Re: Reply to Bennett: Gay AIDS-KS is caused by poppers 3 April 2005
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Jeffrey L Evans,
Layman
USA 81621

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Re: Re: Reply to Bennett: Gay AIDS-KS is caused by poppers

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Relative to both the KS discussion and "No Gold Standard", the lingering questions regarding the source material for the nucleotide sequencing of the "original" HIV genome, the trail seems to lead back to a single KS sufferer (LAI). Following is a cut and paste narrative:

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INSTITUTIONAL RESPONSE TO THE HIV BLOOD TEST PATENT DISPUTE AND RELATED MATTERS

Staff Report of the Subcommittee on Oversight and Investigations

Committee on Energy and Commerce United States House of Representatives

http://www.healtoronto.com/starep1.html

(a) Dr. Popovic's Notes: First, a word about Dr. Popovic's laboratory notes is in order. Dr. Popovic singlehandedly carried out the most important early HIV experiments at the LTCB*, yet his laboratory notes are extraordinarily sparse and fragmentary. Numerous experiments claimed to have been performed are not recorded at all; notably absent are records of the inception of several putative cultures, including the "mystery virus," MOV.

Laboratory records and Gallo/Popovic's retrospective accounts show that the LTCB's seminal experiments, particularly experiments associated with the development of the LTCB HIV antibody blood test and the genetic sequencing of the LTCB's putative "prototype" HIV isolate, were performed with an isolate called "MOV" (for "MO/Variant") and later, with an isolate called "HTLV-IIIb."

The evidence is compelling that "MOV" and "IIIb" were merely different names given to the single readily-usable isolate Popovic/Gallo had in late 1983/early 1984 -- LAI/LAV.

The designation "MOV" appeared without explanation in Dr. Popovic's notes for November 22, 1983, alongside two infected permanent cell lines in which LAI/LAV was growing. No HIV isolate other than LAI/LAV ever grew simultaneously in these two cell lines at the LTCB; neither is there any record of any attempt to infect these cell lines with an isolate other than LAI/LAV.

(b) What the LTCB Records Show: By the LTCB's own records, the materials sent from the IP** to the LTCB included the following:

1. Two shipments (April and July 1983) of DNA from patient "BRU" (the individual who was the source of the original LAV isolate);

2. At least three shipments of BRU serum (July, August, and December 1983). BRU serum was the principal reagent Gallo et al. used, prior to the development of the HIV-specific hyperimmune rabbit antiserum, to test cultures for the presence of the suspected AIDS virus; yet no results from any of the LTCB's experiments with BRU serum were ever reported, neither was any acknowledgement of the serum's use ever made. In fact, during the blood test patent interference proceeding at PTO, attorneys for the Department of Justice submitted a key motion in which they asserted that,

"The receipt of sera by Gallo from Montagnier taken from the patient [BRU] ... is ... of no significance ... there is no evidence to indicate that the sera contained any antibodies to the AIDS virus" (Opposition of Gallo et al. to the Motion for Judgement of Montagnier et al., USPTO interference; p. 13).

3. Two shipments of LAV virus (as cell-free supernatant) -- in July 1983 and September 1983.

The September 1983 shipment of LAV contained two samples with somewhat different identifiers -- "JBB/LAV" and "M2t-/B/LAV." At the time, both samples were believed to contain the same virus, from patient BRU. In reality, as demonstrated in 1991 by Wain- Hobson et al. (Science; 252, pp. 961-965) and confirmed by the Roche analyses, in 1993 (Nature, 363, pp. 466-469), the M2t-/B/LAV LAV/BRU sample had been accidentally contaminated at the Institut Pasteur and overgrown with virus from another patient "LAI." Consequently, in September 1983, Gallo et al. received a sample of BRU (JBB/LAV) and a sample of LAI (M2t-/B/LAV). These samples were both IP HIV isolates. LAI is the source of the LTCB's "HTLV-IIIB."

Following publication of the May 1983 papers, the IP and LTCB scientists continued on their different tracks vis-a-vis the AIDS virus. By mid-summer 1983, the IP scientists had developed an ELISA (enzyme- linked immunosorbent assay) to test human blood samples for antibodies to their newly-discovered virus. The IP scientists also made important discoveries about the characteristics of the virus, including the size of its proteins, its morphology, and, particularly important, its selective tropism for human T-cells. Additional isolates of the virus and results of its further characterization were reported by Dr. Montagnier at the July 1983 meeting of the NCI AIDS Task Force, headed by Dr. Gallo. Particularly important were election micrographs (EMs) of the IP virus, showing its distinctive lentivirus morphology, different from that of the leukemia virus, HTLV-I.

