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

项目1. HIV-1 Env三聚体分选酶A结合纳米粒子的优化及体内评价

• 批准号: 10369069
• 负责人: KEVIN O SAUNDERS
• 金额: $ 113.16万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-17 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10369069
• 关键词: Adopted; Affinity; Antibodies; Antibody Binding Sites; Antibody Formation; Antibody Response; Antigens; Autologous; B-Cell Antigen Receptor; B-Lymphocytes; Binding Sites; Cell Lineage; Cloning; Codon Nucleotides; Cyclic GMP; Development; Effectiveness; Engineering; Enzymes; Evaluation; Failure; Ferritin; Genes; HIV Antigens; HIV-1; Human; Immunization; Immunize; Knock-in Mouse; Link; Macaca; Molecular Conformation; Monoclonal Antibodies; Mutation; Phenotype; Positioning Attribute; Probability; Process; Production; Protein Subunits; Proteins; Reaction; Recombinants; Regimen; Research; Running; Serum; Somatic Mutation; Surface; Technology; Vaccine Design; Vaccines; Virus; cross reactivity; design; expectation; humanized mouse; immunogenicity; in vivo evaluation; innovation; member; mouse model; nanoparticle; neutralizing antibody; next generation sequencing; non-Native; nonhuman primate; novel; phase I trial; sortase; transpeptidation; vaccination strategy

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.

摘要-项目1 以前的疫苗无法在人类体内引发 HIV-1 广泛中和抗体 (bnAb)。两个 这种失败的原因是 bnAb 前体 B 细胞通常很少见，而且这些 B 细胞上的 B 细胞受体 稀有 B 细胞需要广泛的体细胞突变才能进化为高亲和力 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可能性很低 由于密码子偏倚和体细胞突变识别基序的定位而产生的机制。我们的中心假设 为了引发 bnAbs，免疫原必须对 Abs 具有更高的亲和力，并且具有所需的不可能 突变比缺乏这些关键突变的 Ab 更重要。此外，我们假设突变引导疫苗 设计需要结合低亲和力的稀有 bnAb 前体，然后通过顺序免疫 免疫原，选择获得广泛中和表型所需的体细胞变化的抗体。我们 等人观察到，纳米颗粒 (NP) 上的 HIV-1 包膜 (Env) 免疫原多聚化可以 增加 HIV-1 bnAb 谱系的抗体滴度和亲和力成熟。但Env不稳定，可以采用 纳米颗粒表面的非天然构象，导致不良的抗体反应。此外，NP轴承 HIV-1 包膜的蛋白质产量可能较低。为了克服这些缺陷，我们开发了一种创新的、 快速平台，使用酶分选酶 A 将折叠良好、切割的 Env 三聚体共价连接至自 组装铁蛋白纳米颗粒。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 谱系中狂热地选择不可能的突变。在人源化小鼠中， 我们使用 scNP 显示一个名为 CH505.M5 G458Y 的工程环境三聚体来选择一个不可能的关键 8ANC131/CH235 类 CD4 结合位点 (CD4b) bnAb 成熟所需的突变。相同 scNP 在非人灵长类动物 (NHP) 中引发 CD4bs 单克隆中和抗体和血清中和抗体。 为了进一步引导这些 Abs 发展中和广度，我们生成了增强型 Env 三聚体 scNP 免疫原称为 CH505.TF scNP，第三种增强 Env 正在开发中。在CH505.M5 G458Y中 scNP 引发的 NHP、CH505 TF scNP 免疫可提高针对多种 CH505 的中和抗体滴度 病毒。该 IPCVD 将生成 CH505 TF scNP 增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 将确定一个最优 NP 加强方案以扩大人源化小鼠中当前 CD4bs B 细胞谱系的中和能力。 在具体目标 2 中，我们将比较由以下物质组装的 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 成分。最后，具体目标 3 将展示使用初免-加强策略和 cGMP scNP 在 NHP 中诱导 CD4b 谱系 提议进行第一阶段试验。该项目的影响在于它将定义并证明有效性 一种新型、最佳的 NP 疫苗方案，用于扩展和亲和力成熟的 8ANC131/CH235 类 CD4bs bnAbs。