.. al features similar to retroviruses does not constitute sufficient proof that they are retroviruses, that they are infectious particles, even if they are found to band at 1.16 gm/ml.(18) In 1976 Gallo himself pointed out that in human leukemic tissue virus-like particles morphologically and biochemically resembling type-C virus but apparently lacking the ability to replicate, have been frequently observed.(28) Particles with the morphological characteristics of retroviruses were reported in milk, cultures of embryonic tissues and in the majority, if not all, human placentas.(29,30,31) However, they were considered to be an intriguing and important problem that remains to be solved.(32) Evidence from AIDS research shows that: 1. There is no agreement on the precise taxonomic classification of HIV. Initially, HIV was reported as an Oncoviral type-C particle, then a type-D particle,(33) and ultimately as a member of a different Subfamily, a Lentivirus;(34) 2. The T-cell and monocyte HIV infected cultures contain in addition to particles with morphologies attributed to HIV, many other viral particles unlike any of the HIV particles.(35,36,37,38) Non-HIV-infected HT (H9) cells, the cell line from which the Gallo team isolated the first HIV (HTLV-III) and from which most of the published electron micrographs of HIV particles have originated, as well as other cells used for HIV isolation, CEM, C8166, EBV transformed B-cells, and cord blood lymphocytes, express virus-like particles albeit they are somewhat different from the variety of particles accepted as HIV.(39) The above data raises questions not only in regard to the origin and role of the non- HIV particles, but also to the HIV (HTLV-III) particles.
Furthermore, neither Gallo’s team, nor anybody else before or since has published EM micrographs of the material derived from AIDS cultures/co-cultures which bands at 1.16 gm/ml. Thus it is impossible to know which, if any of the particles, band at that density; 3. Most importantly, it is generally accepted that particles reported in the lymph nodes of AIDS patients are HIV. However, in the only EM study(40), either in vivo or in vitro, in which suitable controls were used and in which extensive blind examination of controls and test material was performed, HIV particles were found in 90% (18/20) of patients with persistent generalised lymphadenopathy attributed to HIV, and in 87% (13/15) of patients with non-HIV lymphadenopathies, leading the authors to conclude: The presence of such particles do not, by themselves indicate infection with HIV. Antigen-antibody reactions. One can claim that a given protein is an antigen derived from an exogenous retrovirus if first it is shown that: 1. The protein is a structural component of a particle; 2.
The particle is a retrovirus; 3. The protein is coded exclusively by a viral and not a cellular gene. Once the above are demonstrated, the only way to prove that the antibodies found in AIDS patient sera are directed against the viral antigen is to use the antigen or the isolated virus as a gold standard. The mere finding that a protein from the AIDS cultures bands at 1.16 gm/ml and reacts with sera from AIDS patients cannot be considered to simultaneously prove that: 1. The protein is a viral antigen; 2.
The antibodies in the AIDS patient sera which react with the antigen are specific for that antigen. At present, it is known that about 80% of the proteins which band at 1.16 gm/ml, some of which react with some AIDS sera, do not constitute any of the proteins ascribed to HIV.(41,42,43) Most importantly, prior to the publication of the Science papers, evidence existed, confirmed since, which is at odds with the conclusion that p24 and p41 may therefore be considered viral structural proteins: The p41/45 protein In AIDS research, the p41 and p45 bands are considered to represent one and the same HIV protein. 1. Like Gallo’s group, Montagnier’s team one year earlier, found that AIDS sera reacted with a protein p41/45 from the AIDS cultures and which in sucrose density gradients, banded at 1.16 gm/ml. However, from their data they considered that the p41 band may be due to contamination of the virus by cellular actin which was present in immunoprecipitates of all cell extracts,(5) that is, of HIV infected as well as non-infected cells and cells infected with HTLV-I.
