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Scientific Basis for a Live Attenuated nef – deleted HIV-1 Therapeutic Vaccine 

Note: What follows is for a scientific discussion only and not intended as an offer to sell or a solicitation of an offer to buy anything. Contre Vir™ is in a research and development stage and will not be commercially available until it obtains regulatory approvals such as the ones from US FDA.

Hypothesis

It is postulated by the inventor that a nef deficient live attenuated HIV-1 constructed with a large deletion in the nef and in which the remaining open reading frames, particularly tat, pol, gag, env and vpr are preserved, when injected in an individual infected with a wild-type HIV-1, is therapeutic as a result of one or more of the following mechanisms:
  • By allowing normal interleukin-2 (IL2), gamma interferon (IFNg) and other unknown activating agent/chemokine production in Thelper cells thus restoring signaling and activation of specific cytotoxic (“killer”) T lymphocytes (CTL) recognizing HIV antigen displaying cells in addition to activating B lymphocytes,
  • By preventing anergy of cytotoxic T lymphocytes produced by inhibition of a second chemokine activating signal in specific binding signaling pairs -
  • a. VCAM-1(Vascular Cell Adhesion Molecule-1) : VLA-4 (Very Late integrin Antigen-4),
  • b. ICAM-1/2 (InterCellular Adhesion Molecule –1/2) : LFA-1 (Lymphocyte Function Associated Molecule-1),
  • c. LFA-3 : CD2 (Clusters of Differentiation 2) ,
  • d. B7 (B cell mediated coactivator –7) : CTLA-4 (Cytotoxic T Lymphocyte Activator –4),
  • e. ? : HSA (Heat Stable Antigen) or heretofore unknown lymphokines
  • by the Nef protein. This second signal is required for activation of a T cell that has MHC-I (Major Histocompatibility Complex Class I):TCR (T-Cell Receptor) binding and lack of it creates anergy,
  • By continually activating, stimulating and maintaining a cell mediated immune response to wild-type HIV via lines of specific CTLs which appear to be crucial in controlling and/or eliminating HIV in exposed individuals that didn’t seroconvert, in “non-progressors” and in cross-immunity provided by HIV-2.
  • By eliminating the protection offered by Nef to infected cells mediated via a downregulation of MHC class I on the cell surface.

Using a therapeutic vaccine after exposure is possible only when the disease has a long incubation period before death ensues. One vivid example is use of live attenuated duck-embryo vaccine against rabies. This vaccine is given after exposure or suspected exposure and is extremely effective because rabies has an incubation period of 60 days to 1 year. Since the incubation period in HIV infection leading to AIDS and death is even longer, a post-exposure vaccination is extremely promising.

Scientific rationale behind Contre Vir™

There is currently no specific immunological treatment utilizing specific immunodynamics for the treatment of Human Immunodeficiency Virus (HIV) infection. The two main sub types HIV-1 and HIV-2 are members of a group of closely related human and non-human primate lentiviruses which are RNA retroviruses. Several attempts at prevention and treatment have been made by using virus envelope (gp120, gp160 and gp41) and gag (p24 and p15) proteins to develop humoral (antibody or B-cell mediated immunity) all of which have so far been unsuccessful because the virus engages in cell to cell propagation and largely escapes the neutralizing antibodies. There are other vaccines currently being studied that would use vectors such as canarypox or vaccinia viruses expressing HIV envelope and one or two gag proteins primarily to induce both B and T cell immunity against those proteins or presenting cells strictly for prophylaxis. These are in Phase I studies and their efficacy as a prophylactic is unknown at this time. The HIV also mutates causing different serotypes of antigen in successive generation21. Phenotypic heterogeneity is found in replication kinetics, susceptibility to serum neutralization, anti-viral drug resistance, induction of cytopathicity and host-cell range specificity thus creating the need for a so called “polyvalent” vaccine that will be effective against all the known and unknown clades of HIV-1 . Moreover, some viral proteins inhibit the induction of IL-2 mRNA in infected cells thus defeating the T cell mediated immunity.

Infection by HIV leads to progressive deterioration of cell-mediated immunity via loss of THELPER cells bearing CD4 receptors. This makes the victim susceptible to opportunistic infections such as Pneumocystis carinii pneumonia, cytomegalovirus, Toxoplasma gondii and Mycobacterium tuberculosis infections. Tumors such a Kaposi’s sarcoma also commonly occur. Once the CD4 counts drop to near zero, death ensues rapidly.

