with those of infectious viruses". If the finding of particles and RT activity in the "infected" cultures, and proteins in the "infected" cells which react with sera from AIDS patients is proof of HIV isolation, what does the detection of the same phenomena in non-infected cultures and cells prove?
You also know, in fact you are among the many scientists who have proven, that cultures of non-infected cells, sooner or later, begin to release endogenous retroviruses. Their appearance can be accelerated and the yield increased up to a million fold by stimulating the cultures with mitogens, co-cultivation or by adding to the cultures supernatant from normal, unstimulated cell cultures. Indeed, as far back as 1976 retrovirologists such as George Todaro recognised that "the failure to isolate endogenous virus from certain species may reflect the limitations of in vitro co-cultivation techniques". Montagnier et al (as well as everybody else) employed at least two of the above techniques. In fact, both Montagnier and Gallo concede that not one of the phenomena which they claim prove the existence of HIV can be detected unless the cultures are stimulated. In other words, while the finding of a virus in culture is proof that the virus was introduced from outside (infectious virus, exogenous virus ) the finding of a retrovirus is not. Evidence exists that 70% of AIDS patients and those at risk have antibodies to endogenous retrovirus. This means that even if Barré-Sinoussi, et al Gallo et al and Levy et al, have all obtained proof for the isolated (purification) of a retrovirus, even by your definition, is not possible to claim that the virus is an exogenous (infectious) virus. It could very well be endogenous and, as retrovirologists have suggested, the result and not the cause of a pathogenic process.
In 1997 in an article entitled "Current Problems and the Future of Anti-retroviral Drug Trials", Joep Lange wrote: "There is also a tendency to rapidly and widely publicise positive data and to delay or refrain from publication of studies with negative outcome". In the case of HIV, although both Montagnier's and Gallo's teams widely publicised their claims of isolation (purification) of HIV, neither team published electron micrographs of the material which in isopycnic sucrose gradients bands at 1.16 gm/ml, the "purified HIV". At least in the case of Montagnier's team (and according to Montagnier, Gallo's team as well) this was because their results were negative. In scientific experiments one manipulates the parameter whose effect is in question, executes parallel control experiments with that parameter held constant, holds other parameters constant throughout, replicates both the experimental manipulation and the control experiment and obtains data. Given the fact that retroviral phenomena are expected to arise spontaneously, let alone be induced by the "experimental manipulation" absolutely essential to obtain "HIV", in retrovirology controls constitute the quintessential element. A common thread in "HIV" research is lack of controls. On the rare occasions when authors do claim to use controls they are improper and the experiments not performed blind. For example, "controls" consist of cells cultured from healthy individuals, not sick individuals matched to AIDS patients with respect to "other parameters". To date there is only one exception and this from un-cultured tissue. In 1988 O'Hara and his colleagues from Harvard reported "HIV particles" in 18/20 (90%) of patients with enlarged lymph nodes attributed to AIDS and the identical particles in 13/15 (87%) of patients with enlarged lymph nodes not attributed to AIDS and at no risk for developing AIDS. These data led the authors to conclude, "The presence of such particles does not, by themselves indicate infection with HIV".
Molecular cloning cannot be considered proof for the discovery of a retrovirus. To the contrary, cloning of the retroviral genome can only be performed if and only if one first has a stretch of nucleic acid which a priori has been proven to be the genome of a retrovirus. For this one must first obtain particles in a purified form, or at least in a material where there is not even a remote possibility of the presence of an RNA apart from that contained in such particles. Then the experimenter must show:
1. That the particles have all the morphological characteristics of retroviruses;
2. That the particles are infectious;
3. Contain only RNA and not DNA;
4. The RNA codes for the particles' proteins.
To date there is not one single study proving even one of these conditions, not to mention all of them.
Let us assume that HIV, unlike every other retrovirus or virus is, as you say, a "virus without any proteins, particles etc.", that is, merely a naked stretch of nucleic acid. If a stretch of RNA or DNA (the provirus) is the genome of a unique retroviral particle then the most basic requirement is proof for the existence of a unique molecular entity. That is, all cultures and cells deemed infected must:
1. have a full length of the HIV genome, whatever that length is.
2. the stretch of DNA provirus, the genomes, must be identical.
To date the two "HIV DNA" or HIV DNA" of the same length have been reported. Montagnier and his colleagues reported the "HIV DNA" to be 9 ± 1.5 Kb" whereas Gallo and his colleagues reported that "The overall length of the HTLV-III provirus is approximately 10 kilobases". In the 1984 Levy and colleagues' first study of the "HIV genome", the "broad band (> 15Kb) represents provirus integrated into host cell DNA". In 1995, Pasteur Institute researchers reported that "The complete 9193-nucleotide sequence of the probable causative agent of AIDS, lymphadenopathy-associated virus (LAV) has been determined. The deduced genetic structure is unique; it shows, in addition to the retroviral gag, pol, and env genes, two novel open reading frames we call Q and F". In the same year, Gallo and his colleagues reported their results on the "HIV" nucleotide sequences using clone BH10 but also added, "The sequence of the remaining 182 bp of the HTLV-III provirus not present in clone BH10 (including a portion of R, V5, tRNA primer binding site and a portion of the header sequence) was derived from clone HXB2...Of note is the presence of a fifth open reading frame (nucleotides 8,344-8991) designated 3' orf, present in clone BH8 but truncated in BH10". They concluded, "The complete nucleotide" sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3' terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II...The HTLV-III provirus is 9,749 base pairs (bp) long" In 1990 the HIV genome was said to consist of ten genes. In 1996 Montagnier reported that HIV possesses eight genes and Barre-Sinoussi, HIV has nine genes.
Man and chimpanzee DNA differ by less than 2% but variation in the composition of the "HIV genome" (derived from analysis of "pieces" measuring 2% to 30% of the presumed total) measures between 3-40%. For comparison, two RNA containing viruses (polio and influenza, the latter after 27 years of dormancy,) vary by less than 1% as do RNA molecules self-assembled in test tubes denied the organising influence of living cells.
Given that the DNA sequence determines the composition of a virus's proteins, and the latter the physical, biochemical and biological properties of a virus, how is it possible for such variation to represent one and the same agent? For example, how is it possible that HIV can induce the same antibodies which can be recognised in a universal antibody test containing identical proteins? Since Peter Duesberg reminds us, "there is a range, a small range, in which you can mutate around without too much penalty, but as soon as you exceed it you are gone, and you are not HIV any longer, or a human any longer...then you are either dead or you are a monkey, or what have you", it is evident that whatever the "HIV DNA genome" represents, it cannot be a virus.
If the stretch of "HIV RNA" is the genome of a unique exogenous virus which infects individuals with AIDS or those at risk, then this RNA (or cDNA) should be present in fresh uncultured tissue from all these individuals and in nobody else. Furthermore, if in these individuals there is massive HIV infection, as some of the best known HIV experts claim, Southern/Northern blot hybridisation should be more than sufficient to detect it.
I have always found it extremely difficult to understand why neither Montagnier's nor Levy's groups reported such experiments in 1984 when they claimed to have identified, characterised and cloned the HIV genome. Gallo did and reported "HTLV-III DNA is usually not detected by standard Southern blotting hybridisation ...when it is, the signals are often faint...the observation that HTLV-III sequences are found rarely, if at all, in peripheral blood mononuclear cells, bone marrow, and spleen provides the first direct evidence that these tissues are not heavily or widely infected with HTLV-III in either AIDS or ARC". These studies were confirmed by many other researchers. The finding that when the results were positive the hybridisation bands were "faint", "low signal" was interpreted as proof that HIV seropositive individuals contain HIV DNA in small numbers of cells and at low copy numbers, as interpretation which became generally accepted, although Gallo and his colleagues had an alternative explanation: "Theoretically, this low signal intensity could also be explained by the presence of virus distantly homologous to HTLV-III in these cells". This alternative explanation has been ignored by everybody, including Gallo. However, at a 1994 meeting held in Washington sponsored by the US National Institute of Drug Abuse, Gallo admitted "We have never found HIV DNA in the tumor cells of KS...In fact we have never found HIV DNA in T-cells". Data which has come to light since 1984 suggest that Gallo's and his colleagues' alternative explanation may be a fact:
1. At present there is ample evidence showing that normal human DNA contains sequences related to HTLV-I and HTLV-II;
2. As late as 1994, writing in "Harrison’s Principles of Internal Medicine", Gallo (and Fauci) taught medical students, "...there are no known human endogenous retroviruses". This means that by "virus distantly homologous to HTLV-III" they could have meant none other than the exogenous retroviruses Gallo claimed to have discovered earlier, that is, HTLV-I and HTLV-II. However, at present even Gallo admits that the human endogenous proviral sequences "comprise about one percent of the human genome";
3. Some of the best known HIV experts including Montagnier, Blattner and Gelderblom agree that the pol and gag genes "may be highly conserved between subtypes of virus". In a paper published in 1996 by Reinhart Kurth and his colleagues one reads: "Retrotransposons evolved in a variety of organisms ranging from protozoa to human beings. In these elements, RT genes are linked to genes that code for polyproteins with the potential to self aggregate and to form core particles. These proteins are the equivalents of the retroviral capsid proteins usually designated group-specific antigens (gag)...They [retrotransposons] may be either the derivative or predecessors of retroviruses. Retroviruses differ from retrotransposons by the presence of at least one additional coding region, the envelope (env) gene". In 1984, Gallo group reported that the "HIV genome" hybridised with the "structural genes (gag. pol, and env) of both HTLV-I and HTLV-II. Obviously, the finding of a positive hybridisation "signal" at least with an "HIV" gag or pol probe is no proof for the existence of the "HIV genome".
In fact, at present evidence also exists which shows the presence of "HIV" sequences in non-infected tissues. A .few examples suffice to illustrate this point:
1. Although it is no longer accepted that HIV is transmitted by or is present in insects, in 1986 researchers from the Pasteur Institute found HIV DNA sequences in tsetse flies, black beetles and ant lions from Zaire and the Central African Republic.