Although Gallo’s group did not find such a reaction with p41 in non-infected cells, they did find a p80 protein and concluded that the reaction was non-specific.(8) However, at present it is known that p80 as well as two additional HIV proteins, p120 and p160, are oligomers of p41.(44) Which protein (band), p41, p80, p120 or p160 is detected in a given WB depends on the culture and WB conditions, including temperature and the concentration of sodium dodecyl sulphate used to disrupt the proteins which band at 1.16 gm/ml;(45) 2. Actin is an ubiquitous protein present in all cells including bacteria and several viruses. Well known retroviruses such as the mouse mammary tumour virus have also been shown to contain actin of cellular origin and it has been postulated that this protein plays a key role in both retroviral assembly and budding;(46,47) 3. Platelets from healthy individuals also contain a p41 protein which reacts with sera from homosexual men with AIDS and immune thrombocytopenic purpura (ITP) and which represents non-specific binding of IgG to actin in the platelet preparation.(48) 4. Researchers at the Pasteur Institute have shown that sera from AIDS patients and AIDS risk groups contain high levels of antibody against calf striated muscle actin.(49) The p24/25 protein 1. Apart from a joint publication with Montagnier where they claim that the HIV p24/25 is unique, Gallo and his colleagues have repeatedly stated that the p24s of HTLV-I and HIV immunologically cross-react;(50) 2. Genesca et al.(51) conducted WB assays in 100 ELISA negative samples of healthy blood donors; 20 were found to have HIV bands which did not fulfil the then (1989) criteria used by the blood banks for a positive WB.
These were considered as indeterminate WB, (WBI), with p24 being the predominant band, (70% of cases). Among the recipients of WBI blood, 36% were WBI 6 months after transfusion, but so were 42% of individuals who received WB-negative samples. Both donors and recipients of blood remained healthy. They concluded that WBI patterns are exceedingly common in randomly selected donors and recipients and such patterns do not correlate with the presence of HIV-1 or the transmission of HIV-1, most such reactions represent false- positive results; 3. Antibodies to p24 have been detected in 1 out of 150 healthy individuals, 13% of randomly selected otherwise healthy patients with generalised warts, 24% of patients with cutaneous T-cell lymphoma and prodrome and 41% of patients with multiple sclerosis;(52) 4. Ninety seven percent of sera from homosexuals with ITP and 94% of sera from homosexuals with lymphandenopathy or AIDS contain an antibody that reacts with a 25Kd membrane antigen found in platelets from healthy donors and AIDS patients, as well as a 25 Kd antigen found in green-monkey kidney cells, human skin fibroblasts, and herpes simplex cultured in monkey kidney cells.
This reaction was absent in sera obtained from non-homosexual patients with ITP or non-immune thrombocytopenic purpura;(48) 5. Conversely, the p24 antigen is not found in all HIV positive or even AIDS patients. In one study, the polymerase chain reaction (PCR) and p24 were used to detect HIV in patients at various CDC stages from asymptomatic to AIDS. p24 was detected in 24% patients and HIV RNA in 50%;(53) 6. In another study, In half of the cases in which a subject had a positive p24 test, the subject later had a negative test without taking any medications that would be expected to affect p24 antigen levels..the test is clinically erratic and should be interpreted very cautiously.(54) Thus the finding of viral particles in the AIDS cultures/co- cultures, RT and proteins which react with AIDS related sera in the material from the supernatant or cell lysates which in sucrose density gradients bands at 1.16 gm/ml, cannot be considered synonymous with the isolation or even the detection of a retrovirus. Even if a retrovirus is isolated from in vitro cultures/co-cultures from tissues from AIDS patients, this does not, by itself, constitute proof of the existence of the virus in vivo, (in AIDS patients), and even less that the retrovirus has been exogenously acquired.
