Nucleophilic Antibodies: Characterization and Induction

描述（由申请人提供）：我们寻求：(a)表征具有不可逆特征的结合HIV gp120并催化蛋白质裂解的抗体（Abs）的功能特性，(b)研究这些活性的结构基础，以及(c)研究亲电性gp120类似物在粘膜免疫诱导的中和性IgA反应。不可逆结合的抗体通过全长单体gp120的亲电类似物免疫培养，并识别两个保守的肽区，其中一个是gp120的超抗原位点的一段。蛋白水解抗体是自然产生的人抗体，也能识别超抗原肽区。假设Ab结合位点的亲核氨基酸与gp120或共价反应中间体形成死端不可逆复合物，并进入催化途径，这取决于附属结构元件的存在。由于病毒和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Ab的功能特性。中和研究将使用感染不同原发HIV分离株的外周血单个核细胞进行。结构研究将需要通过晶体学、生化制图和定点诱变来鉴定亲核残基。晶体学还将识别亲核位点的其他成分，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，以及负责抗体表位特异性的非共价结合接触。抗体将在非配体状态下进行研究，并与它们的肽或肽类似表位共价络合。为了研究IgAs切割gp120的先天能力是否可以被招募来防御病毒，我们将尝试使用我们现有的和新的亲电性gp120类似物通过粘膜免疫来扩增中和性蛋白水解IgAs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.