By Valendar Turner
Department of Emergency Medicine, Royal Perth
Hospital, Perth, Western Australia
The real purpose of scientific method is to make sure Nature hasn't misled you into thinking something you don't actually know... One logical slip and an entire scientific edifice comes tumbling down. One false deduction about the machine and you can get hung up indefinitely.
Robert Pirsig, Zen and the Art of Motorcycle Maintenance
Does the currently available evidence prove beyond reasonable doubt that a unique, exogenously acquired retrovirus has been isolated from the tissues of AIDS patients? Perhaps. Perhaps not. This is what I invite you to judge. And in case you are inclined to be assaulted by the opinions of overwhelming majorities, you may take comfort from a most venerated, international scientist who said, "In Science the authority embodied in the opinion of thousands is not worth a spark of reason in one man." I shall reward you with his identity at the end of this talk.
A virus is two things. Number one: It's a microscopic particle of certain size and form. Number two, such particles generate identical progeny by parasitising chemical constituents and energy from a living cell. This is what is actually meant by the term infectious. It is this attribute which justifies a particle being called a virus. This is the property which prevents our calling every particle we see a virus. By definition, a retroviral particle is spherical in shape and has a diameter of 100-120 Nm. On the outside is a shell studded with outwardly projecting knobs, knobs obligatory to latch on to and infect new cells. On the inside there is a core containing RNA as well as some proteins, one of which is an enzyme called reverse transcriptase. The latter gives retroviruses their name and its function is to catalyse the transcription of viral RNA into DNA, that is, to copy information contained in RNA in a direction opposite the customary direction, DNA to RNA. According to virologists, it is the DNA copy of the RNA blueprint, not the original RNA, which hibernates inside the cell nucleus awaiting an opportune time to orchestrate the production of new viruses.
To analyse their constituents and to prove they are truly viruses, retroviral-like particles must first be purified. This is done by a process called density gradient ultracentrifugation, something that may sound complicated but which isn't. A test tube containing a solution of sucrose, ordinary table sugar, is prepared light at the top, but gradually becoming heavier towards the bottom. A drop of fluid from a cell culture is gently placed on top and the test-tube is centrifuged for several hours at extremely high speeds. This generates forces many thousands of times that of gravity and any tiny particles present are gradually forced through the sugar solution until they reach a point where their buoyancy prevents them penetrating further. For retroviral particles, this occurs where the density reaches 1.16 gm/ml, the point where the particles concentrate or, to use virological jargon, band. The 1.16 band can then be selectively extracted and photographed with an electron microscope. So, to prove the existence of a retrovirus one is obliged to:
1. Culture putatively infected cells.
2. Purify a sample in a sucrose density gradient.
3. Photograph the 1.16 band proving there are particles of the right size and form, and there is no other material.
4. Extract and analyse the constituents of the particles and prove they contain reverse transcriptase by showing they can make DNA from RNA.
5. Culture purified particles with virgin cells demonstrating that a new set of particles appears with the same morphology and constituents as the originals.
Now I am going to discuss some of the data from four papers published in Science in May 1984 by Dr. Robert Gallo and his colleagues from the US National Cancer Institute. These papers do not describe the original discovery of what the overwhelming majority regard as HIV, that distinction falls a year earlier to Professor Luc Montagnier and his colleagues from the Pasteur Institute from where, it is important to say, samples were sent to the Gallo laboratory and which later caused Gallo and his colleagues, as well as the US government, quite a number of problems. Neither are the Gallo papers the last word on HIV isolation but there is no doubt they are most important because it was they that led to the famous Washington press conference of April the 23rd 1984, two weeks prior to publication, at which an anxious, waiting world was told that the cause of AIDS had been identified. In fact, as one scrutinises the vast AIDS literature, it is fair to say that of all the papers published on HIV isolation, including the very latest, the Gallo papers are the most rigorous by far. The problem is, are they rigorous enough?
