Fusion peptide directed immunogens that elicit neutralizing and protective antibodies in non-human primates

摘要/项目摘要 HIV-1包膜糖蛋白（Env）是抗体介导的免疫应答的唯一靶标。对感染HIV的患者的研究已经导致针对Env表面上不同表位的抗体的分离，其能够中和广泛的循环HIV毒株，并提供针对非人灵长类动物（NHP）中的反复病毒挑战的被动保护。目前HIV疫苗研究的目标是创造能够在未感染的个体中引发类似抗体反应的免疫原。Env三聚体的可溶性胞外域的基于结构的工程化是大多数当前方法的核心。这种方法的一个主要复杂因素来自Env上的大抗原表面，其中许多脱靶和竞争性表位可用于免疫系统。针对“分散”表位的免疫应答的诱导导致针对感兴趣的靶向bnAb表位的减弱的和不一致的应答。NHP是这项工作的重要模型，因为野生型小鼠不会对在人类中观察到的主要Env nAb表位产生Ab应答。其中使用可溶性Env胞外域三聚体的NHP免疫中的多克隆Ab应答的分析揭示了一组高度免疫原性的表位，其具有发展保护性异源应答并且在某些情况下甚至自体应答的低潜力。这些表位包括Env三聚体胞外域的碱基、N241/N289聚糖孔和可变环（V1-V5）。为了解决这一缺点，我们已经开发了一种合理的方法来一致地引发针对保守融合肽（FP）表位的抗体，对于保守融合肽（FP）表位，存在人bnAb的几个实例。我们已经使用了我们的设计师三聚体和连续抗原释放程序，以产生初步结果，验证这一概念在NHP中的成功。表位靶向的可重复性是这里呈现的最令人印象深刻的初步结果，并且它表明可以容易地实现人中FP nAb的可重复诱导。在这里，我们建议进行一系列的实验，旨在通过最大限度地减少免疫干扰，脱靶表位的可及性，进一步优化这种免疫策略，并最终验证是否可以在NHP中可重复地引发保护性，FP靶向，bnAb。我们还打算用我们设计的SARS-CoV 2 RBD免疫原平行免疫一些动物。RBD免疫将在不同的位点（三角肌），并且不会干扰Env融合肽靶向免疫。这种双重用途的免疫方法通常对未来的HIV疫苗研究很有价值，我们将进行一项试点研究，以证明这种双重用途的免疫策略是有用的，这将对我们未来的HIV疫苗研究有价值。与HIV候选疫苗相比，SARS-CoV 2 RBD还将作为免疫学参数的阴性对照，这具有额外的价值。我们还将我们的免疫原掺入多价纳米颗粒中，以改善免疫原运输，增强B细胞受体亲合力，并阻断非生产性但高度免疫原性的基础表位。此外，我们将使用连续的抗原递送来改善生发中心反应的持续时间，并最终驱动产生能够中和多种HIV毒株的bnAb所必需的高水平体细胞超突变。该项目将通过用异源低剂量重复攻击免疫的NHP来测试我们的疫苗方案的功效而达到高潮。

Abstract/Project Summary HIV-1 envelope glycoprotein (Env) is the sole target for development of antibody-mediated immune response. Studies of patients infected with HIV have led to the isolation of antibodies against different epitopes on the Env surface, capable of neutralizing a broad range of circulating HIV strains and providing passive protection against repeated viral challenges in non-human primates (NHPs). The current goal of HIV vaccine research is to create immunogens capable of eliciting similar antibody responses in naïve individuals. Structure- based engineering of the soluble ectodomain of Env trimer is in the core of most of the current approaches. One major complicating factor with this approach comes from the large antigenic surface on the Env, where many off-target and competing epitopes are available to the immune system. The induction of immune responses against “distracting” epitopes leads to attenuated and inconsistent response against the targeted bnAb epitopes of interest. NHPs are an important model for this work, because wildtype mice do not make Ab responses to major Env nAb epitopes observed in humans. Analysis of the polyclonal Ab response in NHP immunizations where soluble Env ectodomain trimers were used, revealed a set of highly immunogenic epitopes with low potential for the development of protective heterologous, and in some cases even autologous, responses. These epitopes include the base of the Env trimer ectodomain, N241/N289 glycan hole and variable loops (V1-V5). To address this shortcoming we have developed a rational approach to consistently elicit antibodies against the conserved fusion peptide (FP) epitope, for which there are several examples of human bnAbs. We have used our designer trimers and continuous antigen release program to generate preliminary results verifying success of this concept in NHPs. The reproducibility of the epitope targeting is the most impressive preliminary result presented here, and it indicates that reproducible elicitation of FP nAbs in humans may be readily achievable. Here we propose to conduct a series of experiments designed to further optimize this immunization strategy by minimizing the accessibility of immuno-distracting, off-target epitopes, and ultimately verify if protective, FP-targeting, bnAbs can be reproducibly elicited in NHPs. We intend to also immunize some animals in parallel with a SARS-CoV2 RBD immunogen we have designed. The RBD immunizations will be at a different site (deltoid) and will not interfere with the Env fusion peptide targeted immunizations. This dual use immunization approach is generally valuable for future HIV vaccine studies, and we will perform a pilot study to show this type of dual use immunization strategy is informative, which will be of value to use in our future HIV vaccine studies. The SARS-CoV2 RBD will also serve as a negative control for immunological parameters compared to the HIV candidate vaccine, which is of additional value. We will also incorporate our immunogens into multi-valent nanoparticles to improve immunogen trafficking, enhance B cell receptor avidity, and block the non-productive but highly immunogenic base epitope. Further, we will use continuous antigen delivery to improve the duration of germinal center reactions, and ultimately drive the high levels of somatic hypermutation necessary for generating bnAbs capable of neutralizing a wide diversity of HIV strains. This project will culminate by testing the efficacy of our vaccine regimen by challenging immunized NHPs with heterologous low dose repeated challenge.