HIV & AIDS - Chapter 9 - Proposed research projects and studies

VIRUSMYTH HOMEPAGE

PRESIDENTIAL AIDS ADVISORY PANEL REPORT
PROPOSED RESEARCH PROJECTS AND STUDIES
Chapter 9

9.1 General recommendations on research

To undertake a series of immediately doable laboratory, epidemiological and mortality studies on the South African AIDS epidemic to gain better insight into the link between HIV infection and the development of AIDS.

Undertake further studies in South Africa on the virus, to look at the natural history; the rate of disease progression in HIV infected people; the effect of the co-factors in viral loads.

Design questions in research that would seek to understand behaviour within the cultural and the ethnic context.

Research into general anti-AIDS drugs that attack both HIV and the cofactors.

The following research projects and studies were proposed by indicated panelists during the period following the panel meetings.

The proposals that follow (proposals 1,2 and 3) have been put forward by the group that was set up by the Presidential AIDS Advisory panel during their meeting in South Africa in May 2000. The members of the group are Drs H Bialy, P Duesberg, H Gayle and MW Makgoba.

9.2 Proposal 1: Quality assessment of HIV testing: Establishing a Baseline and validating HIV ELISA testing in South Africa.

9.2.1 Rationale

The basic idea in the validation of HIV ELISA Testing in South Africa is to proceed in stages, graded in order of simplicity, and designed so that the results of each stage will determine what, if any, form the next stage will take. This study is based on the fact that:

A virus named Human Immunodefeciency Virus (HIV) has been isolated

The validity and quality assessment of HIV testing is critical for accurate estimates, diagnosis, monitoring and surveillance (mostly for epidemiological data) of the HIV/AIDS epidemic

9.2.2 Establishing a Baseline: Quality Assessment of HIV Testing of five independent sites in South Africa

A random and blinded Quality Assessment study of 2500 samples from different sites will be undertaken. The sites are:

National Institute of Virology

Blood Transfusion Service

South African Institute of Medical research

Department of Virology, University of Stellenbosch

These sites cover the spectrum of high risk and low risk groups as well as high prevalence and low prevalence samples.

The samples will be subjected to time-tested protocols for quality assurance in South Africa and at the Centers for Disease Control in the USA. The correlation between the results in South Africa and the Centers for Disease Control will be analysed to provide the following:

Assist in establishing such baseline parameters as the level of false positives, sensitivity and specificity

Confidence in the validity and accuracy of HIV ELISA Testing done in South Africa

These data should also form the basis of subsequent studies as proposed below in the Preadsorption and Virus isolation experiments.

Time table for the study:

Randomised and coded sample collection was completed in December 2000

Testing is already in progress and results will be obtained shortly

Costs: The MRC and the CDC will cover the costs of shipping and testing, respectively which will add up to US$75 000.00.

9.3 Proposal 2: Determination of the robustness of the current HIV ELISA tests that are being used in South Africa.

9.3.1 Purpose of experiment

To determine the robustness of the current HIV ELISA tests that are being used in South Africa when the sera that is being tested has been treated to remove antibodies that are reactive to a series of known antigens that have been previously reported to interfere under certain conditions with HIV ELISA tests that depend on either recombinant proteins, or recombinant proteins and synthetic peptides such as V3.

9.3.2 Methodology

Blood samples from 100 TB patients that have had no prior HIV serology will be obtained by Professor Mhlongo. An additional 100 blood samples from "HIV/AIDS" patients from the most densely affected region in the country will be obtained by Dr Makgoba.

These blood samples will be taken to the major laboratory in South Africa that does HIV ELISA testing, where Drs Makgoba and Roberto Stock (an investigator from the Institute of Biotechnology in Mexico, an expert on immunodiagnostics of all varieties and protein purification and biochemistry), along with South African colleagues of the panel's choosing (scientists, students, technicians) will have prepared a series of ELISA plates that have been coated to contain:

Antigenic preparations form the most common strains of Mycobacteria in South Africa. If such preparations (we require about 500 micrograms of each) are not available, a detailed protocol is available for their preparation.

A BCG preparation (commercially available).