2. DNA extracted from thyroid glands from patients with Grave's disease hybridised with "the entire gag p24 coding region" of HIV".
3. In 1986 you published a paper entitled "Isolation of a retrovirus from two patients with "common variable hypogammoglobulinaemia" (CVH). Patients with CVH "are prone to certain bacterial and mycoplasma infectious but not to opportunistic viral protozal, and fungal infectious, such as one seen in patients with genetic defects in cellular immunity ..Both patients sera were negative for HTLV-III [HIV] antibodies". I found your paper very interesting for two reasons. Firstly, for what you meant by retroviral isolation and secondly, your Southern blots results. By isolation you meant: "Extensive syncytium formation was seen to polybrene-treated co-cultures, which on electron microscopy showed a retrovirus morphologically indistinguishable from HTLV-III/LAV" (see figure) and animal lentiviruses. Supernatant from this co-culture was positive for reverse transcriptase, and the cells were positive by immunofluorescence with serum from a patient with AIDS and with the anti-HTLV-III monoclonal antibodies, a-p24 and a-a-p19 (from Dr R C Gallo). Southern blots of restricted DNA from infected cells were probed with ABH-10 (from Dr R C Gallo ), indicated that the viral genome showed homology to HTLV-III/LAV but with restriction enzyme sites distinct from the prototype isolates, HTLV-IIIB and LAV. In other words in 1986 by isolation you did not mean propagation but detection of totally non-specific phenomena. More importantly, while Gallo et al using ABH-10 as a probe could not find in T-cells from AIDs patients "HIV DNA" you found it in the T-cells of patients with CVH.
In the second half of the 1980, in order to rescue the concept of an "HIV genome" extensive use was made of the polymerase chain reaction. However, the results with this technique were not much better than with Southern blot hydridisation, and like Gallo in 1984, were interpreted as proof that "HIV infected" individuals contain "HIV DNA" in a small number of cells. A most striking feature of the results "is the scarcity or apparent absence of viral DNA in a proportion of patients" and when found, the "signal is very low". In addition, the specificity of the PCR has never been determined. In the only study in which an attempt was made, using antibody tests as a gold standard (everyone should know that these cannot be used as a gold standard), it was found that in "Seven French laboratories with extensive experience in PCR detection of HIV DNA", the concordance between HIV serology and "HIV DNA" varies between 40-100%. Even if the PCR were specific, with this technique one detects only a small portion of the genome.
In the vast majority of cases the presence of the "HIV genome" is proven by amplifying short" invariant regions" of a "viral gene", usually of the gag gene. However, since it is accepted that a significant proportion of the "HIV genomes" are defective, finding a fragment of a gene is not proof of the existence of the whole gene and even less so for the existence of the whole genome "HIV DNA" or HIV RNA", a fact accepted by many HIV/AIDS researchers.
In yet another effort to rescue the "HIV genome", tests have been developed which claim to measure the "HIV RNA" in plasma which in turn is said to quantify the number of particles in blood, the "viral load" . One does not need to enumerate the many problems of these tests, a glance at the following table suffices to realise that such tests do not exist.
|
HIV-1 STRAIN |
RT-PCR |
BDNA |
NASBA |
|
DJ258 |
<400 |
111,500 |
100,000 |
|
DJ263 |
<400 |
79,800 |
60,000 |
|
SF2 |
225,500 |
38,000 |
240,000 |
|
III-B |
54,000 |
17,000 |
360,000 |
|
ZAM18 |
78,300 |
70,000 |
66,000 |
|
ZAM20 |
178,800 |
125,800 |
420,000 |
|
UG270 |
179,800 |
29,200 |
170,000 |
|
UG274 |
320,000 |
41,400 |
32,300 |
|
CM241 |
18,800 |
72,800 |
35,000 |
|
CM235 |
4,700 |
52,000 |
15,000 |
|
163.3069 |
36,200 |
94,000 |
57,000 |
|
162.307 |
2,800 |
78,100 |
26,000 |
|
G98 |
254,700 |
269,000 |
<400 |
|
LBV21 |
184,500 |
295,000 |
<400 |
|
VI557 |
950,000 |
587,000 |
125,000 |
The three assays frequently used to quantify the "viral load" are reverse transcription-polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA) and branched chain DNA (bDNA). To assess the impact of the assays used and of "genetic variability in HIV-1 RNA quantification", researchers from France "evaluated three commercial kits by using a panel of HIV-1 isolates representing glades A to H...These isolates were expanded in culture. Virus was collected by ultracentrifugation and resuspended in HIV-seronegative plasma. To standardize the quantities of virus to similar levels in each preparation, the p24 antigen was determined and the volume adjusted so that each specimen contained approximately 10pg of p24 antigen per ml". Since all the samples had the same quantity of "HIV", of "HIV RNA", one will expect all the number in the table to be similar if not identical. Looking at the rows the only conclusion one can draw is that at least two of the assays do not measure "HIV RNA". Similarly, from the columns the only conclusion one can draw is that either no one of the test measure "HIV RNA", and/or there is no such thing as "HIV RNA".
Rich et al state, "Plasma viral load ["HIV" RNA] assays are designed for monitoring the effectiveness of antiretroviral therapies and for measuring the quantity of virus in patients with confirmed HIV infection, not for the diagnosis of HIV infection. Their performance in patients who are not infected with HIV is unknown" and their use leads to "Misdiagnosis of HIV infection".
The fact that patients where "viral load decreases to undetectable level still develop AIDS" also means that either the test do not measure "HIV RNA" or the cause of AIDS is not HIV.
In summary, unless one draws conclusion "beyond the validity of the data", it is not possible to conclude that "Barré-Sinoussi et al Gallo and Levy" have proven the isolation of a unique retrovirus, even by your definition of isolation. Neither do the present data prove the existence of a unique molecular entity "HIV DNA", which constitutes the genome of a unique retrovirus, much less of a retrovirus which infects humans.
To answer the Continuum challenge regarding proof for the isolation of HIV, and thus its existence, Edward King from AIDS Treatment Update presented you the challenge. In the April 1996 where your comments were published one reads: "Gene cloning techniques allow researchers to extract the viral genes found in HIV-infected cells. When the complete set of genes is re-introduced into healthy human cells in culture, the cells produce HIV particles". There is no proof for the existence of the "complete set of genes" that is of "HIV DNA" even in one cell of one AIDS patient. The small number of "complete set of genes" reported so far, all of which differ significantly from each other, are from cultured immortal cell lines. Neither is there one single study which proves that, "When the complete set of genes is re-introduced into healthy human cells in culture, the cells produce HIV particles".
QUESTION 5
My question was:
You state: "When you have evidence of infection in culture, purification is not particularly important". These researchers did not know that their cultures were infected. This is what they were attempting to establish. Surely you don't claim that electron micrographs of some budding forms on the cell surface or some cell-free particles in the culture supernatant which do not even have all the morphological characteristics of retroviruses, are proof of infection? Are you further arguing that, without isolation, that is, purification, a scientist can obtain "HIV" proteins and RNA?
Your reply was:
Yes, I do claim that visualization of HIV by electron microscopy was, in 1983/84, an important component of the collective data on virus isolation. Taken together with virus propagation, reverse transcriptase activity and enrichment of particles by isopycnic gradients, it convinced me that HIV is a retrovirus. Even more so, it was Montagnier's electron micrographs published in April 1984 and previously shown at Cold Spring Harbor Laboratory in September 1983 that convinced me that HIV was probably a lentivirus among retroviruses, as they resembled particles of equine infections anaemia virus - a lentivirus first propagated by inoculating a filtrate too small for bacteria to pass through into horses and donkeys and causing disease. So yes, I am definitely arguing that, disregarding your meaning of 'purification', but with my meaning of 'isolation', you can make quite large amounts of HIV proteins and smaller amounts of RNA.
My response:
In AIDS Treatment Update one reads: "other scientists have highlighted the irrelevance of this insistence on purity if the HIV particles themselves are clearly present, for example, it's like saying that it is impossible to identify a German Shepherd dog by its unique appearance, if it happens to be surrounded by a pack of poodles". However, in the HIV cultures in addition to the myriad of other things one could see a "zoo" of particles, looking more or less like one or another type of retrovirus particles but no one having all the morphological characteristics of retroviruses.
The correct analogy for HIV is a person with no knowledge of German shepherds who possesses an aerial photograph of a zoo, expects to see dogs (retroviruses) but all he sees is many objects some of which look like animals (viruses) and decides that one of the objects is a dog, in fact a dog with unique composition and behaviour without first showing the object is (a) an animal; (b) the animal is a dog; (c) the dog is unique.
It is interesting that you now claim in addition to electron micrographs, "virus propagation, reverse transcriptase activity and enrichment of particles by isopycnic gradients" are needed to convince you that "HIV is a retrovirus". However:
1. "Virus propagation" presupposes you already know you have a virus, therefore virus propagation can not be used to prove the existence of a virus.
2. You agree that reverse transcriptase activity is present in all cells (see my response to Answer 4). I will only add here that a decade before Barré-Sinoussi and Jean Claude Chermann claimed proof for the existence of HIV, they wrote:
"Since similar polymerase activity [reverse transcriptase activity] has been found in normal cells, may be mainly ascribed to the cellular enzyme. At the same time Gallo was proving the existence of RT activity in PHA stimulated but not in unstimulated normal, non infected cells. In his interview with Djamel Tahi, Montagnier initially insisted that this activity was "truly specific of retroviruses," but later he said that it was only "characteristic" of retroviruses. Yet while my medical students and residents, and presumably their teachers, have been taught by retrovirologists that reverse transcription is specific to retroviruses, retrovirologists such as Montagnier and Gallo know differently.
