The objective is to understand how the immune system attempts to defend against the disease AIDS, particularly the humoral responses of pre-AIDS (ARC) and AIDS patients against various AIDS viral antigens. The antigenicities of the two most diverse isolates, HTLV-III and ARV-2, will also be compared to determine common antigenic determinants. We propose to clone and express various HTLV-III and ARV-2 antigen genes in E. coli. Bacterial expression vectors using the lac and pR promoters will be used for antigen expressions. Western blot analysis and radioimmunoassays will be used to monitor expression and antigenities. Clones expressing antigenic proteins will be characterized further. Shorter derivatives of these clones will be generated by using restriction enzymes to remove different fragments of the cloned insert. Analysis of these deletion clones will then locate segments of the viral genes that encode the antigenic determinants (epitopes). In vitro will be used to modify individual amino acid within the epitope so that the role of each amino acid in conferring antigenicity will be determined. Patients' antibodies towards individual epitope will be studied immunochemically in terms of specificities, heterogeneity and ability to neutralize viral infectivity. Bacterial expressed antigens will be purified by affinity chromatography using patients' antibodies to absorb the recombinant antigens. Mouse antibody response towards these purified antigens will be compared to that of a typical immune reesponse to the viral antigens in humans. The diversity as well as specificities of the response will be studied by generating monoclonal antibodies. Their ability to neutralize viral infectivity will be studied as well. This work will lead to a better understanding of AIDS virus biology, the viral gene products and identification of the major and conserved antigenic determinants expressed in patients.
