ANTIGENICITIES OF AIDS VIRUS RECOMBINANT PROTEINS

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.