HIV-1 Entry

HIV-1进入

• 批准号: 6762713
• 负责人: Dimiter S Dimitrov
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6762713
• 关键词: AIDS vaccines; CD4 molecule; HIV envelope protein; HIV envelope protein gp120; antiAIDS agent; conformation; cytokine receptors; drug design /synthesis /production; genetic library; helper T lymphocyte; human immunodeficiency virus 1; human tissue; mathematical model; membrane fusion; receptor binding; structural biology; tissue /cell culture; vaccine development; virus infection mechanism; virus receptors

Human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), enters cells by binding its envelope glycoprotein (Env) to receptor molecules and fusing its membrane with the cell membrane. Understanding how receptor molecules mediate HIV entry is critical for elucidating the mechanisms of HIV infection, and design of new antiretroviral treatments and vaccines. We have proposed to identify novel broadly HIV neutralizing human monoclonal antibodies (nhmAbs) by screening antibody phage display libraries (PDLs) against HIV envelope glycoproteins (Envs) complexed with receptor molecules. Last year we reported the identification of a novel hmAb Fab (X5) selected for binding to purified JRFL gp120-CD4-CCR5 complexes by screening of a PDL from an HIV-1-infected long-term nonprogressor, and preliminary data of its characterization. This year we have been extensively characterizing X5, published an article in PNAS (attached), and NCI filed a patent application and is negotiating license agreements with three companies. Fab X5 bound with high (nM) affinity to a variety of Envs including primary isolates from different clades and Envs with deleted variable loops (V1,2,3). Its binding was significantly increased by CD4. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore X5 inhibited cell fusion mediated by Envs from R5, X4 and R5X4 viruses. Of the five broadly cross-reactive HIV nhmAbs X5 is the only one that exhibits increased binding to gp120 complexed with receptors. Our collaborator, Dr. Ji, and his associates have solved the crystal structure of X5 at 1.9 A resolution, and we are currently characterizing the structure of its epitope by site-directed mutagenesis and molecular docking of X5 to known structures of gp120 cores. We also developed a novel methodology based on alternating various Envs (gp140s) and their complexes with CD4 during panning of PDLs. By using this approach we selected four new potent broadly HIV nhmAb Fabs (M12,14,16,18) from a PDL that is different than the one used for selection of X5. Preliminary data suggest that these antibodies potently inhibit cell-cell fusion mediated by Envs of primary isolates from different clades. NCI filed a patent application for these antibodies. These findings suggest that X5, M12, M14, M16 and M18 could be possibly used as entry inhibitors alone or in combination with other antiretroviral drugs for the treatment of HIV-infected individuals, provide evidence for the existence of conserved receptor-induced gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-infected patients, and have important conceptual and practical implications for development of vaccines and inhibitors. The HIV-1 envelope glycoprotein (Env, gp120-gp41) undergoes a series of conformational changes initiated by its binding to receptor molecules. I hypothesized that some of the transient Env conformations on the pathway to entry can be exhibited and retained in fusion proteins of gp120 and gp41 joined by flexible linkers. Two Env89.6 fusion proteins with linkers of 14 and 26 amino acid residues inhibited 10-100-fold more potently entry and cell fusion mediated by Envs from R5 (ADA, JRFL, SF162, Bal), X4 (HXB2, NL4-3) and R5X4 (89.6, DH12) HIV-1 isolates than a fusion protein with a 4 amino acid residue linker obtained under the same conditions, gp120-gp41 without linker or gp120. The high inhibitory activity (50% at about 100 pM - higher than of any currently known HIV-1 entry inhibitor on molar basis) of these proteins against a variety of HIV-1 isolates, including primary isolates, may suggest the existence of broadly conserved structures that are critical for the entry process and can be exposed even in the absence of receptor-mediated activation thus opening new perspectives for elucidation of viral entry mechanisms, and development of inhibitors and vaccines.