3. "enrichment of particles by isopycnic gradients". Neither Barre-Sinoussi, nor Gallo and Levy presented proof for any "isopycnic gradients" including the 1.16 g/ml being "enriched" or even having one retrovirus-like particle. According to Montagnier, such evidence was not published because they (and in his view, Gallo's group as well) could not find any retrovirus like particles in their "isopycnic gradients". This fact alone proves beyond reasonable doubt they did not have a retrovirus and that the RNA and proteins found at the 1.16g/ml band as well as RT activity or any other effect induced by the material in the band have nothing to do with HIV or any other retrovirus. To claim otherwise is no different from me, as a surgeon, claiming to have a patient’s gallstones isolated, that is, removed an in my hands, merely because I have proven he or she has abnormal levels of liver enzymes in his or her blood. May I repeat, to date nobody has published proof for the existence in the "HIV" cultures, not to mention the 1.16 g/ml band showing particles having all the morphological characteristics of retrovirus particles.
You seemed to have misunderstood my question. I did not ask you what amount of "HIV" proteins or DNA you can make. I am solely interested in quality. Not quantity. Allow me to put the question differently: As far as I know, one can claim that a protein or an RNA fragment belongs to an object, for example either a human being or a virus particle, only if proof exists that they come from the object. Do you disagree? In the "isolate" (be it the culture supernatant or the 1.16 gm/ml band) obtained by your "isolation" method, there is proof for the existence of a myriad of non-retroviral objects containing proteins and RNA, but no object which has even the morphological characteristic of retroviruses not to mention the physical characteristics such as their density. How it is possible then to prove that some of the proteins and some of the RNA which banded at 1.16g/ml or present in the culture supernatant of were "HIV" proteins and "RNA" as Montagnier and Gallo did?
QUESTION 6
My question was:
In your view is it scientifically valid to say on the one hand, as Montagnier did, that the 1.16g/ml "material" did not have even particles with the "morphology typical of retroviruses, while on the other hand, asserting that the proteins and RNA were those of a retrovirus, HIV?
Your answer was:
Barré-Sinoussi et al published electron micrographs of early budding forms of virus only, that were not immediately identifiable as retrovirus particles. By September 1983, Montagnier's electron micrographs looked more typical.
My response:
In the very first electron micrograph (published in May 1983), Barré-Sinoussi, Montagnier et al not only immediately identified some particles as retrovirus particles but they stated that: "Electron microscopy of the infected umbilical cord lymphocytes showed characteristic immature particles with dense crescent (C-type) budding at the plasma membrane...This virus is a typical type-C RNA tumor virus". In 1984 Montagnier, Barre-Sinoussi et al reported that their virus was "morphologically similar to D particles such as those found in Mason-Pfizer virus or the virus recently isolated from simian AIDS." (By 1984 researchers from the primate research centres in the United States claimed the existence of AIDS in monkeys and that the cause of AIDS was a type-D retrovirus similar to the Mason-Pfizer virus, a typical type-D retrovirus and suggested that the monkey AIDS and these retroviruses could be helpful in the study of human AIDS and "HIV"). In the same year, in yet another publication, Montagnier et al claimed that the "HIV" particles had "morphology similar to that of equine infectious anaemia virus (EIAV), and D type particles". The EIAV and the visna virus are neither type C nor type D retroviruses but lentiviruses, that is, viruses which have different morphology and said to induce diseases long after infection. (By the time this paper was published it was realised that patients who had a positive "HIV" antibody test did not develop AIDS immediately, that is, there was a delay between the positive test and the appearance of AIDS). It is most astonishing that the one and the same virus is able to change genus from a typical type-C to a typical type-D particle and then to a completely different subfamily, namely a typical lentivirus, apparently at will and according to non-morphological criteria. These taxonomical differences imply that if HIV was a newly discovered mammal, it could have been either human, or a gorilla or an orang-utan.
All this aside, you have not answered my question. Even if Montagnier had electron micrographs typical of retroviruses, they were all from the culture. As Montagnier acknowledged, no retroviruses were seen at the 1.16g/ml band. What I am interested in is how can anybody take some proteins and RNA from the 1.16g/ml band where there are no proof of any retrovirus-like particles and call them "HIV proteins" and "HIV RNA"?
QUESTION 7
My question was:
What possible justification can there be for (a) using these proteins as antigens in an antibody test to prove infection of millions of people by a deadly virus? (b) using this RNA to prove not only infection but also to quantify the viral load?
Your answer was:
There are many different tests for HIV-specific antibodies. Today's commercial test kits are based on oligopeptides and on proteins manufactured from cloned HIV DNA. No biological test for anything is 100% specific and 100% sensitive, but today's HIV tests are as good as tests for any other human viral pathogen. Likewise, today's PCR primers are highly specific and sensitive for the major strains of HIV in developed countries. Some 'outlier' strains, especially in Gabon and Cameroon are not picked up quite as sensitively and therefore estimates of viral load with these 'outlier' infections should be interpreted cautiously.
My response is:
Could it be there is no scientific justification for taking some arbitrary RNA and proteins from a material in which there is no proof for the existence of even retrovirus-like particles and calling these "HIV RNA" and "HIV proteins"?
Above I have discussed the PCR and the "viral load tests". Here I would like to add:
1. If the "viral load" test measures the number of HIV particles in the blood and if there is a high level of such particles as it is claimed, then it should be no problem to detect such particles using the electron microscope. Yet to date, there is not even one electron micrograph proving that such particles do exist in the blood. The only electron micrographs published to date are from lymph nodes. In your 1993 review article in Science entitled "How does HIV cause AIDS?" you state, "It has long been known from electron microscope and immunofluoresence studies (24) that HIV is found in massive amounts in the lymph nodes, even in the asymptomatic phase of infection". In ref. 24 you cite three papers, two by Tenner-Racz et al and one by Armstrong and Horne (the latter authors are from the Royal Perth Hospital where I work). In none of these studies is there evidence for "immunofluoresence studies" of particles. The immunofluoresence studies of Tenner-Racz are of lymph nodes. However, others report similar findings in patients who suffer from a number of non-AIDS related diseases, and also healthy individuals. In the first paper Tenner-Racz et al examined the lymph nodes of 9 patients with persistent generalised lymphadenopathy and two with Kaposi's sarcoma. In fig.2 where their results appear, there are three electron micrographs. In the first two there is one "virus-like" particle in each. The third shows one "Profile suggesting budding". In the second paper there is one electron micrograph showing one particle. It is interesting that without any proof the "virus-like" particles in the first study become "retrovirus particles" and in fact "AIDS-related virus" in the second, although even to a non-electron microscopist it is obvious that the particles lack the appearances of the putative "HIV" particle. In the Armstrong and Horne paper there are a few more particles which are sometimes referred to as "virus-like" and at other times retrovirus-like and whose "morphology conforms" to the type-C retrovirus, not lentivirus as HIV is supposed to be. (Armstrong has retired from the hospital but I contacted Horne who confirmed these were indeed type-C particles). More importantly, particles with morphology ascribed to "HIV" are found in the lymph nodes of non-AIDS patients.
2. The absolutely necessary, but not sufficient condition which one must satisfy before using "PCR primers" in any test is to prove such primers originate from a retrovirus-particle. Since no such proof exists the only conclusion a scientist can draw is that nobody knows what a positive "HIV PCR" is detecting. That this is the case is confirmed by the test manufacturers themselves. For example, Roche states in their package insert, "The AMPLICOR HIV-1 MONITOR Test is not intended to be used as a screening test for HIV-1 or as a diagnostic test to confirm the presence of HIV-1 infection" (Roche Diagnostics, Branchburg, New Jersey, Art. 07 5623 7). HIV/AIDS experts agree. For example, Rich et al state, "Plasma viral load ["HIV" RNA] assays are designed for monitoring the effectiveness of antiretroviral therapies and for measuring the quantity of virus in patients with confirmed HIV infection [positive antibody test], not for the diagnosis of HIV infection. Their performance in patients who are not infected with HIV is unknown" and their use leads to "Misdiagnosis of HIV infection". Thus clinicians like myself are left wondering how to reconcile statements such as "HIV RNA (cDNA) is the RNA of a unique retrovirus HIV" and "today's PCR primers are highly specific and sensitive" with statements such as HIV PCR is "not for the diagnosis of HIV infection" because this will lead to "Misdiagnosis of HIV infection". It does not make any sense that the PCR results are confirmed by the antibody tests whose specificity has never be determined, and vice versa that the specificity of the antibody tests is confirmed by PCR whose specificity is unknown.
3. In my view the antibody tests are the most important aspect in HIV/AIDS. This is because:
(i) you acknowledge that there are "naturally existing viruses" (endogenous, non-infectious viruses) and there is a very high frequency of recombination of the retroviral genomes. "HIV" has only been "isolated" from cultures. As far back as 1988 the CDC researchers pointed out that "the culture technique determines the ability of infected cells to produce virus in vitro but does not necessarily indicate the status of virus expression in vivo." This means that even if one isolates a retrovirus from the culture, and even the isolation is according to our terms, such finding is no proof for the existence of this virus in vivo. The limited number of in vivo electron microscopy studies, failed to prove even the existence of retrovirus particles in AIDS patients, much less of a specific retrovirus. The vast volume of genomic studies, failed to prove the existence in even one single AIDS patient of the full-length "HIV genome". The existence of HIV in vivo then rests with the antigen/antibody reactions.
(ii) the antibody tests are the only tests routinely used to prove "HIV" infection.
(iii) the only way the proponents of the HIV theory of AIDS can defend their position is to reassert the correlation between AIDS and a positive antibody test. In your 1990 Nature publication with Harold Jaffe where you "argued against Duesberg's view" you wrote, "The evidence that HIV causes AIDS is epidemiological and virological, not molecular...HIV is the singular common factor that is shared between AIDS cases in gay men in San Franciso, well nourished young women in Uganda, haemophiliacs in Japan and children in Romania orphanages".
