Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala
Send response to journal:
Eight simple questions and a repeated request to Brian Foley
In his rapid response “Re: A paraphrased request and a question to Brian Foley”, 14th May 2004, Brian Foley wrote: "The Perth group wrote: "…Our request to Brian Foley is: Is it true that
(a) In 1983/85 Montagnier’s and Gallo’s groups claimed to have purified “HIV” and from this “purified” “HIV” to have obtained the “HIV” poly- (A) RNA, that is, the “HIV” genome (and thereby some of the best known and still most often used “HIV” molecular clones). But neither group published electron micrographs (EM) to confirm their purification;
(b) In 1997 Montagnier admitted that his group did not purify “HIV” and in his view neither did Gallo’s group. In fact Montagnier stated that they did not publish any EM because in the “purified” “HIV” they could not find any particles “with the morphology typical of retroviruses”;
(c) In the only EM of “purified” “HIV” published to date the vast majority of the material is microvesicles, that is, cellular fragments amongst which there are a small number of particles which the authors claim to be “HIV” but none of which has all the morphological characteristics attributed to “HIV”.
Yes or no?"
Brian Foley replied: “No; maybe but not relevant; and no:"
We are amazed that Brian Foley’s answer to our question (a) regarding “HIV” purification was “No” since:
(a) in 1983 Montagnier wrote: "That this new isolate was a retrovirus was further indicated by its density in a sucrose gradient which was 1.16 and….when purified, labelled virus from patient 1 was analysed under similar conditions, [reacted with sera from patient 1], three major proteins could be seen: the p25 protein and proteins with molecular weights of 80.000 and 45.000." (1) (emphasis ours);
(b) in the 1984 Science papers Gallo wrote: "The virus was purified from supernatants of cell cultures supporting the continuous production of HTLV-III [HIV]. The virus showed a difference in the makeup of its protein components as revealed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of a sucrose density banded preparation". (2) (emphasis ours)
In answering our first question, (a), Brian Foley wrote: “In the 1983-1985 time period Montagnier's and Gallo's groups did indeed publish electron micrographs of the virus. None of the EMs showed the virus was 100% pure, but that is not necessary. They usually published EMs of virus budding from cells and in the intercellular spaces rather than EMs harvested from the supernatant. They published Southern, northern and western blots of viral RNA, proviral DNA and viral proteins. They showed that antibodies reactive to multiple proteins produced by this virus were found in AIDS patients and a subset of people who were at very high risk for developing AIDS, but not found among the general population.”
We notice that Brian Foley wrote “usually published” instead of “always published” implying that they have indeed published EMs of the 1.16g/ml band despite the fact that in 1997 Montagnier stated that in 1983 he did not publish an EM of the "purified" virus because even after Roman effort they could not see any particles with the "morphology typical of retrovirus" (3). Also when Montagnier was asked if Gallo purified "HIV" he replied: "Gallo?…I don't know if he really purified. I don't believe so" implying that Gallo’s group did not publish EMs of the 1.16g/ml band, their “purified” virus.
So our questions to Brian Foley are:
Q1. Is it true that neither group published EMs of the 1.16g/ml band to confirm purification.
Yes or No?
Q2. Is it true that Montagnier's and Gallo's "viral RNA [and thus] proviral DNA and viral proteins" used for "Southern, northern and western blots" originated from their "purified" virus which may or may not have contained any particles with the morphology of retroviruses
Yes or No?
Q3. Is it true that the only proof they gave for the existence of the “HIV” proteins was a reaction between some proteins in their “purified” virus and antibodies present in the AIDS patients’ sera.
Yes or No?
Q4. Is it true that a reaction between an antigen and antibodies present in an animal or human serum is not proof that the antigen is present in the animal or human and thus that the antibodies are directed against the antigen
Yes or No?
In 1997 Montagnier stated: "….analysis of the proteins [and thus of the nucleic acids] of the virus demands mass production and purification. It is necessary to do that". (3) (emphasis added).
Q5. Since Montagnier and Gallo did not have proof for “purified HIV”, were their “Southern, northern and western Blots” valid.
Yes or No?
