What exactly is wrong with the way HIV has been isolated?

We may ask "Has the "right" way to isolate HIV been followed?" To answer we need to understand the meaning of "isolation" and why "isolation" features so prominently in the virological literature [1]. In everyday use "isolation" refers to an object that is separated from everything else. For example, the moon in the sky, a prisoner placed in solitary confinement, a patient with typhoid fever moved out of the general ward into a single room. Or Marie and Pierre Curie extracting one tenth of a gram of radium chloride from a ton of pitchblende given to them by the Austrian government. Significantly, although we are seldom consciously aware of the fact, we uphold the existence of an object because it is now, or we can render it, separate from the rest of the Universe. Thus isolation is a method of proving the existence of an object including objects hitherto unknown. This is how things were in 1983 two years after AIDS appeared. Scientists were looking for an object that had not been known to exist before which they hypothesised to be a retrovirus but one which was new and infected humans.

It almost goes without saying that in the sense discussed above the laboratory methods used by virologists for "HIV" are not isolation. No virologist has ever achieved with cell cultures from AIDS patients what the Curies did with their ton of pitchblende. However, since virologists believe their methods do lead to proof for the existence of a virus, it has become a tradition to consider these methods "isolation by another name". That is, "isolation" is "shorthand" for this collective of procedures [2]. The problem we must address is not semantics but whether or not the methods applied to cell cultures derived from AIDS patients prove the existence of a retrovirus HIV. Yes or no? That is the question we have addressed in many of our papers and more recently have asked several times over in the BMJ Online debate. (This link takes a long time to load).

According to two randomly selected antibody test kit packet inserts [3] HIV was isolated from AIDS patients in 1983/84 by Montagnier and Gallo and their colleagues. Over the past 20 years their early Science papers have been repeatedly cited as proof of this notion. Gallo himself regards these papers as "clearcut" evidence of the existence of HIV and its causal role in AIDS. Thus an analysis of these seminal papers should confirm that the methods and data reported are proof of "isolation" of a retrovirus HIV. And because Montagnier and Gallo used essentially the same methodology, differing only in the number of patients reported, we can examine their methods and data concurrently.

Table 1 of the second Gallo Science paper [4] defines "Detection and isolation of HTLV-III [HIV] from patients with AIDS and pre-AIDS" cell cultures as "Samples exhibiting more than one of the following":

"...reverse transcriptase [RT] activity in supernatant fluids". (Supernatant is the fluid above the sediment after centrifugation. Retroviral particles are said to be released from infected cells and collect in the supernatant).
"virus observed by electron microscopy".
"intracellular expression [production] of virus-related antigens [proteins] detected with antibodies from seropositive donors or with rabbit antiserum to HTLV-III [HIV]".
"transmission of particles, detected by RT assays or by electron microscopic observations, to fresh human [umbilical] cord blood, bone marrow, or peripheral blood T lymphocytes".

COMMENTS

1. "...reverse transcriptase [RT] activity in supernatant fluids".

Reverse transcriptase is an enzyme characteristic of retroviruses but one which is not confined to retroviruses. Of interest it is the reason for the "retro" in retroviruses. That is, the RT enzyme transcribes (copies) information contained in an RNA sequence into DNA. This is in a direction formerly considered "opposite" to the "normal" flow of information in living matter (DNA → RNA → proteins). Since RT is only a characteristic of retroviruses (as is hair on mammals) it cannot be considered proof of detection or isolation of a retrovirus any more than finding hair proves an animal is a classified species, or a rabbit or the rabbit is the only animal in the zoo. According to the enzyme's discoverer, Howard Temin, RT is present in all cells. Normal lymphocytes stimulated with the same chemical agent obligatory to "isolate" HIV from cell cultures also reverse transcribe RNA into DNA. Gallo himself proved this in 1973 [5]. Normal cells also possess reverse transcribing enzymes which are not RT.

2. "virus observed by electron microscopy".

Visualising particles with the appearances of retroviruses is not proof such particles are retroviruses. Gallo himself knew this in 1976. See Question 3. Referring to these particles as "virus" is putting the cart before the horse. The particles Gallo and Montagnier reported as HIV in their seminal papers are type C retroviral particles, not lentiviral particles which is the taxonomy of "HIV" particles. If neither Gallo nor Montagnier found lentivirus particles how can their papers be considered proof of HIV isolation? And what is the origin and role of the type C particles they did find? Are these a retrovirus? Do these cause AIDS?

3. "transmission of particles, detected by RT assays or by electron microscopic observations, to fresh human [umbilical] cord blood, bone marrow, or peripheral blood T lymphocytes".

