Nucleophilic Antibodies: Characterization and Induction

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DESCRIPTION (provided by applicant): We seek to: (a) characterize the functional properties of antibodies (Abs) that bind HIV gp120 with irreversible character and catalyze the cleavage of the protein, (b) study the structural basis of these activities, and (c) study the neutralizing IgA responses induced by mucosal immunization with electrophilic gp120 analogs. The irreversibly binding Abs were raised by immunization with an electrophilic analog of full- length monomer gp120 and recognize two conserved peptide regions, one of which is a segment of the superantigenic site of gp120. The proteolytic Abs are naturally occurring human Abs that also recognize the superantigenic peptide region. Nucleophilic amino acids in the Ab combining sites are hypothesized to form dead-end irreversible complexes with gp120 or a covalent reaction intermediate that proceeds into the catalytic pathway, depending on the presence of accessory structural elements. As the structures of viral and monomer gp120 are different, we propose further study of Ab functional properties using intact virions as substrates. Neutralization studies will be done using peripheral blood mononuclear cells infected with diverse primary HIV isolates. Structural studies will entail identification of nucleophilic residues by crystallography, biochemical mapping and site-directed mutagenesis. Crystallography will also identify additional constituents of the nucleophilic site, the presence of the accessory catalytic structures (e.g., the oxyanion hole and water interacting residues), and the noncovalent binding contacts responsibe for the epitope specificity of the Abs. The Abs will be studied in unliganded state and complexed covalently to their peptide or peptide analog epitopes. To study whether the innate ability of IgAs to cleave gp120 can be recruited for defense against the virus, we will attempt to amplify the synthesis of neutralizing, proteolytic IgAs by mucosal immunization using our existing and novel electrophilic gp120 analogs. From these studies, we hope to identify the strengths and weaknesses of the Abs and immunogens relevant to HIV vaccination and therapy.