AIDS and HIV infection initially involved homosexual men, intravenous drug users and hemophiliacs in the United States and Europe. However, heterosexual infection has become common and rampant in Africa (particularly Rwanda, Burundi, Zaire, Uganda, Kenya and Tanzania), Brazil, India, Myanmar and Thailand. According to the World Health Organization (WHO), in excess of 40,000,000 people currently harbor HIV infection worldwide.

Just like the viral protein vaccines tried thus far (and research abandoned by the National Institutes of Health due to poor results), pharmacological treatment with AZT (zidovudine), DDC (dideoxycytosine), DDI (dideoxyinosine) and protease inhibitors has also been frustrating due to development of resistant stains of the virus which continue the infection after, at most, a short break. At present, there is no definitive treatment that would effectively eliminate virus harboring cells and restore the cellular immune system. The protease inhibiting drugs have shown great promise and reduced mortality by lowering viral burdens, however, reservoir of pro-viral DNA in the neuroglia, development of resistance and serious sided effects such as diabetes, hypertension and cushingoid fat distribution make them to be far from panacea.

A variety of different approaches have been postulated which do not rely on either the B cell mediated response or on pharmacological intervention in viral synthesis. The late Professor Jonas Salk, in his commentary in Nature noted that as the disease progresses, titers of antibodies to gp41 and virus neutralizing antibody remain constant but the level of antibody which correlates with the presence of antibody dependent cell cytotoxicity (ADCC) and antibody to reverse transcriptase decline. He proposed treatment of symptomatic HIV infected patients with sera from asymptomatic HIV infected patients. He further hypothesized that HIV immunogens given to HIV infected patients would be protective22.

Dead virions have been hypothesized but no researcher has yet tried whole dead virions either for prevention or treatment of HIV infection in humans in a meaningful manner. An inactivated gp120-depleted HIV-1 immunogen (Remune®) has been tried as a therapeutic uplift. While this immunogen increases the CTL activity directed at its own antigens, it cannot improve such activity significantly against other antigens. Further research has revealed that this approach does not work.

Simian Immunodeficiency Virus (SIV) is a primate lentivirus with various strains that affect African green monkeys, macaque monkeys, sooty-mangabee monkeys, rhesus monkeys and chimpanzees. SIV infection in monkeys is widely used to study the physiology and pathology of the primate lentiviruses. A great deal of research as been done by attempting to infect monkeys with artificially created mutants of the SIV to determine their relative infectivity. Many of these studies focused on the role of the nef gene in the physiology of virus life cycle. The nef gene is present in all primate lentiviruses sequenced to date. The gene consists of an open reading frame beginning within or immediately after the 3’ end of the env gene and overlaps the U3 portion of the 3’ long terminal repeat. The gene was previously named F, 3’-orf or B-orf. It is expressed in vivo as determined by antibodies to the nef gene product in infected individuals. Luria et al have shown that at least some nef gene products block the induction of IL-2 mRNA in lymphoid cells triggered by activating agents phorbol myristate acetate (PMA), phytohemeagglutinin (PHA) and/or antibodies against CD3, TCR or CD2. Kestler et al have found rapid reversion of stop codon point mutations in nef to open forms in vivo, demonstrating selective pressure for open, presumably functional forms of nef . It was further shown that nef is necessary for vigorous virus replication in rhesus monkeys, for maintaining normal virus loads and for induction of the disease. Animals inoculated with nef-deletion mutants have remained disease free for at least 3 years while wild-type virus infected animals all developed AIDS and died. This protection also extends to administration of SIV infected cells1. It has also been demonstrated that nef deletion increases viral replication but it is postulated that the responses to nef deletion are different in vivo and in vitro. Additonal evidence, recently published shows a strong role of nef in T-cell signaling defects mediated via SH3, Lck, HLA-B7, NF-kappa pathways etc. Immunization with live attenuated nef- SIV mutants in macaques has shown a strong type 1 THELPER response and beta-chemokine production. It has also been shown that HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages and that the conserved core of HIV-1 Nef is essential for association with Lck and for enhanced viral replication in T-lymphocytes. It has also been shown that the HIV-1 Nef alters Ca++ signaling in myelomonocytic cells through SH3-mediated protein-protein interactions, the significance of which is not fully understood. The HIV-1 nef gene expression affects generation and function of human T cells but not dendritic cells.

