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
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New and repeated questions and requests to Brian Foley
In his rapid response :Re: Twelve simple questions to Brian Foley”, 2nd June 2004, Brian Foley did not answer most of our simple questions. He selected bits and pieces from our rapid response upon which to comment.
Brian Foley wrote: “Everyone agrees that HIV-1 and other lentiviruses are enveloped viruses. The envelope is composed of host cell membrane, containing several host proteins such as HLA-DR, as well as the viral Env protein. These host cell proteins, whether they are present on the viral particles themselves, or in contaminating material, can influence vaccine outcomes, as summarized by David Montefiori in 1995 (1). They do not invalidate all other HIV research. There is no “law” stating that virus preparations must be 100% “pure” to be useful…The paper by George Shaw (2) does use the word “purified”, but they do not state that this means the virus was 100% pure. Likewise, the paper by Mikulas Popovic (4) also used the statement “The yield of virus from H4/HTLV-III cells was assessed by purification of concentrated culture fluids through a sucrose density gradient…” but again I would not interpret that as a claim for 100% purity…None of the papers by Montagnier’s group used the words “pure” or “purified” as far as I can see.”
On page 870, 3rd column, line 8 of their 1983 paper, Montagnier’s group wrote: “When purified, labeled virus from patient 1 was analyzed…”. (1)
Q1. If the virus preparation is not 100% pure but contains extraneous elements such as cellular vesicles which contain proteins and poly(A)-RNA by what means can anyone prove what is cellular and what is viral? For example, how can anyone determine whether a protein with molecular weight of 41000 or a poly(A)-RNA present in the impure virus preparation is cellular or viral?
Note that Brian Foley agreed that the poly(A)-RNA is not specific to retroviruses and that the antibody-antigen reaction is also non-specific.
Brian Foley wrote: “…it is the serological and molecular characterization of the viruses that is critical.”
Q2. Isn’t it true that to do serological and molecular characterisation of the virus first one has to obtain its proteins and genome.
Yes or No?
Brian Foley wrote: “The “critical analysis” of evidence includes repeating the experiments, which is what the Levy lab did to reconfirm the serology of AIDS in the USA (6) and to obtain another HIV-1 M group subtype B genome, from another USA AIDS patient (7,8).”
To “reconfirm the serology of AIDS in the USA”, in Levy’s study, cells were reacted with AIDS patients’ sera. That this reaction is totally non-specific is demonstrated in both Montagnier’s 1983 and Gallo’s 1984 papers. Levy did not confirm Montagnier’s and Gallo’s findings. Unlike Gallo’s and Montagnier’s, his virus was a “type-D” having a provirus of “> 15kb” while Montagnier’s was “9.1 – 9.2 kb” and Gallo’s “approximately 10 kilobases”.
Brian Foley wrote: “Dr. Levy’s clone turned out to be another HIV-1 M group subtype B virus, and the complete genome accession number is K02007. Anyone can look it up and find that it has the Vif, Vpu, Tat, Rev and Nef accessory genes of the HIV-1 M group of lentiviruses. It is not a T-cell leukemia virus at all.”
We would be grateful if Brian Foley would please give us Levy’s reference(s) where the complete genome and its genes were determined.
Brian Foley wrote: “I have already provided the Perth group with many papers which prove that infectious molecular clones of HIV-1 and many other lentiviruses have indeed been created and studied. The Perth group denies that any of them meets their criteria for “proof” and they are correct, because the “proof” they are looking for is impossible. No virus has ever met those criteria, and no lentivirus ever can meet them.”
As we stated in our rapid response “A paraphrased request and a question to Brian Foley” 14th May 2004, “We did not ask for the titles of 90 studies. Especially studies conducted in HIV-2, SIVs, SHIVs, BIV and FIV. Neither for “HIV-1” studies which have no evidence for the existence of “infectious molecular clones”. Let us paraphrase our request: Would Brian Foley please give us a summary of the evidence (not just the title) of a study as well as the evidence from a few confirmatory studies where the existence of an “infectious molecular clone” (as defined by Brian Foley) of “HIV-1” has been proven.”
In Brian Foley’s rapid response “Re: A paraphrased request and a question to Brian Foley” 14 May 2004, he 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 the Fisher, Gallo et al study, the authors “used a transfection technique to investigate the biological properties of molecular cloned HTLV-III DNA.” The clone used was lambda-HXB2 whose ultimate origin is a poly(A)-RNA originating from the 1.16g/ml band. They reported:
(a) “Therefore we have no direct evidence that transfected HTLV-III is integrated in the host cell genome. HTLV-III may integrate randomly and at too low a frequency to be detected by Southern blotting.”
(b) “Expression of the HTLV-III gag-related proteins p15 and p24 by transfected celled was demonstrated using special monoclonal antibodies.” That is, by a totally non-specific reaction.
(c) “Virus particles were detected by electron microscope in all cultures 14-18 days after transfection with pHXB-2D (Fig 4f).”
It is clear that the evidence given in the Fisher et al study does not demonstrate the existence of an “HIV-1 infectious molecular clone” as defined by Brian Foley: “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” (his emphasis). (See Brian Foley’s rapid response “Re: Re-phrasing our two questions to Brian Foley”, 11th May 2004)
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 .”
Nowhere in references 14,15 and 16 is there any evidence that the ultimate origin of the molecular clone, that is, the stretch of DNA called 93IN101, is an RNA which originated from “HIV” particles. In other words, there is no evidence that ultimately 93IN101 is the cDNA of an RNA found in “HIV” particles, the “HIV” genome.
