Design and testing of germline-targeting and boosting immunogens to elicit 10E8-like broadly neutralizing antibodies against HIV

诱导广泛中和抗体（bnAbs）是艾滋病毒疫苗尚未实现的关键目标 发展BnAb 10 E8作为疫苗先导特别令人感兴趣，这是由于其（1）非常高的 宽度，（2）发展广泛中和所需的突变数量少，（3）低 自身反应性，（4）预期的人原始B细胞库中前体的高频率，和（5） 在被动转移研究中提供令人惊讶的有效体内灭菌保护的能力。 诱导10 E8样bnAb的关键挑战是：（a）HIV肽的低种系亲和力， 蛋白质，（B）由凹进的对bnAb接近角的严重限制， 膜近端表位环境和（c）不存在10 E8表位， 天然样三聚体（例如SOSIP或NFL或UFO）。 启动10 E8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10 E8类前体，从而选择BCR 具有在不存在自身反应性的情况下发展广泛中和的潜力。这种方法 还有助于避免与表位的凹陷位置相关的空间问题， 引发具有已知遗传和结构潜力的前体成熟为与以下相容的bnAb： MPER位阻。在这项提案中，我们将设计抗原表位支架免疫原， 使用计算机设计和定向进化激活10 E8样前体。我们将初步 使用人幼稚B细胞分选离体测试免疫原。我们将进一步培育基因敲入小鼠 过表达10 E8样前体的小鼠，我们将用这些小鼠来测试B细胞引发， 体内加强，首先过继转移敲入B细胞至野生型小鼠， 人体中10 E8 bnAb前体和竞争B细胞的模拟频率。 如已知的bnAb是高度突变的，在种系引发后的bnAb的疫苗诱导将使bnAb的免疫原性降低。 可能需要用其他免疫原进行顺序免疫， B细胞受体的成熟。我们将开发不同种类的增强免疫原， 包括具有更多天然表位的表位支架、膜蛋白支架和 膜结合的Env变体稳定在10 E8强烈结合的构象中。我们将 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， 单个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 总之，这些研究旨在开发新的HIV候选疫苗， 将艾滋病毒疫苗研究转向基于战略 bNAb前体的鉴定，最先进的蛋白质工程， 生殖系靶向和加强免疫原，开发人IG基因敲入小鼠模型， 测试人类特异性疫苗，并深入分析疫苗诱导的亲和力 成熟途径，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 10E8 is of particular interest as a vaccine lead due to its (1) very high breadth, (2) low number of mutations required to develop broad neutralization, (3) low auto-reactivity, (4) expected high frequency of precursors in the human na'ive B-cell pool and (5) ability to provide surprisingly potent in vivo sterilizing protection in passive transfer studies. Key challenges for inducing 10E8-like bnAbs are: (a) the low germline affinity by HIV peptides and proteins, (b) the severe restriction on bnAb angle of approach imposed by the recessed, membrane-proximal epitope environment and (c) the absence of the 10E8 epitope from most soluble, native-like trimers (e.g. SOSIP or NFL or UFO). A promising strategy to initiate 10E8-like bnAb induction is germline targeting, in which suitable 10E8-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 proposal, we will engineer epitope-scaffold immunogens that activate 10E8-like precursors, using computational design and directed evolution. We will initially test immunogens ex vivo using human na'ive B cell sorting. We will further generate knock-in mice that over-express 10E8-like precursors, and we will use those mice to test B-cell priming and boosting in vivo, first adoptively transferring knockin B cells to wild- type mice in order to mimic frequencies of 10E8 bnAb precursor and competitor B cells in humans. As known bnAbs are highly mutated, vaccine induction of bnAbs following a germline 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 10E8 binds strongly. We 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. In summary, these studies seek to develop novel HIV vaccine candidates and also to shift HIV vaccine research towards a reductionist approach based on strategic identification of bNAb precursors, 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.