It is known that CTLs specific for epitopes in env, gag, tat and pol proteins can be detected quite early in the infection. It is a commonly held view that these cells, by killing virally infected cells prior to their production of additional infectious virus are responsible for the diminution in viral burden.

Evidence has surfaced from a study of health care workers who had been exposed to HIV contaminated blood but did not seroconvert. When compared to individuals exposed to blood from uninfected individuals, 7 out of 20 persons exposed to HIV infected blood showed CTLs specific for HIV peptides in association with class I major histocompatibility complex (MHC) molecules. None of the individuals exposed to uninfected blood had similar CTLS. The development of class I MHC- restricted and HIV specific CTLs is absolute proof the HIV infection occurred and then stopped. This is evident from the known cell biology of antigen presentation by class I MHC molecules because such a phenomenon generally requires endogenous production of protein from which the peptide is derived. It is therefore quite plausible that the specific CTLs did eliminate the virus.

The recognition of “non-progressors” i.e. a set of HIV-1 infected individuals surviving for a prolonged period of time with essentially normal CD4 counts and with no signs of disease suggest that an immune response is possible that actually control HIV.

No specific information exists to explain this control of HIV in all of the non-progressors but in many cases, they possess CD8 T cells that strikingly limit the capacity of HIV from their CD4 T cells to infect peripheral blood mononuclear cells (PBMCs) that have been activated by phytohemagglutinin (PHA). Pure CD4 cells from non-progressors are quite effective in transmitting their HIV to PHA-activated PBMCs but addition of their CD8 T cells strikingly suppresses such infection. Even supernatants of CD8 T cells can inhibit in vitro infection. It has been postulated that a cell derived soluble factor from CD8s distinct from any know cytokine may be responsible.

Spontaneously (via mutation) attenuated HIVs have been recovered from humans in whom infection has had a very benign course. Kirchhoff et al have described one individual with no sign of disease and a normal CD4 cell count more than 10 years after infection. Genomic analysis of this patient’s HIV-1 virus at various times during this 10 year period has revealed a deletion in the nef gene that was sustained and extended. A set of individuals in Australia was infected from blood products obtained from a single infected donor with a similarly defective HIV-1 virus (The Sydney Blood Bank Cohort). All those infected had a benign, non-progressive course except for one individual who had systemic lupus erythematosus and was on high-dose glucocorticoid treatment14. Additional long-term survivors with defective nef genes in their HIV genome have been reported. A recent study has shown that in the Sydney Blood Bank Cohort, the effect of long-term infection with nef-defective attenuated HIV-1 resulted in an increase in CD45RO+CD4+ T lymphocytes and limited activation of CD8+ T lymphocytes.

Further evidence of CTL mediated immunity to HIV-1, this time via cross-reactivity to antigens of HIV-2, comes from an astounding series of observations. For example, a set of commercial sex workers in Gambia infected with HIV-2 appeared to escape HIV-1 infection altogether in spite of exposure to several HIV-1 infected customers over a period of years. Many of these women have CTLs that recognize peptides shared by HIV-1 and HIV-2 which is suggestive of CTL mediated immune response to HIV–2 infection capable of cross-protection. More support comes from an epidemiological study conducted on a group of commercial sex workers in Dakar, Senegal from 1985-1994. The subjects were segregated into HIV-2+ and HIV-2- groups. It was determined that over an extended period of observation, the risk of subsequent infection with HIV-1 of the HIV-2+ group was about 2/3 less than the HIV-2- women while the rate of other sexually transmitted diseases was virtually identical between the two groups. Other observations have demonstrated similar results.

Evidence suggesting that an established HIV infection may not only be controlled but also actually eliminated comes from the study of one individual. HIV was isolated neonatally from a child but later, at age 5, the child was seronegative, had no clinical symptoms and had no recoverable virus. The infant had positive viral cultures on two separate occasions during the first year suggesting an infection from its mother. So far, nothing is known about either the genomic nature of the virus infecting this child or about the anti-HIV immune response generated by it.