Brian Foley wrote: "Only one of the dozens of molecular clones of the HIV-1 M group subtype B viruses described in the 1983-1985 time period eventually became proven to be an infectious molecular clone, and went on to become one of the best known infectious molecular clones of subtype B. That clone is Lambda-HXB-2, sequenced in 1985 by Ratner et al.  and shown to be infectious by Fisher in 1986 ".
In their paper entitled: "Complete nucleotide sequence of the AIDS virus, HTLV-III" (“HIV”), Ratner, Gallo and their colleagues wrote: "Clone HXB2 was derived from a recombinant phage library or XbaI-digested DNA from HTLV-III-infected H9 cell cloned in lJ1 (ref. 53)”(4).
In ref. 53 by Shaw, Gallo et al, the authors wrote: that the "H9/HTLV-III DNA" [the DNA of the “infected” H9 cells] was screened with an HTLV-III cDNA probe (8) to obtain molecular clones of full-length integrated provirus with flanking cellular sequences.…Figure 1 illustrates the restriction maps of these two clones, designated lHXB-2 and lHXB-3" (5).
Ref. 8 is a paper published by Arya, Gallo and their colleagues. There one reads: "The virus particles were purified from supernatant fluids of HT cells, clone 9 (H9) infected with HTLV-III (HTLV-IIIB) by centrifugation through a sucrose density gradient at equilibrium….The particles were lysed with sodium dodecyl sulfate (SDS), digested with proteinase K, and directly chromatographed on an oligo(dT) cellulose column. The resulting polyadenylate [poly(A)]-containing RNA was used as template to synthesise 32P-labeled complementary DNA (cDNA) [“HIV” genome] in the presence of oligo(dT primers". (6) (emphasis ours).
Q6. Is it true then, that the origin of lHXB-2 "one of the best known infectious molecular clones" of "HIV" is a poly(A)-RNA found in Gallo's "purified" HIV.
Yes or No?
Brian Foley claims that the lHXB-2 "was shown to be infectious by Fisher in 1986 ". We defined infectious molecular clone as follows: "…by infectious molecular clone of a virus is meant the introduction of the viral genome (molecular clone) into suitable cells leading to the appearance of virus particles identical (regarding both appearance and composition) to the ones from which the genome was obtained".
Brian Foley defined infectious molecular clone as follows: "The clone must produce virus particles that are identical by serology, morphology, protein sequences, RFLP, Southern blotting, etc. to the parental virus, and the particles must also be infectious. If a cloned viral genome does not meet these criteria, it is not an INFECTIOUS molecular clone of the virus, be it HIV-1 or any other virus". (emphasis in original).
The evidence in the Fisher, Gallo et al study does not satisfy the criteria of an infectious molecular clone as defined by either Brian Foley or us.
(Fisher AG et al. A molecular clone of HTLV-III with biological activity. Nature 1985;316:262-265).
Brian Foley's answer to our second question, (b), was "maybe but not relevant".
In his rapid response, "Re: A question and a request to Brian Foley", 13th May, Brian Foley agrees that poly(A)-RNA is not specific to retroviruses.
In his rapid response, "Re: Re-phrasing our two questions to Brian Foley", 11th May, Brian Foley agrees that the poly(A)-RNA which Montagnier and Gallo claimed to be the "HIV" genome originated from their 1.16g/ml band, which they claimed was "purified" virus.
Q7. If this is the case, is it not true that to claim the poly(A)-RNA in the 1.16g/ml band (the "purified" virus) is the "HIV" genome, evidence must exist which proves beyond reasonable doubt that the "purified" virus contained nothing else but particles with the morphology of retroviruses. (If impurities existed, they must not contain RNA).
Yes or No?
Brian Foley wrote: "I am not sure what Montagnier meant, if he stated that the material in the 1.16g/ml bands his Lab produced contained no "particles with the morphology typical of retroviruses". He may have only meant that he found the cone-shaped cores of this lentivirus to be unique. Another possibility is that something went wrong between collecting the band and preparing microscope slides, such that the viral particles were disrupted. Any comments on that should be made by the workers in Montagnier's lab who did the work, and not by those of us who can only speculate on the dozens of possibilities".
