HAS
GALLO PROVEN THE ROLE OF HIV IN AIDS?
Emergency
Medicine 1993;5:5-147
Eleni
Papadopulos-Eleopulos, Valendar F. Turner, John M. Papadimitriou
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.
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.
Retroviral
particles.
By definition,
retroviral particles are enveloped infectious particles 100-120nM
in diameter with a core compromising a protein shell and a ribonucleoprotein
complex. RVP are further catergorised according to the site of core
assembly, that is, within the cytoplasm or at the cell membrane,
and by certain other morphological features. Included in this taxonomy
are the Subfamilies Oncoviruses which include Type C and Type D
particles, as well as the Subfamily Lentiviruses.
Prior to the
AIDS era, many retrovirologists showed that the finding of a particle
with morphological features similar to retroviruses does not constitute
sufficient proof that they are retroviruses, that they are infectious
particles, even if they are found to band at 1.16 gm/ml.(18) In
1976 Gallo himself pointed out that in human leukemic tissue "virus-like
particles morphologically and biochemically resembling type-C virus
but apparently lacking the ability to replicate, have been frequently
observed".(28) Particles with the morphological characteristics
of retroviruses were reported in milk, cultures of embryonic tissues
and "in the majority, if not all, human placentas".(29,30,31)
However, they were considered to be "an intriguing and important
problem that remains to be solved".(32) Evidence from AIDS
research shows that:
1. There is
no agreement on the precise taxonomic classification of HIV. Initially,
HIV was reported as an Oncoviral type-C particle, then a type-D
particle,(33) and ultimately as a member of a different Subfamily,
a Lentivirus;(34)
2. The T-cell
and monocyte "HIV infected cultures" contain in addition
to particles with morphologies attributed to HIV, many other "viral
particles" unlike any of the "HIV particles".(35,36,37,38)
"Non-HIV-infected" HT (H9) cells, the cell line from which
the Gallo team "isolated" the first HIV (HTLV-III) and
from which most of the published electron micrographs of "HIV
particles" have originated, as well as other cells used for
"HIV isolation", CEM, C8166, EBV transformed B-cells,
and cord blood lymphocytes, express virus-like particles albeit
they are somewhat different from the variety of particles accepted
as HIV.(39) The above data raises questions not only in regard to
the origin and role of the "non- HIV particles", but also
to the "HIV (HTLV-III) particles". Furthermore, neither
Gallo's team, nor anybody else before or since has published EM
micrographs of the material derived from AIDS cultures/co-cultures
which bands at 1.16 gm/ml. Thus it is impossible to know which,
if any of the particles, band at that density;
3. Most importantly,
it is generally accepted that particles reported in the lymph nodes
of AIDS patients are HIV. However, in the only EM study(40), either
in vivo or in vitro, in which suitable controls were used and in
which extensive blind examination of controls and test material
was performed, "HIV particles" were found in 90% (18/20)
of patients with persistent generalised lymphadenopathy attributed
to HIV, and in 87% (13/15) of patients with "non-HIV lymphadenopathies",
leading the authors to conclude: "The presence of such particles
do not, by themselves indicate infection with HIV".
Antigen-antibody
reactions.
One can claim
that a given protein is an antigen derived from an exogenous retrovirus
if first it is shown that:
1. The protein
is a structural component of a particle;
2. The particle
is a retrovirus;
3. The protein
is coded exclusively by a viral and not a cellular gene.
Once the above
are demonstrated, the only way to prove that the antibodies found
in AIDS patient sera are directed against the viral antigen is to
use the antigen or the isolated virus as a gold standard. The mere
finding that a protein from the AIDS cultures bands at 1.16 gm/ml
and reacts with sera from AIDS patients cannot be considered to
simultaneously prove that:
1. The protein
is a viral antigen;
2. The antibodies
in the AIDS patient sera which react with the antigen are specific
for that antigen.
At present,
it is known that about 80% of the proteins which band at 1.16 gm/ml,
some of which react with some AIDS sera, do not constitute any of
the proteins ascribed to HIV.(41,42,43) Most importantly, prior
to the publication of the Science papers, evidence existed, confirmed
since, which is at odds with the conclusion that "p24 and p41
may therefore be considered viral structural proteins":
The p41/45
protein
In AIDS research,
the p41 and p45 bands are considered to represent one and the same
HIV protein.
