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

项目摘要 一种能产生广谱中和抗体(BNab)的疫苗可以预防艾滋病毒/艾滋病。所有bNAb 必须作用于包埋在膜上的HIV-1包膜糖蛋白(m-env)，不管它有多典型 疫苗使用可溶的、截短形式的Env作为一种不完美的替代品，更容易配制。我们有 一直在开发一种m-env疫苗策略，将纯化的m-env以多价形式呈现在脂质体上， 我们称之为m-Env脂质体(MEL)。MEL在一定程度上是由于开发了稳定、良好的 有序的m-Env和生产细胞，以高产量产生它们。初步研究表明， 异种MEL免疫可引起零星的第2层交叉中和抗体反应。 跨膜Env亚单位gp41的免疫原性在膜的背景下还没有得到充分的研究。使用 Mels，m-Env gp41的免疫原性现在可以在分子上精确地研究，而不需要 与可溶性环境蛋白相关的新表位。在SA1中，我们将使用MEL来免疫兔子，以确定如何 中和抗体反应受gp41免疫聚焦策略的影响，包括创建 Gp41‘葡聚糖孔’，以膜-近端为基础的多价表位特异性免疫原增强 外区(MPER)和融合肽，并通过与异源env的顺序启动。后来, 人免疫球蛋白基因敲门(KI)小鼠将用m-env免疫，包括那些与A 推测一种新的bNab的生殖系前体(IGL)具有直接与MPER结合的能力 并中和HIV-1病毒。在SA2中，我们将使用B细胞从中国献血者样本中分离MPER/gp41 bNAbs 分类和下一代测序生物信息学技术。我们还将审问gp41- MEL免疫人免疫小鼠特异性B细胞应答以确定MPER bNab样IgS 扩大了。在SA3中，我们将部分基于对MPER bNab和MPER的亲和力来筛选来自捐赠者的env 了解同源前体结合的机制，并产生新的免疫原。总的来说， 我们的目标是了解如何诱导gp41的bNAb识别戒备森严的 HIV-1环境病毒尖峰底部的易受攻击的表面。

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.