Initial results of the IP ELISA and more aspects of the characterization of the virus the IP scientists now called "LAV" (for lymphadenopathy-associated virus) were presented by Dr. Montagnier at a September 1983 meeting at Cold Spring Harbor, New York. Simultaneously, in Great Britain, the IP scientists filed a patent application for their virus antibody blood test.

Yet it was not until 1991, in the midst of a three-year series of HHS investigations that Dr. Gallo finally acknowledged publicly what for years he had denied was even physically possible: the virus the LTCB scientists used to make their HIV blood test -- the virus claimed by the LTCB as its "prototype" HIV isolate -- was in fact the IP HIV isolate LAI/LAV.

*(Dr. Robert C. Gallo and his colleagues at the National Cancer Institute's [NCI] Laboratory of Tumor Cell Biology [LTCB])

**(Dr. Luc Montagnier of France's Institut Pasteur)

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We then confirm the story in Montagnier's own words, also a cut and paste for the sake of brevity: ____________________________________________________________

Science Magazine HISTORICAL ESSAY: A History of HIV Discovery Luc Montagnier

http://www.aidscience.org/science/298(5599)1727.html

On 3 January 1983, Françoise Brun-Vezinet obtained a lymph node biopsy from one of Rozenbaum's patients, a young gay man (BRU) with a lymphadenopathy in the neck. I minced the lymph node, dissociated the fragments into single cells, and cultured the T lymphocytes with interleukin-2 and antiserum to human interferon. Fifteen days later, Françoise Sinoussi (by then Barré-Sinoussi) found the first traces of RT in the supernatant of the lymphocyte culture, indicating the presence of a retrovirus. The only retroviruses then known were the human T cell leukemia viruses, HTLV-1 and HTLV-2, identified by Gallo's group. So, we tested whether the viral proteins in the supernatant could be recognized by Gallo's antibodies against HTLV. Surprisingly, our labeled viral supernatant could not be immune precipitated with the HTLV antibodies, but could be precipitated with the patient's own serum (4). A protein with a molecular mass of about 25 kD precipitated by the patient's serum seemed to be the counterpart of the p24 protein of HTLV-1. The virus could not be isolated from blood lymphocytes, a fact that is now explained by the early stage (lymphadenopathy) of this patient's disease when the virus is almost exclusively located in lymphatic tissues. Louis Pasteur's quote that "luck in science smiles on prepared minds" certainly applied to us. We received a biopsy from another young gay male patient (MOI), who was infected with both HTLV and the new lymphadenopathy-associated virus. If MOI had been our first patient, we would have been very confused.

A few months later, I received a blood sample from a young hemophiliac (LOI) with full-blown AIDS, and blood and lymph node samples from a young gay man (LAI) with advanced Kaposi's sarcoma. The LAI virus could be isolated from the patient's blood cells and grew very quickly in the patient's cultured T lymphocytes, killing them as well as killing T lymphocytes from blood donors.

To better characterize the new virus, we tried (unsuccessfully) to grow the BRU isolate in different T cell lines. If we had tried the LAI isolate instead, we would have been able to grow the virus without any trouble. In October 1983, we were finally able to grow the BRU isolate in Epstein-Barr virus-transformed B cell lines, although we discovered later that the LAI virus had contaminated our BRU culture (6). At least six laboratories received the LAI sample (under the name BRU) from our group and experienced the same contamination. We think that the LAI virus readily contaminated the BRU culture because it associates with a mycoplasma species, Mycoplasma pirum, usually present in T cell lines. This physical association makes a fraction of the LAI virus highly infectious, and, in fact, this fraction can be neutralized with antibodies against M. pirum. As mycoplasmas are common contaminants of cultured cells, an infectious pseudotype virus (LAI associated with M. pirum) may have caused several contaminations between 1983 and 1984 in different laboratories.

New evidence that this strange retrovirus was the cause of AIDS came from our team in the fall of 1983 and the winter of 1984 (7). We observed a high frequency of antibodies against the virus in lymphadenopathy patients, and noted the favored tropism of this virus for CD4+ T lymphocytes. Our results were still controversial, however, and we had difficulty in obtaining the funding needed to better characterize the virus and to develop a blood test. The tide only turned in France when Robert Gallo and his group in the United States made a similar discovery. In the spring of 1984, Gallo published more convincing evidence that HIV causes AIDS (8) (see the Viewpoint by Gallo on page 1728), a finding that was confirmed by Jay Levy's group (9). In 1985 came the cloning and sequencing of the HIV genome with identification of new open reading frames specific for lentiviruses (10).