We agree with Brian Foley that neither we nor anybody else including Brian Foley should speculate about what was present in the Gallo and Montagnier "purified" virus. That is why we have been asking for so long for them to publish EMs of their "purified" virus in order that everyone can see what they contain.
Brian Foley's answer to our 3rd question (c), is "no".
Brian Foley wrote: "(c) Although the Perth group continually attempts to mislead people into believing that only one or a very few EMs of HIV exist, there have actually been several electron micrographs of HIV-1 obtained from 1.16g/ml bands [3,4 for examples]. There have been dozens more EMs of HIV-1 pelleted through a sucrose cushion and resuspended [5,6,7 for examples]. None of these shows 100% pure virus particles. The virus particles in those EMs look like HIV-1 or any other lentivirus to me. Whether or not they contain "all" of the morphological characteristics attributed to HIV-1 depends on what list of characteristics you are referring to. I would not expect the envelope glycoprotein spikes often seen on virus as it is budding from cells [8,9,10 for examples] to always survive ultracentrifugation, for example".
We are pleased that Brian Foley accepts that to date nobody has published EMs either from the 1.16g/ml band or “pelleted through a sucrose cushion and resuspended” showing pure virus particles. Brian Foley's reference 3 and 4 are the papers by Bess et al and Gluschankof et al. We have referred many times to these studies in our rapid responses and claimed and still claim that they are the only studies with EMs from the 1.16g/ml (purified “HIV”). Since Brian Foley states that these studies are only “examples” and that “there have actually been several electron micrographs of HIV-1 obtained from 1.16g/ml bands”, would he please give us other references.
Note that Brian Foley’s reference 7 is a study of SIV and thus irrelevant to our debate.
According to Robert Gallo “Retroviruses are enveloped viruses, usually about 100 nm in diameter that form by budding from cell membranes” (7). According to Hans Gelderblom are " viruses with a diameter of 100-120 nm budding at cellular membranes. Cell released virions contain condensed inner bodies (cores) and are studded with projections (spikes, knobs)" (8). According to Pablo Gluschankof, Hans Gelderblom and their colleagues the "HIV" particles "can be identified by the relatively homogenous diameter of about 110nm, the dense cone-shaped core and the "lateral bodies"". (9)
In Brian Foley’s reference 6, “Mature HIV-1 particles were harvested from infected MT-4 cells…Rapid sample preparation minimised deleterious effects on the particle structure.” The diameter of the particles was 145 ±25nm. Not all of the particles had "a central, dense core structure". Many of the particles had a single core which was "cone-shaped". "Surprisingly, a significant fraction of the mature particles contained a second core assembly (32.6%)…which can be conical ["of various sizes"] tubular (C and D arrows) or amorphous…No structures that could correspond to the previously reported lateral bodies were visible". Furthermore, given the large variation of the particles’ contents, it is not possible to say, which, if any, of these particles would satisfy the main physical properties of retroviruses i.e. in sucrose density gradients will band at the density of 1.16g/ml. One would have to conclude then, that either the particles described in Brian Foley's reference 6 are not retroviral particles or the definition of retroviruses is wrong.
If the particles described are retroviruses, since:
(a) they were "harvested" from MT-4 cultures;
(b) according to Montagnier "There are MT cell lines which replicate HIV very well and which at the same time are transformed by HTLV. So, you have a mix of HIV and MTLV. It is a real soup".
It is not possible to say which, if any, are “HIV”. (The authors of Brian Foley's ref. 6 had no controls).
