Emergency Medicine 1993;5:5-147
HAS GALLO PROVEN THE ROLE OF HIV IN AIDS?
Eleni Papadopulos-Eleopulos, Valendar F. Turner, John M. Papadimitriou
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 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.
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
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
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.
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.
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,
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
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
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
2. T4-cell depletion is both necessary and sufficient for the appearance
of the AIDS indicator diseases.
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. *
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.
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
Department of Medical Physics
Royal Perth Hospital
Box X2213 GPO Perth
1. Essex M, McLane MF, Lee TH, et al.
Antibodies to Cell Membrane Antigens Associated with Human T-Cell Leukemia
Virus in Patients with AIDS. Science 1985;220:859-862.
2. Gallo RC, Sarin PS, Gelmann EP, et
al. Isolation of Human T- Cell Leukemia Virus in Acquired Immune Deficiency
Syndrome (AIDS). Science 1983;220:865-867.
3. Gallo RC. The First Human Retrovirus.
Sci Am 1986; 255:78-88.
4. Marx JL. Human T-Cell Leukemia Linked
to AIDS. Science 1983;220:806-809.
5. Barre-Sinoussi F, Chermann JC, Rey
F, et al. Isolation of a T-Lymphotrophic Retrovirus from a patient at Risk
for Acquired Immune Deficiency Syndrome (AIDS). Science 1983;220:868-871.
6. Popovic M, Sarngadharan MG, Read
E, et al. Detection, Isolation,and Continuous Production of Cytopathic
Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS. Science 1984;224:497-500.
7.Gallo RC, Salahuddin SZ, Popovic M,
et al. Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III)
from Patients with AIDS and at Risk for AIDS. Science 1984;224:500-502.
8. Schupbach J, Popovic M, Gilden RV,
et al. Serological analysis of a Subgroup of Human T-Lymphotrophic Retroviruses
(HTLV-III) Associated with AIDS. Science 1984;224:503-505.
9. Sarngadharan MG, Popovic M,Bruch
L, et al. Antibodies Reactive to Human T-Lymphotrophic Retroviruses (HTLV-III)
in the Serum of Patients with AIDS. Science 1984:224:506-508.
10. Culliton BJ. Gallo Inquiry Takes
Puzzling New Turn. Science 1990:250:202-203.
11. Culliton BJ. Inside the Gallo Probe.
12. Hamilton DP. What Next in the Gallo
Case? Science 1991;254:944-945.
13. Palca J. Draft of Gallo Report
Sees the Light of Day. Science 1991;253:1347-1348.
14. Cohen J. HHS: Gallo Guilty of Misconduct.
15. Gallo RC, Sarin PS, Kramarsky B.
et al. First isolation of HTLV-III. Nature 1986;321:119.
16. Rous P. A Sarcoma of the Fowl transmissible
by an agent separable from the Tumor Cells. J Exp Med 1911;13:397-411.
17. Weiss R,Teich N, Varmus H, Coffin
J. RNA Tumor Viruses. Cold Spring Harbor Laboratory. Cold Spring Harbor,
18. Temin HM, Baltimore D. RNA-Directed
DNA Synthesis and RNA Tumor Viruses. Adv Vir Res 1972;17:129-186.
19. Toplin I. Tumor Virus Purification
using Zonal Rotors. Spectra 1973;No. 4:225-235.
20. Bader JP. Reproduction of RNA Tumor
Viruses. In: Fraenkel-Conrat H, Wagner RR, eds. Comprehensive Virology
Vol.4. New York: Plenum Press, 1975:253-331.
21. Sinoussi F, Mendiola L, Chermann,
JC, et al. Purification and partial differentiation of the particles of
murine sarcoma virus (M. MSV) according to their sedimentation rates in
sucrose density gradients. Spectra 1973;No. 4:237-243.
22. Maddox J. More on Gallo and Popovic.
23. Lee MH, Sano K, Morales FE, et
al. Sensitive Reverse Transcriptase Assay to Detect and Quantitate Human
Immunodeficiency Virus. J Clin Microb 1987;25:1717-1721.
24. Gallo RC, Sarin PS, Wu AM. On the
nature of the Nucleic Acids and RNA Dependent DNA Polymerase from RNA Tumor
Viruses and Human Cells. In: Silvestri LG, ed. Possible Episomes in Eukaryotes.
Amsterdam: North-Holland Publishing Company, 1973:13-34.