However, correlation does not prove causation and any "correlation" between "HIV" infection and AIDS is man made. Prior to 1987, one "HIV specific" WB band was considered proof of HIV infection. However, since 15%-25% of healthy, no risk individuals have "HIV specific" WB bands, it became necessary to redefine a positive WB by adding extra and selecting particular bands, otherwise at least one in every seven people would be diagnosed infected with HIV. (Notwithstanding, in the MACS, one band remained proof of HIV infection in gay men until 1990). On the other hand, although AIDS began to decline in 1987,this trend was countered by the addition of more and more diseases and, most recently, mere laboratory abnormalities to each revision (1985, 1987 and 1993) of the first, 1982 CDC definition. The net effect of these changes was to maintain a correlation between "HIV" antibodies and "AIDS" amongst the "risk" groups while the risk of an HIV/AIDS diagnosis outside these groups remained slight. This was further accentuated by avoiding testing outside the risk groups. However, when such studies were performed, for example, (a) amongst 89,547 anonymously tested blood specimens from 26 US hospital patients meticulously chosen to be at no risk of AIDS, between 0.7% to 21.7% of men and 0-7.8% of women aged 25-44 years were found to be HIV WB positive. (It is estimated that approximately 1% of men are gay. Also, at the five hospitals with the highest rates of HIV antibodies, one third of positive tests were in women. Yet men vastly outnumber women as AIDS patients). (b) the US Consortium for Retrovirus Serology Standardization reported that 127/1306 (10%) of individuals at "low risk" for AIDS including "specimens from blood donor centers" had a positive HIV antibody test by the "most stringent" US WB criteria (see below). Thus the correlation between "HIV" antibodies and AIDS, which experts accept as the only proof that HIV causes AIDS, is not a statistic related to the natural, unbridled activity of a virus but is instead a contrivance generated by mankind. Let me repeat, correlation does not prove causation, and the artificiality of this particular "correlation" severely compromises any scientific analysis.
Although the "antigens" used in the HIV antibody tests are said to originate in a unique retrovirus HIV, it is not important if one has a single or "many different tests" or what is used as antigen. Antigens used in serological tests for syphilis and infectious mononucleosis do not originate in the causative agents. However it is absolutely necessary to have tests which are specific. That is, well before introducing the test into clinical practice, the laboratory scientist must prove that the tests are positive only in individuals infected. The specificity of the "HIV" antibodies for proving HIV infection can be determined only by using "HIV" isolation (purification) as a gold standard. This has not been done and presently cannot been done because nobody has purified "HIV". That this is the core, and that there is no proof that "There are many different tests for HIV-specific antibodies" is acknowledged by some of the best researchers in HIV testing and HIV/AIDS such as Philip Mortimer and William Blattner. Even if one uses your definition of isolation and purification it is still not possible for anybody to have determined the specificity of the Western blot, the antibody test used to "confirm" all other antibody tests. This is because the WB is not standardised. The criteria used to define a positive test varied with time (the US Food and Drug Administration have had three so far) and vary between countries or even between laboratories in the same country.
Space does not permit for me to cite the vast volume of data regarding the non-specificity of the 'HIV antibody tests" but I am sure you know where to find the relevant data. Because of this I will limit the discussion to the basic problems associated with the definition of the "HIV" antigens and antibodies. Barré-Sinoussi et al and Gallo et al took the proteins which banded at 1.16g/ml, the "purified" virus and reacted then with the sera from AIDS patients and those at risk. The proteins which were found to most often react with some of the sera sometimes were said to be "HIV" proteins and the reacting antibodies, "HIV" antibodies. From such a reaction it is not possible to determine the origin of one reactant, much less of both.
Let us consider a very ideal situation. Firstly, 75% of the material which bands at 1.16g/ml is in the form of particles ("enriched") which possess all the morphological characteristics of retroviruses. The remaining 25% is cellular microvesicles and other cellular, bacterial and viral constituents. Secondly, all of the many antibodies present in AIDS patients and those at risk are "monospecific", that is, they react with none other than the inducing antigen. A reaction between a protein in the 1.16g/ml band and an antibody in the sera proves that the protein is present in the individual and it is immunogenic. But it does not prove the origin of that protein, that is, whether the protein is retroviral, viral, bacterial or cellular. If we come closer to reality, that is if you accept the proven fact that there are no such things as "monospecific" antibodies, that all antibodies are "polyspecific", that is, they cross-react, then, from such a reaction one cannot ever prove that the protein is present in the individual or that the antibody was directed against it. The reality, the truth, is that:
(a) Barré-Sinoussi et al and Gallo et al did not prove that their 1.16g/ml band contained even one single particle with the morphological characteristics of retroviruses.
(b) The AIDS patients and those at risk have a plethora of antibodies including auto-antibodies, all potentially cross-reactive. This means that the most likely origin of the proteins which band at 1.16g/ml is cellular or maybe bacterial/viral and the least probable retroviral. The same is true for the antibodies. That both the proteins and the antibodies are not retroviral has been proven by well known HIV protagonists. Let us assume that although there is no proof that Barré-Sinoussi's and Gallo's 1.16 g/ml band did contain retroviral particles, it did contain some disembodied retroviral proteins. In this case, if one compares the proteins which band at 1.16g/ml from the "HIV" infected cultures, the "purified" virus, with the proteins which band at the same density from non-infected cultures, the "mock" virus, we would expect the "purified" virus to reveal its "extra" proteins.
In the well known Bess and co-authors 1997 Virology paper, the authors had three HUT-78 (H9) cultures, two infected and one uninfected control. The proteins from the banded material from all cultures including the control (column A), which they called "mock virus", were compared using electrophoresis. They stated that the only difference between the three strips was that the infected strips contained major bands of p24. But these same bands, although weaker, are present in the "mock" virus protein strip whereas, to be HIV proteins, requires them to be present exclusively in the "infected" strips. When asked for proof that p24 etc in the strips B and C were HIV proteins their answer was that the labels were added for the reader's convenience at the suggestion of the reviewers. So Bess and his colleagues have shown that the same proteins are present in the pure HIV and "mock" virus.
In their effort to develop a vaccine, and because humans cannot be injected with either HIV or "mock" virus, Bess and his colleagues first injected macaques with the "mock" virus. (This is culture fluids from the uninfected H9 clone of the human HUT78 cell line "purified" as it would be to obtain "HIV" or "SIV"). After the initial immunisation, the animals were given boosters at 4, 8 and 12 weeks. At fourteen weeks, the monkeys were challenged with intravenous SIV prepared from the same human cells as "mock" virus and then monitored for seroconversion with the SIV Western blot. According to the authors, the animal immunised with "mock virus" "did not seroconvert to viral proteins after intravenous challenge with SIV", and "These results are the first demonstration that immunisation with purified cellular protein can protect from virus infection...It has recently been suggested that immunisation with alloantigens might serve as a vaccine to protect against HIV infection. Our demonstration...support this concept".
The underlying principle of immunisation is its specificity. That is, to protect against microbe 'X', the person or animal must be exposed to material from 'X' in order that the immune system generates specific antibodies. For example, immunisation with hepatitis vaccine does not protect against poliomyelitis. Since monkeys immunised with proteins derived from uninfected human cells are protected from infection with 'SIV' prepared from the same uninfected human cells, "mock" virus and "real" SIV must be identical. If such "mock" virus and "SIV" are one and the same we would expect that when "SIV" is prepared in antigenically different cells, for example, monkey cells, there will not be "protection". This is in fact what Bess and his colleagues proved in another experiment. The only logical explanation of these data is that they reflect immune responses to cellular proteins. Thus SIV proteins, and by inference, HIV proteins, are nothing else but cellular proteins.
All data presented to date is consistent with the "HIV" proteins being cellular. Using "HIV" antibodies as probes, "HIV" proteins have been identified in the tissues of persistently HIV negative, healthy individuals including blood platelet and skin cells, thymus, tonsil and brain. As a mark of the bewildering status of the HIV theory, at least to this clinician, while HIV proteins could not be found in the placentas of 75 HIV positive pregnant women, they could be found in the placentas of 25 healthy, HIV negative women.
Detection of p24 has been also reported in organ transplant recipients. In one kidney recipient (the donor was negative for p24 antigen) who, 3 days following transplantation developed fever, weakness, myalgias, cough and diarrhoea, all "Bacteriological, parasitological and virological samples remained negative [including HIV PCR]. The only positive result was antigenaemia p24, positive with Abbot antigen kits in very high titers of 1000pg/ml for polyclonal and 41pg/ml for monoclonal assays. This antigenaemia was totally neutalizable with Abbot antiserum anti-p24...2 months after transplantation, all assays for p24-antigen became negative, without appearance of antibodies against HIV. Five months after transplantation our patient remains asymptomatic, renal function is excellent, p24 antigenaemia still negative and HIV antibodies still negative". Using two kits, the Abbot and Diagnostic Pasteur, in one study, p24 was detected transiently in 12/14 kidney recipients. Peak titres ranged from 850 to 200 000 pg/ml 7-27 days post- transplantation. Two heart and 5/7 bone marrow recipients were also positive, although the titres were lower and ranged from 140-750 pg/ml. Disappearance of p24 took longer in kidney (approximately 6 months) than in bone-marrow (approximately 4-6 weeks) recipients".
When by 1988 it was realised that by Gallo et al, and thus by your definition of isolation, the frequency of isolation was low, the definition was changed. Since then the detection in the cultures of proteins (since antibodies cross-react, the protein(s) may be other then p24) which react with a p24 antibody is considered proof for "isolation". However, in 1992, Jorg Shupbach, the principal author of the third and co-author of the fourth of the 1984 papers published by Gallo's group on HIV isolation, reported that the whole blood cultures of 49/60 (82%) of "presumably uninfected but serologically indeterminate individuals and 5/5 seronegative blood donors were found positive for p24".
The non-specificity of the p24 antigen test is so obvious that it is accepted by no less an authority on HIV testing than Philip Mortimer and his colleagues from the UK Public Health Laboratory Service, "Experience has shown that neither HIV culture nor tests for p24 antigen are of much value in diagnostic testing. They may be insensitive and/or non-specific".
Interestingly, so far the only evidence of an animal model for AIDS has been obtained by allogenic stimulation, a procedure which leads to the appearance of "type C particles". Since individuals belonging to the AIDS risk groups are repeatedly subjected to alloantigenic insult, one would expect these individuals to have a positive antibody test, and not be surprised if they developed AIDS, without ever coming in contact with a retrovirus, HIV.
