| The human immunodeficiency virus type 1 (HIV-1) has been demonstrated to be highly variable throughout the world and within infected individuals. Much of this variability is localized to the envelope gene (env), especially within five variable regions (V1–V5). We have developed a heteroduplex tracking assay specific to the V1/V2 region (V1/V2-HTA) in an effort to examine how variability evolves in this region and how variability affects virus biology. In one study, the V1/V2-HTA was utilized to analyze longitudinal V1/V2 population changes in a group of late-stage infected subjects (Chapter 2). Sequence analysis of newly emerged V1/V2 variants revealed that these variants could contain small numbers of genetic differences, indicative of recent evolution, or large numbers of genetic differences, indicative of a longer evolutionary history. The genetic mechanisms involved in V1/V2 evolution included point mutations, deletions and/or insertions, and recombination. In a second study, the V1/V2-HTA and a V3-HTA were utilized to address the effect of a drug-induced genetic bottleneck on env populations in a group of late-stage infected subjects who initiated protease inhibitor monotherapy (Chapter 3). The results of this study demonstrated that strong drops in the initial virus load as well as longer durations of virus load reduction have an increased likelihood of altering the V1/V2 and V3 population structure.; The variability associated with env is just one of the reasons why it has proven difficult to develop a useful HIV-1 vaccine. We have utilized the Venezuelan equine encephalitis virus (VEE) vaccine vector to express three different forms of the simian immunodeficiency virus (SIV) Env protein in an attempt to increase the levels of protein expression and immune response in this system (Chapter 4). The Env proteins tested were the full-length Env protein, an oligomerized, soluble Env protein, and a monomeric, soluble Env protein. The results from this study have demonstrated that while all three proteins are expressed in vitro, the levels of neutralizing antibodies are highest for the full-length Env protein. This work demonstrates that protein conformation is important for eliciting neutralizing antibodies. |
