WHAT CAUSES A POSITIVE TEST FOR HIV-ANTIBODIES?

Vladimir Koliadin

April 1998

1. Introduction

In 1993, the widely accepted ideas that a positive test for HIV-antibodies is specific (i.e. means HIV- infection) and that HIV exists as an exogenous retrovirus were challenged [1]. Then, these views were developed by the same [2] and other authors [3,4]. Neither experimental nor even logical arguments were advanced by mainstream AIDS-scientists to disprove the central points in [1]. At present, it is still accepted in mainstream AIDS-science that existence of HIV and high specificity of HIV-antibody tests are ultimately proven scientific facts. Besides conformity of thinking and vested interests, this belief is maintained by the absence of clear-cut alternative explanations to the phenomena which are thought to prove the HIV-causes- AIDS theory. Some important questions have not been answered. If HIV does not exist, what causes a positive test for HIV-antibodies? Why almost all AIDS-patients are seropositive while almost all healthy individuals are not? Why is the positive test strongly associated with high mortality and susceptibility to opportunistic infections?

The heart of any HIV-antibody test is so called HIV-proteins. These proteins are produced by cell- cultures of human lymphocytes. According to the mainstream AIDS-science, these proteins belong to a new retrovirus HIV, by which these cell-lines are presumably infected. HIV-skeptics say that these proteins are of endogenous (intracellular) nature [1]. If to follow facts and basic principles of immunochemistry, the only conclusion which may be drawn from a positive test for HIV- antibodies is that some immunoglobulins in the tested blood-serum have bound to these "HIV- proteins". This is the case for both ELISA and Western Blot (WB) tests, as well as for any other immunochemical test. The point of disagreement is how to interpret such binding. Mainstream science insists that such binding is due to specific antibodies, produced by the immune system after its contact with HIV. The authors of papers [1,2] explain this binding by cross-reaction between "HIV-antigens" and antibodies to other non-HIV antigens, to which AIDS- patients and individuals from high-risk groups are exposed. Such cross-reaction is well known to occur when different antigens share common parts ("antigen determinants"). In this case, antibodies to one of such antigens, are capable to bind selectively to other antigen with the same antigen determinants. Nevertheless, the cross-reactivity hypothesis suffers from one problem: how to explain that almost all AIDS-patients and many people in high-risk groups have been exposed to some antigens bearing common determinants just with "HIV-antigens"? Why almost nobody beyond the risk- groups have not been exposed to such antigens, though anybody in course of his/her life have been exposed to many antigens (vaccinations, infections, etc.)?

2. Why HIV-antibody tests are thought to be specific

In the middle of 1980s, it was shown experimentally that a test for HIV-antibodies is positive in a great proportion of patients with diagnosis of AIDS or pre-AIDS, while it is negative in almost all perfectly healthy individuals. It is essential that AIDS-diagnosis in these patients was established from clinical symptoms, not from HIV-test itself. This undermines the "circular definition" argument advanced by some AIDS-dissidents. Moreover, such studies were carried out according to a blind and randomized protocol. This gave a great credibility to the HIV-causes-AIDS viewpoint. Do such studies actually prove that HIV- positivity is specific to AIDS? Even though it may seem "self-evident" at first sight, it is not so. An important principle of evidence-based science was obviously violated in these studies: the control group was not matched to the AIDS group - perfectly healthy individuals were used as controls [1].

Why is the principle of matched control so important? The following simple example provides some explanation. Let a disease is characterized by some feature, say by elevated body temperature. Let each of 1,000 patients with this disease demonstrates high temperature, whilst each of 1,000 healthy controls - normal (lower) temperature. Does it mean that body temperature is specific to THIS disease? Surely not - simply because the high temperature, though being highly unusual in healthy individuals, is typical to many other diseases. For the same reasons, the ability of HIV-test to discriminate between AIDS-patients and healthy individuals cannot prove specificity of HIV-tests. This obvious flaw in the studies of "specificity of HIV-antibody tests", spotted in paper [1], was ignored by the mainstream science. In respect to which symptoms the control group should be matched to AIDS-patients? It is not an easy question: many symptoms and signs are typical to AIDS. In paper [1] such symptoms were not specified. In the next section an attempt will be made to fill this gap.