Given these data it is no wonder that biotechnology companies such as Abbott Diagnostics include in their package insert the statement: "At present there is no recognised standard for establishing the presence or absence of HIV-1 antibody in human blood".
That antibody tests cannot be used to diagnose infection with a given retrovirus was known before the AIDS era. In 1974 Hans Gelderblom and his colleagues wrote, "While the virus envelope antigens are primarily virus-strain specific, the bulk of internal proteins of the virion with molecular weight (mw) between 10,000 d and 30,000 d are group-specific (gs) for viruses originating in a given animal species (gs-spec. antigens). The major protein constituent of mammalian C-type oncornaviruses with a molecular weight in the range of 30,000 d was found to possess, besides gs spec. antigen, an antigenic determinant that is shared by C-type viruses of many mammalian species including monkeys and was thus termed gs interspecies (gs-interspec.) antigen". The fact that the proteins with molecular weights between 10,000-30,000 and the antibodies which react with them are not specific for retroviruses has been amply demonstrated in the AIDS era. A few examples:
- According to the AID vaccine Clinical Trials Group, "The presence of p24 band was common among low-risk, uninfected volunteers and complicated the interpretation of the Western blot test results".
- If the p24 band in the WB is considered the result of specific reactivity, then approximately 30% of individuals who are transfused with HIV negative blood become infected with HIV as a result.
- A 40 year old, male, HIV antigen negative, heterosexual donor of Rh negative blood was given six 5ml injections of donated Rh positive serum, administered at 4 day intervals. His "wife and child were seronegative on HIV ELISA". The donor serum "was shown to be negative on HIV antibody and antigen ELISA". "Blood taken after the first immunization was shown to be negative on HIV antibody ELISA and immunoblot assay. After the second immunization a weak signal on ELISA, slightly above the cut-off level, was monitored. After the third immunization the signal was strong and immunoblot revealed distinct interaction with p17 and p55 proteins. An even stronger signal was monitored after the fifth immunization. Interaction with p17, p31, gp41, p55 and some other proteins was evident".
- 11/208 (5%) of healthy blood donors and 10/50 (20%) of patients with measles, mumps, herpes simplex, dengue and other viral illnesses had either a p24 or p18 band on the HIV Western blot test.
- The "HIV proteins (p17, p24)" appear in the blood of patients (previously negative for all HIV markers) following "transfusions of HIV-negative blood and UV-irradiation of the autoblood".
- In 1991 Kion and Hoffman injected non-HIV-infected mice with T-lymphocytes from another strain of non-HIV-infected mice. The recipient mice developed antibodies to the HIV gp120 and p24 proteins.
- In 1991, Strandstrom and colleagues reported that 72/144 (50%) of dog blood samples "obtained from the Veterinary Medical Teaching Hospital, University of California, Davis" tested in commercial Western blot assays, "reacted with one or more HIV recombinant proteins [gp120--21.5%, gp41--23%, p31--22%, p24--43%]".
- Last year it was reported that 35% of patients with primary biliary cirrhosis, 39% of patients with other biliary disorders, 29% of those with lupus, 60% of patients with hepatitis B, 35% of hepatitis C, all non-HIV, non-AIDS diseases, have antibodies to the "HIV" p24 "core" protein;
Yet up till the end of 1987, by which time the vast majority of haemophiliacs, gay men and blood recipients who are tested, anybody who was tested and found to have a p24 band on the WB was deemed to be infected with a deadly virus and continued to believe and behave so even after the criteria were changed. In 1987 former US Senator Lawton Chiles of Florida told an AIDS conference how twenty two blood donors were informed they were HIV infected on the basis of an ELISA test whereupon seven then committed suicide.
Before the AIDS era there was proof that the antibodies which reacted with "retroviral envelope proteins", like those which reacted with the "retroviral core proteins" were also non-specific. On the 31st of January 1975 Science published a paper by Gallo and Gallagher entitled: "Type C RNA Tumor virus isolated from cultured Human Acute Myelogenic Leukaemic Cells". This type-C RNA tumour virus, retrovirus, became known as HL23V. The evidence for the isolation of HL23V surpassed that of HIV in at least two aspects. Unlike for HIV, Gallo and Gallagher, (a) reported the detection of reverse transcriptase activity in fresh, uncultured leucocytes; and (b) published an electron micrograph of retrovirus-like particles banding at a sucrose density of 1.16g/ml. This study was followed by several papers in Nature, including one entitled "Infective transmission and characterization of a C-type virus released by cultured human myeloid leukaemic cells", with you as co-author. In this study you reported that the HL23V "C-type particles are infectious. The virus may be propagated to high titre in several cell types and a quantitative bioassay is described. The production of substantial titres of virus has enabled us to characterise the virus in detail and to compare it with and show that it closely resembles the C-type SSV and its associated virus (SSAV) previously isolated from a fibrosarcoma of a woolly monkey. But our data on the relative plating efficiency on human and 1283 marmoset cells, the inhibition of reverse transcriptase activity by specific antisera and the nucleic acid lybridisation studies suggest that, though closely related, the HL23V-1 virus and SSV are not identical....one interesting fact is that the SSV(SSAV) viruses were obtained from a woolly monkey that has been a pet and was therefore in close contact with a human population...If the HL23V-1 virus and SSAV do indeed have a common origin, it is plausible that the monkey was infected by the human virus". Gallo, you and your colleagues concluded, "Preliminary serological studies indicate that antibodies to the virus are widespread in the human population. Moreover, a closely related virus has been isolated several times from normal human embryo cells by Panem et al., and Nooter et al. described elsewhere in this issue the isolation of an SSV-related virus from human leukaemic cells. We should bear in mind, however that isolation of the virus from leukaemic cells does not necessarily mean that the virus is aetiologically related to the leukaemia. Tests are currently in progress to determine whether the virus has oncogenic potential in animals".
The results of your serological studies which indicated that "antibodies to the virus are widespread in the human population", were published in 1977, where you concluded: "The serological studies presented here an by others provide indirect evidence that the infectious mode of transmission remains a real possibility in humans, and suggests that infection with an oncornavirus may be extremely widespread, though perhaps not typically pathogenic."
In 1980 two research group one from the Laboratory of Cellular and Molecular Biology, National Cancer Institute, and another from the Laboratory of Viral Oncology, Memorial Sloan-Kettering Cancer Center found that the antibodies were 'directed against carbohydrate structures' and concluded that "The results are consistent with the idea that the antibodies in question are elicited as a result of exposure to many natural substances possessing widely cross-reacting antigens and are not a result of widespread infection of man with replication competent oncoviruses" (retroviruses). In 1981 Gallo accepted the evidence that the antibodies which reacted with the presumed viral proteins of HL23V were not so directed "but against the carbohydrate moieties on the molecule that are introduced by the host cell as a post-transcriptional event, and which are therefore cell-specific and not virus-specific." This discovery was of such monumental significance that today nobody, not even Gallo, considers HL23V as the first human retrovirus. Or even a retrovirus. While the demise of the HL23V antibodies and thus of HL23V was based on the findings of two research group, that the antibodies were against carbohydrate moieties, as we have pointed elsewhere, there are numerous studies which show this is also the case with the "HIV" antibodies:
1. "One half of the molecular weight of gp120 is represented by oligomannosidic oligosaccharides...Polyclonal antibodies to mannan from yeast also recognize the carbohydrate structure of gp120 of the AIDS virus";
2. "The immunochemical determinants of the antigenic factors of Candida albicans display a high identity with the glycoprotein (gp) 120 of HIV-1: they contain a(1®2) and a(1®3) linked mannose terminal residues";
3. Antibodies to the mannans of Candida albicans "block infection of H9 cells by HIV-1" as well as the binding of lectins to gp120;
4. Recognition of gp120 by antibodies to a synthetic peptide of the same antigen was "partially abolished if it was absorbed with the total polysaccharide fraction of C. albicans" while the antigen recognition by antibodies to "gp120 from human T cell lymphotrophic virus type IIIB", "was totally blocked". From these data the authors concluded: "These results indicate that mannan residues of C. albicans can serve as antigens to raise neutralizing antibodies against HIV infection";
5. "Normal human serum contains antibodies capable of recognizing the carbohydrate moiety of HIV envelope glycoproteins...from 100ml of human serum approximately 200ug of MBIgG was recovered [MBIgG=mannan-binding IgG]...MBIgG bound to HIV envelope glycoproteins gp160, gp120 and gp41";
6. Kashala, Essex and their colleagues have shown that antibodies to carbohydrate containing antigens such as lipoarabinomannan and phenolic glycolipid that constitute the cell wall of Mycobacterium leprae, a bacterium which "shares several antigenic determinants with other mycobacterial species" cause "significant cross-reactivities with HIV-1 pol and gag proteins". This led the authors to warn that among leprosy patients and their contacts there is a "very high rate of HIV-1 false-positive ELISA and WB results", that "ELISA and WB results should be interpreted with caution when screening individuals infected with M. tuberculosis or other mycobacterial species", and furthermore that "ELISA and WB may not be sufficient for HIV diagnosis in AIDS-endemic areas of Central Africa where the prevalence of mycobacterial diseases is quite high";
7. Not only mycobacteria (M. leprae, M. tuberculosis, M. avium-intracellulare) but also the walls of all fungi (Candida albicans, Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, including Pneumocystis carinii), contain carbohydrate (mannans). One hundred per cent of AIDS patients (even those with "No candida clinically") have C. albicans antibodies, leading researchers from St. Bartholomews and St. Stephen's Hospitals to state: "It is possible that candida may act as a cofactor in the development of overt AIDS in HIV infected individuals". It may also be of interest to note that in gay men the only sexual act which is a risk factor for seroconversion is passive anal intercourse (exposure to semen) and that mannose is present in both sperm and seminal plasma.
