Nucleophilic Antibodies: Characterization and Induction

描述(由申请人提供)：我们寻求：(A)表征与HIV gp120结合并催化蛋白质切割的抗体(Abs)的功能特性，(B)研究这些活性的结构基础，以及(C)研究亲电gp120类似物粘膜免疫诱导的中和IgA反应。用全长单体gp120的亲电类似物免疫可识别两个保守的多肽区域，其中一个是gp120超抗原部位的一段，形成不可逆结合抗体。蛋白水解性抗体是自然产生的人类抗体，也识别超抗原肽区域。假设抗体结合部位的亲核氨基酸与gp120或进入催化途径的共价反应中间体形成不可逆的末端络合物，这取决于辅助结构元素的存在。由于病毒和单体gp120的结构不同，我们建议以完整的病毒粒子为底物进一步研究抗体的功能性质。中和研究将使用感染了不同初级艾滋病毒分离株的外周血单核细胞进行。结构研究将需要通过结晶学、生化图谱和定点突变来鉴定亲核残基。结晶学还将鉴定亲核中心的其他成分，辅助催化结构(例如，氧阴离子空穴和水相互作用残基)的存在，以及与抗体表位特异性相关的非共价结合接触。这些抗体将在未连接状态下进行研究，并与其多肽或多肽类似表位进行共价络合。为了研究IGAs裂解gp120的先天能力是否可以被用来防御病毒，我们将尝试利用我们现有的和新的亲电gp120类似物，通过粘膜免疫来扩增中和、蛋白分解的IGAs的合成。从这些研究中，我们希望找出抗体和与艾滋病毒疫苗和治疗相关的免疫原的优点和缺点。

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.