Dissecting the mechanisms of HIV resistance in vivo to broadly neutralizing antibodies

摘要 HIV-1包膜糖蛋白(Env)介导病毒进入宿主细胞，是唯一的中和靶点。 抗体。广谱中和抗体(BNAbs)靶向HIV-1env上高度保守的位置并中和 来自不同支系的各种不同的菌株。尽管如此，bNab免疫疗法旨在抑制 HIV-1复制有时会导致耐bNab的HIV-1毒株的发展，而HIV-1毒株与前 现有的bNab耐药可通过治疗前的预筛选来确定。因此，理解潜在的 BNab耐药机制对于bNAbs未来在免疫治疗和预防中的应用具有重要意义。 导致多重bNAbs抗性的机制和促进bNAbs逃逸的间接机制 不同的群体尤其令人关注公共卫生。 我们的研究旨在为不同水平的bNab耐药性提供重要的见解。在具体目标1中，我们 将研究对多种bNAbs具有耐药性的反弹HIV-1毒株的直接耐药机制。我们会 从bNab治疗的临床研究中筛选样本，确定表现出最高水平的HIV-1毒株的env 对几种bNAbs的抗性程度，研究Env序列、功能、糖基化模式并确定 原子水平分辨率下的抗性包络结构。我们的综合方法将提供独特的配置文件 整合了所有导致bNab抗性的潜在机制的选定的多个抗bNab的环境蛋白。在……里面 在一个平行的方向上，我们将研究反弹的HIV-1毒株通过细胞间传播的能力， 这允许在存在不同组的bNAb的情况下进行有效的病毒复制。我们将检验这一假设 这种对bNab敏感的HIV-1毒株即使在来自 RV397试验可通过细胞间传播有效传播。此外，我们还将研究分子 表现出更高的细胞间传输效率和对bNab抗性的菌株的机制。具体而言 目的2我们将确定最佳的bNab组合以克服bNab抗性，并使用抗体酵母展示 提高对bNab敏感和耐药HIV-1亲和力的重组bNAbs的生物工程技术 菌株。这一方法将使我们能够确认HIV-1对bNAbs的耐药性机制，并测试 假设bNAbs的特定变化可以改善bNab的广度，并允许靶向抗药性的子集 HIV-1毒株。 总体而言，我们的研究将提供高分辨率和全面的观点，对多重耐bNab的HIV-1环境，在 体内HIV-1耐药性的替代途径，以及克服bNab耐药性的可能方法。我们的 这些结果将为制定艾滋病毒-1免疫治疗和预防的新战略奠定坚实的基础 努力。

ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) target highly conserved sites on HIV-1 Envs and neutralize a wide range of diverse strains from different clades. Nevertheless, bnAb immunotherapy aiming to suppress HIV-1 replication sometimes leads to development of bnAb-resistant HIV-1 strains, and HIV-1 strains with pre- existing bnAb resistance can be identified by prescreening before treatment. Thus, understanding the underlying mechanisms of bnAb resistance are critical for the future application of bnAbs for immunotherapy and prevention. Mechanisms that lead to multi-bnAbs resistance and indirect mechanisms that facilitate escape of bnAbs from different groups are of particular public health concern. Our study is structured to provide important insights into bnAb resistance at different levels. In Specific Aim 1 we will study direct resistance mechanisms of rebounded HIV-1 strains that are resistant to multiple bnAbs. We will screen samples from clinical studies of bnAb therapy, identify Envs of HIV-1 strains that exhibit the highest degree of resistance to several bnAbs, study Env sequence, function, glycosylation patterns and determine the structures of resistant Envs at atomic level resolution. Our comprehensive approach will provide unique profiles of selected multi-bnAb resistant Envs that integrate all potential mechanisms contributing to bnAb resistance. In a parallel direction, we will study the ability of rebounded HIV-1 strains to spread through cell-cell transmission, which allows efficient viral replication in the presence of different groups of bnAbs. We will test the hypothesis that bnAb-sensitive HIV-1 strains that replicate despite high levels of bnAb in the serum of participants from the RV397 trial can efficiently spread by cell-cell transmission. Additionally, we will investigate the molecular mechanisms of strains that exhibit increased cell-cell transmission efficiency and bnAb resistance. In Specific Aim 2 we will define optimal bnAb combinations to overcome bnAb resistance and use antibody yeast display technology to bioengineer recombinant bnAbs with improved affinity against bnAb-sensitive and resistant HIV-1 strains. This approach will allow us to confirm mechanisms of HIV-1 resistance to bnAbs and to test the hypothesis that specific changes in bnAbs can improve bnAb breadth and allow targeting of a subset of resistant HIV-1 strains. Overall, our study will provide high-resolution and comprehensive view on multi-bnAb resistant HIV-1 Envs, on alternative pathways of HIV-1 resistance in vivo, and on potential approaches to overcome bnAb resistance. Our results will form a strong basis for the development of new strategies for HIV-1 immunotherapy and prevention efforts.