1. Like Gallo's
group, Montagnier's team one year earlier, found that AIDS sera
reacted with a protein p41/45 from the AIDS cultures and which in
sucrose density gradients, banded at 1.16 gm/ml. However, from their
data they considered that the p41 band "may be due to contamination
of the virus by cellular actin which was present in immunoprecipitates
of all cell extracts",(5) that is, of "HIV infected"
as well as non-infected cells and cells infected with HTLV-I. Although
Gallo's group did not find such a reaction with p41 in non-infected
cells, they did find a p80 protein and concluded that the reaction
was "non-specific".(8) However, at present it is known
that p80 as well as two additional "HIV proteins", p120
and p160, are oligomers of p41.(44) Which protein (band), p41, p80,
p120 or p160 is detected in a given WB depends on the culture and
WB conditions, including temperature and the concentration of sodium
dodecyl sulphate used to disrupt the proteins which band at 1.16
gm/ml;(45)
2. Actin is
an ubiquitous protein present in all cells including bacteria and
several viruses. Well known retroviruses such as the mouse mammary
tumour virus have also been shown to contain actin of cellular origin
and it has been postulated that this protein plays a key role in
both retroviral assembly and budding;(46,47)
3. Platelets
from healthy individuals also contain a p41 protein which reacts
with sera from homosexual men with AIDS and immune thrombocytopenic
purpura (ITP) and which "represents non-specific binding of
IgG to actin in the platelet preparation".(48)
4. Researchers
at the Pasteur Institute have shown that sera from AIDS patients
and AIDS risk groups contain high levels of antibody against calf
striated muscle actin.(49)
The p24/25
protein
1. Apart from
a joint publication with Montagnier where they claim that the HIV
p24/25 is unique, Gallo and his colleagues have repeatedly stated
that the p24s of HTLV-I and HIV immunologically cross-react;(50)
2. Genesca
et al.(51) conducted WB assays in 100 ELISA negative samples of
healthy blood donors; 20 were found to have HIV bands which did
not fulfil the then (1989) criteria used by the blood banks for
a positive WB. These were considered as indeterminate WB, (WBI),
with p24 being the predominant band, (70% of cases). Among the recipients
of WBI blood, 36% were WBI 6 months after transfusion, but so were
42% of individuals who received WB-negative samples. Both donors
and recipients of blood remained healthy. They concluded that WBI
patterns "are exceedingly common in randomly selected donors
and recipients and such patterns do not correlate with the presence
of HIV-1 or the transmission of HIV-1", "most such reactions
represent false- positive results";
3. Antibodies
to p24 have been detected in 1 out of 150 healthy individuals, 13%
of randomly selected otherwise healthy patients with generalised
warts, 24% of patients with cutaneous T-cell lymphoma and prodrome
and 41% of patients with multiple sclerosis;(52)
4. Ninety seven
percent of sera from homosexuals with ITP and 94% of sera from homosexuals
with lymphandenopathy or AIDS contain an antibody that reacts with
a 25Kd membrane antigen found in platelets from healthy donors and
AIDS patients, as well as a 25 Kd antigen found in green-monkey
kidney cells, human skin fibroblasts, and herpes simplex cultured
in monkey kidney cells. This reaction was absent in sera obtained
from non-homosexual patients with ITP or non-immune thrombocytopenic
purpura;(48)
5. Conversely,
the p24 antigen is not found in all HIV positive or even AIDS patients.
