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Effective Vaccines against and Immunotherapy of the HIV:
A preliminary Report.
V. Anomah NGU, Faculty of Medicine and Biomedical Sciences, University of Yaounde I, CameroonDr. Fergus Ambe. Clinique de L'Espoir, Essos Nord, Yaounde, Cameroon
Abstract — The HIV is perceived by the host immune system as partly self because it carries an envelope derived from the host CD4 cell wall membrane. If host immune responses were to kill the virus with its envelope, they would also kill all the host CD4 cells causing a fatal autoimmune disease. To avoid this outcome, the immune system must respond in an ineffective manner that does not kill itself with the virus. The HIV has used its envelope (and CD4) as a hostage to blackmail the immune system. The HIV antigens left after the destruction of its envelope, when injected into an uninfected person as a vaccine are perceived by his immune system as non-self and provoke responses that effectively kill all the antigens of the HIV except its envelope. When the 'vaccinated' person is then infected with the enveloped HIV, the immune responses will kill all its antigens except its envelope. Therefore, an effective vaccine for the HIV consists of all its antigens with only its envelope destroyed. Such a vaccine is safe because, without its envelope, the HIV antigens cannot infect other cells and is effective because the viral envelope has been destroyed. The effectiveness of such vaccines was tested by culturing autologous vaccines prepared from the bloods of HIV infected persons, in vitro, with their respective peripheral leucocytes in a neutral medium. Such vaccinated leucocytes. were then re-injected into the patients concerned and induced some viral destruction in them. When confirmed, vaccines prepared on these principles could provide a simple and cheap form of immuno-therapy of HIV sero-positives and effective vaccines against the HIV/AIDS through out the world. (1) Paper modified from one entitled: Effective Vaccines against and Immuno-therapy of the HIV : Scientific Report and Ethical Considerations from Cameroon. By V. Anomah NGU and Godtrey B Tangwa, first presented at the Fifth World Congress of Bioethics, Imperial College, London, UK, 21-24 September 2000.
Introduction
An effective vaccine for the HIV has long been awaited by the whole world but especially by Sub-Sahara Africa which is being ravaged by AIDS with over 2/3 of all world cases of HIV or nearly 23 million persons in December 1999 (UNAIDS Report, Dec. 1999). Therefore, any proposals that can contribute to finding an effective vaccine against this plague deserve careful study.An ideal vaccine for the HIV should: o Provoke in vaccinated persons, immune responses that actually kill the virus in a natural infection following the vaccination.
Our search for an effective vaccine for the HIV started by trying to understand why the natural infection with the virus provoked immune responses that fail, in practice, to kill and eliminate the virus from the body. Understanding the structure of the virus and its relationship to the host immune system should provide insights into the factor that caused this failure. Removing the incriminating factor should provide a basis for provoking effective immune responses that killed the virus, the sine qua non of an effective vaccine for the HIV.
The structure of the HIV
It had been known, since the discovery of the virus (Sinoussi et al 1983) and a study of its fine structure (McKeating and Willey 1989), that the HIV is an enveloped virus and that, its envelope is derived, by budding, from the cell wall of an infected CD4 cell (see fig 1) as the virus emerges from the cell. This makes the HIV a 'hybrid' virus with elements that are truly viral and specific to it and therefore foreign to the body, but with an envelope that is of host or human origin and therefore not foreign to the body. The presence of host cell wall as the viral envelope has obvious and important immunological implications for the virus and for the body, but the scientific community has, apparently, overlooked these implications or has not taken them into full account.
The immune system and the structure of the HIV
The ability of the immune system of the body to destroy any given virus will depend on how the immune system `perceives' the virus in question: whether as completely foreign to that body i.e. non-self or whether as only partly foreign i.e. partly-self because the virus carries on itself some of the body's structure. The immune response to a virus that is perceived as non-self, all things being equal, will be considerably stronger and more effective in killing such a virus than a response to a virus that is perceived as partly-self. The HIV has an envelope of host origin; it is therefore perceived by the host immune system as partly-self and therefore provokes immune responses that are, so to speak, partial and are considerably less effective in killing the virus than would have been the case had the HIV been without an envelope of host origin i.e. had the HIV been completely non-self.