There are also other studies, carried out with a great number of individuals (applicants for US military service) in which specificity of HIV-tests was reported as 1 false positive reaction in more than 135,000 [5]. Nevertheless, these studies have nothing to do with proofs of existence of HIV or specificity of HIV- antibody tests because just WB test-systems were used as the gold standard of HIV. The only thing shown by these studies definitely is that results provided by some ELISA systems in multistep testing algorithms are well compatible with results of WB tests, at least in a population with a very low prevalence of HIV-seropositivity. Just such question was addressed in these studies, not specificity of WB-tests themselves.

3. Non-specific immunoglobulins as the cause of HIV-seropositivity

The B-lymphocytes (a part of the immune system) normally respond to foreign substances (named "antigens") by increased production of special proteins - immunoglobulins. Some of these immunoglobulins are capable to specific binding to the antigen - that is, they bind strongly to the antigen but not to other substances. Such immunoglobulins are usually named "antibodies". Other immunoglobulins, which are capable to bind to a wide range of antigens, are named "non-specific immunoglobulins". Many authors name all immunoglobulins "antibodies"; to discriminate between the two types they use terms "specific antibodies" and "non-specific antibodies" (or "normal antibodies"). The ability of (specific) antibodies to bind only to the antigen which induced production of these antibodies plays a very important role in biomedical science and serological testing.

If HIV-antibody tests are positive in AIDS-patients and negative in healthy controls, does it mean that blood-serum of AIDS patients contains some factor which is absent in healthy individuals? Most scientists (not specialists in practical immunochemistry), let alone laymen, may believe that at least this fact was proven beyond any reasonable doubt by such studies. Nevertheless, this is not the case: blood-serum from healthy individuals is also capable to provide positive WB. The only difference is that titers of this reaction are higher for AIDS-patients than for healthy controls. The titer is the maximal dilution of the serum for which reaction is still noticeable. For example, R.Gallo and his group managed to obtain "specificity" (in respect to healthy controls) only after 500-fold dilution of the serum [6,7]. Thus, blood serum from healthy controls does provide a positive WB-test, but if diluted less than 1:500. In practical immunochemistry, such a phenomenon is explained by binding of non-specific immunoglobulins, which are present in any individual and bind to almost any antigen. In commercial WB-systems this effect is not noticeable because sensitivity is artificially lowered to avoid positive reactions in healthy individuals.

How to decide whether a test is positive because of binding with specific antibodies or only with non- specific immunoglobulins? In practice of immunochemical testing this problem is apparently resolved by reduction of sensitivity (e.g. by dilution of the tested serum) up to the levels which guarantee negative tests in healthy controls. Such an approach tacitly implies that reactivity and concentrations of non-specific immunoglobulins are about the same in all individuals. Even though being widely accepted, this practice is dubious. If some states of organism are characterized by essentially higher levels of non-specific immunoglobulins or by their higher affinity (ability to bind), blood serum from such individuals can yield a positive test too. How to prove that a positive test for a given sample is caused just by specific antibodies, but not by non-specific immunoglobulins? By definition, specificity of antibodies means they are capable to bind only to one antigen (or very similar ones). Hence, it is necessary to check how this serum reacts with a great many of other antigens. If titers of these reactions are abnormally higher only in respect to the given antigen (as compared with titers of serum from healthy controls), it proves that reaction has been caused by specific antibodies.. On the other hand, if the serum reacts with many other antigens in titers higher than normal, this is a strong reason to suspect that the test is positive due to non-specific immunoglobulins. Unfortunately, such multi-antigen tests are not currently used to confirm specific nature of a positive single-antigen test. This is partially explainable by much higher technical complexity and cost of such verification tests, absence of commercial toolkits, as well as by lack of interest of the mainstream immunology to non-specific phenomena.

