Although considerable progress has been made towards the characterization of functional regions of HIV env proteins and definition of major neutralizing epitopes for laboratory strains of HIV, much less is known about mechanisms of infection and neutralization of clinical isolates. Such information is essential in order to develop an effective vaccine or passive immunotherapy for HIV infection. We will explore the basis for the restricted replication of T cell-tropic and macrophage-tropic viruses in cell lines, PBMCs and macrophages using molecularly cloned strains of HIV- 1 with well-characterized tropisms. The stage in the viral life cycle blocked in nonpermissive cells will be identified, the effect of host cell on the biological properties of virions will be determined, and the specific PBMC targets of T cell-tropic and macrophage-tropic viruses will be characterized. The incorporation of cellular antigens into virions grown in different cell types will be measured, and the neutralizing activities of antibodies to such antigens or to other antigens that have been implicated as accessory receptors will be assayed for different cell types. The sensitivity of cloned primary HIV-1 isolates to neutralization by a variety of MAbs directed against neutralization epitopes in multiple domains of gp120 will be determined. Neutralization assays will be performed in primary blood cells and in various cell lines, and the effects of host and target cell on neutralization sensitivity will be determined. Synergistic neutralization by multiple combinations of MAbs, and the frequency and mechanism of virus escape from multiple MAbs will be determined. Mechanisms of neutralization will be studied, using specific assays for virus binding, fusion, and DNA and protein synthesis. The kinetics of neutralization, the effect of incubation temperature on neutralization and efficiency of gp120 shedding, and the ability of MAbs to different epitopes to induce conformational rearrangements of gp120/gp41 will be determined. Antibodies in human sera that recognize conserved conformational V1/V2 epitopes will be purified and their breadth of reactivity and neutralization potency for laboratory strains and primary HIV isolates determined. These experiments should identify combinations of antibodies and other agents capable of achieving more effective neutralization of clinical isolates of HIV. Recent evidence indicates that the V2 region of gpI20 is a determinant of macrophage tropism and syncytium-inducing activity and that the V1/V2 domain contains potent neutralization epitopes that may be important for protection in vivo. We have recently found that the isolated V1/V2 domain exists as multiple glycoforms and conformeric forms, and have identified sites important for correct folding of this domain. The potential roles of these diverse structural forms on the functions of V1/V2 and gp120 will be explored. Genetic approaches will be utilized to investigate the effects of mutations at sites in V1 and V2 known to be important for expression of conformational epitopes on viral infectivity and tropism. Finally, we will purify homogeneous forms of native V1/V2 and V3 domains in sufficient amounts to allow crystallization studies and spectroscopic analyses to be performed.
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