Retrovirus Pioneer Rejects HIV-AIDS Model
NO RETROVIRUSES CAN
CAUSE AIDS, VIRUS ISOLATION EXPERT ETIENNE DE HARVEN CONCLUDES
Physician Etienne de Harven was a successful cancer researcher and retroviral
expert when he read in 1981 about the first four cases of what would later
be called AIDS. He watched as public hysteria over the possibility of an
infectious cause of AIDS transformed into a palpable demand that scientists
find one.
When National Institutes of Health official Robert Gallo answered the
demand in 1984 by proposing a retroviral cause, de Harven's concern turned
to disappointment. He feels that the near universal acceptance by scientists
and physicians of Gallo's hypothesis occurred only because they did not
properly study it.
Although concerned and disappointed, this did not surprise de Harven.
He had in the 1970s watched Gallo and other retrovirologists undermine
the scientific standards of their profession to make it seem that retroviruses
could cause a non-infectious disease, cancer, in humans. That enabled retrovirologists
to become the principal beneficiaries of federal cancer research dollars.
It also set the stage for their eventual takeover of the emerging AIDS
research budget, which was growing rapidly due to public fear of a contagious
epidemic.
In essays published in the spring issue of Continuum and this issue
of RA, de Harven makes plain his objection to the popular view of AIDS
as a virus-induced condition. He states that retroviruses, including HIV,
lack a capacity to cause immune deficiency, and endorses UC-Berkeley retrovirologist
Peter Duesberg's assertion that such factors as narcotics and HIV drugs
like AZT provide much better explanations for AIDS. He also articulates
support for Australian biophysicist Eleni Papadopulos-Eleopulos' argument
that HIV's existence has never been properly established, and that all
the HIV tests are invalid.
Sterling credentials
De Harven's public alliance with the AIDS Reappraisal movement represents
a major endorsement due to his sterling credentials. In 1953, he graduated
prima cum laude with his MD from the University of Brussels, Belgium. As
a research fellow, first at the Institut Gustave Roussy, in Villejuif,
France, and immediately afterwards at the Sloan Kettering Institute in
New York, he rapidly made two contributions to viral research. In 1958,
he and Sloan-Kettering's Charlotte Friend published the first electron
micrographs (pictures taken with an electron microscope) of the Friend
leukemia virus (FLV), a retrovirus just discovered by Friend in murine
(mouse) leukemia cells. In 1960, he demonstrated with electron microscopy
that the assembly of such retroviral particles occurs on the host cell's
outer membrane. In describing the steps leading to the release of retroviruses
from host cells, he coined the term "budding," which has become a staple
in the vocabulary of freshmen microbiology students.
In 1962, he joined the staff of the Sloan-Kettering Institute as its
chief of Ultrastructural Research Section, in a joint appointment as Professor
of Biology at Cornell. His laboratory became an international center for
ultrastructural studies of retroviruses. In 1981, he moved to Canada, to
lead the Electron Microscope Laboratory at the University of Toronto Pathology
Department, while serving as a staff pathologist at Toronto General Hospital.
He retired from both posts in 1993, and transferred to southern France,
where he continues his affiliation with the University of Toronto as emeritus
professor of pathology.
De Harven spent most of his research career characterizing and isolating
murine retroviruses, using filters and ultracentrifuges to purify the particles,
and electron microscopy to monitor the level of success of purification
and to study viral morphology. His extensive publication record led him
to serve as associate editor of Virology and Cancer Research, and as editorial
board member of Scanning Microscopy, Submicroscopic Cytology, and the Journal
of Electron Microscopy Technique.
Doubting HIV
De Harven doubted the infectious HIV-AIDS model from its inception in
1984, when Gallo presented it in four papers published together in the
May 4 issue of Science. Among the evidence supporting his conclusion, Gallo
claimed to have isolated a new retrovirus, later called HIV, from 34% of
the AIDS patients that he examined. This seemed to be a very week correlation,
since 66% of the patients lacked enough virus to isolate.
As for the patients in whom Gallo did claim to obtain retroviral isolates,
the evidence appeared very weak as well. Although retroviruses infect immune
cells, they don't kill them, so de Harven viewed them as unlikely candidates
for causing AIDS. Was Gallo proposing that his new retrovirus killed host
cells when replicating? No. In the cultures where Gallo claimed replication
of the retrovirus take place, hosts cells survive indefinitely.