There are several reasons to assume that tests for "HIV-antibodies" are positive in AIDS-patients due to non-specific immunoglobulins. First, it is well known that total concentrations of non- specific immunoglobulins are much higher in almost all AIDS-patients than in healthy individuals. This condition, named hypergammaglobulinemia, is highly non-specific and observed in many diseases and abnormal states of organism. (Causes of such a condition will be considered below.) Thus, the principle of matched control is obviously violated in the aforementioned studies - not healthy individuals, but just patients with hypergammaglobulinemia (but without AIDS) had to be used as controls. Second, in many AIDS patients, titers of "HIV-antibodies" are low. Perhaps for these reasons information about titers is conspicuously absent in AIDS-literature. For example, in Russia, due to shortage of test systems so called "pool-method" was frequently used - when blood from several individuals was mixed and tested. It was noted, that even such 2-8 fold dilution of the serum, may have resulted in a false negative test [8]. Thus, titers of putative "HIV-antibodies" in some AIDS patients are about 1:2 - 1:8. In immunology such low titers are usually not considered as significant at all [9]. Third, AIDS patients and individuals from groups with high risk of AIDS are known to be seropositive in respect to many other antigens (e.g. hepatitis-B virus, EBV, etc.). Even though this is usually interpreted as a sign of infection by these pathogens, it is also well explainable by non-specific reactivity of the serum of these individuals [3].

Non-specific reactivity should not be confounded with cross-reactivity [3]. Cross-reactivity is a specific phenomenon, well understood in immunology. Even though cross-reacting antigens differ, they share common determinants, and reaction is specific in respect to these determinants. Normally only a small proportion of antigens cross-react to antiserum against a given antigen. In contrast to the cross-reactions, neither mechanisms of production nor properties of non-specific immunoglobulins are understood in modern immunology. Textbooks of immunology either keep silence or say only a few words about their existence. It is also rarely mentioned in the textbooks, that only a part, maximum 20-30 percent, of the immunoglobulins produced by B-cells in response to an antigen are capable to bind specifically to the antigen; other 70-80% of the immunoglobulins are non-specific [10]. In [3] a hypothesis was advanced that affinity of non-specific immunoglobulins increases radically if the B-lymphocytes are over-stimulated, , and that just this effect leads to positive tests for HIV-antibodies.

The problem of non-specific immunoglobulins is exacerbated in the tests which are based on "sandwich" principle (as in ELISA and WB). In these test-systems, the antigen (e.g. "HIV- antigen") is attached to a surface and forms the first layer in the "sandwich". After exposure to the tested serum, immunoglobulins from the serum bind to the antigen and form the second layer in the "sandwich". To detect these immunoglobulins and estimate their quantity the two-layer sandwich is subject to one more procedure - it is being exposed to antibodies against human immunoglobulins (which are obtained from laboratory animals immunized by human immunoglobulins). These animal antibodies bind to the human immunoglobulins and form the third layer of the sandwich. Just the amount of the animal antibodies bound to human immunoglobulins, not quantity of the human immunoglobulins themselves, is being measured in test-systems of this sort [9]. It is tacitly postulated that affinity (ability to bind) of the animal antibodies to human immunoglobulins is about the same for all individuals and, hence, the amount of the animal antibodies bound is simply proportional to the amount of human immunoglobulins. But this is not the case if non-specific immunoglobulins from some individuals are capable to bind more strongly to various substances than immunoglobulins from healthy controls. Let, for example, such non- specific affinity of non-specific immunoglobulins increased 3 times in respect to various proteins, including both "HIV-antigens" and animal antibodies. Then, the number of molecules of human non-specific immunoglobulins bound per one molecule of the antigen is 3 times higher. After exposure to animal antibodies, 3 times higher number of molecules of the animal antibodies will be bound per one molecule of human immunoglobulins. Thus, the total amount of animal antibodies will be 9 times (3 multiplied by 3) higher, not 3 times. Hence, even a moderate increase in non- specific affinity of immunoglobulins may lead to radical increase in the titers and, hence, to HIV- seropositivity.

