Infection by HIV-1 continues to be a leading cause of death among children and adults throughout the world. Despite recent advances in anti-retroviral therapy, ultimate control of infection will depend upon development of effective vaccines. However, currently available HIV-1 vaccines have failed to elicit broadly reactive antibodies that neutralize significant numbers of primary isolates. In addition, monoclonal antibodies (mAbs) made to date rarely neutralize primary isolates of HIV-1, and the few which do broadly neutralize have little effect on the primary isolate utilization of two of the key HIV-1 co-receptors, either CXCR4 or CCR5. However, sera from highly selected long-term non-progressors broadly neutralize primary isolates more effectively than any available mAb. The applicants hypothesize that these sera contain antibodies that can bind to the conformational determinant(s) on the CD4-gp120 complex that HIV-1 utilizes to bind to the CCR5 co-receptor. The investigators propose that these specific epitopes can be best defined both structurally and genetically by cloning the cells producing mAbs from individuals whose sera show such broad neutralization capability. Since clinical data reveals that this co-receptor is essentially required for infection with HIV-1, further definition of this binding site will be critical in the rational design of an effective HIV-1 immunogen. To test the above hypothesis, sera will be screened from HIV-1 long-term non-progressors (LTNP) for neutralization of R5 isolates in a standard peripheral blood mononuclear cell (PBMC) assay. These sera also will be tested for the ability to block the infection of CD4/CCR5-expressing cell lines by R5 isolates. The three patients whose sera have the highest titers of such activity will be selected for bone marrow biopsy, and a single phage display antibody library (PDL) will be constructed. The PDL will be sequentially panned against multiple R5 gp120/CD4 complexes to positively select for broadly neutralizing antibodies. Negative panning strategies against T cell line tropic gp120 and linear V3 determinants will be employed to remove binding antibodies of lesser interest. Antibodies produced by 100 selected clones then will be screened using neutralization assays and CCR5 binding assays. The best 25 antibodies will be defined carefully for their breadth and degree of neutralization and their ability to be mutated to produce reagents with greater neutralizing capability. These antibodies will be used to define structural, biologic, and genetic features of R5 primary isolates that are critical in the envelope-CCR5 interaction and in the development of an effective immunogen.
