In Aids? Essay, Research Paper
Introduction
In 1982, Robert Gallo from the National Cancer Institute in the USA, put forward the hypothesis that the cause of AIDS is a retrovirus. One year later, Myron Essex and his colleagues (1) found that AIDS patients had antibodies to the Human T-cell Leukemia virus Type-1 (HTLV-I), a virus discovered by Gallo a few years earlier. At the same time, Gallo and his colleagues (2) reported the isolation of HTLV-I from AIDS patients and advocated a role for this retrovirus in the pathogenesis of AIDS. This hypothesis however, was not without a few problems:
1. While HTLV-I was accepted to induce T4-cell proliferation and cause adult T-cell leukaemia,(3) the “hallmark” of AIDS was T4-cell depletion, and the incidence of leukaemia in AIDS patients was no higher than in the general population;
2. The highest frequency of antibodies to this virus was found in Japan, yet no AIDS cases had been reported from that country;(4)
3. In the same month in which Gallo’s and Essex’s groups reported their data, Luc Montagnier and his colleagues from the Pasteur Institute, described the isolation of a retrovirus, later known as Lymphadenopathy Associated Virus (LAV), from the lymph nodes of a homosexual patient with lymphadenopathy.(5) Although this virus was similar to HTLV-I, one of its proteins, a protein with a molecular weight of 24,000 (p24), did not react with monoclonal antibodies to the HTLV-I p24 protein. Samples of this virus were, on several occasions, sent to Gallo’s laboratory.
In May 1984, Gallo, Popovic and their colleagues published four papers in Science in which they claimed to have isolated from AIDS patients, another retrovirus, which they called HTLV-III.(6,7,8,9) On the 23rd of April 1984, before the Science papers were published, Gallo and Margaret Heckler, the then Health and Human Services Secretary called a press conference to announce that Gallo and his co-workers had found the cause of AIDS and had developed a sensitive test to show whether the “AIDS virus” is present in blood.
In 1985, the Pasteur Institute alleged that Gallo had misappropriated LAV in developing the blood test. The ensuing conflict, which reached the American courts, was eventually settled by a negotiated agreement signed in 1987 by Gallo, Montagnier, US President Reagan and French Premier Chirac. The agreement declared Gallo and Montagnier to be co-discoverers of the AIDS virus, presently known as the Human Immunodeficiency Virus (HIV). Nevertheless, the misappropriation conflict drew the attention of John Crewdson, an investigative journalist, and US Senator John Dingell. In November 1989, Crewdson published a lengthy article in the Chicago Tribune newspaper, “With allegations that Robert C. Gallo stole from French scientists the virus he discovered to be the cause of AIDS.”(10) This led to a National Institute of Health (NIH) internal “inquiry” into the allegation with “an outside committee of expert but disinterested parties [led by Yale biochemist Frederic Richards] to oversee the activity of the internal panel”.(11)
Following the inquiry, which was viewed as a fact-finding mission, the Richards committee insisted on a “formal investigation … on suspect data in one of four seminal papers published by Gallo’s lab in Science on 4 May 1984″.(12) In this paper, the first of a series of four, with Mikulas Popovic the principal author, “their appears to be differences between what was described in the paper and what was done”.(10) A draft report of the formal investigation written by NIH Office of Scientific Integrity (OSI), was published in September 1991. In the draft report, Popovic is accused “of misconduct for misstatements and inaccuracies” that appeared in the paper, and that Gallo, as laboratory chief, “created and fostered conditions that give rise to falsified/ fabricated data and falsified reports”. However, Gallo’s actions were not considered to “meet the formal definition of misconduct”.(13)
The final draft report of the OSI, completed in January 1992, was immediately criticised by the Richards Panel as well as Senator Dingell. This led to a review of the OSI report by the Office of Research Integrity (ORI), which found Gallo guilty of scientific misconduct. Nonetheless, the scientific misconduct is said not to “negate the central findings of the [1984 Science] paper”.(13,14) In other words, despite the above findings, at present, it is still accepted, as Gallo and his colleagues concluded, “The results presented in our four papers provided clearcut evidence that the aetiology of AIDS and ARC was the new lymphotrophic retrovirus, HTLV-III”(15) [ARC=3DAIDS related complex]. Although the findings of the Gallo investigation are of considerable importance, in what follows, with few exceptions, we will consider that there were no “differences between what was described in the paper and what was done”. However, the data will be critically analysed with regard to the following:
1. Whether the experimental method described constitutes irrevocable evidence of viral isolation;
2. Whether the authors have presented evidence proving a causal role for HIV in AIDS.
To facilitate this analysis it may be useful to consider what is generally accepted as retroviral isolation.