The first paper begins with cultures made of T-lymphocyte cells from AIDS patients. These cells were chosen because, included amongst their numbers, are the putatively infected cells, a subgroup known as T4 lymphocytes. It is these that are often diminished in AIDS, the hypothesis being that the yet to be discovered retrovirus was infecting and killing them. After an unspecified time, concentrated fluids from these T-cell cultures were subcultured with cells of a stock, leukaemic T-cell line known as HT. In these secondary cultures the Gallo team reported particles in electron microscopic examination of gross, unrefined culture fluids and measured reverse transcriptase activity in both these and banded specimens but without evidence that retroviral- like particles or indeed any particles were present at 1.16 gm/ml. They also reported reactions were seen between culture proteins and some antibodies present in human and animal sera. From these data, the Gallo team claimed to have isolated a new retrovirus, HIV, as well as inducing it to grow in the HT cell line in large enough quantities for use in analysis and diagnosis. In a subsequent third paper, from banded culture fluids obtained from a disrupted HT cell clone, two proteins, and for no other reason than they reacted with antibodies present in human AIDS sera, were deemed to be the HIV proteins. Subsequent papers, published after the Gallo four, using the same logic, increased the number of such proteins to about ten.
Reading these data it is obvious that Gallo and his colleagues had abandoned the traditional method of retrovirus isolation. This is enigmatic when one realises that, in 1976, Gallo himself had stressed that the detection of particles and reverse transcriptase, even reverse transcriptase inside particles, are not proof of the existence of retrovirus because, no matter how remarkably such particles may resemble retrovirus, many such particles are not viruses because they totally lack the ability to replicate (Gallo et al., 1976). You must appreciate the magnitude of the particle problem. Cell cultures contain many and many kinds of particles, some viral-like and some not. The viral-like include retroviral-like. In the 1970s, retroviral-like particles were frequently observed in human leukaemia tissues (Gallo et al., 1976), cultures of embryonic tissues, and "in the majority, if not all, human placentas." (Panem, 1979) One genus of retroviral-like particles, the type-C particle and the one into which Gallo classified his newly discovered retrovirus HIV, is found in "fish, snakes, worms, pheasant, quail, partridge, turkey, tree mouse and agouti" (Grafe, 1991) as well as in "tapeworms, insects...and mammals." (Frank, 1987) This being the case, there seems to be no way of avoiding the rules developed over the decades of research into animal retroviruses, rules that enabled a scientists to sort out this clutter. And there are two more complicating factors. The first is that reverse transcription is not only a property of retroviruses. Normal cells contain enzymes which reverse transcribe RNA and so does hepatitis B virus, a virus that infects T-cells as well as liver cells and is present in a considerable number of AIDS patients. The second is the choice of the HT cell line. It was long known that leukaemic cells theselves can reverse transcribe and, strange as it may seem, although Dr. Gallo was about to look for reverse transcription as a sign of a new retrovirus, the HT cell line originated from a patient who, according to Dr. Gallo, had a disease caused by a retrovirus he discovered called HTLV-I. In fact, in 1983, Gallo reported that the HT parental cell line contained HTLV-I genetic sequences. On this basis alone one would expect to find evidence of reverse transcription in the HT cell line. Given all these data, one would imagine it was impossible for the Gallo team to abandon the need to follow the traditional method and isolate and characterise infectious particles, but abandon it they did. By what reasoning then did the Gallo team claim to have proven the existence of a new retrovirus from AIDS patients?