The appearance of RT activity or particles even in a thousand cultures does not alter the fact neither are specific manifestations of retroviruses. And although Gallo writes "particles, detected by RT assays", this would involve purifying such particles to prove the RT activity was a unique property of those particles. No evidence of such purification of particles was published. In all of HIV research there is no evidence that any particle including that deemed to be HIV has been purified.

4. "intracellular expression [production] of virus-related antigens [proteins] detected with antibodies from seropositive donors or with rabbit antiserum to HTLV-III [HIV]".

"Expression" of "virus-related antigens" [6]. "Expression" is virological jargon that retains the common usage meaning "to transform ideas into words". But in virology what is being transformed is the information ("ideas") stored in viral DNA (or RNA) not into "words" but into viral proteins or particles. Thus in the instance of the fourth criterion "expression" means the production by the cellular biochemical machinery of viral proteins based on the information contained in a segment of viral DNA or RNA. This is the same biochemical process that occurs in normal, healthy cells not infected with a virus but which uses the cellular DNA to produce cellular proteins. By convention, under this circumstance this process is not referred to as "expression". Thus whenever a virologist uses the word "expression" he is asserting the existence of proteins produced by the cell under the direction of a virus. However, in relation to criterion four, since the cell culture experiments were being carried out in order to prove the existence of a virus, it is both unscientific and misleading to refer to such proteins as viral.

"Virus-related antigens" "detected with antibodies" means an antibody test. Given that Montagnier and Gallo were well aware that RT and particles are non-specific findings we must conclude that "HIV isolation" amounts to nothing more than an antibody test. Indeed Gallo confirmed this during an interview with Huw Christie at the 12th International AIDS Conference held in Geneva in 1998. When there is a resolution of the HIV/AIDS debate criterion four will become recognised as the HIV protagonists' greatest mistake. And one that has been made in the past [8]. While these proteins undoubtedly are antigenic in some species of animals, it is impossible to infer from an antibody reaction they are "non-human" or "viral" and thus foreign and antigenic in humans. (Especially since we now know that "HIV" proteins are found in "non-HIV-infected" cells). Neither can one additionally infer they are the antigens responsible for the antibodies which are found in the "seropositive donors" and that those antibodies are directed against these proteins. Those antibodies could arise for reasons which have nothing to do with these proteins or a viral infection but nonetheless cross-react with the proteins. Especially given the fact that despite being "immune deficient" AIDS patients characteristically have high levels of all antibodies. Even if the proteins were "viral" the antibodies could still be cross-reacting. Even if the proteins were normal cellular proteins they could still be cross-reacting or "autoantibodies". The latter are antibodies directed against cellular components including proteins and there is a plethora of such antibodies in AIDS patients. The presumption that the antibodies must be specifically directed against specific "virus-related antigens" cannot be justified by any data in the Gallo and Montagnier papers. In fact to date there are no data in any paper to support the existence of antibodies directed against a retrovirus "HIV" in anyone. See Question 5 and [7-8].

"...rabbit antiserum to HTLV-III" means antibodies directed against HIV proteins present in rabbit serum. Since rabbits do not develop AIDS or HIV infection the only way to produce such antibodies is to immunise (inject) rabbits with HIV. (Just as children are immunised against polio or measles by injecting them with (attenuated or inactivated) polio or measles virus). According to Gallo’s first Science paper the “rabbit antiserum” was made by “repeatedly infecting rabbits with disrupted HTLV-III” [HIV]. However, to obtain the proteins of "disrupted HTLV-III" first one must (a) prove the putative HIV particles are a retrovirus; (b) purify the HIV particles in order to separate the "disrupted HTLV-III" proteins from the cellular proteins; (c) prove the reactions between the "disrupted" proteins and antibodies they induce in rabbits are specific. (The rabbit antibodies could cross-react with "other", "non-HIV" cellular proteins. On the other hand, "non-HIV" cellular proteins injected into rabbits will induce antibodies which react with the same proteins. If the "rabbit antiserum" data is evidence that certain proteins are retroviral then all proteins can be proven retroviral). To date there is no evidence in favour of (a), (b) or (c).

All that Gallo and his colleagues could observe in their experiment resolves the fourth criterion to:

"In cell cultures derived from AIDS patients some proteins react with some antibodies present in the sera of AIDS patients or rabbits which have been immunised with proteins found in these cell cultures".

However, Gallo and his colleagues articulated this criterion as:

"In cell cultures derived from AIDS patients proteins are produced which (a) arise because of a viral DNA present in these cells (this is the meaning of "expressed"); (b) are constituents of this virus or are "related" to this virus; (c) being non-human are foreign and thus antigenic; (d) cause the appearance of serum antibodies which are directed against them and these are the antibodies detected in the "Seropositive donors" ; (f) react with similar antibodies produced by immunising rabbits with the same proteins [9].