The evidence in Brian Foley's ref. 5 raises a few questions regarding the nature of their particles. As far as Brian Foley's claim: "The virus particles in those EMs look like HIV-1 or any other lentivirus to me", suffice to mention:
(1) According to all the "HIV" experts the surface of the “HIV” particles are studded with spikes. According to Montagnier "particles of HIV are shaped like little spheres, each with roughly eighty little rounded projection shaped like pegs [spikes] (see the accompanying figure). Each peg contains three or four molecules of a large protein, gp120, which has a strong affinity for the receptors (now called CD4) of T4 lymphocytes".(10)
According to the authors of Brian Foley's ref. 5: "The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not physically resemble spikes….[the tufts] "are hardly protrusions at all…We have examined many hundreds of these tufts on many hundreds of particles and have found no evidence of even approximate threefold symmetry…some of the protein tufts we observed might represent cellular proteins".(11)
(2) According to Kuznetsov et al, that is the authors of Brian Foley's 5 ref: "The particles recovered from the medium and displayed on the glass substrate appear almost identical to those analysed on the surface of HIV-infected cells and are virtually indistinguishable from virions of MuLV imaged in an earlier study". MuLV is a type-C oncovirus particle not a lentivirus.
(3) Kuznetsov et al used "HIV-infected H9 cell cultures". Thanks to the Gallo enquiry it became clear that the H9 cell line is a clone of the HUT-78 cell line. According to Gallo HUT-78, like MT-4, is infected with HTLV-I, a type-C oncovirus particle. (Wong-Staal F et al. A survey of human leukemias for sequences of a human retrovirus. Nature 1983;302:626-628).
Brian Foley wrote: "In the centre panel of Figure 3 of  which is stated to be virus from the HIV-1(MN)/H9 Clone 4 preparation with a p24-capsid to human HLA ratio of 4.61 to 1 as shown in table 1, there are many more viral particles than cellular vesicles, so I would claim that the Perth group is attempting to mislead people when they state that in the only EM of purified HIV ever published to date, the vast majority of the material is microvesicles".
We have repeatedly asked Brian Foley: "Since "HIV-1(MN)" has been used by laboratories around the world, including Bess' laboratory, in his view would Brian Foley please tell us which of Gallo's evidence proves the existence of "HIV-1(MN)""? (Questions and Answers, 13h October 2003; Basic request which remain unanswered by Brian Foley and Christopher Noble, 24th December 2003; Our basic requests to Brian Foley and Christopher Noble remain still unanswered, 29th April 2004). We are still waiting for an answer. In our rapid response Questions and Answers, 13th October 2003, we wrote: "Note that the only proof which Gallo had given for "the MN isolate" and its transmission and thus for the existence "of the MN virus which has been used by laboratories around the world" is detection in cultures of reverse transcriptase activity (RT), which is non-specific to retroviruses".
In other words "HIV-1(MN)” is nothing more than the supernatant from a stimulated (oxidised) culture having reverse transcriptase activity. The "p24-capsid to human HLA ratio of 4.61 to 1" does not prove that the material which is illustrated in the centre panel of Figure 3 in the Bess et al study contains 4.61 "HIV" particles to 1 microvesicle. This is because:
(1) Bess et al have shown that a p24 is present not only in the "purified" "HIV" but also in the microvesicle. Montagnier was the first to claim proof for the existence of the "HIV" p24. However, he obtained the proteins from material which did not contain even retrovirus particles. This is as good a proof as any that p24 is not an "HIV" protein;
(2) Even if p24 is an "HIV" protein, since both p24 and HLA were determined by an antibody-antigen reaction, and since all antibodies, including monoclonal, cross-react, such a reaction cannot be used to quantify or even identify the proteins;
(3) The p24/HLA ratio in table 1 and the EMs in the centre panel of figure 3 were obtained from different preparations. As far as the arrowed particles in the centre panel of figure 3 said to be "HIV" particles suffice to repeat what we said in our rapid response, "The request remains the same and is still pure and simple", 12th June 2003: “Furthermore, none of the particles arrowed as being HIV have the morphological characteristics attributed to HIV or even to retroviruses. There is no correlation even in their diameters. Retroviruses have a diameter of 100-120nm while the diameters of the particles in the two electron micrographs assumed to be HIV have an average diameter of 234nM with none of these particles having a diameter of less than 160nM. By just this criterion alone it is impossible to claim this material contains even a retrovirus, pure or impure".
Brian Foley wrote: “If one person says HIV has not been properly isolated and another (or 50,000 others) say that is has, one side or the other must not be telling the truth. Asking each of them to repeat their assertions is no substitute for looking at the raw data”.