Retroviral Isolation
Peyton Rous (16) is credited with the discovery and isolation of the first retrovirus. In 1911 he was able to repeatedly induce tumours in a particular breed of chickens by means of tumour derived, cell free filtrates. It is instructive to repeat Rous’ own thoughts on his observation: “The first tendency will be to regard the self-perpetuating agent active in this sarcoma of the fowl as a minute parasitic organism. Analogy with several infectious diseases of man and the lower animals, caused by ultramicroscopic organisms, gives support to this view of the findings, and at present work is being directed to its experimental verification. But an agency of another sort is not out of the question. It is conceivable that a chemical stimulant, elaborated by the neoplastic cells, might cause the tumour in another host and bring about in consequence a further production of the same stimulant”.
The tumour inducing filtrates became known as “filterable viruses” or oncoviruses and, more recently, exogenous retroviruses and infectious retroviruses.(17) In the 1950s, in animal cultures and in fresh tissue, especially tumour tissue, particles later attributed to retroviruses, were readily detectable with electron-microscopy (EM). In 1970, the enzyme reverse transcriptase (RT), which transcribes RNA into DNA, was discovered in oncoviruses. Because of this, in the 1970’s, oncoviruses became known as retroviruses. In the preceding decade, density gradient centrifugation was introduced to separate and isolate sub-cellular particles including viruses. Because some cellular constituents were found to have the same buoyant density as viruses, when viruses were isolated from cell cultures, the best results could be obtained with supernatant fluids which had high viral concentration and low cellular contaminants. This was best satisfied by non-cytopathic viruses and by culture conditions which maintained maximum cellular viability. All retroviruses isolated prior to HIV satisfy the above conditions.(19)
Taking advantage of the above retroviral properties, by repeated suspensions and sedimentation in sucrose density gradients, one could obtain, at a density of 1.16 gm/ml, a relatively pure concentration of retroviral particles-that is, obtain retroviral particles separate from everything else, and thus isolate them.(19) Nonetheless, as many eminent retrovirologists point out, contamination of the viral preparation with particles which contain RT, but could be nothing more than “cellular fragments”, microsomes from disrupted cells, “membraneous vesicles which may enclose other cellular constituents including nucleic acids”, especially when “inadvertent lysis of cells” was induced, could not be avoided.(17,18,19,20) Because of this, to prove that the material which banded at 1.16 gm/ml contained nothing else but particles with “No apparent differences in physical appearances”, and that the particles were indeed retroviruses, every retrovirus preparation was further analysed using the following assays:
(a) physical-EM for virus count, morphology and purity;
(b) biochemical-RT activity, viral and cellular RNA, total protein, gel analyses of viral and host proteins and nucleic acids;
(c ) biological-infectivity in vivo and in vitro.(19,20)
In other words, the first step in the effort of isolation of a retrovirus is the demonstration that:
1. The particles seen in the cultures band at 1.16 gm/ml;
2. In the 1.16 gm/ml band there is little present but the particles;
3. “No apparent differences in physical appearances” between particles are seen.
Isolation of HTLV-III (HIV).
In the first, seminal paper on HIV isolation, entitled “Detection Isolation and Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS”,(6) Popovic, Gallo and their colleagues first described a leukaemic T-cell line, HT. This cell line was exposed “to concentrated culture fluids harvested from short-term cultures of T-cells… obtained from patients with AIDS or pre-AIDS. The concentrated fluids were first shown to contain particle-associated RT”. The finding in the HT cell line as well as in 8 clones derived from it including H4, H9 and H17, of: (a) RT; (b) cell immunofluoresence with serum from a haemophilia patient with pre- AIDS, and “Rabbit antiserum to HTLV-III”, was considered evidence for the existence in these cultures of a retrovirus which was named HTLV-III. “Both virus production and cell viability of the infected clone H4 (H4/HTLV-III) were monitored for several months. Although virus production [RT activity] fluctuated (Fig. 2a), culture fluids harvested and assayed at approximately 14-day intervals consistently showed particulate RT activity [RT activity in the material which banded at 1.16 gm/ml] which has been followed for over 5 months… Thus the data show that this permanently growing T-cell population can continuously produce HTLV-III”. EM examination of the H4 clone culture showed “the presence of extracellular viral particles”.
Some of the findings of the Gallo investigation are relevant to the above experiments:
1. The HT cell line was not cultured with concentrated fluids originating from individual AIDS patient T-cell cultures as is implied in the paper but from fluids pooled, first from the individual cultures of 3 patients and ultimately from the individual cultures of 10 patients. The Gallo investigation found this procedure to be “of dubious scientific rigor”. One scientist described it as “really crazy”.11
2. According to the OSI inquiry, “the statement in the published papers that the samples were “first” shown to be secreting RT, “is contradicted by the evidence of the notebooks that only one of the three [initial cultures] was tested”.22 In evidence which Popovic gave to the inquiry he said that he had pooled the supernatant fluids from the ten cultures because none “individually was producing high concentrations of reverse transcriptase”. (The levels of RT are not given).