Also, Gallo attempted to isolate his retrovirus only from supernatants
(cell-free culture fluid) of stimulated cell cultures, rather than from
fresh patient's plasma. Why was this? Could it be that there wasn't enough
virus in the plasma to isolate it in the first place? After all, culturing
causes viral populations to increase beyond the levels found in vivo. "If
there was enough virus to isolate directly from the plasma, as the current
concept of alleged 'viral load' suggests," de Harven says, "Gallo could
have done so very easily, and it would have made his fabulous story much
more convincing."
Then there were the replication data Gallo used to demonstrate that
these samples — which he called retroviral isolates — indeed contained
viruses. Gallo added the primary "isolates" (the ones made from supernatants
of cultures to which he had added patient tissue) to virgin cultures, which
he then spiked with powerful stimulants. He showed that from the resulting
secondary supernatants he could obtain a continuous supply of "retroviral
isolates" identical to the originals, indicating that something in them
had the capacity to replicate, a fundamental feature of viruses.
But since stimulants can induce culture cells to release all sorts of
material and particles, and since both the primary and secondary "isolates"
resulted only from stimulated cultures, de Harven wondered if these "isolates"
were merely artifacts of culture stimulation.
There were other reasons to doubt that these samples were viral isolates.
A retroviral isolate should contain a single species of RNA molecule, and
it should code for the proteins in the sample.
Although Gallo showed that his "isolates" contained RNA molecules and
several proteins, including the enzyme reverse transcriptase, he didn't
show that all the RNA molecules were identical, or that any one of them
coded for the proteins. Instead, he concluded that the molecules were viral
simply because some of the proteins reacted to antibodies in the fresh
(uncultured) plasma of 88% of his AIDS patients, and because he assumed
— incorrectly, de Harven says — that reverse transcriptase is an exclusive
feature of retroviruses.
Gallo's electron micrography data particularly intrigued de Harven,
as this involved his specialty. He says that a convincing and proper demonstration
of the existence of a retrovirus should contain two sorts of electron micrographs.
One should show retrovirus-looking objects of identical size and appearance
in uncultured patient tissue samples; the other should show isolates of
those objects, meaning samples that exclusively contain objects having
identical size and appearance to each other and the objects observed in
the tissue. Instead, Gallo presented micrographs of stimulated cultures
to which he had added AIDS patient immune cells or plasma, but no micrographs
of his "isolates." The micrographs of the cultures showed cells along with
spherical objects nearby of various sizes which Gallo claimed where representatives
of his new retroviral species.
To this day, de Harven says he can find no studies that have improved
upon Gallo's electron microscopy evidence. No one has claimed to have produced
electron micrographs of retrovirus-looking objects in the uncultured plasma
of a single AIDS patient, or to have obtained "HIV isolates" from uncultured
plasma. De Harven calls this "a totally unacceptable contradiction with
the alleged PCR measurements of 'viral load' in these patients."
Only in 1997 did researchers — two teams, in fact — finally publish
electron micrographs of so-called "HIV isolates" (Virology 230:125 and
134) made from stimulated cultures derived from AIDS patients' blood. As
de Harven suspected, the published pictures showed mostly non-viral material,
i.e. a heterogeneous mixture of debris plus, he says, "particles that resembled
cellular fragments but had no resemblance to retroviral particles."
The authors labeled some of these particles HIV and stressed that control
cultures — cultures unexposed to AIDS patients' blood — produced no such
particles. "But those 'control' cultures had not been stimulated and, therefore,
are questionable," de Harven says.
The Good Old Days
De Harven says when he began his career in 1953, claims of viral isolation
and causation were much more convincing, and had been so since the birth
of virology, in 1892.
That year a Russian bacteriologist discovered he could induce mosaic
disease in healthy tobacco plants by injecting them with fluid from sick
plants. Since the fluid didn't contain any of the diseased tissue, he concluded
that there was some exogenous causative agent. He showed that the agent
was microbial rather than molecular, since microbes can sustain their populations
within an organism via replication, whereas exogenous molecules can do
so only via regular replenishment from an outside source. He also established
that this microbe was invisible with the light microscope and smaller than
any known bacteria or fungi — the only known microbes at that time — by
reproducing the results after passing the fluid through special filters
with ultramicroscopic pore sizes (a process called ultrafiltration).
In 1898, a Dutch scientist found that this filterable infectious agent
was an unknown type of microbe, since it remained active even when subjected
to challenges that would destroy bacteria and fungi. He called it a virus.