We agree that there “is no substitute for looking at the raw data.”.
Although Brian Foley has failed to tell us in his view who is the discoverer of “HIV”, in his rapid response “Re: Out of all the Responses Four Arguments Have Emerged. Three of These Things are not Like the Other...” 24th March 2004, he wrote: “Gallo and Montagnier are credited with "discovering" HIV-1”.
Q8. Doesn’t it follow that we must start with Montagnier’s and Gallo’s groups 1983-1985 “raw data”.
Yes or No?
Brian Foley wrote: "I have previously cited more than 50 papers which describe clones meeting those criteria and more, but I will go through one of them  step-by-step as requested by the Perth group. In this paper the authors describe the creation and preliminary analyses of an infectious molecular clone of a HIV-1 M group subtype C isolate named 93IN101, isolated in India in 1993 from a cohort of individuals described in , and deposited in the NIH AIDS Reagent Repository . The infectious molecular clone was named plasmid-Indie-C1. Virions produced from the cloned virus are shown in figure 2B".
We asked for references which contain scientific proof for the existence of "HIV" infectious molecular clones, not for a reference containing "preliminary analyses". Ref 16 and 17 contain no scientific information regarding either 93IN101 or the plasmid-Indie-C1. Nowhere in ref. 16, 17 or 15 is there any evidence that 93IN101 or the primers used to obtain plasma-Indie-C1 either directly or indirectly originated from a retrovirus particle.
In figure 2B there are some extracellular, but not budding, particles. We have only a photocopy of the study, and maybe because of this, it is not clear if the particles have the morphological characteristics of retroviruses. Nowhere in ref. 15 is there any evidence that these particles satisfy either ours or Brian Foley's definition of an infectious molecular clone.
So we repeat our request: “Would Brian Foley please give us a study and a few confirmatory studies where the existence of an “infectious molecular clone” of “HIV” has been proven.”
1. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioun C, Rozenbaum W, Montagnier L (1983) Isolation of a T-Lymphotrophic Retrovirus from a patient at Risk for Acquired Immune Deficiency Syndrome (AIDS). Science 220:868-871.
2. Sarngadharan M, G., Popovic M, Bruch L. (1984). Antibodies Reactive to Human T-Lymphotrophic Retroviruses (HTLV-III) in the Serum of Patients with AIDS. Science 224:506-508.
3. Tahi D. (1998) Did Luc Montagnier discover HIV? Text of video interview with Professor Luc Montagnier at the Pasteur Institute July 18th 1997. Continuum 5:30-34.
4. Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, Gallo RC, Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277-284.
5. Shaw GM, Hahn BH, Arya S, Groopman JE, Gallo RC, Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. (1984) Science 226: 1165-1171.
6. Ayra SK, Gallo RC, Hahn BH, Shaw GM, Popovic M, Salahuddin SZ, Wong-Staal F. Homology of genome of AIDS-associated virus with genomes of human T-cell leukemia viruses. (1984) Science 225: 927-929.
7. Gallo RC, Fauci AS. The human retroviruses. In: Isselbacher KJ, Braunwald E, Wilson JD, Martin JB, Fauci AS, Kasper DL, editors. Harrisons Principles of Internal Medicine. 13 ed. New York: McGraw-Hill Inc., 1994:808-814.Gelderblom HR, Özel M, Hausmann EHS, Winkel T, Pauli G, Koch MA. (1988). Fine Structure of Human Immunodeficiency Virus (HIV), Immunolocalization of Structural Proteins and Virus-Cell Relation. Micron Microscopica 19:41-60
8. Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ. Cell Membrane Vesicles Are a Major Contaminant of Gradient-Enriched Human Immunodeficiency Virus Type-1 Preparations. (1997) Virology 230: 125-133.
9. Montagnier, L (2000) Virus. page 88. W.W. Norton & Company, New York, London.
10. Kuznetsov YG, Victoria JG, Robinson WE, Jr., McPherson A. Atomic force microscopy investigation of human immunodeficiency virus (HIV) and HIV-infected lymphocytes. J Virol 2003;77:11896-909.
Competing interests: None declared