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

诱导广泛中和抗体 (bnAb) 是 HIV 疫苗开发中一个未实现的关键目标。 BnAb DH511 作为疫苗先导物受到高度关注，因为它具有非常高的广度和高体内保护作用 MPER bNAb 的效力。诱导 DH511 样 bnAb 的主要挑战是：(a) DH511-的低亲和力 像 HIV 肽和蛋白质的前体一样，(b) 对 bnAb 的接近角度的限制 凹进的近膜表位环境和 (c) 大多数细胞中不存在 DH511 表位 可溶的、类似天然的三聚体。 启动 DH511 样 bnAb 诱导的一种有前景的策略是种系靶向，其中合适的 DH511 类前体使用工程免疫原特异性激活，从而选择具有 在没有自身反应性的情况下发展广泛中和的潜力。这种方法也将有助于 通过用以下物质引发前体，避免与表位凹陷位置相关的空间问题 已知的遗传和结构潜力可成熟为与 MPER 空间限制兼容的 bnAb。在这个 项目，这是多项目协作提案的项目1，我们将设计表位支架 使用计算设计以高亲和力结合并激活 DH511 样前体的免疫原 以及定向进化。 由于已知 bnAb 具有高度突变性，因此在种系靶向启动后疫苗诱导 bnAb 可能需要使用其他旨在促进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、膜蛋白支架和膜结合 Env 变体的支架 稳定在 DH511 牢固结合的构象中。可溶性和膜性的结构研究 与 DH511 谱系成员复合的结合免疫原将指导免疫原的发育。 该合作提案的动物核心将产生表达 DH511 样蛋白的敲入小鼠 前体细胞，项目 2 将使用这些小鼠来测试 B 细胞体内启动和增强。项目2将 在敲入小鼠中进行连续初免/加强免疫实验，并使用 ELISA、细胞计数法、 单 B 细胞分选、测序和中和测定，以跟踪和优化亲和力成熟， 向项目 1 提供实验反馈，以迭代改进免疫原。 总之，这些研究旨在开发新型艾滋病毒候选疫苗，并改变艾滋病毒疫苗 研究基于最先进的蛋白质工程的还原论方法来开发 种系靶向和增强免疫原，开发人类 Ig 敲入小鼠模型以实现 人类库特异性疫苗的测试，以及疫苗诱导的亲和力成熟的深入分析 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.