4. What increases the levels of non-specific immunoglobulins

As far as elevated levels of non-specific immunoglobulins and their higher affinity may lead to positive tests for "HIV-antibodies" it is reasonable to consider the causes of such conditions. It is widely accepted that hypergammaglobulinemia (which is typical to AIDS and many other conditions) results from polyclonal activation of B-lymphocytes caused by intensive stimulation by multiple foreign antigens. Individuals in groups with high risk of AIDS are abnormally frequently exposed to such multiple antigens [1]: multiple infections, including STD and AIDS- defining opportunistic infections, anal reception of sperm, contaminants of injected recreational drugs, foreign blood received either in course of sharing syringes for injection of such drugs or as transfusions, contaminants of the blood factors received by hemophiliacs as well as these factors themselves. Thus, the significantly higher frequency of positive tests for "HIV-antibodies" observed in the groups with high risk of AIDS is highly likely to be nothing but an indicator of such over-stimulation by multiple foreign antigens and, in many cases, an indicator of some diseases. There are no any factual reasons to consider the antigen overload itself as a life- threatening factor.

Along with the above mentioned factors, one more factor of antigen overload deserves a closer attention - prolonged use of broad spectrum antibiotics. Such an antibiotic abuse is closely associated with high promiscuity (frequent change of sexual partners): antibiotics are used to treat frequent STD as well as permanent prophylaxis of STD (a fashion popular among promiscuous individuals). Moreover, permanent use of broad spectrum antibiotics is prescribed to many HIV- seropositive individuals and to most AIDS-patients - as an apparent prophylaxis against opportunistic infections. Antibiotics cause antigen overload indirectly. These drugs suppress the friendly bacterial flora of the gut. These flora are of vital importance for digestion of food as well for stifling various opportunistic pathogenic microorganisms. Suppression of the friendly flora of the gut by antibiotics results in serious problems with digestion of food and in development of opportunistic intestinal infections [14]. Such intestinal abnormalities frequently cause increased permeability of the gut walls ("leaky gut syndromes") [15]. Proteins are essential parts of our diet, and they are very powerful foreign antigens. Why don't they cause antigen overload in healthy individuals? Normally, proteins are digested into short fragments which are not antigens (cannot induce immune response), and only these non-antigen short permeate the gut walls and run into the blood stream. Abnormally high permeability of the gut walls makes it possible for molecules of proteins themselves to run into the blood stream and to become a powerful factor of antigen overload. This mechanism provides a plausible explanation for the epidemiological association between promiscuity, diagnosis of AIDS, and HIV-seropositivity, mediated by a common factor - antibiotic abuse. It also explains the unusually high rate of HIV-seropositivity in Africa, where intestinal infections are rampant.

Besides the role of foreign antigens, there is another plausible hypothesis advanced in [11]: it holds that both typical signs of AIDS - hyperactivation of B-cells and T4-lymphocytopenia - are intrinsic features of the classical stress-syndrome. Stress-syndrome is a highly non-specific reaction of organism to various adverse factors: diseases, intoxication, psychological trauma, etc. This reaction is mediated by elevated concentrations of corticosteroids ("stress-hormones"), and it may be induced by direct injection of corticosteroids. (Incidentally, corticosteroids are frequently used to treat inflammatory conditions in AIDS-patients.) If to combine this hypothesis with the aforementioned one (that hyperactivation of B-cells results in increased affinity of non-specific immunoglobulins), it provides a coherent explanation of why people with severe opportunistic infections (named AIDS) usually demonstrate T4-lymphocytopenia and HIV-seropositivity.

