NOVEL MONOCLONAL ANTIBODIES FOR INVESTIGATING GP120

描述（改编自申请人摘要）：HIV-1感染 仍然是儿童和成人死亡的主要原因 在全世界都有。 尽管抗逆转录病毒疗法最近取得了进展， 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疫苗未能引起 广泛反应的抗体，中和大量的初级 分离株 此外，迄今为止制备的单克隆抗体（mAb）很少 中和HIV-1的主要分离株，以及少数广泛中和HIV-1的分离株， 中和对两个主要分离物的利用几乎没有影响， 关键的HIV-1共受体，CXCR4或CCR5。 然而，来自高度 选定的长期非进展者广泛中和原发分离株， 比任何可用的mAb更有效。 申请人假设这些 血清含有可结合构象决定簇的抗体 HIV-1利用CD4-gp120复合物与CCR5结合， 共受体 研究人员提出，这些特定的表位可以是 通过克隆细胞， 从其血清显示如此广泛中和的个体产生mAb 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，进一步定义 结合位点在合理设计有效的HIV-1 免疫原 为了检验上述假设，将对血清进行HIV-1长期筛查， 非进展者（LTNP），用于中和标准品中的R5分离株 外周血单核细胞（PBMC）测定。 这些血清也将是 测试阻断表达CD4/CCR5的细胞感染的能力 R5分离株。 血清滴度最高的三个病人 将选择具有这种活性的噬菌体用于骨髓活检， 构建抗体库（PDL）。 PDL将是 依次淘选多种R5 gp120/CD4复合物， 选择广泛中和抗体。 消极的淘选策略 将使用针对T细胞系嗜性gp120和线性V3决定簇 以除去不太感兴趣的结合抗体。 100人产生的抗体 然后使用中和试验和CCR5筛选选定的克隆 结合测定。 最好的25种抗体将被仔细定义， 中和的宽度和程度以及它们被突变成 生产具有更强中和能力的试剂。 这些抗体 将用于定义R5的结构，生物学和遗传特征 主要分离株，是至关重要的，在case-CCR 5的相互作用， 开发有效的免疫原。

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.