Using similar techniques based on ultrafiltration, scientists found viral
causes for other diseases, including many animal and even human diseases.
In 1956, Friend demonstrated the existence of a mouse leukemia (immune
cell cancer) virus by inducing the disease in adult Swiss mice with injections
of ultrafiltered tissue extracts from leukemic laboratory mice.
Scientists learned to approximate the size of viruses by using filters
of different and relatively well-calibrated pore diameters. This enabled
Friend to demonstrate that her virus, like all other viruses then shown
to cause cancer in lab animals, probably had a diameter close to 100 nanometers
(nm).
Such techniques also enabled scientists to purify viral samples according
to size, to remove entities that are much larger or smaller than their
virus, in attempts to produce viral isolates. De Harven and Friend followed
this standard procedure to obtain purified samples of their retrovirus.
The next step of the standard procedure called for them to produce micrographs
of their purified samples, to see how close they had come to ideal isolation,
and to document the size and morphology of the objects in their sample.
Their micrographs showed very little else but spherical objects of the
same size (100 nm) and morphology. The objects looked exactly like those
in electron micrographs of the leukemic mouse tissue from which they'd
abstracted their filtrates. The objects also closely resembled the viruses
shown to induce cancer in other laboratory animals.
In the 1970s, purification by ultrafiltration gave way to purification
by density. This process involves placing a sample on a sucrose gel that
has a graduated density, meaning that from top-to-bottom the gel's density
increases from light to heavy. By spinning the gel in a high-speed centrifuge
— a process called ultrafugation — the sample's contents settle at their
characteristic densities, forming bands that constitute density-purified
samples.
De Harven and Friend showed that Friend's virus, like all those associated
with cancers in lab animals, banded at 1.16 grams per millimeter (gm/ml).
"Collecting the 1.16 band is a very efficient method of concentrating
retroviruses," de Harven explains. "Retroviruses concentrated by this method
look similar, under the electron microscope, to those concentrated by the
old ultrafiltration method. The degree of purity of these retroviral concentrates
depends on the amount of cellular microvesicles and debris contaminating
the initial sample which may very well sediment at the same density of
1.16 gm/ml."
Other methods came along to help scientists to study viruses.
Growing cells in cultures and infecting them with viral isolates permitted
virologists to distinguish two general groups of viruses. When some viruses,
like those associated with polio, influenza, and herpes, replicate inside
culture cells, the cells quickly die. Other viruses, including those associated
with cancer, lack this cytolytic capacity, and replicate without killing
their hosts. The viruses associated with cancerous lab animals, including
Friend's, actually make the culture cells become cancerous, a process called
transformation or immortalization, adding support to the contention that
they cause cancer in vivo.
Virologists also developed a way to study the proteins and gene molecules
— RNA or DNA — of viruses. By using chemicals that break viruses apart,
and a process called electrophoresis that separates molecules according
to molecular weight, scientists can turn a 1.16 gm/ml retroviral isolate
band into several new bands, each one representing an isolate of a different
molecular species that composes the virus.
Logically, this process applied to a retroviral isolate should produce
a single band with RNA molecules in it, and those molecules should be identical
to each other and code for the proteins that form the other bands.
Even before the advent of this technology, Friend showed that her virus,
like the others associated with cancer, contained RNA, which is why these
viruses were called "RNA Tumor Viruses."
In 1970, scientists found that the protein forming one of the electrophoresis
bands from retroviral isolates was an enzyme capable of reverse-transcribing
RNA molecules into DNA molecules. They named this enzyme reverse transcriptase,
and gave RNA tumor viruses the new name retroviruses.
That same year Duesberg showed that these viruses caused cancer because
their RNA molecules contained a special extra onc-gene. This contrasted
with the cyctolytic viruses, which other scientists found contained a gene
that coded for a cytolytic enzyme responsible for destroying the host cell's
membrane.
Isolation standards
De Harven says that the collective lessons of retrovirology by 1970
led to an important fundamental conclusions: a 1.16 gm/ml band qualified
as a retroviral isolate only after electron microscopy showed that it looked
like one, and unstimulated cultures showed that it behaved like one. This
is because on many occasions virologists obtained 1.16 gm/ml bands that
flunked the electron microscopy test, including bands that contained nothing
at all that even vaguely resembled viruses. And some 1.16 gm/ml bands that
did resemble retroviral isolates failed to demonstrate any replicative
capacity at all, and therefore qualified as isolates of non-viral objects
that resembled retroviruses.