For their 1984 papers, they reiterated the limitations of particles and reverse transcriptase and made three assumptions which, taken together, constitute a precept known as specific reactivity. (Gallo et al., 1986) The first assumption was that AIDS patients are infected with a replicating retroviral particle, a virus which could be grown in cell cultures to yield unique, virus-specific proteins. Second, being foreign, the virus would stimulate the production of a number of distinctive antibodies directed against the viral proteins. Third, the proteins and the antibodies react specifically, that is, only with each other and with no other agent. Let us take a very careful look at this paradigm. First, when the Gallo team began their experiments, the existence of specific viral proteins as constituents of a replicating viral particle which could infect humans was entirely hypothesis, not fact. Second, antibodies and proteins are not monogamous, even the purest of each take on other partners. Third, even if they were monogamous, we know that AIDS patients contain antibodies to many different agents, many with which they are infected, for example hepatitis B and cytomegalic inclusion viruses, mycoplasma, fungi and mycobacteria. Unless Gallo further hypothesised that all these agents or parts of them, or their respective antibodies, disappear from cultures or sera, when blood from an AIDS patient is mixed with cell cultures of the same or another AIDS patient, how can anyone tell what is reacting with what, let alone define precisely where each of the reactants originated? As far as the reactions are concerned, it's no different from mixing up milk from six species of animals, adding a mixture of a dozen different acids and claiming to know which acid is curdling which milk. So, although the term specific HIV proteins conjures up visions of proteins being extracted from retroviral-like particles proven to be a unique virus, this is not how it was done. It was done by breaking up cells of the HT cell line, not a virus particle, and observing unknown proteins reacting with unknown antibodies. From these data both the proteins and the antibodies were deemed viral, and not just any virus, but HIV. That's all. Logic or magic? And as an aside, similar to the proteins, the origin of what is called the HIV genome, the HIV RNA, is also based on circumstance, not on purification and dissection of particles proven to be infectious. The Gallo team may have claimed isolation of a new retrovirus but what they actually did was weave a nexus between reverse transcriptase, particles, and certain proteins under the dubious imprimatur of specific reactivity. Is this virus isolation? Is this even virus detection?
There are also a number of unsolved mysteries in the Gallo papers.
Mystery number one:
Reading the first paper one gets the impression that the HT cell line was cultured with individual AIDS patient cultures. However, the National Institutes of Health enquiry instigated after allegations of misappropriation of the French specimens found that the HT cell line was cultured with concentrated fluids pooled initially from individual cultures of three patients and ultimately from the individual cultures of ten patients. (Maddox, 1992) In evidence given to the enquiry, the reason given was because none of the individual cultures "was producing high concentrations of reverse transcriptase." That means not enough to convince the Gallo team of scientists or anybody else there actually was a virus in any of the individual specimens in the first place. The fact that pooled specimens produced reverse transcription is not proof of a retrovirus. The conditions may have merely changed in favour of the action of one of the cellular enzymes that performs the same trick. Or it could have been due to the HT cell line, unaided or at the behest of its HTLV-I retrovirus. The Gallo investigation found the pooling of specimens "of dubious scientific rigor." One scientist described the procedure as "really crazy." In essence, it is no different from investigating an outbreak of pneumonia by having all patients spit in separate pots and, when nothing turns up, getting them all to spit in the same pot.
Mystery number two:
The method of specific reactivity required a source of antibodies to the putative viral proteins. The logical place to obtain these was from AIDS patients -- after all, that is what the hypothesis required. The antibodies reported in the first paper appeared from two sources, a haemophiliac patient known as E.T. who had pre-AIDS and rabbits. Yes, rabbits. What precisely constituted E.T's pre-AIDS we are not told, but pre-AIDS is often generalised enlargement of the lymph nodes, a condition not invariably followed by AIDS and which is not AIDS. Thus, according to the paradigm of specific reactivity, we cannot be sure that E.T. actually had the right kind of antibodies. Rabbits do not develop AIDS and if specific antibodies to a retrovirus were to exist, they could only be produced by immunising rabbits with pure virus or, as the first Gallo group paper reported, from rabbits infected repeatedly with disrupted HIV. I hope you are beginning to see the problem. To make antibodies just to HIV, one has to inject rabbits with pure HIV. Pure virus means isolated virus and if rabbits were injected with pure virus, why should it be necessary to produce antibodies to define the isolation of virus that had already been isolated?