5. HIV and mortality in Africa: another look at the facts

In a study conducted in a rural population of Uganda (Lancet 1994, 343, 1021-23), about 10,000 individuals were tested for "HIV-antibodies". In those aged 13-44 years, 9.6% were found HIV- seropositive. During follow up, mortality was estimated as 96/1000 man-years (=96 man out of 1000 in 1 year) in the HIV-positive group, and as only 1.4/1000 m.-y. in the HIV-negative group. Thus, mortality in HIV-seropositives was 60 times higher than in their HIV-negative counterparts. At first sight, these results seem to be a reliable proof for causal role of HIV in AIDS, specificity of HIV-tests, and reality of HIV. For this reasons these results are frequently referred to by proponents of the HIV-AIDS theory (see for example [12,13]).

Let us look at the data from another standpoint, and consider an alternative hypothesis - that a positive "HIV-test" is only a non-specific marker of various infectious diseases. Such a "marker- effect" is likely to be caused by the elevated concentrations of non-specific immunoglobulins and increase in their non-specific affinity associated with infectious diseases, especially with multiple infections and intestinal ones (see section 4). Such diseases are known to be the main cause of mortality in this region, at least in relatively young ages. If HIV-seropositivity is actually only a marker of these diseases, most sick individuals in this population have to fall into the HIV- seropositive group. Hence, mortality in HIV-seronegative group has to be much lower than the usual mortality rate in this region. On the other hand, if HIV-tests actually detect a new pathogen (HIV) and HIV causes additional mortality (as the mainstream AIDS science holds), mortality in the HIV-negative group should not decrease in course of the HIV-epidemic. Thus, comparison of mortality in the HIV-negative group with its usual rate, observed before the hypothetical HIV- epidemic, can easily discriminate between the two hypotheses.

What is the usual mortality in this population? Even though any reliable information about mortality in Uganda is absent, it is well known that a great proportion of population there usually die from various diseases in relatively young ages, and it was the case far before the hypothetical HIV-epidemic. Is mortality rate 1.4/1000 in HIV-negative group compatible with such information? If to compute the proportion of deaths in 30-year period (say, since age 13 to 44) from such an annual rate of mortality, it gives only 4 percent of deaths in 30 years. It is not compatible with the above information that a great proportion of Africans die and died young. Thus, mortality in the HIV-negative group (1.4/1000 m-y) is significantly lower than the usual mortality in this region. In other words, HIV-test is capable to select a great proportion of the individuals who were to die even without the hypothetical "HIV-epidemic". It is in perfect agreement with the "HIV-is-only-a-marker" hypotheses.

Proponents of the HIV-AIDS theory [12] insist that the 9.3/1000 m-y mortality in the 13-44 age group at large is significantly higher than in other parts of the country, and this cannot be explained by causes other than HIV. Such argumentation is flawed - simply because the region was selected for this study just because mortality was higher than in other regions of Uganda. There are many other possible causes for the increase in morbidity and mortality from infectious diseases in some areas of an African country. Irrespective to the actual causes of this increase, both hypotheses predict much higher mortality in the HIV-seropositive group than in the HIV- negative one, and this difference cannot discriminate between the two hypotheses. Nevertheless, mainstream AIDS-scientists try to present this difference as a proof of the official HIV-causes- AIDS hypothesis. For some reasons, they also "forget" to pay attention to the mortality rate in the HIV-negative group, which is obviously lower than the usual rate in this region - what is in perfect agreement with the "HIV-is-only-a-marker" hypothesis and contradicts to the "HIV-causes- AIDS" one.

