Does HIV Exist (forwarded from sci med aids) 1/3

weissm at rockvax.rockefeller.edu weissm at rockvax.rockefeller.edu
Thu Mar 9 16:32:40 EST 1995


Date:         Thu, 9 Mar 1995 14:05:46 -0600
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Subject:      Does HIV exist?
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From: tmiller at newssun.med.miami.edu (Todd Miller - Pharmacology)
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Subject: Does HIV exist?
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Date: Thu,  9 Mar 1995 09:17:43 CDT
Organization: University of Miami, School of Medicine

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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 HAS GALLO PROVEN THE ROLE OF HIV IN AIDS?

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Eleni Papadopulos-Eleopulos
Physicist
Department of Medical Physics
Royal Perth Hospital

Valendar F. Turner
Staff Specialist
Department of Emergency Medicine
Royal Perth Hospital
  John M. Papadimitriou
Professor of Pathology
Department of Pathology
University of Western Australia

Correspondence to:
Eleni Papadopulos-Eleopulos
Department of Medical Physics
Royal Perth Hospital
Box X2213 GPO Perth

ABSTRACT
The evidence that Robert Gallo and his colleagues presented on 4th May 1984
regarding HTLV-III (HIV) isolation and the role of HIV in the pathogenesis
of AIDS is critically analysed. It is concluded that the evidence does not
constitute proof of the isolation of a retrovirus, that the virus is
exogenous or that the virus is causally related to AIDS.
 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 colleagues1 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
colleagues2 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 Rous16  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.18 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.19,21
 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.22  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:

1. 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);
2. 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



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