NOVEL MONOCLONAL ANTIBODIES FOR INVESTIGATING GP120

描述(改编自申请人的摘要)：感染艾滋病毒-1 仍然是儿童和成人死亡的主要原因 在世界各地。尽管最近在抗逆转录病毒治疗方面取得了进展， 感染的最终控制将取决于开发有效的 疫苗。然而，目前可用的HIV-1疫苗未能诱导 广泛反应的抗体，中和大量的原发 分离株。此外，迄今为止制造的单抗很少。 中和HIV-1的主要分离物，以及少数广泛起作用的分离物 中和剂对两个菌株的初生菌株利用率影响不大 关键的HIV-1共受体，CXCR4或CCR5。然而，来自高水平的血清 选定的长期非进展者广泛中和初级分离株 比任何可用的单抗都有效。申请者假设这些 血清中含有能与构象决定簇结合的抗体(S) HIV-1与CCR5结合的CD4-gp120复合体 共同受体。研究人员提出，这些特定的表位可以是 通过克隆细胞在结构和遗传上都得到了最好的定义 从血清表现出如此广泛的中和性的个体中生产单抗 能力。由于临床数据显示，这种辅助受体是 感染HIV-1所必需的，对此的进一步定义 结合部位将是合理设计有效的HIV-1病毒的关键 免疫基因。 为了验证上述假设，将对长期感染HIV-1的血清进行筛查 标准中和R5分离株的非进化子(LTNP) 外周血单个核细胞(PBMC)测定。这些血清也将是 检测对表达CD4/CCR5的细胞的感染阻断能力 用R5分离物进行筛选。血清滴度最高的三名患者 将这种活性的单噬菌体选作骨髓活组织检查 将构建展示抗体库(PDL)。PDL将是 先后对多个R5 gp120/CD4复合体进行了摇摄，以确定 选择此选项可广泛中和抗体。消极的淘金策略 针对T细胞系的趋化gp120和线性V3决定簇 去除不太感兴趣的结合抗体。100产生的抗体 然后将使用中和试验和CCR5对选定的克隆进行筛选 有约束力的分析。最好的25种抗体将被仔细定义为他们的 中和的广度和程度以及它们被突变为 生产中和能力更强的试剂。这些抗体 将用于定义R5的结构、生物和遗传特征 在外膜-CCR5相互作用中和在 一种有效免疫原的开发。

DESCRIPTION (Adapted from the applicant's abstract): Infection by HIV-1 continues to be a leading cause of death among children and adults throughout the world. Despite recent advances in anti-retroviral therapy, ultimate control of infection will depend upon development of effective vaccines. However, currently available HIV-1 vaccines have failed to elicit broadly reactive antibodies that neutralize significant numbers of primary isolates. In addition, monoclonal antibodies (mAbs) made to date rarely neutralize primary isolates of HIV-1, and the few which do broadly neutralize have little effect on the primary isolate utilization of two of the key HIV-1 co-receptors, either CXCR4 or CCR5. However, sera from highly selected long-term non-progressors broadly neutralize primary isolates more effectively than any available mAb. The applicants hypothesize that these sera contain antibodies that can bind to the conformational determinant(s) on the CD4-gp120 complex that HIV-1 utilizes to bind to the CCR5 co-receptor. The investigators propose that these specific epitopes can be best defined both structurally and genetically by cloning the cells producing mAbs from individuals whose sera show such broad neutralization capability. Since clinical data reveals that this co-receptor is essentially required for infection with HIV-1, further definition of this binding site will be critical in the rational design of an effective HIV-1 immunogen. To test the above hypothesis, sera will be screened from HIV-1 long-term non-progressors (LTNP) for neutralization of R5 isolates in a standard peripheral blood mononuclear cell (PBMC) assay. These sera also will be tested for the ability to block the infection of CD4/CCR5-expressing cell lines by R5 isolates. The three patients whose sera have the highest titers of such activity will be selected for bone marrow biopsy, and a single phage display antibody library (PDL) will be constructed. The PDL will be sequentially panned against multiple R5 gp120/CD4 complexes to positively select for broadly neutralizing antibodies. Negative panning strategies against T cell line tropic gp120 and linear V3 determinants will be employed to remove binding antibodies of lesser interest. Antibodies produced by 100 selected clones then will be screened using neutralization assays and CCR5 binding assays. The best 25 antibodies will be defined carefully for their breadth and degree of neutralization and their ability to be mutated to produce reagents with greater neutralizing capability. These antibodies will be used to define structural, biologic, and genetic features of R5 primary isolates that are critical in the envelope-CCR5 interaction and in the development of an effective immunogen.