Project 1. Optimization and in vivo evaluation of HIV-1 Env trimer sortase A-conjugated nanoparticles

摘要-项目1 以前的疫苗无法在人体中引发HIV-1广泛中和抗体（bnAb）。两 这种失败的原因是bnAb前体B细胞通常很罕见， 稀有的B细胞需要广泛的体细胞突变以进化成高亲和力bnAb。这些体细胞的一个子集 突变--称为不可能突变--由体细胞突变产生的概率很低 由于密码子偏好性和体细胞突变识别基序的定位，我们的核心假设 引发bnAb的关键是免疫原必须对Ab具有更高的亲和力， 相比于缺乏这些关键突变的Ab。此外，我们假设突变导向疫苗 设计将需要采用低亲和力的罕见bnAb前体，然后通过免疫接种， 免疫原，选择获得广泛中和表型所需的体细胞变化的Ab。我们 和其他人已经观察到，在纳米颗粒（NP）上多聚化HIV-1包膜（Env）免疫原可以 增加HIV-1 bnAb谱系抗体滴度和亲和力成熟。然而，Env是不稳定的，可以采用 NP表面上的非天然构象，导致不期望的Ab应答。此外，NPs轴承 HIV-1包膜蛋白产量低。为了克服这些缺陷，我们开发了一种创新的， 快速平台，其使用酶分选酶A将良好折叠的、切割的Env三聚体共价连接至自 组装铁蛋白质NP。这些分选酶A缀合的NP（scNP）保持接近天然的Env三聚体 构象，允许在进化的bnAb谱系中对不太可能的突变进行狂热选择。在人源化小鼠中， 我们使用了一个展示了一个被称为CH 505.M5 G458 Y的工程化Env三聚体的scNP来选择一个不太可能的关键， 8ANC 131/CH 235类CD 4结合位点（CD 4 bs）bnAb成熟所需的突变。相同的 scNP在非人灵长类动物（NHP）中引发CD 4 bs单克隆中和Ab和血清中和Ab。 为了进一步引导这些Ab发展中和宽度，我们产生了增强Env三聚体scNP， TF scNP，并且具有正在开发的第三加强Env。在CH505.M5 G458 Y scNP引发的NHP，CH 505 TF scNP免疫增强针对多种CH 505的中和Ab效价 病毒该IPCAVD将产生CH 505 TF scNP加强免疫原和第二个连续Env 用于I期试验的三聚体scNP增强免疫原。在具体目标1中，项目1将确定最佳NP 加强方案以扩大人源化小鼠中当前CD 4 bs B细胞谱系的中和能力。 在具体目标2中，我们将比较由以下组装的scNP的缀合效率和免疫原性： 来自项目2的研究级或开发运行的铁蛋白和分选酶A组分。具体目标3 将证明使用初免-加强策略和cGMP scNP在NHP中诱导CD 4 bs谱系 在第一阶段试验中。本项目的影响是，它将确定和证明有效性 一种新的、最佳的NP疫苗方案，用于扩增和亲和力成熟8ANC 131/CH 235类CD 4 bs bnAb。

ABSTRACT-PROJECT 1 Previous vaccines have been unable to elicit HIV-1 broadly neutralizing antibodies (bnAbs) in humans. Two of the reasons for this failure are that bnAb precursor B cells are often rare and that the B cell receptors on those rare B cells require extensive somatic mutation to evolve into high-affinity bnAbs. A subset of these somatic mutations—termed improbable mutations—have a low probability of being made by the somatic mutation machinery due to codon bias and positioning of somatic mutation recognition motifs. Our central hypothesis for eliciting bnAbs is that immunogens must have a higher affinity for Abs with the required improbable mutations than for Abs lacking these key mutations. Moreover, we hypothesize that mutation-guided vaccine design will need to engage low-affinity rare bnAb precursors and, then, through immunization with sequential immunogens, select for Abs acquiring the somatic changes requisite for a broad neutralization phenotype. We and others have observed that multimerizing HIV-1 envelope (Env) immunogens on nanoparticles (NPs) can increase Ab titers and affinity maturation of HIV-1 bnAb lineages. However, Env is unstable and can adopt non-native conformations on the surface of NPs, leading to undesired Ab responses. Additionally, NPs bearing HIV-1 envelope can be of low protein yield. To overcome these pitfalls, we have developed an innovative, rapid platform that uses the enzyme sortase A to covalently link well-folded, cleaved Env trimers to self- assembling ferritin protein NPs. These sortase A-conjugated NPs (scNPs) maintain near-native Env trimer conformation, allowing avid selection of improbable mutations in evolving bnAb lineages. In humanized mice, we used a scNP displaying an engineered Env trimer called CH505.M5 G458Y to select for a key improbable mutation required for maturation of the 8ANC131/CH235 class of CD4 binding site (CD4bs) bnAbs. The same scNP elicited CD4bs monoclonal neutralizing Abs and serum neutralizing Abs in nonhuman primates (NHPs). To further guide these Abs to develop neutralization breadth, we have generated a boosting Env trimer scNP immunogen called CH505.TF scNP and have a third boosting Env under development. In CH505.M5 G458Y scNP-primed NHPs, CH505 TF scNP immunization boosts neutralizing Ab titers against multiple CH505 viruses. This IPCAVD will generate a CH505 TF scNP boosting immunogen and a second sequential Env trimer scNP boosting immunogen for a Phase I trial. In Specific Aim 1, Project 1 will determine an optimal NP boosting regimen to broaden the neutralization capacity of current CD4bs B cell lineages in humanized mice. In Specific Aim 2, we will compare conjugation efficiency and immunogenicity of scNPs assembled from research-grade or development-run ferritin and sortase A components from Project 2. Lastly, Specific Aim 3 will demonstrate the induction of CD4bs lineages in NHPs using the prime-boost strategy and cGMP scNPs proposed for the Phase I trial. The impact of this Project is that it will define and demonstrate the effectiveness of a novel, optimal NP vaccine regimen to expand and affinity mature 8ANC131/CH235 class CD4bs bnAbs.