人类免疫缺陷病毒 (HIV) 是获得性免疫缺陷综合征 (AIDS) 的病原体，通过将其包膜糖蛋白 (Env) 与受体分子结合并将其膜与细胞膜融合而进入细胞。了解受体分子如何介导 HIV 进入对于阐明 HIV 感染机制以及设计新的抗逆转录病毒治疗和疫苗至关重要。 我们建议通过针对与受体分子复合的 HIV 包膜糖蛋白 (Envs) 筛选抗体噬菌体展示文库 (PDL) 来鉴定新型广泛 HIV 中和性人单克隆抗体 (nhmAbs)。去年，我们报道了通过从 HIV-1 感染的长期非进展者中筛选 PDL 来鉴定一种新型 hmAb Fab (X5)，该 Fab 被选择用于与纯化的 JRFL gp120-CD4-CCR5 复合物结合，并提供了其表征的初步数据。今年我们对X5进行了广泛的描述，在PNAS上发表了一篇文章（见附件），NCI也提交了专利申请，并正在与三个公司谈判许可协议。 Fab X5 以高 (nM) 亲和力与多种 Env 结合，包括来自不同进化枝的初级分离株和具有删除的可变环 (V1,2,3) 的 Env。 CD4 显着增加其结合。 X5 通过选择来自 A、B、C、D 和 G 分支的代表性 HIV-1 初级分离株来抑制外周血单核细胞的感染，其效率与广泛中和抗体 IgG1 b12 相当。此外，X5 抑制 R5、X4 和 R5X4 病毒的 Envs 介导的细胞融合。在五种广泛交叉反应的 HIV nhmAb 中，X5 是唯一一种与受体复合的 gp120 结合力增强的抗体。我们的合作者季博士和他的同事已经以 1.9 A 的分辨率解析了 X5 的晶体结构，目前我们正在通过 X5 的定点诱变和与 gp120 核心已知结构的分子对接来表征其表位的结构。 我们还开发了一种基于在 PDL 淘选过程中交替使用各种 Env（gp140）及其与 CD4 的复合物的新颖方法。通过使用这种方法，我们从 PDL 中选择了四种新的有效的广泛 HIV nhmAb Fab (M12、14、16、18)，该 PDL 与用于选择 X5 的 PDL 不同。初步数据表明，这些抗体可有效抑制由来自不同进化枝的初级分离株的 Env 介导的细胞-细胞融合。 NCI 为这些抗体提交了专利申请。这些发现表明，X5、M12、M14、M16 和 M18 可能单独用作进入抑制剂或与其他抗逆转录病毒药物联合用于治疗 HIV 感染者，为存在保守的受体诱导的 gp120 表位提供了证据。可以作为艾滋病毒感染患者中有效的广泛交叉反应中和抗体的靶标，并对疫苗和抑制剂的开发具有重要的概念和实际意义。 HIV-1 包膜糖蛋白（Env，gp120-gp41）通过与受体分子的结合而引发一系列构象变化。我假设进入途径上的一些瞬时 Env 构象可以在由柔性接头连接的 gp120 和 gp41 融合蛋白中展示和保留。两个带有 14 和 26 个氨基酸残基接头的 Env89.6 融合蛋白可更有效地抑制来自 R5 (ADA、JRFL、SF162、Bal)、X4 (HXB2、NL4-3) 的 Env 介导的进入和细胞融合 10-100 倍和 R5X4 (89.6, DH12) HIV-1 分离物，而不是获得具有 4 个氨基酸残基接头的融合蛋白在相同条件下，没有连接子的gp120-gp41或gp120。这些蛋白质对多种 HIV-1 分离株（包括初级分离株）的高抑制活性（约 100 pM 时为 50%，以摩尔计高于任何目前已知的 HIV-1 进入抑制剂），可能表明存在广泛保守的对进入过程至关重要的结构，即使在没有受体介导的激活的情况下也可以暴露，从而为阐明病毒进入机制以及抑制剂和疫苗的开发开辟了新的视角。