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 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. 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 SIVsm-INFECTION OF MACAQUES: ? To investigate the role of host factors in SIV-infection of macaques, we used a well-defined molecularly cloned virus (SIVsmE543-3). Susceptibility to SIV infection of primary PBMC in vitro varies significantly between individual macaques and the susceptibility phenotype correlates with the extent of in vivo viral replication following in vivo inoculation. However, although macaques with low susceptibility phenotype initially maintained low levels of viremia for variable periods of time, eventual progression to AIDS was observed coincident with increasing viremia, suggesting viral and/or immune escape in these animals. ? ? Immune responses were evaluated in four macaques that progress rapidly to AIDS in under 6 months. These animals developed transient SIV-specific CTL and antibody responses and failed to develop antibody to either tetanus toxoid or Hepatitis A, consistent with a global immune defect. In collaborative studies with the Martin laboratory, the phenotype of CD4+ T cells was evaluated in SIVsm-infected RP macaques. SIVsmE543-3-infected RP macaques showed profound and early depletion of memory CD4+ T cells explaining the immune deficits in these animals. Virus from RP macaques was molecularly and biologically characterized. In a parallel study, 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. Full length infectious molecular clones of various RP viruses were derived from plasma of RP RhH635. This macaque was inoculated with virus isolated from tissues of one of the initial RP study animals. The 635 viruses replicated less efficiently in primary macaque PBMC and macrophages than the parental SIVsmE543-3 strain. Pilot studies with three selected viruses were performed in two rhesus macaques per virus. Macaques inoculated with one of these viruses, H635-FC developed moderate to high primary and setpoint viremia and have exhibited CD4+ T cell depletion, consistent with progression to AIDS. Sequence analysis of plasma virus in these animals demonstrated rapid loss of RP-specific env mutations as well as progressive development of heterogeneity. Coinoculation of rhesus with 543 and FC clones resulted in pathogenic infection with rapid disease in one of 3 macaques. Interestingly, 543 and FC viruses were equally represented in plasma during primary infection but 543 predominated later.? ? Previous studies in this lab demonstrated that infection of PT macaques with SIVagm9063 results in AIDS. We assessed the pathogenicity of the SIVagm9063 and two primary SIVagm isolates in PT macaques. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+ T cell subsets and remain healthy. The plateau levels among SIVagm90 and SIVagm9063-inoculated macaques varied widely from 100 to a million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4+ T cell loss at 6 months post infection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (p values < 0.01). Considering all the macaques, the degree of CD4+ T cell loss by 6 months post infection, correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac-infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. ? ? ASYMPTOMATIC INFECTION OF NATURAL HOST SPECIES? A second 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, SIVagm and SIVlhoest are capable of inducing AIDS in macaques but are not virulent for their natural host. These studies have utilized a molecularly cloned, pathogenic SIVagm9063-2 that induces AIDS in pigtailed macaques but results in asymptomatic infection of African green monkeys. ? ? Natural and experimental infection of vervets (Chlorocebus pygerythrus) 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. We recently expanded these studies by evaluating plasma viremia in a cohort of 12 naturally infected vervets from Tanzania. Plasma viral load varied from 1000 to >100,000 copies/ml in these animals. PCR and sequence analysis of portions of the envelope gene of these novel SIVagm isolates revealed wide genetic diversity, although all isolates clustered phylogenetically with previously characterized SIVagm isolates from vervets, confirming that these source animals were indeed vervet AGM. The kinetics of viremia of one of these viruses in naive vervets was evaluated in a pilot experiment using intravenous inoculation of 1 ml of blood from one naturally-infected donor, AG1. These animals exhibited high levels of primary viremia. The range of setpoint viremia was similar to that observed in the naturally-infected cohort. These animals have remained asymptomatic for 1 year of follow-up. ? ? We evaluated the viral kinetics of a natural SIVagm isolate in AGM. Two species of AGM were evaluated, vervet monkeys (the species of origin of SIVagm90) and sabaeus monkeys from the Barbados. The virologic outcome in sabaeus and vervet AGM was surprisingly divergent. Inoculation of sabaeus AGM with SIVagm90 resulted in low and variable levels of primary viremia and low setpoints (<100 to 10,000 copies/ml). In contrast, inoculation of vervet AGM resulted in high primary viremia and establishment of moderate plateau levels (10,000 to 100,000). Regardless of the extent of viremia, CD4+ T lymphocytes remained stable throughout infection of AGM with no significant relationship between viral load and CD4+ T cell loss.
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