Mystery number three:
In the second paper, the Gallo team attempted what they called HIV isolation from 72 AIDS patients. Again, they cultured cells and detected particles and reverse transcriptase in unrefined culture fluids, and observed some protein/antibody reactions, but also added a fourth category, transmission, by which was meant finding particles or reverse transcriptase in bone marrow and other cells cultured with fluids, but not purified banded, photographed fluids, from one of the 72 starting cultures. What is enigmatic about the second paper is that HIV isolation was defined merely as detecting at least two of any of these four phenomena. The same criticism applies as in the first paper. Nothing was isolated and detection of unspecific phenomena is not surrogate isolation of a retrovirus. Even it were, this peculiar definition leads to some rather bizarre possibilities, for example, instances of virus isolation without the need to see particles or measure reverse transcriptase, for a retrovirus about as convincing as trying to sell a car without a body and an engine. Even so, loose as these criteria were, isolation was successful in only 26 of the 72 patients, that is, in only 36%. And, in case you think things have improved, there is a recent, international cooperative study reported by the World Health Organisation. In this study, by HIV isolation was meant detection of a single protein, p24, in culture fluids using a single antibody. Not only is p24 not specific for HIV (Agbalika et al., 1992; Mortimer et al., 1992), but from 224 HIV positive individuals, the success rate was a mere 37%, not significantly better than Gallo's figures a decade earlier. (WHO, 1994)
Even if the Gallo team had proved the existence of a new retrovirus, on what basis did they claim it was the cause of AIDS? Even if virus had been isolated from all patients and all patients had antibodies, which they didn't because in the fourth paper, the data showed only 88% of AIDS patients had antibodies (and on a single ELISA test which no one now regards as specific), is this sufficient proof that HIV causes AIDS? If the bank manager and his constant, faithful offsider are present at the bank robbery, is this proof that the manager robbed the bank? The Gallo papers provide no evidence whatsoever that HIV kills T4-cells or that low numbers of T4-cells is necessary and sufficient for the development of the AIDS infections and cancers and, I might add, there is still no such evidence. (Papadopulos-Eleopulos et al., 1994)
Let me finish by summarising the problem. The method of retrovirus isolation presented at the beginning flows logically from the definition of a virus. It is model of intelligibility, it is the only method, and was used for decades of research into animal retroviruses. (Sinoussi et al., 1973; Toplin, 1973) The problem is that, to date, nobody in the world has reported use of this method in AIDS patients. Without it, for example, how can one resolve the dilemma imposed by the numerous particles of stunning morphological variability present in cell cultures of AIDS patients? Even so, although some particles are the right diameter, there are no particles with the right diameter AND the projecting knobs, both integral to the definition of a retroviral particle and the latter essential to infect new cells. (Gelderblom et al., 1988; Layne et al., 1992; Levy, 1996) Yet, as I speak, there is still not even one published electron micrograph from a density gradient to tell us which, if any constituents of this zoo of particles, presents itself to be proved an infectious retrovirus. Perhaps, if someone were to look, there might not be any. Reading the literature, it is obvious that scientists everywhere have abandoned the traditional method of isolation and, under the assumed aegis of specific reactivity, claimed that two unknowns, antibodies and proteins, interact in specific pairs simultaneously betraying each other's genesis from a virus. In other words, what is the guts of what is called HIV isolation is actually no more than a chemical reaction, an antibody test, and from an antibody test, one cannot claim proof of isolation of anything. If an antibody test is isolation of a virus then the pregnancy test, which uses an antibody to detect the placental hormone beta HCG, must be regarded as placental isolation. Of course, there may be instances of specific reactivity involving viral proteins and antibodies, but the only way to prove this is to compare reactions in the test-tube with the virus of interest. Nature would then reveal specific reactivity by the fact that reactions, the virological equivalent of curdling milk, show up only when there is virus and never if there is no virus. This is crux of the matter and where the evidence for the existence of HIV begins to fall apart. To prove specific reactivity, one must first isolate the virus for use as a gold standard for comparison. One cannot adopt specific reactivity as a premise to prove the existence of a virus if one must first isolate the virus to prove the premise upon which isolation is contingent. Try as you will but the cart does not go before the horse and the Gallo argument is reductio ad absurdum.