25. Whitkin SS, Higgins PJ, Bendich
A. Inhibition of reverse transcriptase and human sperm DNA polymerase by
anti-sperm antibodies. Clin Exp Immunol 1978;33:244-251.
26. Varmus H. Reverse Transcription
Sci Am 1987;257:48-54.
27. Hart DJ, Gogu SR, Agrawal KC, et
al. Inhibition of RNA-dependent DNA polymerases (reverse transcriptase)
of normal cells by activated azidothymidine:a possible basis for drug toxicities?
In: Vol I, Abstracts VII International Conference on AIDS,Florence, 1991:110.
28. Gallo RC, Wong-Staal F, Reitz M,
et al. Some Evidence For Infectious Type-C Virus in Humans. In: Baltimore
D, Huang AS, Fox CF, eds. Animal Virology. New York: Academic Press Inc.,1976:385-407.
29. Panem S, Prochownik EV, Reale FR,
et al. Isolation of Type C Virions from a Normal Human Fibroblast Strain.
30. Panem S, Prochownik EV, Knish WM,
Kirsten WH. Cell Generation and Type-C Virus Expression in the Human Embryonic
Cell Strain HEL-12. J Gen Virol 1977;35:487-495.
31. Panem S. C Type Virus Expression
in the Placenta. Curr Top Pathol 1979;66:175-189.
32. Sarngadharan MG, Robert-Guroff
M, Gallo RC. DNA Polymerases of Normal and Neoplastic Mammalian Cells.
Biochim Biophys Acta 1978;516:419-487.
33. Klatzmann D, Barr=82-Sinoussi F,
Nugeyre MT, et al. Selective Tropism of Lymphadenopathy Associated Virus
(LAV) for Helper-Inducer T Lymphocytes. Science 1984;225:59-63.
34. Montagnier L. Lymphadenopathy-Associated
Virus:From Molecular Biology to Pathogenicity. Ann Int Med 1985;103:689-693.
35. Gendelman HE, Orenstein JM, Martin
MA, et al. Efficient Isolation and Propagation of Human Immunodeficiency
Virus on Recombinant Colony-Stimulating Factor 1-Treated Monocytes. J Exp
36. Gelderblom HR, Ezel M, Hausmann
EHS, et al. Fine Structure of Human Immunodeficiency Virus (HIV), Immunolocalization
of Structural Proteins and Virus-Cell Relation. Micron Microsc 1988;19:41-60.
37. Lecatsas G, Taylor MB. Pleomorphism
in HTLV-III, the AIDS virus. S Afr Med J 1986;69:793-794.
38. Hockley DJ, Wood RD, Jacobs JP,
et al. Electron Microscopy of Human Immunodeficiency Virus. J Gen Virol
39. Dourmashkin RR, O'Toole CM, Bucher
D, Oxford JS. The presence of budding virus-like particles in human lymphoid
cells used for HIV cultivation. In: Vol I, Abstracts VII International
Conference on AIDS,Florence,1991:122.
40. O'Hara CJ, Groopmen JE, Federman
M. The Ultrastructural and Immunohistochemical Demonstration of Viral Particles
in Lymph Nodes from Human Immunodeficiency Virus-Related Lymphadenopathy
Syndromes. Hum Pathol 1988;19:545.
41. Henderson LE, Sowder R, Copeland
TD, et al. Direct Identification of Class II Histocompatibility DR Proteins
in Preparations of Human T-Cell Lymphotropic Virus Type III. J Virol 1987;61:629-632.
42. Lundberg GD. Serological Diagnosis
of Human Immunodeficiency Virus Infection by Western Blot Testing. JAMA
43. Edwards VM, Mosley JW and the Transfusion
Safety Study Group. Reproducibility in Quality Control of Protein (Western)
Immunoblot Assay for Antibodies to Human Immunodeficiency Virus. Am J Clin
44. Pinter A, Honnen WJ, Tilley SA,
et al. Oligomeric Structure of gp41, the Transmembrane Protein of Human
Immunodeficiency Virus Type 1. J Virol 1989;63:2674-2679.
45. Zolla-Pazner S, Gorny MK, Honnen
WJ. Reinterpretation of Human Immunodeficiency Virus Western Blot Patterns.
46.Damsky CH, Sheffield JB, Tuszynski
GP, et al. Is there a role for Actin in Virus Budding? J Cell Biol 1977;75:593-605.
47. Stanislawsky L, Mongiat F, Neto VM, et al. Presence of Actin in Oncornaviruses.
Biochem Biophys Res Com 1984;118:580-586.
