HIV-1 vaccine design emphasizing bnAb targets on membrane Env liposomes

项目摘要 一种能激发广泛中和抗体（bnAb）的疫苗可以预防艾滋病毒/艾滋病。所有bnAb 必须作用于HIV-1包膜糖蛋白（m-Env）的膜包埋形式，然而典型的 疫苗使用可溶的截短形式的Env作为更容易配制的不完全替代物。我们有 一直在开发将纯化的m-Env以多价形式存在于脂质体上的m-Env疫苗策略， 我们称之为m-Env脂质体（MEL）。在某种程度上，MEL是通过开发稳定、良好的 有序的m-Env和高产的生产细胞。初步研究表明， 异源MEL免疫引发散发性2级交叉中和抗体应答。 跨膜Env亚基gp 41的免疫原性在膜环境中的研究不足。与 m-Env gp 41的免疫原性现在可以用分子精确度进行研究，而不需要 与可溶性Env相关的新表位在SA 1中，我们将使用MEL免疫兔子，以确定如何 中和抗体应答受gp 41免疫聚焦策略的影响，包括产生 gp 41“聚糖孔”，用基于膜近端的多价表位特异性免疫原加强 外部区（MPER）和融合肽，并通过用异源Env.后来， 人IG基因座敲入（KI）小鼠将用m-Env免疫，包括与免疫球蛋白紧密结合的那些。 我们发现，一种新型bnAb的推断生殖系前体（iGL）具有直接结合MPER的能力 中和HIV-1在SA 2中，我们将使用B细胞从中国供体样品中分离MPER/gp 41 bnAb 分类和下一代测序生物信息学技术。我们也会审问gp 41- MEL免疫的hu IG KI小鼠中特异性B细胞应答，以确定MPER bnAb样iGL是否 扩大在SA 3中，我们将部分基于对MPER bnAb的亲和力筛选来自供体的Env， 同源前体，以了解结合机制，并产生新的免疫原。总的来说， 我们的目标是了解如何诱导针对gp 41的bnAbs，这些bnAbs识别一个受到良好保护的，但尽管如此， HIV-1 Env刺突底部的脆弱表面。

Project Summary A vaccine that elicits broadly neutralizing antibodies (bnAb) could protect against HIV/AIDS. All bnAbs must act on the membrane embedded form of the HIV-1 envelope glycoprotein (m-Env), however typical vaccines use soluble, truncated forms of Env as an imperfect surrogate that is simpler to formulate. We have been developing a m-Env vaccine strategy that presents purified m-Env in multivalent form on liposomes, which we term, m-Env liposomes (MELs). MELs have been enabled in part by development of stable, well- ordered m-Env and producer cells that generate them in high yield. Preliminary Studies show that heterologous MEL immunization elicited sporadic tier 2 cross neutralizing antibody responses. Immunogenicity of the transmembrane Env subunit, gp41, is underexplored in the membrane context. With MELs, immunogenicity of m-Env gp41 can now be studied with molecular precision, and without the neoepitopes associated with soluble Env. In SA1, we will immunize rabbits using MELs to determine how neutralizing antibody responses are affected by gp41 immunofocusing strategies, including the creation of gp41 `glycan holes', boosting with multivalent epitope-specific immunogens based on membrane-proximal external region (MPER) and fusion peptide, and by boosting sequentially with heterologous Envs. Later, human Ig locus knockin (KI) mice will be immunized with m-Envs, including those that bind tightly to an inferred germline precursor (iGL) of a novel bnAb that we show has capacity to directly bind to the MPER and neutralize HIV-1. In SA2, we will isolate MPER/gp41 bnAbs from Chinese donors samples using B cell sorting and next generation sequencing bioinformatics techniques. We will also interrogate the gp41- specific B cell response in MEL immunized hu Ig KI mice to determine whether MPER bnAb-like iGLs expanded. In SA3, we will screen Envs from donors in part on the basis of affinity for MPER bnAb and cognate precursors to understand the mechanism of binding, and to generate novel immunogens. Overall, we aim to understand how to elicit bnAbs to gp41 that recognize a well guarded, but nevertheless vulnerable surface at the base of the HIV-1 Env spike.