The HIV-1 envelope (env) glycoprotein (gp120-gp41) plays critical roles during virus entry into the target cell mediating binding to cellular receptors and the fusion of viral and cellular membranes. The biologically relevant form of env is a heavily glycosylated oligomeric complex. The location of th eenv on the viral membrane renders it an important target for vaccine development. Indeed, it is the only viral component accessible to antibodies and is the viral antigen to which neutralizing antibodies are directed. Unfortunately, most neutralizing antibodies to env exhibit limited cross-reactivity with other HIV-1 isolates, notaly, primary isolates. However, that much more broadly neutralizing antibodies to conformation- dependent epitopes canbe developed has been indicated, and an effective vaccine should elicit potent, broadly neutralizing antibodies to a number of epitopes. Thus, it is important to understand the determinants in env that stimulate the production of antibodies that are broadly reactive. We have found that immunizationwithsoluble, oligomeric env (BH8) effectively generates monoclonal antibodies (Mabs) that are broadly cross- reactive. The majority of these Mabs reacted with conformational epitopes, many reacting preferentially or exclusively with env oligomer. Several novel neutralizing Mabs to oligomer dependent, and specific,epitopes in the fp41 ectodomain were also identified. These studies have indicated the importance of investigating the oligomeric antigenic structure of env from primary isolates. In addition, evaluation of glycosylation contribution in epitope masking on oligomeric env will be important in determining if simple changes can be made to enhance cross-reactive neutralizing antibody production. One way neutralizing antibodies could work is to perturb some betastable property of env required in fusion. This kind of activity could inactivate virus andplay a role in preventing cell-cell transmission. It is like that these antibodies willreact with epitopes that are dependent on oligomeric structure andreact with highly conserved determinants. The overall goal of this project is to fully evaluate the consequences of env oligomerization and glycosylationon antigenic structure in primary isolates, and to understand how oligomeric env modulates during interactions with cellular receptors. Specifically, we will: 1) Develop and characterize soluble oligomeric and glycosylation-altered envs from primary HIV-1 isolates; 2) Assess the effects of oligomerization and glycosylationon antigenic structure in small animals, developing Mabs, and identify highly conserved and neutralizing epitopes; and 3) Characterize the env-mediated fusion process using a novel video fluorescence microscopy technique - monitoring receptor-induced conformation changes in situ in env, analyzing these changes with a variety of exisitng and newly developed Mabs as probes, and identify important functional domains as env interacts with cellular receptors.
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