8. Since antibodies to mannans react with the "HIV proteins" then, as Essex and his colleagues15 have pointed out for mycobacterial infection in Africa, one would expect the sera of all people infected with fungi and mycobacteria to cross-react with the "HIV-1 glycoproteins" as well as to cause "significant cross-reactivities with HIV-1 pol and gag proteins".
9. Researchers from the University of Rome injected healthy mice with an E. coli lipopolysaccharide (LPS) and reacted their sera with two synthetic peptides, one encompassing gp120 V3 loop of "HIV-1 MN" and the other "representing a gp41 immunodominant epitope". The "LPS-treated mice showed a significant antibody reactivity" with the two peptides. (V Colizzi et al., personal communication).
10. In the same study, the authors reported data from the sera of HIV-negative subjects with autoimmune disorders. Recombinant gp120 and a panel of synthetic peptides derived from the amino acid consensus sequences of the HIV gp120, gp41, p24 or several unrelated proteins were tested by specific ELISA. "The first set of experiments performed on four patients with Sjorgern syndrome (SjS) and four patients with systemic lupus erythematosus (SLE) revealed a significant anti-gp120 reactivity compared to healthy HIV-negative controls. Moreover, such binding could be almost completely inhibited by preincubation with free gp120. A significant anti-p24 reactivity was observed in 18 out of 29 [62%] sera from SjS patients and in 13 out of 25 [52%] from SLE patients, while anti gp41 was observed only in 3 out of 14 [21%] SjS and in 2 out of 20 [10%] SLE affected patients. Similar analyses were performed in the murine model of autoimmunity, showing that sera from MRL/lpr mice were able to bind all HIV related peptides in age-dependent manner. The analysis of a panel of HIV unrelated peptides showed that SLE as MRL/lpr sera bind both HIV related and unrelated peptides while SjS sera failed to do so". In other words, sera which contain autoantibodies react with the principal "HIV proteins" gp120, gp41 and p24.
11. The same authors also reported similar results from (i) experiments where "Two month old male CBA mice were immunized for 6 weeks with 50x106 allogenic lymphoid cells obtained from either BALB/c or B6 male mice"; (ii) "Sera from 62 polytransfused (at least 10 tranfusions/year) patients with thalassemia".
12. Similarly, in 1991, Kion and Hoffman reported "Mice of the autoimmune strains MRL-lpr/lpr and MRL-+/+ made antibodies against gp120". Mice that have been exposed to T-lymphocytes from another murine strain were shown to make antibodies against gp120 and p24 of HIV.
These data pose the following crucial questions:
1. Given the fact that individuals with fungal and mycobacterial infections have antibodies which may react with "HIV proteins" in the absence of "HIV", and that E. coli is an intestinal commensal and a potential bacterium in all of us, how can one assert that:
(a) reactions between antibodies in the sera of AIDS patients and proteins present in cultures derived from the tissues of AIDS patients is proof that the reacting proteins are constituents of a unique retrovirus HIV and the antibodies are specific to these proteins?;
(b) PCP, candidiasis, cryptococcosis, coccidioidomycosis, histoplasmosis, tuberculosis or Mycobacterium avium-intracellulare disease, that is, the vast majority of the opportunistic infections (88% of AIDS cases diagnosed between 1988 and 1992 had one or more fungal or mycobacterial infections) which signify AIDS are caused by HIV on the basis of a positive antibody test?
(c) a positive antibody test in individuals with fungal and mycobacterial infections proves HIV infection?
Since:
(a) mice and patients with autoimmune diseases (SjS and SLE) and AIDS patients share many clinical and immunological (autoantibodies) manifestations;
(b) patients polytransfused with allogenic blood and mice injected with foreign cells and foreign proteins develop "HIV antibodies" but are not infected with HIV; why should gay men, IV drug users and haemophiliacs, who are all exposed to foreign cells and/or foreign proteins, not also develop "HIV antibodies" and not be infected with HIV?
In my view your proof that HIV exists depends on your belief in a nexus between a series of non-specific phenomena (particles, reverse transcription, antigen/antibody reactions and PCR). I contend this does not constitute proof any more than claiming a cigarette smoking patient with weight loss, fever, sweats and coughing up blood has to be suffering from a hitherto unknown and undiscovered variety of lung cancer. I further argue that the epidemiological data, far from "proving" these data are due to the effects of a unique retrovirus, do exactly the opposite. Indeed, as I suggested in my introduction, these data prompt an urgent reappraisal of how and why such non-specific data have been interpreted as proving the existence of HIV. The mistake that has been made is the assumption that finding antibodies that react with culture proteins proves (a) the proteins are constituents of a retrovirus; (b) the antibodies specifically react with these proteins; (c) isolation of a virus. That this is not the case follows from an appreciation of basic science and was, for retrovirologists, the lesson of the HL23V era. It is uncanny that, by changing a few dates, nouns and adjectives in the papers of the HL23V era, it is possible to arrive in the HIV era.
In conclusion I thank you for your contribution and cordially invite you to continue the debate in the spirit Robert Oppenheimer who wrote, "One thing we find throughout the house [of science]: there are no locks; there are no shut doors; wherever we go there are signs and usually the words of welcome. It is an open house, open to all comers". I also must ask that you refrain from comparisons between my views on HIV isolation and David Irving’s on the Holocaust. I find the comparison not only irrelevant but distasteful.
Yours sincerely,
VF Turner MD
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121. Matthews R, Smith D, Midgley J, et al. (1988). Candida and AIDS: Evidence for protective antibody. Lancet ii:263-266.
122. Caceres CF, van Griensven GJP. (1994). Male homosexual transmission of HIV-1. AIDS 8:1051-1061.
123. Mann T, Lutwak-Mann C. (1981). Male Reproductive Function and Semen. New York: Springer-Verlag, 1981.
124. Fraziano M, Montesano C, Lombardi VR, et al. (1996). Epitope specificity of anit-HIV antibodies in human and murine autoimmune diseases. AIDS Res. Hum. Retroviruses 12:491-496.
125. Hu DJ, Fleming PL, Castro KG, et al. (1995). How important is race/ethnicity as an indicator of risk for spefific AIDS-defining conditions? J. Acq. Immune Def. Syndr. Hum. Retrovirol. 10:374-380.
126. Calabrese LH. (1989). The rheumatic manifestations of infection with the human immunodeficiency virus. Semin. Arthritis Rheum. 18:225-239.
127. Nakamara M, Burastero SE, Ueki Y, Larrie JW, Notkj AL, Casali P. (1988). Probing the normal and autoimmune B cell repertoire with Epstein-Barr virus: Frequency of B cells producing monoreactive high affinity autoantibodies in patients with Hashimoto's disease and systemic lupus erythematosus. J. Immunol. 141:4165-4172.
ADDITIONAL DATA RE PROTEASE INHIBITORS/HAART
*According to one Australian expert, the success of the protease inhibitors is "the nail in the coffin" of dissident theories. In this regard, the findings of a recent study are interesting. "To assess the long-term effects of highly active anti-retroviral therapy, we examined 3O4 anti-retroviral-experienced patients who were placed on HAART for a period of 18 months. The baseline CD4 count was 385 X lO6/1 and HIV RNA level was 3.2 log10 copies/ml. At baseline, 39 percent were classified as asymptomatic, 33 percent were symptomatic and 28 percent had an AIDS defining illness. The HAART regimens included 3-5 anti-retroviral agents at least one of which was a protease inhibitor. After 18 months, 14 percent of the population remained asymptomatic, 10 percent of which had an undetectable viral load. 39 percent were symptomatic and 47 percent of the population had an AIDS defining illness. The average CD4 count after 18 months on HAART was 301.79 X 106/1 and HIV RNA level of 3.2 log10 copies/ml.
Christina M. Ramirez and Michael S. Gottlieb. California Institute of Technology. Long-term Highly Active Anti-Retroviral Therapy in an Anti-Retroviral Experienced Population. Keystone Symposia HIV Vaccine Development: Opportunities and Challenges and AIDS Pathogenesis. January 7-13th, 1999, Keystone, Colorado. www.newsfile.com
ADDENDUM I
LETTER REJECTED BY NATURE
NO REASON GIVEN
26/2/99
Dear Sir,
Please find a small contribution to your Correspondence columns. We look forward to an answer to this vitally important question.
Yours sincerely,
VF Turner
Department of Emergency Medicine
Royal Perth Hospital
Perth, Western Australia
In news and views (Nature 4th February), Robin Weiss and Richard Wrangham stated "the retrovirus that is the main cause of AIDS has been a puzzle ever since it was discovered by Barre-Sinoussi and here colleagues in 1983".
The senior, most quoted author of the 1983 Barré -Sinoussi et al paper (1) entitled: "Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS)"1 is Luc Montagnier. To isolate HIV, the authors used the long accepted method for retroviral isolation, that is, banding, (ultracentrifugation) in density gradients. They claimed to have shown that the density of 1.16g/ml, the sucrose density at which retroviral particles band, contained "purified, labelled virus", that is, the 1.16g/ml band contained nothing else but retrovirus particles isolated from everything else.
In an interview published late last year,2 given to the French journalist Djamel Tahi, Montagnier was asked why they did not publish electron micrographs proving that the 1.16g/ml band contained isolated HIV particles. Montagnier answered: No such proof was published, because, even after a "Roman effort", at the density of 1.16g/ml they could see no particles with "morphology typical of retroviruses". He gave similar answers to repeated questions, including "I repeat, we did not purify", that is, isolate HIV.
Given the important and highly significant differences in the consequences of these contradictory claims, it is a matter of urgency for the other 11 authors of the 1983 study, and especially Barré -Sinoussi and her co-workers from the Pasteur Institute, to either confirm or refute Montagnier's statements?
