The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV. The proposed studies will use the well-characterized equine infectious anemia virus (EIAV) model to identify virus factors important in the evolution and selection of immune escape variants during progression of lentiviral disease. The lifelong persistence of virus in vivo is a function of its ability to evade immune recognition and elimination as well as its ability to replicate, i.e. its replicative capacity. The overall goal of these studies is to determine if changes in the host immune environment during progression of disease reproducibly select for virus variants with altered replicative capacity. Specifically, we propose test the hypothesis that genetic changes in Rev and SU during progression of disease contribute to evasion of broadly neutralizing antibody and CTL at a cost in virus replicative capacity.
The first aim will determine if env/rev genotypes that predominate at sequential stages of EIAV disease differ in replication phenotype. Infectious clones containing dominant env/rev genotypes representative of each stage of disease will be tested for replicative capacity in growth kinetic and growth competition assays, and the relative fitness of pairs of variants will be estimated.
The second aim will determine the impact of variation in Rev and SU on replicative capacity and immune evasion. The replication phenotype of Rev will be quantified as nuclear export activity, and used to infer an immune evasion phenotype based on sensitivity to CTL killing. The SU replication and immune evasion phenotypes will be measured as infectivity and sensitivity to neutralizing antibody, respectively. These values, together with the replication fitness score obtained in Specific Aim 1, will be used in statistical models to determine which env/rev phenotypes affect changes in virus load during progression of disease. The third specific aim will use EIAV-based pseudovirus to identify specific molecular determinants of immune evasion and replication phenotypes. This will establish if there is a genetic link between escape from broadly neutralizing antibody and replicative capacity. In the fourth aim, in vivo competition assays will directly test the predictions of the statistical model developed in the second aim. Viruses containing env/rev genotypes that differ in immune evasion and/or replication phenotype will be inoculated into horses that have distinct immune environments with respect to EIAV, including: immunodeficient, naove, and convalescent. Following infection, dominant genotypes will be identified by quantitative RT-PCR and sequence analysis. These results will identify the relationship between immune evasion and replication phenotype and determine the respective roles of SU and Rev phenotype in EIAV selection under different immune environments in vivo. This detailed, integrative analyses will identify critical virus determinants of immune evasion and identify new vaccine targets that limit virus escape from broadly reactive immune responses. ? ?
Persistent virus infections are the etiological agents of AIDS and some cancers. The proposed studies will identify the genetic mechanisms that enable lentiviruses to modify their replication and thereby escape elimination by the host immune system. This strategy allows the virus to persist in vivo and, over time, acquire new mutations that leads to progression of clinical disease. The results of this study will identify new vaccine strategies for HIV-1 and persistent viruses that inhibit immune escape and prevent progression to AIDS and cancer. ? ? ?
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