Uncleaved prefusion-optimized trimers on nanoparticles as HIV vaccines

项目摘要 人类免疫缺陷病毒1型（HIV-1）的包膜糖蛋白（Env）具有广泛的表位， 中和抗体（bNAbs）是HIV-1疫苗开发的主要目标。高分辨率结构 BG 505 SOSIP.664三聚体的研究为疫苗设计提供了合理的依据。虽然目前的GP 140设计 在优化后，可以为选定的菌株产生天然样三聚体，这是Env亚稳定性的根本原因 仍然是未知的，适用于所有HIV-1毒株和亚型的通用设计平台仍然是一个未实现的目标。 挑战.此外，尽管越来越多的人认识到病毒样颗粒（VLP）在治疗中的优势， 在HIV-1疫苗中尚未严格探索bNAb诱导、天然样三聚体的纳米颗粒展示 发展在R 01中，我们将通过结合三聚体设计，晶体学， 电子显微镜（EM），纳米颗粒展示，免疫，血清学测定，抗体分离，库 测序和抗体谱系分析。在目标1中，我们将研究HIV-1 Env的根本原因， 亚稳定性，并开发未切割的，融合前优化的（UFO）三聚体平台。我们假设gp 41 其包含具有无序N-末端弯曲（N-HR 1）的结构应变的七肽重复1（HR 1）， 亚稳定性的来源。我们已经设计了第一代UFO三聚体与修改N-HR 1和解决他们的问题。 结构.我们将开发一个优化的UFO平台，适用于不同的Env，具有高纯度，高产量， 稳定性和选择代表性UFO三聚体用于疫苗开发。我们将评估三聚体抗原性， 一组bNAb和非NAb，并确定它们的原子结构。在目标2中，我们将设计稳定的gp 140 作为疫苗候选物的三聚体呈递纳米颗粒。我们假设，自组装纳米粒子与 下面的3重轴可用于显示天然样GP 140三聚体。我们已经报道了gp 140纳米颗粒 基于24-mer铁蛋白（12 nm）和60-mer E2 p（23 nm）。我们将优化基于UFO gp 140纳米颗粒 铁蛋白E2 p和超稳定60聚体I3-01（25 nm），用于Aim 3中的体内测试。我们将验证纳米粒子 通过EM组装并使用一组抗体评估纳米颗粒抗原性。在目标3中，我们将评估 优化的UFO三聚体和纳米颗粒在WT小鼠、兔 和非人灵长类动物（NHP）。我们假设UFO三聚体可以在所有动物模型中诱导2级NAb， 而展示8-20个UFO三聚体的gp 140纳米颗粒可以比单独的三聚体更有效地引发二级NAb。 我们将首先在WT小鼠中筛选UFO三聚体和纳米颗粒，并使用纯化的抗体评估血清中和作用。 IgGs我们将选择一小部分免疫原用于兔的免疫，并提出最有前途的免疫原 NHP。在我们的初步研究中，gp 140纳米颗粒在小鼠中诱导了针对BG 505.T332N的自体2级NAb 和兔子在6-8周内，支持我们的方法。我们将分离疫苗诱导的2级NAb， 和结构研究，并进行下一代测序（NGS），以研究B细胞库和跟踪NAb 谱系发育我们的目标是为未来的临床研究确定最有效的候选疫苗。

Project Summary The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) harbors the epitopes of broadly neutralizing antibodies (bNAbs) and is the main target of HIV-1 vaccine development. High-resolution structures of BG505 SOSIP.664 trimers have provided a rational basis for vaccine design. Although current gp140 designs can produce native-like trimers for selected strains after optimization, the underlying causes of Env metastability are still unknown and a general design platform applicable to all HIV-1 strains and subtypes remains an unmet challenge. Furthermore, despite the increasing appreciation for the advantages of virus-like particles (VLPs) in bNAb elicitation, nanoparticulate display of native-like trimers has not been rigorously explored in HIV-1 vaccine development. In this R01, we will develop HIV-1 vaccine candidates by combining trimer design, crystallography, electron microscopy (EM), nanoparticle display, immunization, serological assays, antibody isolation, repertoire sequencing, and antibody lineage analysis. In Aim 1, we will investigate the fundamental causes of HIV-1 Env metastability and develop the uncleaved, prefusion-optimized (UFO) trimer platform. We hypothesize that gp41, which contains a structurally strained heptad repeat 1 (HR1) with a disordered N-terminal bend (N-HR1), is the source of metastability. We have designed 1st-generation UFO trimers with a modified N-HR1 and solved their structures. We will develop an optimized UFO platform applicable to diverse Envs with high purity, yield, and stability and select representative UFO trimers for vaccine development. We will assess trimer antigenicity using a panel of bNAbs and non-NAbs and determine their atomic structures. In Aim 2, we will engineer stable gp140 trimer-presenting nanoparticles as vaccine candidates. We hypothesize that self-assembling nanoparticles with underlying 3-fold axes can be used to display native-like gp140 trimers. We have reported gp140 nanoparticles based on the 24-mer ferritin (12 nm) and 60-mer E2p (23nm). We will optimize UFO gp140 nanoparticles based on ferritin, E2p, and a hyperstable 60-mer, I3-01 (25nm) for in vivo testing in Aim 3. We will validate nanoparticle assembly by EM and evaluate nanoparticle antigenicity using a panel of antibodies. In Aim 3, we will assess the immunogenicity and B-cell response for a subset of optimized UFO trimers and nanoparticles in WT mice, rabbits, and nonhuman primates (NHPs). We hypothesize that UFO trimers can induce tier-2 NAbs in all animal models, while gp140 nanoparticles displaying 8-20 UFO trimers can elicit tier-2 NAbs more effectively than trimers alone. We will first screen UFO trimers and nanoparticles in WT mice and assess serum neutralization using purified IgGs. We will select a small set of immunogens for rabbit immunization and advance the most promising ones to NHPs. In our preliminary study, gp140 nanoparticles induced autologous tier-2 NAbs to BG505.T332N in mice and rabbits within 6-8 weeks, supporting our approach. We will isolate vaccine-elicited tier-2 NAbs for functional and structural studies and perform next-generation sequencing (NGS) to study B-cell repertoires and trace NAb lineage development. Our goal is to identify the most effective vaccine candidates for future clinical studies.