N-glycan baiting to target the highly effective HIV Env shield

诱导交叉中和抗体（cNAb），以抑制Env后不同临床HIV分离株的进入 疫苗接种仍然是开发广泛有效的疫苗的高度优先事项，但是针对 交叉保守的Env决定簇是难以捉摸的。然而，最近我们完成了一项重要的 在这个过程中的第一步。我们用天然柔性连接（NFL）三聚体脂质体免疫家兔 含有高度保守的CD 4结合位点（CD 4 bs）附近的靶向N-聚糖缺失， 激活对该区域的B细胞反应。N-聚糖屏障中的工程化缺口逐渐恢复， 异源连续加强。我们在血清中引发了交叉中和活性，并纯化了选定的IgG。 动物，并随后克隆了两个cNAb。与该提案最相关的cNAb E70提供了证据， 一个从根本上不同的方法的原则。CD 4 bs导向的E70相对有效地对抗多层次的免疫缺陷。 2分离株，重要的是，识别约50%的N-聚糖作为其表位的一部分，以及相邻的 保守的多肽是CD 4 bs的一部分。(The 2nd mAb，1C 2，是一种广泛中和抗体（bNAb）， 中和80%的40个分离物组，并被引导至GP 41：GP 120界面）。高分辨率冷冻电镜 NFL天然样三聚体上E70表位的结构提供了关于如何训练E70表位的关键初步数据。 免疫系统识别邻近CD 4 bs的保守蛋白表面的其他N-聚糖，以介导 2级分离株的交叉中和。我们称这种方法为“N-聚糖诱饵”。对于这种方法，我们将 保留一个选定的邻近CD 4 bs的N-聚糖作为“诱饵”，同时去除所有其他邻近的N-聚糖， 在初始引发免疫原上的聚糖。将在6个单独的N-聚糖位点进行预充， 保守的CD 4 bs蛋白表面。这允许给定的细胞受体识别包含在细胞中的嵌合表位。 “锚聚糖”和周围的保守多肽。随着增强，所有N-聚糖最终都 以逐步的方式恢复，限制接近给定N-聚糖诱饵的角度，以再生完整的N-聚糖。 聚糖屏蔽，同时仍然驱动导向原始锚聚糖和蛋白质作为嵌合体的B细胞亚群， 非自身表位。这种策略与我们最初的靶向受体结合的方法有根本的不同。 通过完全缺失所有近端N-聚糖来定位。因此，该补助金的主要目标是产生新的 HIV三聚体Env免疫原通过基于结构的设计，在CD 4 bs处含有N-聚糖缺失，但 保留单个“锚聚糖”。我们将首先通过以下方式表征N-聚糖锚定的引发免疫原： 最先进的生物物理学方法（目标1）。接下来，我们将在目的2中确定小动物的免疫原性 和3，包括新生儿。在小动物中靶向N-聚糖屏障后，我们将进行非- 人灵长类动物（NHP）免疫原性和攻击（目的4）。如果我们成功地在 CD 4 bs，我们可以将这种方法应用于HIV Env上的其他位点，基本上将保护性N-聚糖 作为中和靶点的抗病毒屏障，这通常由显著的HIV引发的bNAb完成。

Elicitation of cross-neutralizing Abs (cNAbs) to inhibit entry of diverse clinical HIV isolates following Env vaccination remains a high priority to develop a broadly effective vaccine, but the elicitation of cNAbs against cross-conserved Env determinants has been elusive. However, very recently we accomplished an important initial step in this process. We primed rabbits with immunization of native flexibly linked (NFL) trimer-liposomes containing targeted N-glycan deletions proximal to the highly conserved CD4 binding site (CD4bs) to better activate B cell responses to this region. The engineered gaps in the N-glycan shield were gradually restored by heterologous sequential boosting. We elicited cross-neutralizing activity in the serum and purified IgG of selected animals and, subsequently, cloned two cNAbs. The cNAb most relevant to this proposal, E70, provides proof-of- principle for a fundamentally different approach. The CD4bs-directed E70 is relatively potent against multiple tier 2 isolates and, importantly, recognizes approximately 50% of N-glycan as part of its epitope as well as adjacent conserved polypeptide that is part of the CD4bs. (The 2nd mAb, 1C2, is a broadly neutralizing antibody (bNAb), neutralizing 80% of a 40 isolate panel and is directed to the gp41:gp120 interface.) The high-resolution cryoEM structure of the E70 epitope on the NFL native-like trimer provides critical preliminary data on how to train the immune system to recognize other N-glycans proximal to the conserved protein surface of the CD4bs to mediate cross-neutralization of tier 2 isolates. We call this approach “N-glycan baiting”. For this approach, we will leave one selected N-glycan proximal to the CD4bs intact as “the bait” while removing all other proximal N- glycans on the initial priming immunogens. The priming will be performed at 6 individual N-glycan sites that ring the conserved CD4bs protein surface. This allows a given cell receptor to recognize chimeric epitopes comprised of the “anchor glycan” and surrounding conserved polypeptide. With boosting, all N-glycans are eventually restored in a step-wise manner, limiting angles of approach to a given N-glycan bait to regenerate the intact N- glycan shield while still driving a subset of B cells directed to the original anchor glycan and protein as a chimeric, non-self-epitope. This strategy fundamentally differs from our original approach in targeting the receptor binding site by full deletion of all proximal N-glycans. Accordingly, the major objective of this grant is to generate novel HIV trimeric Env immunogens by structure-based design, containing N-glycan deletions at the CD4bs but to retain individual “anchor glycans”. We will first characterize the N-glycan-anchored priming immunogens by state-of-the-art biophysical methods (Aim 1). Next, we will determine immunogenicity in small animals in Aims 2 and 3, including neonates. Following targeting of the N-glycan shield in small animals, we will perform non- human primate (NHP) immunogenicity and challenge (Aim 4). If we are successful at N-glycan baiting at the CD4bs, we can apply this approach to other sites on the HIV Env, essentially turning the protective N-glycan shield against the virus as a target of neutralization as is often done by the remarkable HIV-elicited bNAbs.