HTLV-I and HTLV-II antigents (also commercially available).

Antigenic preparations from common parasitic infective agents in the parts of SA that are heavily HIV/AIDS infected and from which your samples are drawn.

Antigenic preparations from the most common (non-TB) bacterial and viral infections in these populations. (Sufficient quantities of d and e are available either through WHO/TDR or commercially.)

Sera from each of the 200 samples will be added to the ELISA wells of these plates and incubated for two hours, after which the contents of the wells will be transferred to HIV ELISA plates and treated as is normally done. Everything will be done in duplicate.

9.4 Proposal 3: Molecular beacons

South African HIV researchers need to be assisted to gain even greater awareness of the power and usefulness of the beacon technology as a general diagnostic tool, but particularly with reference to Multiple Drug Resistant Tuberculosis. It is not being proposed that the beacon assay be used as any form of gold standard.

The first proposed step is to set up and calibrate the ABI Prism Machine as well as teach South African researchers how to operate the machine and how to synthesise, purify and use molecular beacons. Once this is done, decisions will then be taken as to what degree and how the use of the beacon technology on the samples collected for the Quality Assessment of HIV Testing (as in Proposal 1 above) would be productive.

The proposals that follow were suggested by members of the Presidential AIDS Advisory Panel either during the panel meetings in May and July 2000 or during the Internet debate between the two meetings.

9.5 Proposal 4: Do most people with HIV infection show signs of AIDS within five (5) to ten (10) years?

Proposer: Prof Peter Duesberg

One Thousand and Five Hundred (1500) healthy HIV-positive and 1500 matched healthy HIV-negative men from the South African army and/or mining industry, or some other governmental institution would be required for this study. This experiment will exclude people who suffer poverty, malnutrition, poor sanitation. Since the time of infection of these men is not known, and since they are currently healthy, their times to AIDS would be randomly distributed from a maximum of 5-10 years to a minimum of one day to AIDS. On average they are half way into their HIV to AIDS latent period of 5-10 years, or 750-1500 days (1/2 of 5-10 years) from getting AIDS. Therefore in the HIV positive group there should be 1 or 2 AIDS cases per day, and in the negative group there should be no AIDS cases. We would know much of the answer in a few months and certainly within a year if we had 1500 men in each group. It would take longer if the groups are smaller. The cost would be one conventional HIV test per person, and perhaps a second one if a AIDS disease co occur and a phone call per person or to their supervisor every 2 months to find out how they are.

9.6 Proposal 5: Preadsorption and Virus Isolation Experiments - The need for a Gold Standard in the diagnosis of HIV infection.

The proposal on the preadsorption studies has strong similarities with proposal 2 above, but is included in this document as it appears as a package with the proposed experiments on virus isolation.

Proposers: Dr Eleni Papadopoulos-Eleopoulos and Dr Val Turner

9.6.1 Importance of the Proposed HIV Experiments

At present, all the HIV experts admit that:

Agents other than HIV can cause decrease in T4-cells (Acquired Immune Deficiency, AID).

The diseases, which are said to indicate AIDS (the syndrome, that is, the "S" in AIDS, can manifest in the absence of HIV infection.

Since the late 1970s, all the HIV experts claim that:

The main cause of AID is a new agent, HIV.

AID leads to the appearance of S, the syndrome.

Any patient who has AIDS and is infected with HIV, the cause of AIDS in that patient is defined to be HIV.

This means that once the existence of HIV is accepted, it is not possible to refute the HIV theory of AIDS by claiming that:

HIV does not fulfil the Koch postulates.

HIV does not fulfil the Farr law.

AIDS is caused by recreational drugs and sex plays no role in the causation of AIDS.

AIDS is caused by antiretrovirals.

HIV antibodies neutralise HIV.

Each of the above arguments against the HIV theory can be easily refuted :

Regarding the Koch postulates, if the existence of HIV is accepted and if the antibody tests are considered to prove HIV infection, then the Koch postulates have been fulfilled.

There are many examples (TB, malaria, hepatitis B) showing that the Farr law which stimulates that the epidemics of infectious diseases have a "bell-shaped" epidemiological curve does not apply.