However: It is important to note that RT is determined by estimation of the incorporation of [3H] labelled nucleotides into DNA and is reported as counts per minute (cpm), and it is acknowledged that background radioactivity, that is, radioactivity in the absence of infection, can be as high as 0.4 X 104 cpm.(23)
The above findings give rise to additional questions: If the first HTLV-III was isolated from HT cell cultures with the pooled supernatants, then how was the “Rabbit antiserum to HTLV-III” obtained for the immunofluoresence studies? How was it possible to ensure the specificity of rabbit antisera to a virus before the virus has been isolated? Similarly, how was it possible, before viral isolation, to ascertain that patient serum used to test material from the cultures did indeed interact specifically with the same virus?
(c ) The OSI found the claim that “the culture” was continuously producing HTLV-III (RT activity), was incorrect since the culture was “reinoculated on at least two occasions” with more supernatant.(11,22)
In the second paper,(7) the authors describe their attempt to isolate HTLV-III from mitogenically stimulated T-cell cultures obtained from 115 patients with AIDS, pre-AIDS and clinically normal homosexual men. In Table I entitled “Detection and Isolation of HTLV-III from patients with AIDS and pre-AIDS”, they state: “Samples exhibiting more than one of the following were considered positive: repeated detection of a Mg2+- dependent 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. In further experiments (8,9):
1. Lysates of the H4/HTLV-III and H17/HTLV-III “infected” cell lines were tested with patient sera using the Western blot (WB) technique.[Footnote 1];
2. “The specificity of these reactions [for HTLV-III] was studied by comparing lysates of H4/HTLV-III and H17/HTLV-III with lysates of the same clones, H4 and H17, before viral infection (Fig.2A). No antigen from uninfected clones reacted with the sera, with the exception of a protein with a molecular weight 80,000 in H17 which bound antibodies from all of the human samples tested”. They concluded: “These results show clearly that the antigens detected after virus infection are either virus-coded proteins or cellular antigens specifically induced by the infection”.
3. The reaction with patient sera of the H4/HTLV-III cells was then compared with the reaction of the material from the H4/HTLV-III culture fluids which in sucrose density gradients banded at 1.16 gm/ml. Of the proteins which banded at 1.16 gm/ml, two, p41 and p24, were found to react with some patient sera. They concluded: “p24 and p41 may therefore be considered viral structural proteins”;
4. Finally, they used the ELISA [Footnote 2] technique to test for HTLV-III antibodies. 88% (43/49) of patients with AIDS, and 79% (11/14) patients with pre-AIDS but “less than 1 percent of heterosexual subjects”, had antibodies “reactive against antigens of HTLV-III”. “To understand the molecular nature of the antigens recognized by ELISA”, the sera were analysed by WB. “…the antigen most prominently and commonly detected among all of the sera from AIDS patients had a molecular weight of 41,000 (p41)…Reactivity to p24 of the virus was generally very weak and was clear only in two cases”.
From the above data it is obvious that by HTLV-III (HIV) isolation was meant detection of more than one of the following phenomena:
1. RT, either in the culture fluids, or in the material from these fluids or cellular lysates which in sucrose density gradients band at 1.16 gm/ml;
2. In culture fluids, but not in the material which bands at 1.16 gm/ml, particles with morphological characteristics of retroviruses (RVP);
3. Proteins, (p41, and in some cases, p24), which, in sucrose density gradients, band at 1.16 gm/ml, (but without proof that they are unique constituent parts of the particle), and react with patient sera.
However, isolation is defined as separating an object, (HIV), from everything else, and not the detection of some phenomena attributed to it (RT, WB), or similar to it, (RVP). Phenomena can only be used for retroviral detection, not isolation, and even then if, and only if, it is first shown that each is specific for the virus by use of the only valid gold standard, HIV itself, “HIV isolation”. It is important to note that in the earlier (1983) report by Montagnier’s group on HIV (LAV) isolation, the same experimental procedures and findings as those described by Gallo were reported. The only exception was that Montagnier’s group did not “infect” an immortalised cell line, yet Gallo’s group considered that Montagnier and his colleagues had not described “true isolation”.(6) In fact, in 1984, evidence existed that RT, antigen-antibody reactions (WB), and RVP, are non- specific for retroviruses. The indirect evidence, that is, evidence that has been obtained without a gold standard from recent AIDS research, has confirmed the above.
Reverse transcriptase
Although Gallo has described the enzyme reverse transcriptase as “unique to retroviruses”, this is not the case, a fact stressed by its discoverers, (both Nobel laureates).(17) Reverse transcription can be found in leukaemic T-cells,24 (HT and its clones including H9, from which the first “HTLV-III (HIV) virus was isolated”, is a leukaemic cell line), normal spermatozoa,25 and, according to Harold Varmus, another Nobel laureate, more recently, in the uninfected cells of yeasts insects and mammals.(26) As far back as 1973, Gallo himself was the first to show that RT can be found in “PHA stimulated (but not unstimulated) normal human blood lymphocytes”.(24) Confirmation of this was reported at the 1991 Florence AIDS conference where evidence was presented that the drug AZT can inhibit the action of normal cellular RT,27 and this was postulated as a mechanism for drug toxicity.