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

诱导广谱中和抗体(BNAbs)是HIV疫苗开发中一个关键的未实现的目标。 由于bNab DH511具有很高的广度和很强的体内保护性，因此作为疫苗的先导引起了人们的高度兴趣 MPER bNAbs的效价。诱导DH511样bNAb的关键挑战是：(A)DH511的低亲和力- 类似于艾滋病毒多肽和蛋白质的前体，(B)对bNab接近角度的限制 凹陷、膜-近端表位环境和(C)MOST缺乏DH511表位 可溶的，天然的三聚体。 启动类似DH511的bNab诱导的一个有前途的策略是种系靶向，在这种策略中，合适的 DH511类前体是使用工程免疫原专门激活的，因此选择具有 在没有自身反应性的情况下发展广泛中和的潜力。这种方法也会有所帮助。 避免与表位凹陷位置相关的空间位阻问题，通过用 已知的遗传和结构潜力成熟为符合MPER空间限制的bNAbs。在这 项目，这是一个多项目协同提案的项目1，我们将工程表位-脚手架 免疫原与类DH511前体高亲和力结合并激活，使用计算设计 并引导进化。 众所周知，bNAbs是高度突变的，疫苗诱导的bNAbs遵循种系靶向启动 可能需要用其他旨在引导亲和力成熟的免疫原进行顺序免疫 B细胞受体。我们将开发不同类别的增强免疫原，包括表位- 具有更多天然表位的支架、膜蛋白支架和膜结合环境变异体 稳定在DH511强烈结合的构象中。可溶性和膜的结构研究- 与DH511谱系成员结合的免疫原将指导免疫原的发展。 这项合作计划的动物核心将培育出表达类似DH511基因的转基因小鼠 项目2将利用这些小鼠在体内测试B细胞的启动和增强。项目2将 在转基因小鼠中进行连续的启动/加强免疫实验，并使用ELISA法、细胞术、 单个B细胞分选、测序和中和分析以跟踪和优化亲和力成熟， 向项目1提供实验反馈，以允许反复改进免疫原。 总而言之，这些研究寻求开发新的艾滋病毒候选疫苗，并改变艾滋病毒疫苗 基于最新蛋白质工程发展的简化论方法研究 生殖系靶向和增强免疫原，开发人Ig敲入小鼠模型，以使 测试人类特异谱疫苗，并深入分析疫苗诱导的亲和力成熟 体内途径指导迭代疫苗优化。

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.