Basic scientific and epidemiological evidence shows that sex, namely high frequencies of passive anal intercourse, plays a role in the acquisition of both a positive antibody test and AIDS.

Since AIDS was diagnosed five years before antiretrovirals started to be used, the antiretrovirals cannot be considered as an argument against the HIV hypothesis.

Since 1935 evidence existed which shows that antibodies to infectious agents do not neutralize them. At present, immunologists are trying to find a mechanism that excludes neutralization to explain the effect of vaccines.

The only way to prove or disprove the HIV theory is by experiments. Several experiments have been proposed including the following:

Test a number of African patients who clinically have AIDS for HIV antibodies and/or perform PCR tests.

Identify young American military recruits who have been found to be HIV positive 10 years ago and determine how many have progressed to AIDS.

Study the relationship between retroviral particles in the plasma as determined by electron microscopy (pictures) and the viral load test.

Although these experiments are useful, they never can prove or disprove the HIV theory of AIDS. Regarding the first experiment listed above, finding for example that only 50% of African patients who satisfy the clinical definition of AIDS in Africa have a positive antibody test will show that the African clinical syndrome has a poor positive predicting value for HIV infection and will reduce the number of AIDS cases by 50% but will not disprove the HIV theory. With respect to the second experiment listed above, even if a very small proportion, for example 10%, of military recruits had developed AIDS in ten years, it does not prove that HIV is not the cause of AIDS. There are many infectious diseases in which only a small proportion of infected patients end up with the clinical manifestation. Finally, the third experiment listed above, even if it shows that no correlation exists at all between retroviral-like particles observed in the plasma, it does not prove that HIV is not the cause of AIDS or even that the patients are not infected with HIV.

From the very beginning when the HIV theory was introduced, we wanted to perform pre-adsorption experiments. These experiments like the ones outlined above even if they show that all the antibodies present in AIDS patient's sera can be adsorbed by antigens other than HIV are not going to disprove the HIV theory or that the patients are infected with HIV. However, they will show that it is not possible to claim that a positive antibody test proves HIV infection unless HIV isolation (purification) is used as a gold standard to prove the specificity of this test.

The reasons why we have been proposing, again from the beginning of the HIV era, for the HIV isolation (purification) experiments are:

The necessity of HIV isolation as a gold standard.

The absolute necessity of isolation to prove the existence of a unique retrovirus, HIV.

The lack of such proof.

Claims for HIV isolation (purification) have been made by Montagnier's group in 1983 and by Gallo's group in 1984. However, in 1997 Montagnier acknowledged that his group did not obtain proof for isolation (purification) and in his view neither did Gallo's group. In the same year, some of the best known retrovirologists noted that no one had presented proof for isolation (purification) of a unique retrovirus, HIV.

The isolation experiments, as proposed by us, will prove once for all if HIV has been isolated (purified) and thus if there is such a thing as a human retrovirus, HIV. There are several indications that this has not been achieved so far:

According to Montagnier, what he called "purified HIV" did not even have particles with the morphology typical of retroviruses.

In 1997, for the first time, two groups of researchers published pictures showing the results of their efforts to obtain HIV isolation (purification). Both groups accept that their pictures show that most of the material that was supposed to be "purified HIV" in fact are non-retroviral-like particles (cellular microvesicles, "mock virus"). These particles are also present in the pictures of material obtained in the same way as the "purified HIV" from cell cultures which were said not to be infected. Nonetheless, these researchers did claim that although they could not obtain "purified HIV" particles, the material obtained from "infected" cultures did contain some particles which were "HIV". However as we have repeatedly pointed out elsewhere including at the Johannesburg meeting of the Presidential AIDS Advisory Panel :

The particles did not have even the most basic characteristic of retroviruses, the dimensions. A fact which has not been denied by the principal author of one of the publications.

If some of the particles in the "purified HIV" material were indeed "HIV" then this material will have at least some proteins which were not present in the "mock virus" which originated from the non-infected cultures.