Finally, an Australian study of non-progressors has conclusively shown that an HIV-1 infected blood donor and a cohort of six recipients (Sydney Blood Bank Cohort or SBBC) infected from this donor were infected with an HIV-1 with deletions in the nef gene. This nef deficient strain has not caused disease even in the members affected by the immunosuppressive effects of age, drug therapy and systemic lupus erythematosus (SLE).

It has been shown that the regulation of the quality of the immune response is controlled mainly by the cytokines available at the time of priming in addition to the antigen dose. Specifically, interleukin-4 (IL-4) appears to inhibit priming for IFNg production by T cells and IL-12 appears to strikingly enhance priming for IFNg production.

Several researchers have been sounding cautionary notes based upon their own observations and those concerns must be addressed here. Dr. Ruth Ruprecht of Dana-Farber Cancer Institute, a highly respected and leading researcher first showed that a nef deleted SIV can cause disease in newborn infants in 1995, followed by a publication in 1999 in which her group conclusively showed that a multiply deleted construct of SIV caused AIDS in six out of eight newborn macaques and in 2 out of 16 adult macaques one of which died of simian AIDS. Another concerning note comes from a follow-up of the Sydney Blood Bank Cohort. Even though other researchers have found that even a small 12 base pair deletion in the SIVmac nef can protect from a simultaneous infection by wild-type SIV in cynologus monkeys followed for 68 months without causing disease, we concur with the warning issued by Dr. Ruprecht and her colleagues regarding using a gene-deficient viral construct as a prophylactic vaccine in healthy individuals and are not proposing a prophylactic use in healthy subjects. However, as a therapeutic vaccine, we are actually encouraged by the above findings. It must be noted that 25% of the newborns and 88% of the adults did not develop the disease. In fact, 75% of the adult macaques that were injected did not show any signs of the disease at all, despite a long-term follow-up. Moreover, even the adult macaques that developed signs of disease progression only did so after a considerably longer period than with a wild type SIV. As a therapeutic vaccine, if the proposed Phase I research reveals high levels of immunogenicity in 75% of the subjects it would be extremely encouraging indeed! In fact, a group from Stanford University led by Dr. Douglas Owens suggests that even a therapeutic vaccine that is effective in 75% of the recipients would extend lives, prevent deaths and save money. The Sydney Blood Bank Cohort had a relatively minor mutation in the nef and we believe that a substantial deletion of the nef as proposed here will have a more significant impact on reduction in the pathogenicity of the virus than seen in the Sydney Blood Bank Cohort (SBBC).

It is therefore postulated by the inventor that influencing the quality of the immune response might enhance its protective value. Since CTLs seem to be the most important factor thus far in control and/or elimination of HIV in instances where such control/elimination have been observed, control of the balance of cytokines that are generated and enhancement of the degree of expression of immunity at a cellular level are extremely important. It is very likely that the Nef protein prevents a second signal required for activation of T cells (in addition to the MHC-11:TCR binding) such as pairs VCAM-1:VLA-4, ICAM-1/2:LFA-1, LFA-3:CD2, B7:CTLA-4,?HSA or heretofore unknown lymphokines. The exact mechanism of how the Nef protein accomplishes this is unknown but there is evidence to suggest that there is interference in protein-tyrosine phosphorylation and the IL-2 transcription via interdigitating leucine hydrophobic bonds (“leucine zipper”) between Fos and Jun protooncogene coded proteins. Promoting induction of HIV-1 specific IFNg producing T cells and CTLs as opposed to T cells that produce other cytokines such as IL-4 and related cytokines such as IL-5 and IL-10 while eliminating probable hindrance to IL-2 production by deletion of the nef gene is a new approach for significantly increasing the potency and quality of the immune response in infected individuals. This is particularly true regarding development of potent specific CTL lines, which seem to have played a major role in non-progressors and HIV-2 infected commercial sex workers. Another study has recently shown that Nef protected HIV-1 infected cells by reducing the epitope density on their surfaces allowing evasion of CTL lysis by them. This mechanism appears to be mediated via Nef driven down-regulation of MHC class I and could be overcome by adding an excess of the relevant HIV-1 epitope as a soluble peptide.

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