Eleni Papadopulos-Eleopulos1 Valendar F.Turner2 John M. Papadimitriou3 David Causer1 Bruce Hedland-Thomas1 Barry Page1 Charles Geshekter4
(1) Department of Medical Physics, Royal Perth Hospital, Perth, Western Australia. email vturner@cyllene.uwa.edu.au (2) Department of Emergency Medicine, Royal Perth Hospital, Perth, Western Australia; (3) Department of Pathology, University of Western Australia. (4) Department of History California State University, Chico Chico, California USA
REFERENCES
1. Barré-Sinoussi, F., et al. Science 220, 868-71 (1983).
2. Tahi, D. Continuum 5, 30-34 (1998).
The complete Continuum reference is: "Did Luc Montagnier discover HIV? Text of video interview with Professor Luc Montagnier at the Pasteur Institute July 18th 1997" http://www.virusmyth.com/aids/data/dtinterviewlm.htm
ADDENDUM II
REJECTED BY NATURE
NO REASON GIVEN
HIV TRANSMISSION FROM CHIMPANZEES
In their letter to Nature (February 4th), Gao et al claimed to have proven;
(i) the identification in a chimpanzee of the genome of a new simian immunodeficiency virus (SIVcpz), SIVcpz US;
(ii) that their chimpanzee, Marilyn, as well as two of the other 3 chimpanzees in which to date a SIVcpz was reported, GAB1 and GAB2, belonged to a chimpanzee subspecies, P.t. troglodytes;
(iii) "the natural range of P.t. troglodytes coincides uniquely with areas of HIV-1 group M, N and O endemicity";
(iv) "HIV-1 infection of humans occurred as a result of cross-species transmission of SIVcpz from P.t. troglodytes".
(v) "P.t. troglodytes is the primary reservoir for HIV-1";
A close analysis of the evidence on which they are basing their claims raises several questions:
(a) Marilyn was "wild-caught in Africa (country of origin unknown), exported to the United States as an infant".
Two of the chimpanzees, GAB1 and GAB2, originated in Gabon. GAB1, who was 4 years old when reported HIV-1 positive, was caught when she was "about 6 months" and was kept with another 49 wild-caught animals at the International Centre of Medical Research (CIRM) in Gabon. GAB2, who was also reported HIV-1 positive, was about 2 years old when she was shot in the wild, kept in a village for 2 days and then was brought to CIRM, "where it died of its wounds one week later".
In the 1989 study where the "HIV-1 seropositivity" of GAB1 and GAB2 was reported, the authors (which included one of Gao's co-authors, Peeters) concluded: "...on examination, none of the people caring for the animals and none of those living in the village showed antibodies to HIV/SIV. Furthermore, the region where the chimpanzee was captured is known to have a low seroprevalence rate...It has been suggested that human AIDS retroviruses originated from monkeys in Africa. However, this study and other previous studies on SIV do not support this suggestion". In other words, by the time when "HIV infection" and AIDS already reached their peak in the US, Europe and Australia, the number of individuals proven HIV seropositive in Gabon were few if any.
In a 1990 study published in Nature by researchers from CIRM and the Pasteur Institute, including Wain-Hobson, where the authors described "the molecular cloning and sequencing" of SIVcpzGAB1, one reads: "In Gabon, only 2 out of 83 chimpanzees tested were seropositive, indicating that SIVCPZ is not widely dispersed in this region...Of more than 250 chimpanzees caught over the last 15-20 years in West Africa, none were seropositive. This might explain the absence of naturally infected chimpanzees in captivity in the United States as virtually all are of West African origin".
How is it possible to espouse "prevalence in the natural host "geographic coincidence", and "plausible routes of transmission" as evidence to substantiate the claims that HIV-1 originated in P.t. troglodytes and that this sub-species is the natural reservoir for HIV-1?
(b) The three P.t. troglodytes, GAB1, GAB2 and Marilyn were said to be infected with HIV-1/SIVcpz on the basis of an antibody test.
However given that:
(i) as Philip Mortimer points out, "…it may be impossible to relate an antibody response specifically to HIV-1 infection".
(ii) when the blood was collected none of the animals was perfectly healthy although none had AIDS.
(iii) the only way to prove the specificity of an antibody test is to use the virus isolation as a gold standard. Although no effort has been spared, no SIVcpz could be isolated either from GAB2 or Marilyn (see comments below for GAB1).
How is it possible to claim proof for infection on the basis of an antibody test?
(c)
(i) If GAB1 and Marilyn were infected then, given that the animals were brought to the colony as infants where no other animals or humans working there were infected and, according to Weiss, "Chimpanzees in captivity are mostly taken from the wild before they become sexually active and so rarely harbour SIV", how did these two chimpanzees become infected?
(ii) Since the three chimpanzees found positive were all female, and since HIV/SIV is acquired following sexual maturity, how did they become infected?
(iii) If the animals were infected with a virus SIVcpz and this was transmitted to humans, why was this not transmitted to any other of the 49 animals at CIRM where GAB1 was kept or to the 93 animals in the colony where Marilyn was kept, not even to her 6 living offspring or her mates? (By the age of 26 she had a total of 14 pregnancies).
(d) The additional "lines of evidence" that Gao et al use to substantiate transmission are based on genomic studies. Gao et al claimed to have shown that "All HIV-1 strains known to infect man, including HIV-1 groups M, N and O, are closely related to just one of these SIVcpz lineages, that found in P.t. troglodytes". Indeed, if all these HIV-1 and SIVcpz strains represented one and the same virus, then their genomes will have to be "closely related". In fact they should represent a unique molecular entity. Even in the genomes of RNA viruses, including influenza, which are considered to be most variable, a 1% sequence difference is considered to represent "extreme variability". This is because small genetic differences lead to significant phenotypic differences. For example the difference between the human and the chimpanzee genome is less than 2%.
In the 1989 study of SIVcpzGAB1 Peeters et al wrote: "Nucleic acid hybridisation experiments appear to indicate that the virus is different from HIV-1 and HIV-2". In the 1990 Nature paper by researchers from CIRM and the Pasteur Institute, including Wain-Hobson, one reads: "Several regions of the chimpanzee sequences were more than 50% divergent with respect to HIV-1BRU. Some parts of the gag gene were almost as varied as the hypervariable regions in env...The vpu gene found only in the type 1 viruses was particularly different (64% divergent to HIV-1BRU)…It is also apparent that the SIVCPZ genome was not simply a more diverged HIV-1 isolate…It is not possible to conclude that SIVCPZ was the precursor to HIV-1, if indeed infection ever passed in that direction. Even given this premise the vpu data indicates that SIVCPZ was not the immediate precursor".
In a 1994 study of the SIVcpzGAB2, Peeters and his colleagues wrote: "The genetic distance between SIVcpz-gab [SIVcpz GAB1) and SIVcpz-gab2 is 14.1%. Genetic distances to the HIV-1 genotypes A, B and D strains are 13.7 to 16.3%, whereas distances to group O HIV-1 strains are 15.4 to 18.5%". Contrary to Gao, in 1994 Peeters and his associates concluded: "On the basis of their respective distances to each other and to the HIV-1 strains SIVcpz-gab and SIVcpz-gab2 can be assigned as representative for two distinct genetic lineages of HIV-1-related chimpanzee lentiviruses". By 1993 it was reported that "in the A-G HIV-1 genotypes the intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%...The maximum level of variability in gag is still well below that observed for the envelope region of HIV-1".
The HIV-1 group O has "65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 [HIV-1 group O] had similarities to HIV-1 and HIV-2 of 53 and 49% respectively...Comparison of the MVP-5180 amino acid sequences with that of the Gabon chimpanzee virus showed similarities of 70, 78 and 53% in the gag, pol and env genes, respectively".
As far as the genomic differences between HIV-1 group N, on the one hand, and group M and O on the other is concerned, it suffices to quote from the 1998 study where its existence was first reported. "Proviral DNA amplification with several sets of HIV-1 group M and O primers was attempted on pelleted end-cultured cells. Amplification was negative with eight different group M env, gag or pol primers and five group O env or gag primers". How is it possible to claim proof for the existence of a unique molecular entity which constitutes the genome of a unique retrovirus HIV-1/SIVcpz?
(e) The only way to prove that an RNA (and its cDNA) is the genome of a retrovirus is to demonstrate that it comes from a retrovirus particle and such RNA codes for its proteins. This can be done only by obtaining the particles separate from everything else, purifying, isolating them.
In the 1989 study where Peeters et al reported the isolation of SIVcpzGAB1, stimulated peripheral blood lymphocytes "from healthy human donors" were cocultured with the same type of cells from the chimpanzees. Supernatant from the coculture was centrifugated for 10 mins at 400.000g. Detection in the pellet of reverse transcriptase activity, using An(dT)12-18 as template primer was considered proof for SIVcpzGAB1 isolation. Such a method for viral isolation is no different from claiming that elevations in serum liver enzymes proves the existence of gallstones and moreover, that the gallstones have been isolated from the patient and are in the surgeon’s hands separate from everything else. The SIVcpzGAB1 "genome" was obtained either by hybridising the RNA present in the pellet (they presented no proof that the pellet contained even retrovirus-like particles), where one would expect to find ample cellular RNA, with probes "from HIV-1oyi, a Gabonese HIV-1 strain", or from "SIVCPZ-infected human lymphocytes" again using HIV-1oyi as a probe. The "genome" thus obtained was compared with the genome of HIV-1BRU.
We could find no details as to how the HIV-1oyi "genome" was obtained. HIV-1BRU is the "HIV-1" which according to Weiss was "discovered by Barre-Sinoussi and her colleagues in 1983". The senior author of the 1983 Barre-Sinoussi study was Luc Montagnier. However, in 1997 Montagnier not only acknowledged that they did not isolate HIV-1BRU, but their "pure" virus from where they chose some RNA and called HIV RNA, did not even contain particles with "morphology typical of retroviruses".