9.6.2 Principles of the Proposed Experiments

9.6.2.1 Pre-adsorption experiment

Serum is taken from patients who have a positive "HIV" ELISA and divided into two parts.

One part is used to repeat the "HIV" ELISA and the intensity of the reaction (optical density, OD) is noted.

The other part is incubated for at least one hour with non-"HIV" proteins. These non-"HIV" proteins can originate from lymphocytes, semen (sperm), E.coli, M.tuberculosis, or other infectious agents which are relevant to a diagnosis of AIDS.

Following incubation with the non-"HIV" proteins, the serum is tested with the "HIV" ELISA and the OD noted.

If the OD in 4 is lower than the OD in 2, it will mean that the antibodies present in the patient's original serum react both with "HIV" proteins and non-"HIV" proteins. There are two reasons for this, either:

The antibodies are "HIV" antibodies but they cross-react with non-"HIV" proteins; or

The antibodies are non-"HIV" antibodies.

From this, it follows that the antibody test cannot be used to prove "HIV" infection unless "HIV" isolation (purification) is used as a gold standard to prove (a) is the reason and not (b).

9.6.2.2 "HIV" Isolation (purification)

One of the physical characteristics of retroviruses is their density. In sucrose density gradients, they band at the density of 1.16gm/ml. So for this experiment:

Bands of sucrose of different densities are layered in a centrifuge tube, the lighter at the top and the heavier at the bottom.

Establish cell cultures by taking cells from patients who are said to be "HIV" infected (test cultures) and patients who are said not to be "HIV" infected (control cultures).

Take the supernatant, that is, the culture fluid, (specimen) and place it at the top of the centrifuge tube.

Spin the centrifuge tube for many hours at very high speed.

If the specimen contains retroviral particles, they will aggregate (band) at 1.16gm/ml.

Extract the 1.16gm/ml band and examine it with an electron microscope (EM).

Compare the EM pictures obtained from the test and control cultures. Particles with the morphology of retroviruses should be present only in the EMs from the test cultures. To claim that the 1.16gm/ml band is pure then the EM should show no other material but retroviral-like particles.

If there is no difference in the EMs of the test and control cultures no matter what is seen, then it is not possible to claim that the test cultures are infected with a retrovirus.

Extract the proteins from the 1.16 gm/ml band obtained from both test and control cultures and compare them. If no difference exists then there is no proof that the test cultures contain "HIV" regardless what the EMs show.

Extract the nucleic acids from the 1.16gm/ml band obtained from both test and control cultures and compare them. If no difference exists then there is no proof that the test cultures contain "HIV" regardless what the EMs show.

9.7 Proposal 6: Questionable African AIDS /HIV Statistics - Epidemiology

Proposers: Prof. Gordon Stewart, Prof. Sam Mhlongo, Dr. Christian Fiala, Prof. Charles Geshekter and Dr. Roberto Giraldo

The proposers will need to spend some 2 - 3 days in Geneva re: UNAIDS data.

9.8 Proposal 7: Proposed investigation of the diagnosis of HIV/AIDS

Proposers: Prof. Gordon Stewart, Dr. Roberto Giraldo, Dr. Harey Bialy and Prof. Sam Mhlongo

Principal Proposer: Prof Gordon T. Stewart

9.8.1 Current procedure

The investigations should be arranged in consultation with Professor Schoub or Dr Gray, and performed in the laboratory or laboratories responsible for routine serological tests for HIV by the ELISA method or Western Blot or both.

For clinical purposes and for surveillance, a diagnosis of AIDS (AIDS/HIV, HIV Disease) is made by the demonstrations of antibodies to antigens of HIV obtained from original LAV-BRU, HTLV3 or similar complex cellular co-cultures. This is usually done by the ELISA method of immunofluorescence with or without "Confirmation" by a chromatographic test for identification of the same reacting antibodies by Western blot.

