Viral Immunology Core

病毒免疫学核心

• 批准号: 7904638
• 负责人: Tchavdar P. Kratchmarov
• 金额: $ 31.57万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904638
• 关键词: Acquired Immunodeficiency Syndrome; Adsorption; Affinity; Antibodies; Antibody Formation; Binding; Biological Assay; Cell Line; Cells; Chimeric Proteins; Collaborations; DNA; Data; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Epitopes; Evaluation; Fractionation; Generations; Genome; Goals; HIV-1; Human; Immunology; Infection; Knowledge; Laboratories; Learning; Maps; Molecular Biology; Monkeys; Monoclonal Antibodies; Mutate; Mutation; Patients; Peptides; Preparation; Procedures; Property; Protein Inhibition; Proteins; Publications; Reporting; Reproducibility; Research; Research Personnel; Role; Serum; Specificity; Standard Preparations; Structure; Teleconferences; Testing; Variant; Viral; Virus; Virus Diseases; Work; comparative; design; env Gene Products; env Genes; meetings; member; mutant; neutralizing antibody; novel strategies; pathogen; polyclonal antibody; programs; prophylactic; simian human immunodeficiency virus; therapeutic vaccine

The overall role of the Viral Immunology Core is to provide a standard platform to characterize the properties of new Env genes generated by this HIVRAD program and to evaluate the epitope specificity and neutralization breadth and potency of sera and monoclonal antibodies generated by the other projects. This will include the following activities: 1) Preparation of standard stocks of HIV-1 and SHIV viral supernatants for neutralization assays. Single-round replication-competent HIV-1 pseudotyped viruses and functional SHIV genomes will be generated by transfecting HEK293 cells. The titers of these viruses will be determined and stocks will be stored for use by this Core and provided to other members of the Program as needed. 2) Performing neutralization assays with human and monkey mAbs and pAbs generated by this program. These antibodies will be tested against parental and modified HIV-1 and SHIVs produced by the Molecular Biology Core. Envs neutralization breadth will be determined against panels of 24 tier 2 subtype B and C pseudoviruses. 3) Mapping the antibody binding epitopes in the envelope proteins of infected monkey sera and monoclonal antibodies. This will be done by performing: a) ELISAs to peptides, fusion proteins and rgp120 proteins, b) neutralization assays with existing (over 200) and newly constructed chimeric or mutant HIV-1 pseudoviruses, c) FACS analysis of cells transfected with selected Envs, d) fractionation of human and monkey sera by affinity adsorption on protein columns. The data generated by this Core will be evaluated and provided to Dr. Pinter and other co-investigators in this program in graphical and tabular forms and analytical reports. These data will be presented on internal teleconferences, scientific meetings and submitted for publications. The experimental procedures to be performed in this core have been routinely used in this laboratory for a number of years, as confirmed by an established record of publications.

病毒免疫学核心的总体作用是提供一个标准平台来表征病毒免疫学核心 HIVRAD 程序生成的新 Env 基因的特性，并评估表位特异性和 其他项目产生的血清和单克隆抗体的中和广度和效力。这 将包括以下活动： 1) 制备 HIV-1 和 SHIV 病毒上清液的标准储备液 用于中和测定。单轮复制能力的HIV-1假型病毒和功能 SHIV 基因组将通过转染 HEK293 细胞产生。这些病毒的滴度将被确定 库存将被存储以供该核心使用，并根据需要提供给该计划的其他成员。 2） 使用该程序生成的人和猴 mAb 和 pAb 进行中和测定。 这些抗体将针对亲本和修饰的 HIV-1 和 SHIV 进行测试，这些抗体是由分子 生物学核心。 Envs 中和宽度将根据 24 个 2 级亚型 B 和 C 的面板确定 假病毒。 3) 绘制受感染猴血清包膜蛋白中抗体结合表位的图谱 和单克隆抗体。这将通过执行以下操作来完成：a) 对肽、融合蛋白和 rgp120 蛋白，b) 使用现有（超过 200 个）和新构建的嵌合体或突变体进行中和测定 HIV-1 假病毒，c) 对用选定的 Env 转染的细胞进行 FACS 分析，d) 人类细胞的分级分离 和猴血清通过蛋白质柱上的亲和吸附。该Core生成的数据将是 评估并以图形和表格形式提供给品特博士和本项目的其他共同研究人员 和分析报告。这些数据将在内部电话会议、科学会议和 提交出版物。在此核心中执行的实验程序已按惯例进行 正如既定的出版物记录所证实的那样，该实验室已使用该实验室多年。