Grouppe Kurosawa: HIV - Background of the Science (print)

Background of the Science

An increasing number of studies have shown that many individuals at high risk for becoming infected with the HIV virus have remained free of infection. In some cases, the reasons are genetic. Mutations in the membrane receptors that bind the virus inhibit its entry into susceptible cells. However, in other cases there are no clear explanations how some people can defend themselves against the HIV virus, while others cannot. If we had a simple, clear answer to this question, a vaccine could be designed to mimic the body's natural strategy for clearing the HIV virus. The vaccine protocol Grouppe Kurosawa has adopted is based on the assumption that the intrinsic defect in the immune response to the HIV virus is the virus's innate ability to stimulate its own expression while simultaneously suppressing the immune response to its very presence.

For many years, the reduced CD4 T cell count that characterizes AIDS was difficult to explain because very few peripheral blood T cells contained virus. This observation lead to wild speculations that the true cause of AIDS was not the HIV virus. Since the asymptomatic period of AIDS can be 10 years or longer, it was generally assumed that the primary immune response against the virus successfully controlled its re-expression for many years. We now know this is not true. The initial immune response against the virus is incapable of completely controlling the virus, because the viral particle contains factors that impair optimal immunological recognition of the virus. The reduction of the viral titer after infection can be attributed to a massive die-off or apoptosis of infected T cells, induced primarily by the HIV accessory protein VPR and anti-gp120 immune complexes. Viruses mutated at VPR can escape the apoptosis and continue to produce virus, although at a 10 fold reduced rate.

In 1995, two studies simultaneously published in Nature showed that HIV protease inhibitors dramatically decreased the amount of HIV RNA in the blood while simultaneously increasing the numbers of CD4 T cells. These findings were unexpected. Cells infected with HIV "should" continue to produce virus after treatment with protease inhibitors. The viral particles would be non-infectious, but they should still contain viral RNA. Therefore, there should have been no dramatic immediate fall in the amount of HIV RNA in the blood. The authors concluded that the spread of HIV was absolutely dependent on the infection of fresh cells, and that viral particles and virally infected cells have half-lives of only a few days. Protease inhibitors prevented the infection of new cells, while infected cells died within days to be rapidly replaced in the blood by newly generated CD4 cells. This interpretation of the experimental data is now considered incorrect.

There is no direct evidence that high rates of production and destruction of CD4 T cells are occurring during an HIV infection. When the teleomere lengths of CD4 T cells from infected and non-infected individuals were compared, there were no differences. Since teleomere length shortens during every cell cycle, this suggests the CD4 T cell population in infected individuals is turning over relatively normally. In addition, an analysis of HIV-infected lymph nodes found a 10 fold lower turnover of HIV infected cells than the rate "predicted' in the 1995 studies. The drop in CD4 T cells which occurs during HIV infections could be secondary to a reduced synthesis and/or maturation of CD4 T cells in the thymus, apoptosis (programmed cell death) of infected and neighboring cells, and a redistribution or movement of T cells out of the blood. Protease inhibitors may decrease the amount of HIV RNA in the blood by inhibiting the ability of viral proteases to degrade the cytoskeletal proteins that negatively regulate viral secretion. Viral proteases are pathogenic when over expressed. The HIV protease, in particular, is known to degrade actin, vimentin, alpha-actinin, desmin, myosin and tropomyosin. The viral protein VIF is known to bind vimentin. The vimentin intermediate filament network links the plasma membrane to the nuclear membrane and is considered to play a role in gene transcription. The collapse of the intermediate filament network induces nuclear changes characteristic of apoptosis. Reverse transcriptase, on the other hand, binds beta-actin. When virally infected T cells bind fibronectin, or other cells, the actin filaments depolymerize and form a thick ring underneath the plasma membrane. This results in a 40% reduction in the secretion of HIV within 48 hours. When the same cells are cultured on polystyrene, a plastic used in laboratory culture dishes, actin filaments do not depolymerize and viral secretion is not impaired. In the lymph nodes and thymus, organs that harbor active viral infections, infected cells are by definition associated with other cells and exposed to fibronectin. These cellular contacts could impair or modulate the release of virus. Viral proteases may not be necessary for the packaging and release of virus in a culture dish, but they probably play a fundamental role in rearranging the cellular cytoskeleton so virus can be release in the body. Therefore, it could be argued that protease inhibitors reduce the amount of viral RNA in the body by impairing the ability of virally infected cells to release virus.