This is because: 1. At present, it is generally accepted that one of the most striking features that distinguish retroviruses from all other animal retroviruses is the presence, in the chromosomes of normal uninfected cells, of genomes [proviruses] closely related to, or identical with those of infectious viruses. The human genome, in addition to other proviral sequences, is known to contain both HTLV-I (55,56) and HIV (57) sequences. Depending on conditions, the proviral genome remains unexpressed or part or all of it may be expressed. The latter may or may not lead to the assembly of viral particles (endogenous retrovirus).(17) In animal cultures, healthy non-virus producing cells sooner or later spontaneously release retroviruses.(20) The appearance and yield can be increased by (i) mitogenic stimulation;(58) (ii) co-cultivation techniques;(59) (iii) cultivation of cells with supernatant from non-virus producing cultures.(60) According to one eminent retrovirologist, George Todaro, the failure to isolate endogenous viruses from certain species may reflect thelimitation of in vitro cocultivation techniques;(61) 2. Gallo’s team, like everybody else: (i) isolated HTLV-III (HIV) from cell cultures; (ii) isolated HTLV-III from mitogenically stimulated, activated cell cultures; 3. In addition, Gallo and his colleagues also used co-cultivation techniques; 4.
The first HTLV-III isolation was from the HT (H4, H9, H17) cell line. Reading Gallo and his colleagues’ first paper, one surmises that the HT cell line was established in Gallo’s laboratory. The Gallo inquiry revealed that the HT cell line is in fact HUT78, a cell line established in another laboratory from a patient with mature T4-cell leukaemia, a disease which Gallo claims is caused by the exogenous retrovirus, HTLV-I.(3) If so, then all HT cell cultures, and the clones derived from it, infected with HTLV-III or non-infected, and the material from these cultures which bands at 1.16 gm/ml, should contain HTLV-I, and thus RT and retroviral particles. Furthermore, because about 25% of AIDS patients have antibodies to HTVL-I,(1) and the immunogenic proteins of HTLV-I and HIV have the same molecular weights, then approximately 25% of the non-infected HT (H4, H9, H17) cultures in addition to RT and particles, should have, in the Western blot, the same bands as those of the HTLV-III infected cultures. Thus, these WBs will erroneously appear positive for HTLV-III. Proof that HTLV-III (HIV) is causally linked to AIDS. Gallo claims, a claim accepted by the vast majority of AIDS researchers, that in the May 1984 Science papers he and his colleagues presented unambiguous evidence that this [virus] and this alone was the cause of AIDS.(62) A minimum requirement for making such a claim should be presentation of the following evidence: 1.
That all AIDS patients are infected with HTLV-III; 2. Infection with HTLV-III leads to T4-cell depletion, given the assumption that HTLV-III leads to the clinical syndrome by its T4 cytotoxicity. The evidence for the existence of HTLV-III was viral isolation and ELISA antibody tests. Even if one assumes that the data presented represents true isolation, the virus was isolated from less that half (10/21) of AIDS patients with opportunistic infections, and in less than one third (13/43) with Kaposi’s sarcoma, then and now the two most characteristic AIDS diseases. Even if the virus could have been isolated from all patients, given the nature of retroviruses and the method used for HTLV-III isolation (cultures, mitogenic stimulation, co- cultivation) the possibility cannot be excluded that the virus did not exist in vivo (in AIDS patients), and that it was a provirus whose expression was facilitated by the culture conditions. The only method used to prove HIV infection in vivo was the antibody tests.
Such a test can only be used only after its specificity has been proven by use of the only possible gold standard, the virus itself. This has not been done. Furthermore, the antibody test used by Gallo was ELISA, at present known to be non-reproducible and non-specific. In a study of 1.2 million healthy military applicants conducted by Colonel Donald Burke and his colleagues,(63) it was found that although approximately 1% of all individuals had an initial positive HIV ELISA, only 50% of repeat ELISAs were positive. Of the latter, only approximately one third were associated with two subsequent positive WBs.
In Russia, in 1990, out of 20,000 positive ELISAs only 112 were confirmed using the WB as a gold standard. In 1991, of approximately 30,000 positive ELISAs, only 66 were confirmed.(64) Nowhere in the four Science papers was HTVL-III cytotoxicity mentioned. The only reference to any cellular abnormalities or pathology in general is in the first paper where one reads: The virus positive cultures consistently showed a high proportion of round giant cells containing numerous nuclei (Fig. 1a). These cells resemble those induced by HTLV-I and -II except that the nuclei exhibit a characteristic ring formation. (Fig.