This leaves us in a perilous quandary. What are these unknown antibodies to unknown proteins which we call being HIV positive? They could represent a virus, but that remains to be proven by isolating a retroviral-like particle and proving it is a retrovirus. It is certainly not cogent to argue that the conjunction of a number of unspecific phenomena makes one possibility a definite outcome any more than claiming that ten men, all dressed in white, hitting a ball around a paddock, must be playing cricket. They might just as well be Ku Klux Klaners playing baseball. If not a virus, then what? If someone tests positive, is this proof that a virus has been transmitted? Or is it something altogether different? Whatever these reactions mean, they do seem to be a marker for AIDS in the high risk groups, but are they just as significant in those at low or at no risk? Does just knowing you're HIV positive affect your health? Does your doctor knowing you're positive lead to treatments for a virus you may not have? Could such treatments themselves cause harm? We now know that antibodies to the germs that cause the diseases present in 90% of AIDS patients also react with the so called HIV proteins. (Muller et al., 1991; Kashala et al., 1994) Are we being fooled by antibodies that have nothing to do with a retrovirus? Are we seeing curdle from a different milk? Why, in one study, did 10% of 1300 individuals at low risk for AIDS including blood donors have antibodies to a sufficient number of HIV proteins to deem them HIV infected by the most stringent United States criteria? (Lundberg, 1988) Why do 30% of individuals transfused with HIV negative blood develop antibodies to the same p24 protein nearly every HIV researcher uses to "isolate" HIV? (Genesca et al., 1989) Why do 50% of dogs have antibodies to one or more of these same proteins? (Strandstrom et al., 1990) How come healthy, non-HIV-infected mice injected with blood from similar mice, or mice injected with extracts of a common human bowel bacterium, develop some of the same antibodies? Why does transfusion of one's own, irradiated blood produce the same antibodies? (Kozhemiakin & Bondarenko, 1992) If these data do not mean that HIV antibodies are non-specific, then there must be some completely unknown as well as very peculiar ways for men, dogs, and mice to acquire HIV infection. On the other hand, if some humans, injected with their own or someone else's blood, or mice injected with foreign cells and foreign proteins develop "HIV antibodies" but are not infected with HIV, why should gay men, IV drug users, and haemophiliacs, who are all exposed to foreign cells and/or foreign proteins, not also develop "HIV antibodies" and not be infected with HIV? Is it possible that we been misled by non-retroviral phenomena altogether? This would not be the first time. Over the mid- to late-1970s, Gallo and his colleagues claimed to have isolated the first human retrovirus, HL23V, from patients with various types of leukaemia and their evidence included a picture from a density gradient. (Gallagher & Gallo, 1975; Gallo et al., 1976) Soon enough antibodies to the HL23V proteins were found to be widespread, even amongst normal people and there was great excitement that a cause of leukaemic was at last in the offing. However, two groups of researchers then found that the antibodies were in reality directed against a wide range of naturally occurring substances, thus destroying that particular notion of specific reactivity. (Barbacid et al., 1980; Snyder & Fleissner, 1980) Overnight, HL23V vanished from the scientific literature, so much so that Gallo now never mentions it. Does a similar fate await HIV? Neville Hodgkinson (Hodgkinson, 1996), the former science and medical correspondent for the London Sunday Times, has suggested that HIV is the greatest scientific blunder of the twentieth century. If so, there are alternative theories and therapies for AIDS we would do well to consider.
Now, are you ready for that scientist? His name is Galileo Galilei, a man no stranger to heresy. Perhaps we should heed his counsel and begin to trust our own sparks. I say the sooner the better. *
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Friday, March 14, 2008
Posted by David G. Collins, B.Msc. at 3/14/2008 07:20:00 PM