Being aware that:

The enzyme RT is not unique to retroviruses.
The process of reverse transcription is not unique to RT or to retroviruses.
Particles with the appearances of retroviruses are not unique to retroviruses and neither Montagnier nor Gallo found a retroviral lentivirus particle in AIDS patients.
A scientist cannot assert "expression" of "virus-related antigens" in cell cultures without prior proof (a) that virus exists; (b) the cell is infected with that virus; the proteins are viral and not cellular.
An antibody test is not virus isolation.
The fact that proteins and antibodies react is not proof that (a) the proteins are viral; (b) the proteins are foreign and induce the appearance of the antibodies; (c) the antibodies are found in AIDS patients; the antibodies are directed against the proteins.
Because of the statement in Table 1, "Samples exhibiting more than one of the following..." — proof for the existence of the new retrovirus may be fulfilled in the absence of RT or particles or proteins in cell cultures of AIDS patients. Yet RT, particles and proteins are necessary properties of retroviruses.

we leave it up to the reader to contemplate "What is actually wrong with the way HIV has been isolated" [9-13].

ENDNOTES

1. A PubMed search on [virus AND isolation] brings up 92,314 papers. A search on [isolation AND bacteria] produces 241,851 papers.

2. Both basic and specialised Virology texts rarely define "isolation". When they do such attempts are non-illuminating. For example, Levy defines isolation as a "sample of a virus from a defined source"(1) and White as the ability to "identify a totally unforeseen virus, or even discover an entirely new agent" (2). Under the heading "virus isolation" are listed methods of culturing specimens in tissue and chick embryo cells, as well as live animals, following by documentation therein of cytopathic and pathological effects, haemoabsorption, immunofluorescence, antigen/antibody reactions and "characterisation of the viral genome" (2-5) HIV experts, including Luc Montagnier and Robin Weiss define "virus isolation" as "propagating them [viruses] in cells in culture" (6, 7). If "virus isolation" is to "take a sample of a virus from a defined source", or "propagating them in cells in culture", then first one must have prior knowledge that a virus exists in "a defined source" or "in cells in culture". However, proof that virus exists in a "defined source" or "in cells in culture" or can be "propagated" is contingent upon isolation of the virus in order there can be a virus in a "defined source" or "in cells in culture" which can be "propagated" . Thus the term "virus isolation" is not separation (purification) and subsists as a circular definition.

Levy JA, Fraenkel-Conrat H, Owens RA. Virology. 3rd ed. London: Prentice-Hall; 1994.
White DO, Fenner FJ. Medical Virology. 4th ed. San Diego: Academic Press; 1994.
Fields BN, Knipe DM, Howley PM, editors. Fundamentals of Virology. Philadelphia: Lippincott-Raven; 1996.
Dimmock NJ, Primose SB. Introduction of Modern Virology. 4th ed. Oxford: Blackwell Science; 1996.
Timbury MC. Notes on Medical Virology. Edinburgh: Churchill Livingstone; 1994.
Papadopulos E, Turner V, Weiss R. Email debate with Professor Robin Weiss on the existence of HIV. 1999.
Tahi D. Did Luc Montagnier discover HIV? Text of video interview with Professor Luc Montagnier at the Pasteur Institute July 18th 1997. Continuum 1998;5:30-34.

3. Genelabs Diagnostics "HIV Blot 2.2 Western blot assay and the Abbott Laboratories Diagnostics Division ELISA

4. Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan M, Haynes BF, et al. Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and at Risk for AIDS. Science 1984;224:500-503.

5. 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, editor. Possible Episomes in Eukaryotes. Amsterdam: North-Holland Publishing Company; 1973. p. 13-34.

6. The meaning of "related" in the term "virus-related antigens" is unclear. Does Gallo mean the proteins are made under the direction of viral DNA but are not constituents of the virus? Or does the presence of the virus stimulate cellular DNA to make cellular proteins? What if the fate of these proteins? If cellular are they "inserted" into the virus? If "viral" and not inserted into the virus are they "lost" when the virus particle leaves the cell?

7. The only way to verify the specificity of the antibody tests it to compare the test with HIV itself. That is, to use HIV isolation as the gold standard. If HIV experts ever were to do this (which so far they have not), such "verification" would amount to testing the antibody tests against an antibody test. That would be like claiming a photograph proves the identity of a particular person because it is identical to another photograph of the same person.