6. Other explanations for correlation between HIV-seropositivity and AIDS

The central goal of this summary is to show that there are no any proofs that a positive test for HIV-antibodies is caused by any specific antibodies, and that non-specific immunoglobulins is highly likely to be the actual cause of HIV-seropositivity. Let us imagine that it will be shown experimentally that HIV-seropositivity is caused by specific antibodies - that is, blood serum from HIV-seropositive individuals reacts (in significantly higher titers than serum from healthy controls) only with the "HIV-proteins", but not with any other antigens. Surely, this would refute the hypothesis described here. Will such refutation be a proof that HIV-seropositivity is actually caused by HIV?

The main problem with the official HIV-causes-AIDS theory is that it has never been shown that "HIV-proteins" are related to a virus. The only thing known for sure is that these proteins are produced by human lymphocytes in certain conditions in vitro (in a test tube). To the same extent, they may be produced by the lymphocytes in vivo (in living organism). The "HIV-proteins" are likely to be of cellular (endogenous) nature [1] - that is, to be encoded in human genome by normally inactive genes ("inactive gene" means that the protein encoded by this gene is not produced by the cell). It is well known that only a small proportion of genes in human genome are active, and different sets of genes are active in different types of cells (even though genome is the same for all cells of a given organism). Even in cells of the same type some genes can be switched on (or off) if conditions change. The mechanisms which switch genes on and off are poorly understood in modern biology. If in some abnormal conditions lymphocytes start to produce the "HIV-proteins" (the corresponding genes are switched on), these proteins will be "foreign" for the immune system and specific antibodies will be produced against these proteins. Naturally, blood serum of such individuals will yield specific reaction to the "HIV-proteins". In other words, specific antibodies against "HIV-proteins" may be only a marker that corresponding genes have switched on due to some metabolic changes. Thus, existence of HIV cannot be proved by immunochemical and serological methods - irrespective to whether HIV-seropositivity is caused by non-specific immunoglobulins or by specific antibodies.

Even if to prove rigorously that HIV exists as an exogenous transmissible agent (this has never been done [1-4]) and that HIV-seropositivity is caused by HIV-infection, it still cannot prove that HIV is the cause of AIDS. Correlation between HIV-infection and AIDS-defining diseases is equally explainable by the "HIV-is-a-marker" hypotheses, which hold that HIV is a benign passenger virus [***] and can easily infect only individuals with some deviations from normal health status, thus, being only a marker of such deviations. In this case, morbidity and mortality may be much higher in HIV-positive individuals than in their HIV-negative counterparts, but not because the HIV-infection. Critical reevaluation of the studies carried out in Africa (see section 5) demonstrates that the results support "HIV-is-only-a-marker" hypotheses, and contradict to HIV- causes-AIDS one irrespective to the actual causes of HIV-seropositivity.

Conclusions

Specificity of antibodies cannot been established in serological studies with one antigen: it is necessary to prove that titers of the antigen-antibody reaction are significantly higher (than in healthy controls) only for this antigen, but not in respect to other antigens. As far as such multi- antigen serological studies have never been carried out, increase in non-specific affinity of immunoglobulins is the most likely cause of HIV-seropositivity. The problem is exacerbated by the use of "sandwich" tests (like ELISA and WB) - because they are abnormally sensitive to any increase in affinity of non-specific immunoglobulins. Besides the non-specific immunoglobulins, there are several other mechanisms which can lead to correlation between HIV-seropositivity and diseases, while not being compatible with the official "HIV-causes-AIDS" hypothesis. The main flaw of the mainstream AIDS-science is that no experimental or epidemiological studies have been designed and carried out to rule out such alternative explanations. There are no any reasons to consider HIV-seropositivity as a reliable marker of a deadly disease. The main danger of HIV- seropositivity is of iatrogenic (caused by medicine) nature: such individuals are at high risk of administering abnormally severe and prolonged "prophylactic" treatment (antibiotics, antiretrovirals). This iatrogenic effect is a direct consequence of the uncritical acceptance of the HIV-causes-AIDS theory.

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