In other words, everything that looks like a retrovirus is not necessarily
a retrovirus, and everything that looks like a retroviral isolate is not
necessarily a retroviral isolate.
De Harven says this conclusion holds even for 1.16 gm/ml bands that
contain reverse transcriptase, which virologists have "found not to be
unique for retroviruses, but instead a normal constituent of many cells."
No retroviral isolates from humans
The retrovirus-cancer link demonstrated among special inbred lab animals
caused scientists to wonder if retroviruses might explain some forms of
human cancer.
This idea initially interested de Harven. But he and some other retrovirologists
began to doubt that retroviruses could cause human cancer.
For one thing, the retrovirus-cancer link in animals was reserved for
special cases of inbred mice, chickens, and cats, and even then the link
was far from perfect. Among these peculiar laboratory animals, scientists
could usually isolate retroviruses from the subjects that had cancer, but
sometimes they could not. And sometimes they could isolate retroviruses
even in the absence of cancer. Furthermore, these retroviral isolates would
not induce cancer when injected into wild mice and chickens. Nor could
anybody isolate retroviruses from any wild animals, or even other laboratory
animals, besides special inbred cats.
Nonetheless, the entire profession in 1955 set its sights on isolating
retroviruses from humans suffering from various types of cancers. By 1970,
the examination of many cancerous human tissues resulted in some electron
micrographs that showed — mixed in with the cells and other material —
a few particles "with a vague resemblance to retroviruses," but no micrographs
of isolates. "Unfortunately," de Harven says, "the term 'virus-like particles'
kept being described in the literature, wrongly perpetuating the notion
that actual retroviruses were occasionally associated with some human cancers."
Typical retroviruses — objects of identical size and appearance, isolates
of which contain a single RNA species that codes for the proteins in the
sample — were never conclusively demonstrated for humans. "This was in
sharp contrast to the highly reproducible demonstration, by electron microscopy,
of typical retroviruses in a variety of mouse and chicken leukemias and
tumors."
Gallo lowers the standards
Nonetheless, prior to HIV's official birth in 1984, Gallo published
the only three claims for isolation from human subjects of retroviruses,
each one of which he said caused a different form of human leukemia. His
claims flunked the strict criteria established previously by scientists
demonstrating the existence of retroviruses in lab animals, and causally
linking those retroviruses to cancer.
Gallo's "evidence" resembled what he'd later use to demonstrate HIV's
existence and causal role in AIDS: the presumption that all 1.16 gm/ml
bands that contain reverse transcriptase are "retroviral isolates"; the
presumption that anything that vaguely resembles a retrovirus is a retrovirus;
the use of stimulated cultures; micrographs of those cultures, but not
of the density-purified bands; the failure to obtain them from uncultured
plasma; the failure to use unstimulated cultures; the failure to obtain
these bands from most of the patients examined; the failure to demonstrate
that these bands affect virgin cultures in a way that explains the disease
(rather than transforming the culture cells, Gallo's 1.16 gm/ml bands required
that the culture cells already be cancerous); the failure to demonstrate
that his bands contained a single RNA species and that it coded for the
proteins in the band; the failure to demonstrate that the RNA contained
a gene — in this case, an onc-gene — that could explain the disease.
"Why no micrographs of the 1.16 gm/ml bands themselves?" de Harven asks.
"Most probably, Gallo's electron microscopy results from his 'isolates'
were negative and swiftly ignored."
Yet a large segment of the biomedical profession, eager to consolidate
the hypothetical role of human retroviruses in human diseases, accepted
Gallo's new standards. Though one of his first claims was eventually discarded,
the pump was primed for the retroviral hunt among AIDS patients.
"The faith in retroviruses as pathogens assumed quasi-religious proportions,"
de Harven laments. "Since electron microscopy could not demonstrate viruses
in the 1.16 bands from human subjects, we forgot about microscopy and started
relying on 'markers.' Microscopy is time-consuming and skill-demanding.
Who has time for that? Not when research funding was getting difficult
and when major pharmaceutical corporations were starting to finance 'crash
programs' for speedy answers.
"When retroviruses are legion, molecular markers provide a useful approach
to quantification probably better than direct particle counting under the
electron microscope (which I always found difficult). But without isolates,
the use of markers is methodological nonsense. 'Markers' of what? We know
that all of the so-called 'HIV markers' are totally non-specific."
-- by Paul Philpott