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

诱导交叉中和抗体(CNAbs)抑制HIV不同临床分离株在环境中的进入 疫苗接种仍然是开发广泛有效疫苗的高度优先事项，但cNAbs的诱导对抗 交叉保守的环境决定因素一直难以捉摸。然而，就在最近，我们完成了一项重要的 这一过程的第一步。我们用天然柔性连接(NFL)三聚体脂质体免疫兔子 在高度保守的CD4结合位点(CD4bs)附近包含靶向N-糖链缺失，以更好地 激活B细胞对该区域的反应。N-葡聚糖屏障上的工程缝隙通过 异源顺序增压。我们在血清和纯化的免疫球蛋白中诱导了交叉中和活性 动物，并随后克隆了两个cNAb。与这项提案最相关的cNAb E70提供了以下证据： 一种根本不同的方法的原则。CD4bs导向的E70相对有效地对抗多层 2个分离物，重要的是，识别大约50%的N-糖链作为其表位的一部分以及邻近的 作为CD4b的一部分的保守多肽。(第二单抗1C2是一种广谱中和抗体(BNAb)， 中和40%隔离板的80%，并定向到gp41：gp120接口。)高分辨率低温电子显微镜 NFL天然类三聚体上E70表位的结构为如何训练 免疫系统识别CD4b保守蛋白表面附近的其他N-糖链参与介导 2级分离株的交叉中和。我们称这种方法为“N-葡聚糖诱饵”。对于这种方法，我们将 保留CD4b近端的一个选定的N-糖链作为“诱饵”，同时去除所有其他近端的N-糖链- 葡聚糖对最初的免疫原起作用。将在环上的6个单独的N-葡聚糖位置进行启动 保守的CD4bs蛋白表面。这允许给定的细胞受体识别包括 “锚定多聚糖”和周围的保守多肽。有了助推剂，所有的N-糖链最终都 以循序渐进的方式恢复，限制接近给定N-糖链诱饵的角度，以再生完整的N-糖链 糖链屏蔽的同时仍然驱动B细胞的子集作为嵌合体定向到原始锚链糖链和蛋白质， 非自身表位。这种策略与我们最初以受体结合为靶点的方法有根本的不同 完全删除所有近端的N-糖链。因此，这笔赠款的主要目标是产生新的 基于结构设计的HIV三聚体Env免疫原，在CD4b处含有N-糖链缺失，但 保留个别的“锚定糖链”。我们将首先通过以下方式表征N-葡聚糖锚定的启动免疫原 最先进的生物物理方法(目标1)。接下来，我们将在AIMS 2中确定小动物的免疫原性 包括新生儿在内的3例。在小动物体内靶向N-葡聚糖盾之后，我们将进行非 人类灵长类(NHP)免疫原性和挑战(目标4)。如果我们在N-葡聚糖诱饵上成功 CD4bs，我们可以将这种方法应用于HIV环境的其他部位，基本上将保护性的N-糖链 屏蔽病毒作为中和的目标，这通常是由艾滋病毒引起的bNAbs所做的。

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.