Brian Foley wrote: “The infectious molecular clone was named plasmid-Indie-C1… Virions produced from the pIndie-C1 infectious molecular clone were proven to infect cells… Production of viral proteins from cells infected with virus particles derived from the pIndie-C1 infectious molecular clone was similar to the production of viral proteins produced by another, well-characterized infectious molecular clones of HIV-1 (pNL43 as shown in figure 2D).
Cells transfected with the pIndie-C1 plasmid showed marked cytopathic effects (figure 2A). The gp120 Envelope protein produced by the pIndie-C1 clone reacted more strongly with sera from individuals infected with HIV-1 M group subtype C viruses (figure 3A lane d, bottom two blots) than with sera from individuals infected with HIV-1 M group subtype B viruses (figure 3A lane d, top two blots), whereas gp120 produced from subtype B infectious molecular clones pNL43 and pSF2 reacted more strongly with the sera from the individuals infected with subtype B (figure 3A lanes b and c). No reactivity was detected from the uninfected control cells (figure 3A lane a).
As shown in figure 3B, top frame (note that the lanes were loaded in a different order; a-control, b-pNL43, c-pSF2, d-pIndie-C1), the 4H4 monoclonal antibody, which recognizes the Nef epitope shown below , reacted strongly with the Nef proteins produced from the pIndie-C1 and pNL43 clones, but not with the pSF2 clone, as might be expected by the many differences in this region of the pSF2 epitope:
4H4 Epitope MGGKWSKSSVVGWPTVRERMRRAPTVRERMRRAEPAADGVGAA
As shown in figure 3B, bottom frame, a mixture of three monoclonal antibodies which each recognize a different Vpr epitope, reacted equally strongly with the Nef proteins produced by all three clones.
“Production of viral proteins” and of “virions” was determined by a reaction between antibodies to p24, gp120, Nef proteins or patients’ sera and “infected” cells (not the virus particles). In addition, the reaction is totally non-specific.
Brian Foley wrote: “Virions produced from the cloned virus are shown in figure 2B.”
Figure 2B shows extra-cellular virus-like particles.
Nowhere in reference 14 is there any evidence that the particles satisfy Brian Foley’s definition of an “infectious molecular clone”: “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.” (See Brian Foley’s rapid response “Re: Re-phrasing our two questions to Brian Foley”, 11th May 2004)
So let us repeat our request to Brian Foley once again:
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.
In response to two of our questions:
Brian Foley wrote: “Briefly, retroviruses carry two single-stranded RNA genomes in the virion. The retroviral reverse transcriptase uses a cellular transfer RNA primer (the Lys-3 tRNA for all lentiviruses) (12). The retroviral integrase enzyme then inserts the proviral DNA into the genome of the host cell, where it can remain dormant for the life of the cell, or can become activated to produce new virions, if it is not defective. The ultimate origin of the proviral genomes in the Lamba-HXB-2 and Lamda-XHB-3 was not any 1.16 g/ml band. The probe used to screen the genomic library WAS derived from one or more 1.16 g/ml bands.”
We are pleased that Brian Foley agrees with us that the “probe used to screen the genomic library WAS derived from one or more 1.16 g/ml bands.”. Both these questions were prefaced by quoting from three of Gallo’s papers to make to clear how the lambda-HXB-2 and the probe were derived. It appears that more explanation is necessary.
Gallo’s group performed two series of experiments. In the first, H9 cells were cultured with tissue derived from AIDS patients. The supernatant from these cultures was banded in sucrose gradients. A poly(A)-RNA was extracted from the 1.16g/ml band and this RNA was defined as being the “HIV” genome. The cDNA of this poly(A)-RNA was used as a probe in the second series of experiments.
In the second series of experiments, an H9 cell culture was “infected” with “HIV”. According to all HIV experts, including Brian Foley, when “HIV” enters a cell its RNA, which by definition is a poly(A)-RNA identical to the poly(A)-RNA obtained from the 1.16g/ml band in the first series of experiments, is reverse transcribed into cDNA (“HIV” proviral DNA). “The retroviral integrase enzyme then inserts the proviral DNA into the genome of the host cell”.
Following the “infection” of the H9 cells, Gallo and his associates reported the following: “Preliminary analyses of Southern digests of H9/HTLV-III DNA revealed that the virus was present in this cell line both as unintegrated DNA and as proviral DNA integrated into the cellular genome at multiple different sites. Since the HTLV-III provirus was found to lack Xba restriction sites, a genomic library was constructed by using Xba I-digested H9/HTLV-III DNA, and this was screened with an HTLV-III cDNA probe (8) to obtain molecular clones of full-length integrated provirus with flanking cellular sequences. Fourteen such clones were obtained from an enriched library of 106 recombinant phage, and two of these were plaque-purified and characterized. Figure 1 illustrates the restriction maps of these two clones, designated ëHXB-2 and ëHXB-3.” (2)
Summarizing, the ëHXB-2 and ëHXB-3 originated from the provirus. The provirus (cDNA) is obtained by reverse transcription of the “HIV” genome, that is, of the “HIV” RNA which is an RNA identical to the poly(A)-RNA found to band at the 1.16g/ml band in the first series of experiments. So we rephrase our questions:
Furthermore, we would be grateful if Brian Foley would please answer all our previous questions and well as those asked here.
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. 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.
Competing interests: None declared