48. Stricker RB, Abrams DI, Corash
L, et al. Target Platelet Antigen in Homosexual Men with Immune Thrombocytopenia.
49. Matsiota P, Chamaret S, Montagnier
L. et al. Detection of Natural Autoantibodies in the serum of Anti-HIV
Positive-Individuals. Ann Inst Pasteur/Immunol 1987;138:223-233.
50. Wong-Staal F, Gallo RC. Human T-lymphotropic
retroviruses. Nature 1985;317:395-403.
51. Genesca J, Jett BW, Epstein JS,
et al. What do Western Blot indeterminate patterns for Human Immunodeficiency
Virus mean in EIA-negative blood donors? Lancet 1989;II:1023-1025. 52.
Ranki A, Johansson E, Krohn K. Interpretation of Antibodies Reacting Solely
with Human Retroviral Core Proteins. NEJM 1988;318:448-449.
53. Delord B, Ottmann M, Schrive MH,
et al. HIV-1 expression in 25 infected patients:A comparison of RNA PCR,
p24 EIA in Plasma and in situ Hybridization in mononuclear cells. In: Vol.
I, Abstracts VII International Conference on AIDS,Florence, 1991:113.
54. Todak G, Klein E, Lange M, et al.
A clinical appraisal of the p24 Antigen test. In:Vol. I, Abstracts VII
International Conference on AIDS,Florence,1991:326.
55. Mager DL, Freeman JD. Human Endogenous
Retroviruslike Genome with Type C pol Sequences and gag Sequences Related
to Human T-Cell Lymphotropic Viruses. J Virol 1987;61:4060-4066. 56. Banki
K, Maceda J Hurley E, et al. Human T-cell lymphotropic virus (HTLV)-related
endogenous sequence, HRES-1 encodes a 28-kDa protein: A possible autoantigen
for HTLV-I gag- reactive autoantibodies. Proc Natl Acad Sci 1992;89:1939-1943.
57. Horwitz MS, Boyce-Jacino MT, Faras
AJ. Novel Human Endogenous Sequences Related to Human Immunodeficiency
Virus Type 1. J Virol 1992;66:2170-2179.
58. Aaronson SA, Todaro GJ, Scholnick
EM. Induction of murine C- type viruses from clonal lines of virus-free
BALB/3T3 cells. Science 1971;174:157-159.
59. Hirsch MS, Phillips SM, Solnik
C, et al. Activation of Leukemia Viruses by Graft-Versus-Host and Mixed
Lymphocyte Reactions In Vitro. Proc Nat Acad Sci 1972;69:1069-1072.
60. Toyoshima K, Vogt PK. Enhancement
and Inhibition of Avian Sarcoma Viruses by Polycations and Polyanions.
61. Todaro GJ, Benveniste RE, Sherr
CJ. Interspecies Transfer of RNA Tumour Virus Genes: Implications for the
search for "Human" Type C Viruses. In: Baltimore D, Huang AS,
Fox CS, eds. Animal Virology. New York: Academic Press Inc.;1976:369-384.
62. Connor S. AIDS: Science Stands
On Trial. New Scientist 1987; Feb 12:49-54.
63. Burke DS, Brundage JF, Redfield
RR, et al. Measurement of the False Positive Rate in a Screening Program
for Human Immunodeficiency Virus Infections. NEJM 1988;319:961-964.
64. Voevodin A. HIV screening in Russia.
65. Gazdar AF, Carney DN, Bunn PA,
et al. Mitogen Requirements for the In Vitro Propagation of Cutaneous T-Cell
Lymphomas. Blood 1980;55:409-417.
66. Rubinstein E. The Untold Story
of HUT78. Science 1990;248:1499-1507.
67. Poiesz B, Ruscetti FW, Mier JW,
et al. T-cell lines established from human T-lymphocytic neoplasias by
direct response to T-cell growth factor. Proc Natl Acad Sci 1980;77:6815-6819.
68. Chiodi F, Albert J, Olausson E,
et al. Isolation Frequency of Human Immunodeficiency Virus from Cerebrospinal
Fluid and Blood of Patients with Varying Severity of HIV Infection. AIDS
Res Hum Retrovirol 1988;4:351-358.
69. Learmont J, Tindall B, Evans L,
et al. Long-term symptomless HIV-1 infection in recipients of blood products
from a single donor. Lancet 1992;340:863-867.
(Accepted: 2 April, 1993)