The HIV blackmails the host immune system
If the host immune system were somehow to ignore the presence of its own cellular membrane on the viral envelope and were to perceive the HIV as non-self (instead of partly-self), the corresponding immune responses provoked would destroy not only the virus with its envelope but also all the CD4 cells from which the viral envelope was derived. This would constitute a serious, and a rapidly fatal autoimmune disease. If the immune system is to avoid such a fatal outcome and preserve itself, it would 'be obliged to' that is, `blackmailed into' producing immune responses that do not destroy the virus in order not to destroy itself with the virus. If, in fact, the immune system has not killed the patient with the virus, it is because of this 'blackmail' exercised by the HIV on the host immune system using the viral envelope and the CD4 cell wall membrane as 'hostage'. The viral envelope is unquestionably the most powerful secret weapon of the HIV with which it frustrates and blocks those immune responses that could effectively kill the virus in the body. It is the viral envelope, more than any other factor, that ensures the survival of the HIV in the body and blackmail is its modus operandi.It should be pointed out that this blackmail is not total. If it had been total, there would be no immune responses present in infected persons and the virus would then have a free hand and its rapid and undisturbed action in the body would kill' the patient in a relatively short time. The relatively long natural history of this infection and the presence of immune responses in patients is evidence that there is partial immune control of the virus but this is greatly hampered by the presence of the envelope.
The ideal solution for the body
If the immune system had a choice in the matter, for its own survival, it would prefer the situation whereby the responses kill only the virus without any damage to the viral envelope or to the CD4 cells. To provoke, in an uninfected person, such 'ideal' immune responses that kill only the virus sparing its envelope, one must start with HIV anti-gens from which the viral envelope has been deliberately destroyed before hand, transforming it into non-self antigens. Non-self antigens, perceived as completely foreign to the body, will provoke immune responses that effectively kill the virus concerned and in an uninfected person, will therefore kill an invading HIV, sparing its envelope and CD4. These will be spared because the envelope was absent, from the start, from the viral antigens used to inoculate or vaccinate the uninfected person. Such antigens should be effective as a vaccine because they provoke responses directed exclusively against the non-self part of the virus only, with no risk to its envelope. The empty envelope that remains after this has no biological significance.Therefore all HIV antigens, minus its envelope, constitute an effective vaccine for the HIVI As there is, as yet, no established hierarchy for HIV antigens, it is prudent to have all its true antigens present in the vaccine. (It is significant to note that in HIV infected persons, there are no immune responses to the viral envelope itself because of its host origin. Its presence on the virus is simply to give the virus the status of partly self in relation to the host immune system and so ensure the survival of the virus).
Preparing an effective vaccine for the HIV
To prepare an effective vaccine for the HIV, therefore, one destroys the viral envelope with lipid solvents acting on the lipid part of the phospho-lipo-proteins of the envelope. The solvent is removed and the residue constitutes the vaccine. It is a safe vaccine because without its envelope, the HIV cannot infect other cells. Such a vaccine would thus consist of non-self, non-life and all the non-enveloped HIV antigens only.A vaccine produced on the above principles, including any further refinements and purification that may be later carried out, will be relatively easy and cheap to produce and, being a non-life vaccine, should not be very demanding on the cold chain for its distribution. Above all, it should be effective because it is based on the correct understanding of the role of the viral envelope in the life and structure of the virus.
Pre-testing the vaccine as an autologous vaccine
It had been proposed that this simple vaccine be considered for the 3-phase trial to which other HIV vaccines are being subjected. Its production in sufficient quantifies for such a trial should pose minimal logistic problems. Lackingthe scientific `credibility to persuade competent organisations, funding bodies, the media and the public to accept such a trial, we were obliged to take the unprecedented step of pre-testing autologous vaccines on the infected persons concerned. The purpose was to show that the vaccine did indeed provoked immune responses that actually killed the HIV. The immune killing of the HIV, whether it occurred in normal or in infected persons, is the ultimate sine qua non of an effective vaccine against the virus. Convinced that the scientific basis of this approach was sound, we were quite prepared to accept any criticisms that this new and unusual approach might provoke among traditional immunologist!The scientific basis
The scientific and immunological basis for pre-testing this vaccine on infected persons was given in a previous paper (Ngu 1997). It is summarised as follows: circulating in the peripheral blood of all persons, are various groups of immune cells or immunocytes - macrophages, monocytes, lymphocytes including CD4 and CD8 - assigned to deal with the various pathogens -viruses, bacteria, parasites, fungi etc. that invade the body from time to time. In an immune competent person, there will be other groups of immunocytes fresh from the bone marrow, on 'stand by' As it were, as yet uncommitted to any specific pathogen, but ready to deal with any new pathogen that may invade the body at that moment That such uncommitted immunocytes exist in HIV infected persons can be deduced from the fact that, at the start of the HIV infection, most other pathogens are held well in check. The progressive destruction of the immune system by the HIV reduces, in time, the pool of uncommitted immunocytes available to deal with new pathogens, which, as opportunistic infections, eventually overwhelm the patient The available uncommitted immunocytes in an immune competent HIV patient would perceive the vaccine as a new pathogen (antigen) as they would any other new pathogen (antigen) and should deal with it as such. The vaccine is new because it is completely non-self whereas the HIV is partly self.The following is a preliminary report on the outcome of testing autologous vaccines in HIV infected persons most of whom had clinical AIDS.
