Project 1. Design of immunogens that bind to the UCA and to IAs and mature DH511 bnAbs in optimal affinities

广泛中和抗体（bnAb）的诱导是HIV疫苗开发的一个关键的未满足的目标。 BnAb DH 511由于其非常高的宽度和高的体内保护性而作为疫苗先导物受到高度关注。 MPER bNAb的效力。诱导DH 511样bnAb的关键挑战是：（a）DH 511-BnAb的低亲和力， 类似于HIV肽和蛋白质的前体，（B）由所述前体施加的对bnAb接近角度的限制， 凹陷的近膜表位环境和（c）大多数表位缺乏DH 511表位 可溶的天然样三聚体。 启动DH 511样bnAb诱导的一种有前景的策略是种系靶向，其中合适的靶向方法包括： 使用工程免疫原特异性激活DH 511类前体，从而选择具有 在不存在自身反应性的情况下产生广泛中和的潜力。这种方法也将有助于 避免与表位的凹陷位置相关的空间问题， 已知的遗传和结构潜力成熟为与MPER空间限制相容的bnAb。在这 项目，这是一个多项目合作建议的项目1，我们将工程师的缩影-脚手架 使用计算设计以高亲和力结合并激活DH 511样前体的免疫原 和定向进化 如已知的bnAb是高度突变的，在种系靶向引发后bnAb的疫苗诱导 可能需要用其他免疫原进行顺序免疫， B细胞受体。我们将开发不同种类的增强免疫原，包括表位- 具有更多天然表位的支架、膜蛋白支架和膜结合Env变体 稳定在DH 511强烈结合的构象中。可溶性和膜的结构研究- 与DH 511谱系成员复合的结合免疫原将引导免疫原的发育。 这项合作提案的动物核心将产生表达DH 511样的基因敲入小鼠。 项目2将使用这些小鼠来测试体内B细胞引发和加强。项目2将 在基因敲入小鼠中进行顺序初免/加强免疫实验，并使用ELISA，细胞计数， 单B细胞分选和测序以及中和测定以跟踪和优化亲和力成熟， 为项目1提供实验反馈，以允许免疫原的迭代改进。 总之，这些研究旨在开发新的HIV候选疫苗，并改变HIV疫苗 基于最先进的蛋白质工程的还原论方法的研究， 生殖系靶向和加强免疫原，开发人IG基因敲入小鼠模型， 测试人类库特异性疫苗，并深入分析疫苗诱导的亲和力成熟 体内途径，以指导迭代疫苗优化。

Induction of broadly neutralizing antibodies (bnAbs) is a critical unmet goal of HIV vaccine development. BnAb DH511 is of high interest as a vaccine lead due to its very high breadth and the high in vivo protective potency of MPER bNAbs. Key challenges for inducing DH511-like bnAbs are: (a) the low affinity of DH511- like precursors for HIV peptides and proteins, (b) the restriction on bnAb angle of approach imposed by the recessed, membrane-proximal epitope environment and (c) the absence of the DH511 epitope from most soluble, native-like trimers. A promising strategy to initiate DH511-like bnAb induction is germline targeting, in which suitable DH511-class precursors are specifically activated using engineered immunogens, thus selecting BCRs with the potential to develop broad neutralization in the absence of autoreactivity. This approach will also help circumvent steric problems associated with the recessed location of the epitope, by priming precursors with known genetic and structural potential to mature into bnAbs compatible with MPER steric restraints. In this project, which is Project 1 of a multi-project collaborative proposal, we will engineer epitope-scaffold immunogens that bind with high affinity to and activate DH511-like precursors, using computational design and directed evolution. As known bnAbs are highly mutated, vaccine induction of bnAbs following a germline-targeting prime will likely require sequential immunization with other immunogens designed to shepherd affinity maturation of the B-cell receptor. We will develop different classes of boosting immunogens, including epitope- scaffolds with more native epitopes, membrane-protein scaffolds and membrane-bound Env variants stabilized in a conformation to which DH511 binds strongly. Structural studies of soluble and membrane- bound immunogens in complex with DH511 lineage members will guide immunogen development. The Animal Core of this collaborative proposal will generate knock-in mice that express DH511-like precursors, and Project 2 will use those mice to test B cell priming and boosting in vivo. Project 2 will conduct sequential prime/boost immunization experiments in knock-in mice and use ELISA, cytometry, single B cell sorting and sequencing and neutralization assays to track and optimize affinity maturation, providing experimental feedback to Project 1 to allow for iterative improvement of immunogens. In summary, these studies seek to develop novel HIV vaccine candidates and also to shift HIV vaccine research towards a reductionist approach based on state-of-the-art protein engineering to develop germline-targeting and boosting immunogens, development of human Ig knock-in mouse models to enable testing of human-repertoire-specific vaccines, and in-depth analysis of vaccine-induced affinity maturation pathways in vivo to guide iterative vaccine optimization.