1a is a light microscopic examination of clone H4/HTLV-III). The H4 clone was obtained from the HT cell line using irradiated mononuclear cells from peripheral blood of a healthy blood donor as a feeder. At present, it is known that the HT cell line and thus H4 are HUT78, derived in 1980 from a patient with mature T4-cell leukaemia,(65,66) However, other cell lines derived from patients with the same clinical syndrome are known to exhibit similar morphologies including multinucleated giant cells.(67) Thus the cellular morphological characteristics observed in the first paper may have been an intrinsic property of the HT cell line, or the result of the culture conditions, or both, and not due to HTLV-III. Finally, Gallo and his colleagues did not provide any data on the immunological status of those individuals from whom viral isolation was attempted, and no data was presented proving that: 1. HTLV-III (HIV) is both a necessary and sufficient cause of T4- cell depletion; 2.
T4-cell depletion is both necessary and sufficient for the appearance of the AIDS indicator diseases. Conclusions The data and arguments that have been presented by Gallo and his colleagues do not constitute proof of HIV isolation or an unambiguous role for HIV in the pathogenesis of AIDS. Although some researchers currently use methods of viral isolation essentially the same as that described by Gallo’s group, most use less rigorous methods including singleton detection of p24 (by antibody techniques), or RT. Notwithstanding, with all of these techniques, including that described by Gallo and his colleagues, which itself seen to be greatly problematic, HIV cannot be isolated from 20%-70% of HIV positive and AIDS patients(68,69) Thus we are faced with a problem of considerable importance. The HIV antibody tests, both ELISA and WB, the only routinely used tests proving the existence in vivo of HIV, have yet to be verified against the only suitable gold standard, viral isolation. The available evidence suggests that this long overdue but most basic requirement of test evaluation is likely to prove an immense problem, and while the HIV antibody tests are useful prognostic markers in the high risk groups, their use as diagnostic and epidemiological tools for HIV infection is questionable. References 1. Essex M, McLane MF, Lee TH, et al.
Antibodies to Cell Membrane Antigens Associated with Human T-Cell Leukemia Virus in Patients with AIDS. Science 1985;220:859-862. 2. Gallo RC, Sarin PS, Gelmann EP, et al. Isolation of Human T- Cell Leukemia Virus in Acquired Immune Deficiency Syndrome (AIDS). Science 1983;220:865-867.
3. Gallo RC. The First Human Retrovirus. Sci Am 1986; 255:78-88. 4. Marx JL.
Human T-Cell Leukemia Linked to AIDS. Science 1983;220:806-809. 5. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-Lymphotrophic Retrovirus from a patient at Risk for Acquired Immune Deficiency Syndrome (AIDS). Science 1983;220:868-871. 6. Popovic M, Sarngadharan MG, Read E, et al. Detection, Isolation,and Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS.
Science 1984;224:497-500. 7.Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and at Risk for AIDS. Science 1984;224:500-502. 8. Schupbach J, Popovic M, Gilden RV, et al. Serological analysis of a Subgroup of Human T-Lymphotrophic Retroviruses (HTLV-III) Associated with AIDS. Science 1984;224:503-505. 9. Sarngadharan MG, Popovic M,Bruch L, et al. Antibodies Reactive to Human T-Lymphotrophic Retroviruses (HTLV-III) in the Serum of Patients with AIDS.
Science 1984:224:506-508. 10. Culliton BJ. Gallo Inquiry Takes Puzzling New Turn. Science 1990:250:202-203.
11. Culliton BJ. Inside the Gallo Probe. Science 1990;248:1494-1498. 12. Hamilton DP.
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Gallo RC, Sarin PS, Kramarsky B. et al. First isolation of HTLV-III. Nature 1986;321:119. Bibliography The evidence that Robert Gallo and his colleagues presented on 4th May 1984 regarding HTLV-III (HIV) isolation and the role of HIV in the pathogenesis of AIDS is critically analysed.
It is concluded that the evidence does not constitute proof of the isolation of a retrovirus, that the virus is exogenous or that the virus is causally related to AIDS. Medicine Essays.