The patients:
A sample of 20 patients seen between January 1996 and June 2000 because they had had CD4 cell counts done (CD4 were first done in Yaounde in 1996 and few patients could afford this test). Based on the counts, patients' immune status was graded as shown on the table. Clinically, all persons except the first two had symptoms of AIDS of varying degree of severity. There were 10 males and 10 females and their' ages varied from 23 to 55 years. We had received ethical clearance from the Ministry of Health and patients were informed of what was being done. There was no formal control group of patients for the study who received placebos instead.
We prepared the autologous vaccine as described in 8 above from the blood of patients concerned. Some vaccines were prepared using ether as the solvent and others used chloroform. In 2 patients vaccines prepared with ether and chloroform were used in the same patient on different occasions. (See table 1 last column for more information). The vaccines were to be tested as autologous vaccines in the very persons providing the viruses for the vaccines. They were used to inoculate or 'vaccinate', in vitro, cultures of peripheral leucocytes of the patients concerned and these cultures were then re-injected into them.
The culture of peripheral leucocytes with autologous vaccine:
20 ml of venous blood was withdrawn from the patient into a syringe with heparin and allowed to stand in an incubator at 37 C for 30 -45 minutes in a long sterile glass tubes. The leucocytes rich plasma above the red blood cells sediment was transferred into sterile centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant plasma was discarded and the cell deposit re-suspended in 5 ml of normal saline. After thorough mixing, the cell mixture was again centrifuged and the supernatant discarded. The procedure was repeated 3 times in all to remove all traces of the patient's serum from the cell deposit (No attempt was made to separate the lymphocytes from the other leucocytes using ficoll) The final cell deposit was re-suspended in 5 ml of a tissue culture medium (RPMI 1640 GIBCO Scotland) to which 1% antibiotics (Penicillin, Streptomycin, Neomycin) and 1% L Glutamine had been added. Human cord serum screened for HIV, hepatitis and syphilis was added to enrich the medium. To this cell mixture, lml of 1 in 1000 dilution of the autologous vaccine was added. The leucocytes vaccine culture, LVC, was incubated at 37 'C for 3 to 4 hours. The mixture was then re-injected subcutaneous into the patient concerned. The rest of the autologous vaccine was kept frozen until required 3 -4 weeks later when the procedure was repeated 2 -3 times in all. This was to reinforce the immunisation or vaccination of the patient by exposing his/her peripheral leucocytes in vitro to their autologous vaccines in a medium free from the patient's serum.
Preliminary result
The leucocytes vaccine culture, LVC, when injected under the skin of the forearm of the person concerned caused slight local pain at the site of injection lasting 2 to 24 hours with some induration in some patients. Seven to 10 days later, most patients felt some slight fever of about 37.5 degrees Cent Those with low CD4 had fevers that were slightly higher, - about 38 to 39 degrees Cent The fevers were accompanied by body aches and loss of appetite. Such reactions to the vaccines passed by the third or fourth week.
Attention was focused mainly on the degree of immune depression graded as proposed by Centre Pasteur Cameroun, on the pre-treatment CD4, the post treatment CD4, pre-treatment weights and post treatment weights. The parameters of CD4 and weights were singled out because at the time, apart from clinical and haematological parameters which all showed improvements but could not be included in this table, they were the best objective indicators of the response, of the patients to their vaccines. There were then no facilities for viral load estimates.