The only evidence ever presented as proving the existence of the SIVcpzGAB2 genome was reported by Peeters and his associates and is as follows: "From this chimpanzee we have been unable this far to isolate a lentivirus, but some of the primary peripheral blood mononuclear cells (PBMCs) have remained available in a frozen state. To investigate the genetic relationship to the SIVcpz-gab isolate [SIVcpzGAB1], proviral DNA was extracted from these primary PBMCs [no mention is made how it was possible to extract the proviral DNA from the chimpanzee DNA], and a 280-base pair (bp) fragment of the pol gene was amplified by a nested polymerase chain reaction (PCR). Subsequently PCR fragments were cloned and sequenced". No mention is made as to how they obtained the PCR primer. Gao et al used "consensus sequences" as primers and the following method: "Here we used the polymerase chain reaction (PCR) to amplify HIV- or SIV-related DNA sequences directly from uncultured (frozen) spleen and lymph-node tissue obtained at autopsy in order to characterise the infection responsible for Marilyn's HIV-1 seropositivity. Amplification and sequence analysis of subgenomic gag (508 base pairs (bp)) and pol (766 bp) fragments revealed the presence of a virus related to, but distinct from, known SIVcpz and HIV-1 strains. Because virus isolation from the autopsy tissues was unsuccessful, we used PCR to amplify and sequence four overlapping subgenomic fragments that together comprised a complete proviral genome, which we termed SIVcpzUS".
However,
(i) The specificity of the PCR for HIV has never been proven. The only way to obtain such proof is to use virus isolation as a gold standard. Even if one accepts the claims for SIVcpzGAB1 isolation, it is agreed that although no effort has been spared SIVcpz could not be isolated from the other two animals. This means that the PCR results obtained for the genomes of SIVcpzGAB2 and SIVcpzUS are false.
(ii) Even if they were specific for retroviruses; given that:
(a) The genome of all human beings and animals contain retroviral proviruses i.e. genomes of the endogenous retroviruses.
(b) There are homologies between the genomes of different retroviruses especially in the gag and pol genes. In fact according to Montagnier and Wain-Hobson the gag and pol genes "are generally conserved among retroviruses".
(c) In not one of the studies which claimed proof for the existence of the SIVcpz genomes did the authors use controls.
(iii) How is it possible to claim that the sequences detected in the DNA "of SIVcpz-infected human lymphocytes", the PBMCs of GAB2 and in Marilyn's "spleen and lymph-node tissue" were those of an exogenous retrovirus which is transmitted from one chimpanzee to another and from chimpanzee to humans and not those of an endogenous retrovirus?
(iv) Since there is no proof that the three chimpanzees ever came in contact with HIV-1 infected humans or animals or that they transmitted such a virus to other humans or animals, is it not more "plausible" to conclude that if these animals did harbour a retrovirus, the retrovirus was endogenous?
In analysing the "SIVcpz" molecular biology one cannot help reflecting upon the words of Sir John Maddox, "Is there a danger, in molecular biology, that the accumulation of data will get so far ahead of its assimilation into a conceptual framework that the data will eventually prove an encumbrance? Part of the trouble is that excitement of the chase leaves little time for reflection. And there are grants for producing data, but hardly any for standing back in contemplation".
Eleni Papadopulos-Eleopulos1 Valendar F.Turner2 John M. Papadimitriou3 David Causer1 Bruce Hedland-Thomas1 Barry Page1 Charles Geshekter4 Etienne DeHarven5
1Department of Medical Physics, Royal Perth Hospital, Perth, Western Australia. email bruce.hedland-thomas@rph.health.wa.gov.au 2Department of Emergency Medicine, Royal Perth Hospital, Perth, Western Australia; 3Department of Pathology, University of Western Australia. 4Department of History California State University, Chico Chico, California USA 5Professor (Emeritus) of Pathology, University of Toronto, Toronto, Canada.
REFERENCES
1. Gao, F., et al. Nature 397, 436-441 (1999).
2. Peeters, M., et al. Aids 6, 447-51 (1992).
3. Huet, T., Cheynier, R., Meyerhans, A., Roelants, G. & Wain-Hobson, S. Nature 345, 356-9 (1990).
4. Mortimer, P.P. Med. Internat. 56, 2334-2339 (1989).
5. Weiss, R.A. & Wrangham, R.W. Nature 397, 385-386 (1999).
6. Gilden, R.V. et al. Lancet i, 678-679 (1986).
7. Steinhauer, D.A. & Holland, J.J. Annual Review of Microbiology 41, 409-433 (1987).
8. Janssens, W., et al. AIDS Research and Human Retroviruses 10, 1191-2 (1994).
9. Louwagie, J., et al. AIDS 7, 769-780 (1993).
10. Gurtler, L.G., et al. Journal of Virology 68, 1581-5 (1994).
11. Simon, F., et al. Nature Medicine 4, 1032-7 (1998).
12. Toplin, I. Spectra , 225-235 (1973).
13. Tahi, D. Continuum 5, 30-34 (1998).
14. Lower, R., Lower, J. & Kurth, R. Proceedings of the National Academy of Sciences of the United States of America 93, 5177-5184 (1996).
15. Wain-Hobson, S., Alizon, M. & Montagnier, L. Nature 313, 743 (1985).
16. Maddox, J. Nature 335, 11 (1988).
Reference 13 is also available at http://www.virusmyth.com/aids/data/dtinterviewlm.htm
ADDENDUM III
19/7/99
Dear Mr Ranieri
I have been thinking about your e-mail of 8 April, and I realise that I cannot have made myself clear when I first responded to your query. So let me take up the two points you made, even though nearly 2 months have elapsed.
1. You understand that Dr Turner and colleagues have not questioned whether other viruses besides HIV exist, and cause disease. You are, of course, absolutely right that a scientist does not prove that a particular virus exists by pointing to the existence of others. That is the inverse of the point I was trying to make. It is precisely because Val Turner and his colleagues in Perth have not queried the existence of other viruses that I find it difficult to take their ideas on HIV seriously. All the 'failings' they attirbute to HIV could equally well, according to their own stringent criteria, be levelled against any virus with a lipid envlope, eg small pox, influenza, measles, mumps or yellow fever. The evidence is as good, or rather, in Dr Turner's eyes as bad, as for HIV. No virologist in the 101 year history of the discovery of viruses ever demanded for any virus the definition of 'purification' that they and the magazine Continuum claim are the standard criteria of virus isolation. So we end up talking at cross-purposes.
2. You find it difficult to accept that I can bow out of this discussion. Let me explain by analogy. Do you expect today's physicists to spend time in prolonged e-mail 'discussion' with members of the flat earth society? Or do you think modern astronomers are merely speculating to hold, as Copernicus, Keppler and Galileo bravely did, that the planets orbit around the sun rather than the earth? Would you think it irresponsible for an astronomer in 1999 to bow out of such a discussion if a believer in a flat, planar universe was ignored and then claimed his view was deliberately suppressed by the scientific orthodoxy of the day? So who do you think is the Galileo on HIV - Turner or Montagnier?
Now let's come back to my argument under item 1, that the evidence the existence of viruses like measles or yellow fever is no better than that for HIV. Even if as a non-scientific journalist you did not wish to take sides, would you think it logical to hold that Jupiter, say, orbits the earth while Mars, Venus, Saturn, etc, orbit the sun?
As to bowing out, perhaps I should not have bowed in my replying to Dr Turner in the first place. Dr Turner e-mailed me after I co-authored a short commentary in Nature on SIV in chipanzees being a possible source of human HIV, based on other scientists' work. He contacted me, to clarify my views on the existence of HIV and on a quote of Montagnier, because his patients were anxious about it (do surgeons treat AIDS?). After he said he passed his reply to you (are you his patient?), [Ranieri is not a patient] he told me that any communication he receives from me is his property. I now realise that Dr Turner was spoiling for a fight. He knew my views already, but wished to engage in argument in a manner that to me seems strange to open, cordial scientific dispute. So I chose to bow out.
However, it would be difficult to accuse me of supposing the debate about whether HIV causes AIDS. Together with Dr Harold Jaffe I have stated my views succinctly and cogently nine years ago (Nature 345: 659-660, 1990). I have nothing more to add, or subtract, from what we put on record then, except that what new, reliable data have come to light since then confirm our views [Duesberg never took anyone to task over the isolation/existence of HIV. The Perth group fare still the only scientists to publish these arguments]. So why should I go on repeating myself? If I were a radio or television producer, I think I might seek to explore a programme on latter day flat-earthers as a sociological phenomenon. What lies behind HIV and AIDS denial? Why do such people feel persecuted by scientific orthodoxy when in reality they are simply ignored? Why do quasi-fascist concepts keep slipping in, like the holy grail of 'purification'? Doesn't HIV denial resemble holocaust denial?
First, I would take a vacation on the beach with the writings of two Italian Nobel Laureates who had suffered under fascism: Enrico Fermi the physicist who discovered nuclear fusion, and Primo Levi, the chemist who won the Nobel Prize for Literature, but later took his own life. These two thinkers would help me to place the issues of scientific controversy and society in proportion. [Why not Giordano Bruno?]
If all that seemed too strong to my director of programmes for family viewing, I would propose a documentary to mark the centenary of the discovery of viruses, ie distinguishing them from other microbes. How influenza killed more people in 1918/19 than all who died in the Great War, whether it will happen again, why the Hong Kong chicken flu didn't spread. How WHO conquered smallpox, controlled yellow fever and is on its way to eradicating polio. Why HIV, cancer viruses and herpes viruses cannot be eliminated in the same way. Where HIV came from and where it may be going. Will new viruses appear? Should we be scared about Ebola and Lassa fever spreading to Western Australia? Why did a virus related to measles transfer from fruit-bats to horses and thence to humans in Queensland three years ago, and in Malaysia three months ago. Are the fruit-bats in the surburbs of Australian cities therefore a threat to human health? There's plenty of good material for responsible, entertaining and lively debate about viruses waiting for an enterprising reporter.
Kind Regards
Robin A Weiss
(1) Dr. VF Turner
Consultant Emergency Physician
Royal Perth Hospital
Perth, Western Australia
Voice +618 92242693
Fax +618 92247045
Email vturner@cyllene.uwa.edu.au |
(2) Robin A Weiss
Professor of Viral Oncology
Wohl Virion Centre
Windeyer Institute of Medical Sciences
University College London
46 Cleveland Street
London W1P 6DB, UK
Tel: +44 207 679 9554
Fax: +44 207 679 9555
email: r.weiss@ucl.ac.uk |