8. Unfortunately, the historical caveat against “antibody proven isolation” provided by HL23V, the world’s “first” human retrovirus, has remained unknown, unheeded, or forgotten. HL23V was “discovered” by Gallo in 1975 (1) and promoted as a “real possibility” for an infectious cause of human leukaemia (2). In fact, the evidence for the "isolation" of HL23V surpassed that of HIV in at least two aspects. Unlike HIV, Gallo's group reported the detection of reverse transcriptase activity in fresh, uncultured cells (3) and published an electron micrograph of virus like particles banding at a sucrose density of 1.16 gm/ml (1). As with HIV, the existence of HL23V was also predicated on antibody reactivity and, on the basis that 55/72 (76%) health humans were found seropositive, the authors concluded infection with HL23 “may be widespread in humans” (4). However, in 1980 researchers from the National Cancer Institute and the Sloan Kettering Cancer Center published data that proved the “HL23” antibodies are non-specific on the basis that preadsorption of sera with “substances as diverse as normal components of serum, extracts of bacteria, and even nonprotein molecules such as glycogen” removed reactivity and “The results are consistent with the idea that the antibodies in question are elicited as a result of exposure to many natural substances possessing widely crossreacting antigens and are not a result of widespread infection of man with replication-competent oncoviruses [retroviruses]” (5,6). In 1981 Gallo accepted the evidence that the antibodies which reacted with proteins of HL23V were directed not against the proteins "but against the carbohydrate moieties [molecules] on the molecule that are introduced by the host cell as a post transcriptional [after production from DNA and RNA] event, and which are therefore cell specific and not virus specific" (7). This discovery was of such significance that today nobody, not even Gallo, considers HL23V as being the first human retrovirus, or even a retrovirus. Yet despite the facts that the “envelope” proteins of HIV are also glycoproteins and mycobacterial (8) and fungal antigens (9) also adsorb “HIV” antibodies, HIV is still regarded bona fide. At least one expert has attempted to respectfully dispose of HL23V as a "blind alley". However, if HL23V is a blind alley then HIV is an even blinder alley. Investigative journalist John Crewdson's extremely interesting and informative account of this topic can be found here.

Gallagher RE, Gallo RC. Type C RNA Tumor Virus Isolated from Cultured Human Acute Myelogenous Leukemia Cells. Science 1975;187:350-353.
Kurth R, Teich NM, Weiss R, Oliver RTD. Natural human antibodies reactive with primate type-C antigens. Proceedings of the National Academy of Sciences of the United States of America 1977;74:1237-1241.
Gallo RC, Wong-Staal F, Reitz M, Gallagher RE, Miller N, Gillespie DH. Some evidence for infectious type-C virus in humans. In: Balimore D, Huang AS, Fox CF, editors. Animal Virology. New York: Academic Press Inc.; 1976. p. 385-405.
Aoki T, Walling MJ, Bushar GS, Liu M, Hsu KC. Natural antibodies in sera from healthy humans to antigens on surfaces of type C RNA viruses and cells from primates. Proceedings of the National Academy of Sciences of the U S A 1976;73(7):2491-5.
Barbacid M, Bolognesi D, Aaronson SA. Humans have antibodies capable of recognizing oncoviral glycoproteins: Demonstration that these antibodies are formed in response to cellular modification of glycoproteins rather than as consequence of exposure to virus. Proceedings of the National Academy of Sciences of the United States of America 1980;77:1617-1621.
Snyder HW, Fleissner E. Specificity of human antibodies to oncovirus glycoproteins: Recognition of antigen by natural antibodies directed against carbohydrate structures. Proceedings of the National Academy of Sciences of the United States of America 1980;77:1622-1626.
Kalyanaraman VS, Sarngadharan MG, Bunn PA, Minna JD, Gallo RC. Antibodies in human sera reactive against an internal structural protein of human T-cell lymphoma virus. Nature 1981;294:271-273.
Kashala O, Marlink R, Ilunga M, Diese M, Gormus B, Xu K, et al. Infection with human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic viruses among leprosy patients and contacts: correlation between HIV-1 cross-reactivity and antibodies to lipoarabinomannan. Journal of Infectious Diseases 1994;169:296-304.
Papadopulos-Eleopulos E, Turner VF, Papadimitriou JM, Causer D. HIV antibodies: Further questions and a plea for clarification. Current Medical Research and Opinion 1997;13:627-634.

9. Is a positive Western blot proof of HIV infection?

10. Has Gallo proven the role of HIV in AIDS?

11. Christine Johnson interview with Eleni Papadopulos-Eleopulos

12 . A great future behind it: The Yin and Yang of HIV

13. The Last Debate