A more powerful interpretation of the protease inhibitor data is that these compounds are inhibiting the chymotrypsin activity of the proteasome complex, thereby inducing apoptosis in virally infected cells. This is now known to be true. In the absence of an active proteasome complex, the endogenous inhibitors of the NF-kB transcription factor cannot be degraded, therefore they remain capable of inhibiting the activation of NF-kB dependent genes, such as pro-inflammatory hormones, inhibitors of apoptosis, and the HIV virus itself. Infected cells die, while free infectious virus cannot infect new T cells in the absence of NF-kB activation. The proteasome is also now known to process HIV gag polyproteins, resulting in the maturation and release of viral particles from infected cells. Highly specific proteasome inhibitors, such as epoxomicin, reduce HIV release resulting in a decrease in viral RNA in the blood. If commercial HIV protease inhibitors were truly specific to the HIV viral protease, this would not occur. Protease inhibitors may have been designed to specifically inhibit the HIV protease, but that is not why they are effective inhibitors of HIV RNA synthesis.

Understanding the dynamics of CD4 T cell turnover in AIDS is fundamental to vaccine design. Does the T cell count decline because infected cells are rapidly dying and/or because replacement cells are not developing properly? If T cells are being sequestered out of the blood in different tissue compartments, how does this occur? If neutralizing antibodies against gp160/120 were effective in removing HIV as soon as it entered the body, the answer to these questions would not be the priority that it is today. The neutralizing antibodies would block viral infectivity, activate complement, destroy the virus, and there would be no disease. Unfortunately, HIV is a complicated virus and an effective vaccine strategy requires that certain assumptions be made about the methods by which HIV becomes established in the body. Classically, an effective immune response against a virus involves a period of inflammation followed by a feedback period of immunosuppression that terminates the ongoing immune response from becoming excessive, and, in some cases, fatal. Picture in your mind a prairie fire that started small but continued to spread.

In the absence of pro-inflammatory hormones, a virus cannot be cleared from the body. The presence of TNF-alpha during the acute phase of viral infection plays a key role in destroying virally infected cells. In AIDS, the periods of inflammation and immunosuppression appear to be reversed. The asymptomatic stage of AIDS may exist because a vigorous immune response against the virus cannot be mounted. At the same time, the spread of the virus is held "in check" by numerous immunological and hormonal factors that ultimately collapse over time. It is the argument of Grouppe Kurosawa that multiple viral proteins contribute to this state of immune inactivity or immunosuppression, and that an effective vaccine must neutralize these proteins as soon as they are freed from viral particles or virally infected cells. If the virus cannot create a state of immunosuppression, the body's natural defenses, both humoral and cell-mediated, would be able to clear it from the body.

In this essay, we will argue that the viral proteins NEF, VPR, TAT and p24/p17 are immunosuppressive and contribute to the spread of HIV. Vaccine protocols that do not target these proteins may be unable to completely control the spread of the virus. Contrary to current dogma, we believe that the viral gp160/120 protein should not be incorporated into the design of an HIV vaccine. Although there is no doubt that gp160/gp120 contributes substantially to the defective immune response associated with HIV infections, we believe targeting this protein in a vaccine protocol does more harm than good. The humoral immune response to the gp160/120 membrane protein contributes substantially to the establishment of an HIV infection and the progressive decline in CD4 T cells. Anti-gp160/120 immune complexes, bound to the surface of macrophages and dendritic cells by complement receptors, crosslink CD4 on uninfected T and B cells and induce apoptosis or programmed cell death. The scientific literature is VERY clear on this point. The most unambiguous data on the dangers of anti-gp160/gp120 immune complexes was collected by American Red Cross scientists who constructed human CD4 transgenic mice. After immunization and boost with purified gp120, these mice suffered a 7 fold reduction CD4 T cells and a 2-3 fold reduction in B cell numbers 6 days after the booster immunization. The authors concluded that gp160/gp120 immune complexes not only sensitized non-infected CD4 T cells to programmed cell death, but that they directly induced it. The only immune response to the gp160/120 protein that should be encouraged is one of immunological tolerance.