Our explanations above clearly show the central role that the viral envelope plays in the life and survival of the virus in the body by presenting the virus to the host immune system as partly self. The immune responses provoked, although quantitatively abundant, are qualitatively partial and ineffective in completely eliminating the virus. They are partial in the sense that they are directed against both the non-self and the self parts of the virus at the same time, (this is how the virus is perceived) the self part or the viral envelope thwarting so to speak, the responses which should have been effective against the non-self parts of the HIV. It is evident from the foregoing that the only immune responses that can effectively kill the HIV are those provoked against the non-self part of the virus alone (in the absence of the viral envelope). This is the basis for the proposal to produce an effective vaccine for the HIV simply by destroying the viral envelope of the virus. The viral antigens left after this constitute a safe vaccine because without its envelope the HIV antigens cannot infect other cells and it is effective because the envelope has been removed from the antigens.
As indicated above, we were obliged to, show that the vaccine was effective by testing it as auto-vaccines in the HIV infected persons concerned. The preliminary study reported above had several defects of design and reporting. Patients numbers were small, selection was poor (sero-positives only would have been preferred to AIDS patients). The intervals for reporting the CD4 were irregular. Viral load estimates would have been very useful but were not available at the time.
These shortcomings caused in part, by logistic problems that (will be addressed in a follow-up study) do not, invalidate the results. These show in all cases, improvements in the clinical status of patients as judged by physical and haematological indicator (not recorded) and were accompanied by a rise of CD4 in all cases. In the absence of viral load measurements, these changes strongly suggest that the virus was, indeed, being killed by the immune response to the auto-vaccines in the presence of some immune deficiency.
All patients showed clinical improvements - diarrhoea stopped, haematological parameters improved and patients' gained weight All patients showed a rise of the CD4 (compare columns marked PRE-RX CD4 and POST-RX CD4). This rise was slight in the first 2 patients with no objective immune depression, was moderate in most other patients and was quite high in case No. 6 for reasons that we were unable to explain.
Discussion
the scientific community has known for some time that the HIV has an envelope taken from the wall of the infected CD4 as it emerges from the infected cell and that the virus is therefore a kind of `hybrid' being partly foreign to the body and partly self. It is remarkable that the evident and important immunological implications of this fart have not been taken into account in the various attempts to produce a vaccine against it.When confirmed, vaccines prepared on the above basis should have 2 very important applications. First, they can be used in the manner indicated above as a form of immunotherapy for early HIV sero-positives and for patients with functional immune systems. (One of our AIDS patients received immunotherapy in 1994, not included in this study, has remained in good health, and viral particles were not detected in his blood in a New York Laboratory in May 2000).
The second and most important application for this vaccine is obviously to provide complete immune protection for non-infected persons. As they are easy and cheap to produce, the vaccines could be produced on a regional or sub-regional basis using the viral types or sub-types prevalent in the region concerned. There should be no need to await the discovery of a single vaccine that covers the whole world. The simultaneous production and use of this simple vaccine in the various regions could protect uninfected persons through out the world in a relatively short time and so break the further spread of this virus! These are proposals worthy of serious consideration by those who are engaged in the fight against the HIV/AIDS.
Acknowledgements
Deo Omnis Gloria. We wish to thank the patients who participated in this study for their collaboration. We thank the Ministry of Public Health and the Ethical Committee for permission to carry out this study. We thank the Minister of Higher Education for financial support We dedicate this paper to the late Honore Tchakountie for valuable technical services. Ms Catherine Nkeh provided competent secretarial services.
References
- UNAIDS Report December 1999.
- McKeating, J A. and Willey R L. (1989), `Structure and function of the HIV envelope", AIDS, 3: S35 - S41.
- Ngu V A. (1997), "The viral envelope in the evolution of HIV: a hypothetical approach to inducing an effective immune response to the virus", Medical Hypotheses, 48; 517 - 521.
- Barre-Sinoussi F, Charmann J C. Rey F., et al. (1983), "Isolation of a T-lymphotropic retrovirus from a patient at high risk for acquired immune deficiency syndrome AIDS." Science, 220: 868 - 871. The Paper was first published September 2001 in the "Journal of the Cameroon Academy of Science, Vol. 1, pp 1 - 68. Received: 17/02/2001 Accepted: 30/05/2001
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VANHIVAX, HIV/AIDS: A Summary Update Before setting out against an enemy as formidable as the HIV, it is prudent and even very important to understand thoroughly the enemy; (1)to know as much as possible about the structure and any significant or unusual elements of the structure of the virus, (2) to interpret correctly the role played by such elements, (3) if possible the evolution of the virus and (4) to fully understand the biological consequences or effects of the presence of the virus in the body because a virus that persists must have some effects on the body.
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