In one study, the polymerase chain reaction (PCR) and p24 were used
to detect HIV in patients at various CDC stages from asymptomatic
to AIDS. p24 was detected in 24% patients and HIV RNA in 50%;(53)
6. In another
study, "In half of the cases in which a subject had a positive
p24 test, the subject later had a negative test without taking any
medications that would be expected to affect p24 antigen levels...the
test is clinically erratic and should be interpreted very cautiously".(54)
Thus the finding of viral particles in the AIDS cultures/co- cultures,
RT and proteins which react with AIDS related sera in the material
from the supernatant or cell lysates which in sucrose density gradients
bands at 1.16 gm/ml, cannot be considered synonymous with the isolation
or even the detection of a retrovirus. Even if a retrovirus is isolated
from in vitro cultures/co-cultures from tissues from AIDS patients,
this does not, by itself, constitute proof of the existence of the
virus in vivo, (in AIDS patients), and even less that the retrovirus
has been exogenously acquired. This is because:
1. At present,
it is generally accepted that "one of the most striking features
that distinguish retroviruses from all other animal retroviruses
is the presence, in the chromosomes of normal uninfected cells,
of genomes [proviruses] closely related to, or identical with those
of infectious viruses". The human genome, in addition to other
proviral sequences, is known to contain both HTLV-I (55,56) and
HIV (57) sequences. Depending on conditions, the proviral genome
remains unexpressed or part or all of it may be expressed. The latter
may or may not lead to the assembly of viral particles (endogenous
retrovirus).(17) In animal cultures, healthy non-virus producing
cells sooner or later spontaneously release retroviruses.(20) The
appearance and yield can be increased by (i) mitogenic stimulation;(58)
(ii) co-cultivation techniques;(59) (iii) cultivation of cells with
supernatant from non-virus producing cultures.(60) According to
one eminent retrovirologist, George Todaro, "the failure to
isolate endogenous viruses from certain species may reflect thelimitation
of in vitro cocultivation techniques";(61)
2. Gallo's
team, like everybody else: (i) "isolated HTLV-III (HIV)"
from cell cultures; (ii) "isolated HTLV-III" from mitogenically
stimulated, activated cell cultures;
3. In addition,
Gallo and his colleagues also used co-cultivation techniques;
4. The first
"HTLV-III isolation" was from the HT (H4, H9, H17) cell
line. Reading Gallo and his colleagues' first paper, one surmises
that the HT cell line was established in Gallo's laboratory. The
Gallo inquiry revealed that the HT cell line is in fact HUT78, a
cell line established in another laboratory from a patient with
mature T4-cell leukaemia, a disease which Gallo claims is caused
by the exogenous retrovirus, HTLV-I.(3) If so, then all HT cell
cultures, and the clones derived from it, "infected with HTLV-III"
or non-infected, and the material from these cultures which bands
at 1.16 gm/ml, should contain HTLV-I, and thus RT and retroviral
particles. Furthermore, because about 25% of AIDS patients have
antibodies to HTVL-I,(1) and the immunogenic proteins of HTLV-I
and HIV have the same molecular weights, then approximately 25%
of the non-infected HT (H4, H9, H17) cultures in addition to RT
and particles, should have, in the Western blot, the same bands
as those of the "HTLV-III infected" cultures. Thus, these
WBs will erroneously appear positive for HTLV-III.
Proof that
HTLV-III (HIV) is causally linked to AIDS.
Gallo claims,
a claim accepted by the vast majority of AIDS researchers, that
in the May 1984 Science papers he and his colleagues presented "unambiguous
evidence that this [virus] and this alone was the cause of AIDS".(62)
A minimum requirement for making such a claim should be presentation
of the following evidence:
1. That all
AIDS patients are infected with HTLV-III;
2. Infection
with HTLV-III leads to T4-cell depletion, given the assumption that
HTLV-III leads to the clinical syndrome by its T4 cytotoxicity.
The evidence
for the existence of HTLV-III was "viral isolation" and
ELISA antibody tests. Even if one assumes that the data presented
represents "true isolation", the virus was isolated from
less that half (10/21) of AIDS patients with opportunistic infections,
and in less than one third (13/43) with Kaposi's sarcoma, then and
now the two most characteristic AIDS diseases. Even if the virus
could have been isolated from all patients, given the nature of
retroviruses and the method used for HTLV-III isolation (cultures,
mitogenic stimulation, co- cultivation) the possibility cannot be
excluded that the virus did not exist in vivo (in AIDS patients),
and that it was a provirus whose expression was facilitated by the
culture conditions. The only method used to prove HIV infection
in vivo was the antibody tests. Such a test can only be used only
after its specificity has been proven by use of the only possible
gold standard, the virus itself. This has not been done.
Furthermore,
the antibody test used by Gallo was ELISA, at present known to be
non-reproducible and non-specific. In a study of 1.2 million healthy
military applicants conducted by Colonel Donald Burke and his colleagues,(63)
it was found that although approximately 1% of all individuals had
an initial positive HIV ELISA, only 50% of repeat ELISAs were positive.