(4) Science. 1983 May 20;220(4599):868-71.

Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L.

A retrovirus belonging to the family of recently discovered human T- cell leukemia viruses (HTLV), but clearly distinct from each previous isolate, has been isolated from a Caucasian patient with signs and symptoms that often precede the acquired immune deficiency syndrome (AIDS). This virus is a typical type-C RNA tumor virus, buds from the cell membrane, prefers magnesium for reverse transcriptase activity, and has an internal antigen (p25) similar to HTLV p24. Antibodies from serum of this patient react with proteins from viruses of the HTLV-I subgroup, but type- specific antisera to HTLV-I do not precipitate proteins of the new isolate. The virus from this patient has been transmitted into cord blood lymphocytes, and the virus produced by these cells is similar to the original isolate. From these studies it is concluded that this virus as well as the previous HTLV isolates belong to a general family of T- lymphotropic retroviruses that are horizontally transmitted in humans and may be involved in several pathological syndromes, including AIDS.

(10) Cell. 1985 Jan;40(1):9-17.

Nucleotide sequence of the AIDS virus, LAV. Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M.

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. Remarkably, Q is located between pol and env and F is half-encoded by the U3 element of the LTR. These data place LAV apart from the previously characterized family of human T cell leukemia/lymphoma viruses.

(10) Cell. 1985 Aug;42(1):369-82.

Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus. Sonigo P, Alizon M, Staskus K, Klatzmann D, Cole S, Danos O, Retzel E, Tiollais P, Haase A, Wain-Hobson S.

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.

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Super Condensed Version from both sources:

Laboratory records and Gallo/Popovic's retrospective accounts show that the LTCB's seminal experiments, particularly experiments associated with the development of the LTCB HIV antibody blood test and the genetic sequencing of the LTCB's putative "prototype" HIV isolate, were performed with an isolate called "MOV" (for "MO/Variant") and later, with an isolate called "HTLV-IIIb."

The evidence is compelling that "MOV" and "IIIb" were merely different names given to the single readily-usable isolate Popovic/Gallo had in late 1983/early 1984 -- LAI/LAV.

Montagnier:

A few months later, I received a blood sample from a young hemophiliac (LOI) with full-blown AIDS, and blood and lymph node samples from a young gay man (LAI) with advanced Kaposi's sarcoma. The LAI virus could be isolated from the patient's blood cells and grew very quickly in the patient's cultured T lymphocytes, killing them as well as killing T lymphocytes from blood donors.

To better characterize the new virus, we tried (unsuccessfully) to grow the BRU isolate in different T cell lines. If we had tried the LAI isolate instead, we would have been able to grow the virus without any trouble. In October 1983, we were finally able to grow the BRU isolate in Epstein-Barr virus-transformed B cell lines, although we discovered later that the LAI virus had contaminated our BRU culture (6). At least six laboratories received the LAI sample (under the name BRU) from our group and experienced the same contamination.

In 1985 came the cloning and sequencing of the HIV genome with identification of new open reading frames specific for lentiviruses.

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BONUS:

In contrast to the bold assertion above (10), Wain-Hobson was later co-author of less certain assessments of the molecular identity of HIV, perhaps contributing to the discussion regarding confusion with HERVs and self assembling RNA:

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Cell. 1989 Sep 8;58(5):901-10.

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Meyerhans A, Cheynier R, Albert J, Seth M, Kwok S, Sninsky J, Morfeldt- Manson L, Asjo B, Wain-Hobson S. Laboratoire de Biologie et Immunologie Moleculaires des Retrovirus, Institut Pasteur, Paris, France.

A genetic study has been made of the HIV tat gene from sequential HIV -1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.

J Acquir Immune Defic Syndr. 1989;2(4):344-52.

HIV-1 isolates are rapidly evolving quasispecies: evidence for viral mixtures and preferred nucleotide substitutions. Goodenow M, Huet T, Saurin W, Kwok S, Sninsky J, Wain-Hobson S. Laboratorie de Biologie et Immunologie Molecularies des Retrovirus, Institut Pasteur, Paris, France.

RNA viruses are renowed for their genetic variability. The human immunodeficiency viruses (HIV) are no exception. A rapid method has been established for the genetic identification and differentiation of viral strains based on the sequencing of many M13 clones of gene-amplified products. Some isolates are internally relatively homogeneous while others are heterogeneous. There was no correlation between virus complexity and disease stage. One isolate was in fact a mixture of two distinct strains. A strong preference for G----A base substitutions was observed. These data indicate that HIV isolates cannot be described in simple molecular terms and should rather be considered as quasispecies.

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Competing interests: None declared