Pathogenesis Studies Of Simian Immunodeficiency Virus In

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Abstract

An experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease is critical for the study of the pathogenesis of human AIDS. Simian immunodeficiency virus (SIV) infection of macaques satisfies this criterion and is therefore a relevant model. SIV induces an immunodeficiency syndrome in infected macaques that is remarkably similar in pathogenesis to human AIDS. An important use of this animal model system is the detailed study of pathogenesis and viral determinants of disease since many studies of this type are not feasible in humans. Such investigations should allow us to determine how primate lentiviruses destroy the immune system of their hosts, and facilitate the development of more rational therapeutic antiviral strategies. The purpose of this project is to investigate host and viral factors involved in variable disease progression in SIV-infected macaques and the lack of disease in African primates infected with their own strains of SIV. PATHOGENESIS OF SIV-INFECTION OF MACAQUES: To investigate the role of host factors in SIV-infection of macaques, we used well-defined molecularly cloned viruses (SIVsmE543-3). Susceptibility to SIV infection of primary PBMC in vitro varies significantly between individual macaques. Interestingly, the susceptibility phenotype correlates with the extent of in vivo viral replication following inoculation of these animals. To investigate this phenomenon, we characterized a cohort of six rhesus macaques for susceptibility phenotype and then inoculated them with SIVsmE543. Susceptibility of PBMC in vitro was highly predictive of subsequent viremia. The two highly susceptible macaques exhibited a high level of plasma viremia; one failed to develop a transient antibody and CTL response and was euthanized due to AIDS by 16 weeks. Two macaques of the intermediate phenotype had significant viremia and two macaques with a low degree of susceptibility had very low viremia. We have also evaluated the immune responses, both SIV-specific and to unrelated antigens in macaques that progress rapidly to AIDS in under 6 months. Four rapid progressors (RP) from three separate studies were evaluated for antibody and CTL responses. These animals developed transient CTL and antibody responses. These animals were also immunized with tetanus toxoid and Hepatitis A (Haverix) to evaluate their ability to respond to memory and recall antigens. RP macaques failed to develop antibody to either antigen, consistent with a global immune defect in rapid progressor macaques. To characterize viral factors involved in pathogenesis, virus from RP macaques was molecularly and biologically characterized. Sequence analysis of env genes cloned from three SIVsm-infected animals exhibited common substitutions in the env gene. These substitutions were unusual in that they involved residues that were generally conserved and that were known to affect binding of env to CD4 or coreceptor. Fusion assays revealed that the majority of envelope genes from such rapid progressor macaques utilize CCR5 as a receptor in the absence of CD4. Studies are ongoing to evaluate the pathogenesis of one such isolate in a naive cohort of rhesus macaques in parallel with the parental virus, SIVsmE543-3. We performed two related studies of the pathogenic determinants of the acutely lethal SIVsmPBj6.6 molecular clone. One study evaluated which of five specific substitutions in the minimally pathogenic PBj6.9 clone were responsible for its decreased virulence as compared to PBj6.6. The D119G substitution in envelope of SIVsmPBj6.9 was associated with a marked reduction in infectivity of this virus relative to SIVsmPBj6.6. An associated processing defect in gp160 of SIVsmPBj6.9 and chimeras expressing the D119G substitution suggests that a reduction in virion envelope incorporation is the mechanistic basis for reduced virion infectivity. In vivo studies revealed that substitution of the 6.9 amino acid into PBj6.6 (D-119) abrogated the pathogenicity of this previously pathogenic virus. Introduction of the PBj6.9 G-119, however, did not confer full virulence to the parental PBj6.9, implicating one or all of the other four substitutions in the virulence of SIVsmPBj6.6. We also evaluated the role of the tyrosine at position 17 of SIVsmPBj Nef in the acute pathogenesis of this virus by introducing this unique residue into AIDS-inducing clones, SIVsmE543-3 and SIVagm9063-2. The introduction of Tyr-17 conferred the ability to replicate in resting macaque PBMC and altered the pathogenesis of the AIDS-inducing viruses. Thus these animals exhibited some of the acute symptoms and pathology of macaques inoculated with SIVsmPBj although the disease was less severe. However, the Tyr-17 did not alter pathogenesis by increasing viral replication in the host since the kinetics and extent of viral replication were similar in macaques inoculated with either Y+ or Y- clones. ASYMPTOMATIC INFECTION OF NATURAL HOST SPECIES A secondary but equally important goal of this project is to study the mechanisms underlying the apparent lack of pathogenicity of SIV for their natural host species with emphasis on SIVagm from vervet monkeys. SIVsm from sooty mangabeys, SIVagm from African green monkeys and SIVlhoest are capable of inducing AIDS in macaques but are not virulent for their natural host. To more extensively study the underlying mechanisms for differential virulence of SIV, we derived the molecularly cloned, pathogenic SIVagm9063-2 that induces AIDS in pigtailed macaques and Asian species but results in asymptomatic infection of African green monkeys. Most recently we have derived primary isolates of SIV from naturally-infected vervets that will be used in subsequent animals studies. A quantitative realtime RT-PCR assay for measuring viral RNA in plasma samples was developed as well as assays for identifying target cells of SIV in such animals by confocal microscopy. Natural and experimental infection of vervets with SIVagm was evaluated by in situ hybridization, plasma viral load assays and limiting dilution coculture to identify the numbers and distribution of infected cells in tissues. Virus was found in lymphoid tissues and the gastrointestinal tract and in the lung macrophages of one animal. Plasma viral load varied widely from undetectable (<1000 copies/ml) to 800,000 copies/ml. This wide range in viral load in naturally-infected AGM was unexpected since naturally-infected sooty mangabey monkeys exhibit very high levels of plasma viremia. We have initiated a study to evaluate the viral kinetics of a primary SIVagm isolate in AGM and pigtailed monkeys. These studies will be the basis for proceeding on to intervention studies with chemical immunosuppression or depletion of CD8 T cells with specific monoclonal antibodies. Preliminary results show that the primary plasma viral load in inoculated AGM ranges from undetectable to 100,000 copies/ml, whereas viremia in the pigtailed macaques was significantly higher (10 to 100 million copies/ml). Pigtailed macaques exhibited early signs of SIV-induced disease (lymphadenopathy, and early CD4 depletion) whereas no clinical or pathologic changes have been observed in the AGM.