Nucleophilic Antibodies: Characterization and Induction

亲核抗体：表征和诱导

• 批准号: 8015976
• 负责人: Sudhir Paul
• 金额: $ 45.44万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015976
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Amino Acids; Antibodies; Antibody Specificity; Antigens; B-Lymphocytes; Binding; Binding Sites; Biochemical; CD4 Antigens; Cleaved cell; Complex; Crystallography; Dependence; Distant; Elements; Epitopes; HIV; HIV Envelope Protein gp120; Human; Immunization; Immunoglobulin A; In Vitro; Individual; Length; Libraries; Link; Lupus; Maps; Membrane; Monoclonal Antibodies; Mus; Pathway interactions; Patients; Peptides; Peripheral Blood Mononuclear Cell; Phage Display; Preparation; Property; Proteolysis; Reaction; Reagent; Receptors, Antigen, B-Cell; Recruitment Activity; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Superantigens; Testing; Vaccination; Viral; Virion; Virus; Water; analog; base; interest; monomer; neutralizing antibody; novel; peptide analog; response; stem; synthetic peptide

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.

描述（由申请人提供）：我们寻求：（a）表征将HIV GP120与不可逆性结合的抗体（ABS）的功能特性，并催化蛋白质的裂解，（b）研究这些活动的结构性基础，以及（c）研究中和的IgA响应用电磷酸化的中和IgA响应用电磷酸GP gp120诱导的中和IgA反应。通过用全长单体GP120的亲电类似物免疫来升高不可逆转的结合ABS，并识别两个保守的肽区域，其中一个是GP120的超抗原位点的一部分。蛋白水解ABS是自然存在的人类ABS，也识别超抗原肽区域。假设AB组合位点中的亲核氨基酸与GP120或共价反应中间体形成死端不可逆的复合物，该反应取决于辅助结构元素的存在，该反应中间会进入催化途径。由于病毒和单体GP120的结构不同，我们建议使用完整的病毒体作为底物对AB功能特性进行进一步研究。使用外周血单核细胞感染了多种原发性HIV分离株，将进行中和研究。结构研究将需要通过晶体学，生化图和定向诱变来鉴定亲核残基。晶体学还将确定亲核位点的其他成分，辅助催化结构的存在（例如，氧孔和水相互作用的残基）以及非共价结合触点是ABS的表位特异性。 ABS将以非配合的状态进行研究，并共价为其肽或肽模拟表位。为了研究IGAS裂解GP120的先天能力是否可以募集用于防御病毒，我们将尝试使用我们现有和新型的亲电GP120类似物来通过粘膜免疫来扩增中和中和，蛋白水解IGA。从这些研究中，我们希望确定与HIV疫苗接种和治疗相关的ABS和免疫原子的优势和劣势。