Project 1

项目1

• 批准号: 10339443
• 负责人: Richard Thomas Wyatt
• 金额: $ 115.59万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-04 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339443
• 关键词: Adolescent; Adult; Animal Model; Animals; Antigens; Autologous; B-Lymphocytes; Binding; Binding Sites; Cavia; Cell surface; Clinic; Complex; Coupled; Cryoelectron Microscopy; Crystallography; Cysteine; Development; Disulfides; Epitope Mapping; Epitopes; Goals; HIV; HIV Envelope Protein gp120; HIV-1 vaccine; Human; Immune Sera; Immunity; Immunization; Immunoglobulin G; Infant; Keyhole Limpet Hemocyanin; Length; Liposomes; Macaca; Maps; Membrane; Messenger RNA; Modification; Negative Staining; Oryctolagus cuniculus; Peptides; Polysaccharides; Primates; Process; Protomer; Public Health; Regimen; Resolution; Serum; Site; Specificity; Structure; Tail; Techniques; Technology; Testing; Time; Translating; Vaccination; Vaccines; Validation; Wild Type Mouse; alpha helix; base; biophysical techniques; compare effectiveness; design; env Gene Products; experimental study; immunogenicity; improved; in vivo; lipid nanoparticle; neutralizing antibody; nonhuman primate; novel; real time monitoring; response; restoration

The elicitation of cross-neutralizing or broadly neutralizing Abs (bNAbs) to diverse HIV strains by Env vaccination remains a high priority for a broadly efficacious vaccine. The elicitation of bNAbs against conserved Env determinants remains elusive; however, the recent isolation of bNAbs to the fusion peptide and other sites of vulnerability demark promising leads in this process. Using N-glycan deleted NFL trimer-liposome priming and heterologous boosting/restoration, cross-neutralizing responses in rabbits were elicited with isolation of a CD4 binding site (CD4bs)-directed bNAb, E70, and 1C2 (87% breadth) directed toward the gp120:gp41 interface, as delineated by high resolution cryoEM (Dubrovskaya et al., Immunity 2019). More recently, we have also elicited cross-neutralizing responses in guinea pigs using this approach with novel, full length stabilized “MIF” trimers as well as autologous tier 2 neutralizing responses in wild type mice following mRNA lipid nanoparticle (LNP) vaccination. Accordingly, the major objective of Project 1 will be to leverage these initial promising small animal results to elicit bNAbs in non-human primates (NHPs) using an “epitope-targeted” approach against the CD4bs and the gp120:gp41 trimer interface. As both sites are conserved Env protein determinants ringed by glycans, the N-glycan deletion priming and restoration regimen will be further optimized. The NFL trimers will be modified to enhance presentation of the targeted sites, while improving trimer stability and homogeneity by tail-anchoring on covalently coupled trimer-liposomes, the cell surface from mRNA, or with a heterologous trimer motif (MIF). The three presentation platforms will be cross-compared for effectiveness and translatability. Further, based on studies indicating human infants and adolescents more readily develop bNAbs compared to HIV-infected adults, immunization responses will be compared between juvenile and adult macaques. As a secondary objective, the same regimens will be tested in guinea pigs to cross-validate the animal models. Guided approaches monitoring “real-time” serum IgG responses by EM polyclonal epitope mapping (EMPEM; Core C) as well as rapid monoclonal Ab (mAb) isolation (Project 2/VRC) will be utilized to inform boosting from a select, diverse panel of structure-based, stabilized and homogeneous NFLs, iterative redesign and subsequent experiments. All NFL trimers will be produced in Project 1 for the entire P01, validated by biophysical methods, including DSC, EM and crystallography (Core C). Following elicitation of Env serum responses, isolated mAbs will be screened to confirm elicitation and neutralization specificity. By these integrated processes and comprehensive analysis, we will elicit and preferentially drive neutralizing antibodies to cross-neutralizing sites in primates in anticipation of human testing in the clinic.

通过 Env 疫苗接种对多种 HIV 毒株产生交叉中和或广泛中和抗体 (bNAb) 仍然是广泛有效的疫苗的高度优先事项。针对保守 Env 的 bNAb 的引发 决定因素仍然难以捉摸；然而，最近将 bNAb 分离到融合肽和其他位点 脆弱性标志着这一过程中有希望的线索。使用 N-聚糖删除的 NFL 三聚体脂质体引发和 通过分离 CD4 引发兔子的异源增强/恢复、交叉中和反应 结合位点 (CD4bs) 导向的 bNAb、E70 和 1C2（87% 宽度）导向 gp120:gp41 界面，如 由高分辨率冷冻电镜描绘（Dubrovskaya 等人，Immunity 2019）。最近，我们还引出了 使用这种方法与新颖的全长稳定“MIF”三聚体在豚鼠中产生交叉中和反应 以及野生型小鼠中 mRNA 脂质纳米颗粒 (LNP) 后的自体 2 级中和反应 疫苗接种。因此，项目 1 的主要目标是利用这些最初有前途的小动物 使用针对 CD4b 的“表位靶向”方法在非人类灵长类动物 (NHP) 中引发 bNAb 的结果 和 gp120:gp41 三聚体接口。由于这两个位点都是由聚糖环绕的保守 Env 蛋白决定簇， N-聚糖缺失启动和恢复方案将进一步优化。 NFL三聚体将被修改 增强目标位点的呈现，同时通过尾部锚定提高三聚体稳定性和同质性 共价偶联的三聚体脂质体、来自 mRNA 的细胞表面或异源三聚体基序 (MIF)。 三个演示平台的有效性和可翻译性将进行交叉比较。进一步，基于 研究表明，与感染艾滋病毒的成年人相比，人类婴儿和青少年更容易产生 bNAb， 将比较幼年猕猴和成年猕猴的免疫反应。作为次要目标， 相同的方案将在豚鼠中进行测试，以交叉验证动物模型。引导方法监控 通过 EM 多克隆表位作图（EMPEM；Core C）以及快速 单克隆抗体 (mAb) 分离（项目 2/VRC）将用于从精选的多样化组中获得增强信息 基于结构的、稳定且同质的 NFL、迭代重新设计和后续实验。所有 NFL 三聚体将在项目 1 中为整个 P01 生产，并通过生物物理方法验证，包括 DSC、EM 和晶体学（核心 C）。引发 Env 血清反应后，将筛选分离的 mAb 确认诱导和中和特异性。通过这些集成的流程和综合分析，我们 将引发并优先驱动中和抗体到达灵长类动物的交叉中和位点，以预期 在诊所进行人体测试。