Nucleophilic Antibodies: Characterization and Induction

描述（由申请人提供）：我们寻求：（a）表征以不可逆特征结合HIV gp120并催化蛋白质裂解的抗体（Ab）的功能特性，（B）研究这些活性的结构基础，和（c）研究由亲电子gp120类似物粘膜免疫诱导的中和伊加应答。通过用全长单体gp120的亲电类似物免疫产生不可逆结合Ab，并且识别两个保守肽区，其中一个是gp120的超抗原位点的区段。蛋白水解Ab是也识别超抗原肽区的天然存在的人Ab。在抗体结合位点的亲核氨基酸被假设为形成死端不可逆复合物与gp120或共价反应中间体，进入催化途径，这取决于存在的辅助结构元件。由于病毒和单体gp120的结构不同，我们建议使用完整的病毒粒子作为底物进一步研究抗体的功能特性。将使用感染不同原代HIV分离株的外周血单核细胞进行中和研究。结构研究将需要通过晶体学、生物化学作图和定点诱变鉴定亲核残基。结晶学还将鉴定亲核位点的其它成分、辅助催化结构的存在（例如，含氧阴离子空穴和水相互作用残基），以及非共价结合接触，其响应Ab的表位特异性。将在未配体状态下研究Ab，并与其肽或肽类似物表位共价复合。为了研究IgA切割gp120的先天能力是否可以被招募用于防御病毒，我们将尝试使用我们现有的和新的亲电子gp120类似物通过粘膜免疫来扩增中和、蛋白水解IgA的合成。通过这些研究，我们希望确定与HIV疫苗接种和治疗相关的抗体和免疫原的优势和劣势。

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.