There is a consensus among expert advisers and health authorities internationally supporting this procedure and indeed denying, as in the Durban Declaration, that there is any reason to doubt the reliability of diagnoses, surveillance or clinical decisions made on this basis. But, irrespectively of the state of health of an individual or community, a positive result by either method supports and in many cases mandates a diagnosis of AIDS. The latest revision of the ICD assumes that all seropositive persons are at risk of AIDS and that the majority will proceed to develop signs, sooner or later. Although false-positive, false-negative, cross-reactive and indeterminate results frequently occur for various reasons or for no obvious reasons, it is further assumed that a "True" positive result can be identifies as a reliable indicator of infection with live HIV and therefore of active disease which will progress to AIDS or AIDS-related conditions (ARC’s). This belief prevails despite the fact that direct isolation of HIV as proof of infection is difficult or impracticable, and that other surrogate tests such as the PCR/RNA and lymphocyte counts are not appropriate for routine diagnosis.

Presymptomatic screening of all pregnant females is deemed to be necessary for prevention or treatment of HIV disease in them and in their infants. However, it is known that many results are indeterminate, that false positive and negative results can occur and that many other disorders and physiological changes can give indeterminate or non-specific results. In situations where the incidence of "True" seropositivity is low, the likelihood of false or indeterminate results may be higher. This leads to uncertainties, anxieties, and unnecessary interventions like cessation of breast-feeding or termination of pregnancy. Errors in either direction are frequent and can cause catastrophes in relationships, families and communities, especially in countries where stigmatisation, social exclusion and expulsions occur when positive results are known or suspected

Reliability of sero-diagnosis is therefore the critical element in the identification and management of all forms of HIV/AIDS, and for assessment and prevention of vertical as well as horizontal transmission. To improve quality control in diagnosis and surveillance, it is suggested that the following method and precautions be adopted This will measure the overlap between HIV/AIDS and other prevalent disorders, give ongoing estimates of sensitivity and specificity of serological results, and provide a data-base for checking projections. Since the object of the exercise, is to check and improve the reliability of diagnosis and prognosis by these tests, which often precede clinical diagnosis or development of disease, especially in pregnant women and infants, the test is regarded as the independent and the outcome as the dependent variable.

9.8.2 Investigation of reliability of serological tests for HIV

Under present procedure, a person who is seropositive to HIV (i.e. whose blood contains antibodies to HIV) is diagnosed as having AIDS or a related condition (ARC, or AIDS-defining Disease (ADD)), or being at risk of it. But it is known that many conditions unrelated to HIV/AIDS can also give positive or indeterminate results for shorter or longer periods. These conditions include tuberculosis and malaria, recent vaccinations, certain tumours, pregnancy and other altered states of health which are commonplace in populations where AIDS is prevalent, especially in Africa. For accurate diagnosis and to enable appropriate advice to be given to patients, contacts and families, it is important to recognise this overlap. For doctors and health authorities, it is essential to know the full implications and extent.

Since seropositivity in itself mandates a diagnosis of AIDS, the overlap has to be ascertained by recording details of any other conditions present at the time in samples of blood sent to designated laboratories from clinics, hospital wards and surveys. Results, whether positive, negative or indeterminate, are then tabulated for comparison with the information held by the senders, each set of data being "Blinded". This produces data sets in which the frequencies of these three grades of results are shown in relation to clinical diagnosis of presumed AIDS, at risk of AIDS from behaviour or contact, AIDS-defining diseases (ADD’s), AIDS or ADDS plus other named diseases or conditions, and other diseases or conditions without AIDS.

With thousands of samples of blood being routinely tested as at present, this procedure will yield data sets from which the frequency of true positive (AIDS only) results can be measured against those in the other categories. If, for example, 10 out of a hundred samples are true positive but 10 positive results are obtained also from those with other diseases without unequivocal clinical signs of AIDS, a person with a positive result in that sample is as likely to have some other condition, and so on according to the alternates indicated in the text of the full proposal.

Because direct identification of HIV itself is not required and is indeed impracticable at present for routine diagnosis, indirect serological tests are the measures used for decisions about all aspects of HIV/AIDS, and especially for assessing and controlling vertical, perinatal and puerperal transmission. Failure to detect false positive and false negative reactions leads to errors not only in diagnosis, treatment and other interventions, but also to erroneous projections and fear – or alternatively irresponsible disregard – of dangers to persons, families and entire communities. These dangers apply to underestimates no less than to overestimates of AIDS and also to risks of overlooking other diseases submerged in the over-riding classification of HIV/AIDS. The present proposal, which should be discussed and implemented co-operatively with existing clinical and laboratory services, is designed to minimise these dangers.

