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

项目概要 产生广泛中和抗体（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) 的膜嵌入形式，无论其典型情况如何 疫苗使用可溶性、截短形式的 Env 作为不完美的替代品，更容易配制。我们有 正在开发一种 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 的部分实现是通过开发稳定、良好的 有序的 m-Env 和产生高产量的生产细胞。初步研究表明 异源 MEL 免疫引发了零星的 2 级交叉中和抗体反应。 跨膜 Env 亚基 gp41 的免疫原性在膜环境中尚未得到充分研究。与 MEL、m-Env gp41 的免疫原性现在可以通过分子精度进行研究，并且无需 与可溶性环境相关的新表位。在 SA1 中，我们将使用 MEL 对兔子进行免疫以确定如何 中和抗体反应受到 gp41 免疫聚焦策略的影响，包括产生 gp41“聚糖孔”，用基于膜近端的多价表位特异性免疫原增强 外部区域 (MPER) 和融合肽，并通过使用异源 Env 顺序增强。后来， 人类 Ig 位点敲入 (KI) 小鼠将使用 m-Env 进行免疫，包括那些与 我们证明新型 bnAb 的推断种系前体 (iGL) 具有直接与 MPER 结合的能力 并中和 HIV-1。在 SA2 中，我们将使用 B 细胞从中国捐赠者样本中分离 MPER/gp41 bnAb 排序和下一代测序生物信息学技术。我们还将询问 gp41- MEL 免疫的 hu Ig KI 小鼠中的特异性 B 细胞反应，以确定 MPER bnAb 样 iGL 是否存在 扩大了。在 SA3 中，我们将部分根据与 MPER bnAb 的亲和力筛选来自捐赠者的 Env， 同源前体以了解结合机制并产生新的免疫原。总体而言， 我们的目标是了解如何引出 bnAbs 到 gp41 上，以识别受到严密保护的，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.