Project 1

交叉中和抗体(BNAbs)对不同HIV毒株的环境免疫诱导作用 对于广泛有效的疫苗来说，仍然是一个高度优先的问题。BNAbs对保守环境的诱导作用 决定因素仍然难以捉摸；然而，最近分离到的融合多肽和其他位点的bNAbs 漏洞标记有望在这一过程中提供线索。用N-葡聚糖缺失的NFL三聚体-脂质体引发 分离CD_4诱导兔异源增强/修复、交叉中和反应 结合位点(CD4bs)导向gp120：gp41界面的bNAb、E70和1C2(87%宽度)，AS 由高分辨率低温电子显微镜描绘(Dubrovskaya等人，《免疫2019》)。最近，我们还引发了 以新型、全长稳定的“MIF”三聚体为供体的豚鼠交叉中和反应 以及野生型小鼠对信使核糖核酸脂纳米粒(LNP)的自体第2层中和反应 接种疫苗。因此，项目1的主要目标将是利用这些最初有希望的小动物 使用针对CD4bs的“表位靶向”方法在非人灵长类动物(HAP)中诱导bNAbs的结果 和gp120：gp41三聚体界面。由于这两个位点都是由糖链组成的保守的环境蛋白决定簇， N-糖链缺失引发和修复方案将进一步优化。NFL三聚体将被修改 通过尾部锚定来增强目标位点的呈现，同时提高三聚体的稳定性和同质性 在共价偶联的三聚体-脂质体上，细胞表面从mRNA或带有异源三聚体基序(MIF)。 这三个演示平台将在有效性和可译性方面进行交叉比较。此外，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bNAbs， 将对幼年猕猴和成年猕猴的免疫反应进行比较。作为次要目标， 同样的方案将在豚鼠身上进行测试，以交叉验证动物模型。指导性方法监测 EM多克隆表位映射(EMPEM；Core C)和快速检测血清免疫球蛋白 将利用单抗(MAb)分离(项目2/vrc)从精选的、多样化的 基于结构的、稳定的和同质的NFL、迭代重新设计和后续实验。全美橄榄球联盟 将在项目1中为整个P01生产三聚体，并通过生物物理方法进行验证，包括DSC、EM 和结晶学(核心C)。在环境病毒血清反应激发后，分离的单抗将被筛选以 确认启发式和中和性。通过这些综合流程和综合分析，我们 会诱导并优先驱动中和抗体到灵长类动物体内的交叉中和部位 在诊所进行人体测试。

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.