9.8.3 Extensions

This investigation could be extended to Sentinel surveillance and all cases of AIDS (with controls) admitted to hospitals. Samples giving positive and indeterminate results should be, as often as is practicable, subjected to tests for antibodies to other agents, for example: CMV, HSV, VZ, EBV, and to tests for auto-immune and non-specific antibodies, in such conditions as pregnancy, disseminated lupus erythematosis and other auto-immune disorders to see if patterns of cross reactions can be identified. These data might then be used for more critical analysis of the hypothesis that HIV is the essential cause of AIDS.

9.8.4 Interpretation of findings

Measure sensitivity as % of cases detected in one or two tests, and also consistency and reproducibility between the tests.

Assess sero-positivity in relation to general state of health.

Relate B to ADD's.

D. and E. will identify conditions associated with non-specific positive results.

F. will indicate the weighting that might be attached to a given result.

This proposal is submitted in outline so that it can be circulated for comment and revision.. It is accepted that further detail will be required for implementation which should be arranged if possible with the National Institute of Virology, with those responsible for testing and surveillance, and for compilation of registration data in South Africa.

Although Sentinel Surveillance as organised by the WHO requires serodiagnosis by Elisa, using recombinant antigens prepared from co-cultures of HIV, it should be noted that the Bangui definition of AIDS agreed by the WHO and member States in 1987, is regarded as sufficient to warrant a diagnosis of AIDS or AIDS-related conditions without any serological test. National and international data do not normally indicate the proportion or location of diagnoses or projections made on this basis but it is obviously important to include in the programme described above some provision for identifying this proportion.

9.9 Proposal 8: Study to find out the real meaning of HIV Tests

Principal Proposers: Dr Harvey Bialy and Dr Roberto Giraldo

To take blood from four groups of people and run the tests highly diluted, undiluted and at a wide spectrum of dilutions in between.

The first group would be a group of healthy people of many different age groups.

The second group would be a group of people from the AIDS risk groups.

The third group would be a group of people with clinical conditions unrelated to AIDS; and

The fourth group would be a group of patients with full manifestations of AIDS.

All groups would be subjected to both ELISA and Western blot tests. Additionally, all plasma samples will be subjected to the viral load test for HIV.

The result of such experiment could determine whether these tests measurements bear any relationship to an individual's level of exposure to stressor or oxidizing agents. If so, the tests could be salvaged as a measure of individual's level of intoxication.

Bases and references for these experiments can be seen in my postings "Tests for HIV are highly inaccurate" and "Everybody is HIV-positive".

9.10 Proposal 9: To test the reliability of one of the main laboratory methods currently used to quantify HIV in the blood of seropositive individuals - using the Electron Microscope.

Proposer: Prof. Etienne de Harven:

9.10.1 Aim of the experiments.

To test the reliability of one of the main laboratory method currently used to quantify so-called HIV in the blood of sero-positive individuals. More specifically, to use electron microscopy (EM) to verify that the blood plasma of patients identified as having a high "viral load" by PCR does indeed contain retroviral particles, and that, therefore, such samples could be used to isolate and purify HIV, free from cell debris and adventitious material from co-cultures.

9.10.2 Materials and Methods

Using the Roche Diagnostics Corporation "Amplicor HIV-1" monitor test:

Readily access to 5 patients with very high PCR counts. (Group A)

Readily access to 5 patients with undetectable PCR counts. (Group B)