Of the latter, only approximately one third were associated with
two subsequent positive WBs. In Russia, in 1990, out of 20,000 positive
ELISAs "only 112 were confirmed" using the WB as a gold
standard. In 1991, of approximately 30,000 positive ELISAs, only
66 were confirmed.(64)
Nowhere in
the four Science papers was HTVL-III cytotoxicity mentioned. The
only reference to any cellular abnormalities or pathology in general
is in the first paper where one reads: "The virus positive
cultures consistently showed a high proportion of round giant cells
containing numerous nuclei (Fig. 1a). These cells resemble those
induced by HTLV-I and -II except that the nuclei exhibit a characteristic
ring formation". (Fig. 1a is a "light microscopic examination
of clone H4/HTLV-III"). The H4 clone was obtained from the
HT cell line "using irradiated mononuclear cells from peripheral
blood of a healthy blood donor as a feeder". At present, it
is known that the HT cell line and thus H4 are HUT78, derived in
1980 from a patient with mature T4-cell leukaemia,(65,66) However,
other cell lines derived from patients with the same clinical syndrome
are known to exhibit similar morphologies including multinucleated
giant cells.(67) Thus the cellular morphological characteristics
observed in the first paper may have been an intrinsic property
of the HT cell line, or the result of the culture conditions, or
both, and not due to HTLV-III. Finally, Gallo and his colleagues
did not provide any data on the immunological status of those individuals
from whom viral isolation was attempted, and no data was presented
proving that:
1. HTLV-III
(HIV) is both a necessary and sufficient cause of T4- cell depletion;
2. T4-cell
depletion is both necessary and sufficient for the appearance of
the AIDS indicator diseases.
Conclusions
The data and
arguments that have been presented by Gallo and his colleagues do
not constitute proof of HIV isolation or an unambiguous role for
HIV in the pathogenesis of AIDS. Although some researchers currently
use methods of "viral isolation" essentially the same
as that described by Gallo's group, most use less rigorous methods
including singleton detection of p24 (by antibody techniques), or
RT. Notwithstanding, with all of these techniques, including that
described by Gallo and his colleagues, which itself seen to be greatly
problematic, HIV cannot be "isolated" from 20%-70% of
HIV positive and AIDS patients(68,69) Thus we are faced with a problem
of considerable importance. The HIV antibody tests, both ELISA and
WB, the only routinely used tests proving the existence in vivo
of HIV, have yet to be verified against the only suitable gold standard,
viral isolation. The available evidence suggests that this long
overdue but most basic requirement of test evaluation is likely
to prove an immense problem, and while the HIV antibody tests are
useful prognostic markers in the high risk groups, their use as
diagnostic and epidemiological tools for HIV infection is questionable.
*
Appendices
Appendix
1. In the Western Blot test, proteins are electrophoretically
separated according to molecular weight and charge. The separated
proteins are then transferred on to nitrocellulose strips by a process
known as electroblotting. When sera are added and the strips developed,
coloured bands appear representing sites of protein/antibody reactions.
Each band is designated by a small "p" for the protein
followed by its molecular weight in thousands.
Appendix
2. In the ELISA (Enzyme Linked Immunosorbent Assay), unseparated
proteins are attached to a solid base such as the walls of plastic
tubes or microplates. The serum being tested is incubated in these
containers where antibody is fixed to the solid phase antigens.
After washing, enzyme-labelled anti-human immunoglobulin is added
and also incubated. The containers are again washed and a substrate
specific for the enzyme is introduced. The resulting colour change
is proportional to the amount of antibody present and is read by
eye, or with a spectrophotometer.
Acknowledgements
We wish to
thank all our colleagues and especially Udo Sch=81klenk, Barry Page,
Bruce Hedland-Thomas, David Causer, Richard Fox, John Peacock, David
Prentice, Ronald Hirsch, Patricia Shalala, Keith Jones, Alun Dufty,
June Rider Jones, Coronary Barrow, Dorothy Davis, Julian Smith,
Mark Strahan, Vincent Turner, Wallace Turner, Gary James and Graham
Drabble for their continued support and assistance.
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
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(Accepted:
2 April, 1993)
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