Low speed centrifugation to separate and discard erythrocytes, leukocytes and platelets. Plasma samples (10 ml) diluted 1/1 with cold heparinized Ringer solution. Filtration by aspiration through a Millipore 0.6u membrane. Collecting filtrate #1, and filtering it this time using a Millipore 0.2u membrane. Collecting filtrate #2 and placing it in appropriate Beckman tubes for ultracentrifugation in either a fixed angle or a swinging bucket rotor, using the refrigerated ultracentrifuge to spin the sample under 30.000g for 2 hours. Inspect the tubes for the likely presence of extremely small pellets. Avoiding any risk of resuspending the pellets, cover them with 1.5 % glutaraldehyde in 0.1M cacodylate buffer (pH7) overnight at 0-4°C, rinsed with buffer and post-fixed with 1% osmium tetroxide for 90 min. After rinsing, the pellets will be kept for several hours in 0.5% uranyl acetate at 0-4°C, dehydrated in ethanol and propylene oxide and embedded in Epon. Thin sectioning with diamond knives, staining with uranyl acetate and lead citrate will be followed by examination under the transmission electron microscope at initial magnification ranging between 10.000 and 40.000x.

If the current interpretation of PCR "viral load" result is correct, EM should show plenty of retroviruses in Group A, none in Group B. Otherwise, the existence of a viremia in "high viral load" patients will have to be fundamentally re-appraised.

Samples from the same patients will be used to perform a classical PCR test by the Roche Amplicor HIV-1 routine method, following rigorously the test kit manufacturer’s recommendations.

The pellets obtained in the microfuge, with the 5 samples from Group A, will be analyzed by EM using a methodology comparable to the one described above. This could, eventually, confirm the presence of retroviral particles and will certainly permit to evaluate the presence and the amounts of cell debris.

The absence of HIV particles from the discarded supernatant should be verified, simply by submitting it to high speed centrifugation at 30.000xg for 2 hours and subsequent analysis of the resulting pellet, as described above.

Control samples should be prepared by the double Millipore filtration method that is known to eliminate most cell debris and to concentrate retroviruses in an almost pure form. The final viral pellet will be compared to a "microfuge" pellet from the same patients, both pellets being then processed identically for RNA amplification.

9.10.3 Technical assistance needed:

Technical assistants, trained in routine virology, knowledgeable in ultrafiltration and ultracentrifugation methods, and fully trained in routine Roche Amplicor PCR method.

Technical assistant knowing routine transmission EM procedures, with special proficiency in chemical fixation, plastic embedding, and ultrathin sectioning.

Major equipment needed:

Laminar flow hood, Millipore filtration equipment, refrigerated ultracentrifuge, PCR test kit, ultramicrotome fitted with diamond knives, high resolution transmission electron microscope.

Estimated cost:

If trained technical assistance and all the major equipment are available, the cost of running these experiments can be anticipated to be approximately 500 US dollars for each patients. Total cost for 10 patients would run around 5,000 US dollars. If the results of the proposed experiment are clear-cut and unambiguous, the n umber of 10 patients should suffice to make the point. If they are not, another group of 10 patients should be considered for an extension of the study.

Time required:

If the appropriate patients can be reached without delay, the total time frame of these experiments should probably not exceed a matter of 4 to 8 weeks. My presence would be essential in setting up the sampling procedures and to perform all the EM examinations.

At this time, priority should be given to the identification of ZA labs were this type of work could efficiently be performed, efficiency depending on 3 main factors: 1) a friendly welcome, 2) satisfactorily trained and available assistants, and 3) appropriate equipment. If this would appear difficult, we should then consider doing part of the work either in Europe or in New York.

Finally, it would be of considerable importance for me to be informed of the experimental proposals presented by Dr. Peter Duesberg and by the Perth group, in order to coordinate the entire project in a coherent fashion.

9.11 Proposal 10: To determine which is more harmful - HIV or Anti HIV drugs?

Proposers: Dr. David Rasnick and Dr. Claus Köehnlein

Brief Description:

Chimpanzees are known to be susceptible to HIV infection (i.e., develop antibodies to HIV), however, none to date has come down with AIDS. We propose to take 60 chimpanzees divided into three groups of 20 each as follows:

Group A is the HIV negative controls.

Group B is infected with HIV but otherwise treated exactly as Group A.

Group C is treated with HIV and is put on a life-time course of the three drug anti-HIV cocktail known as HAART.

There are two outcomes of the study:

which animals come down with AIDS-defining and other diseases?

which live longer?

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