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

诱导广谱中和抗体(BNAbs)是HIV疫苗的一个关键的未实现的目标 发展。由于bNab 10E8的(1)含量很高，因此作为疫苗的先导特别有兴趣 广度，(2)产生广泛中和所需的突变数量低，(3)低 自身反应性，(4)人类原始B细胞库中预期的高频率前体和(5) 在被动转移研究中提供惊人有效的体内灭菌保护的能力。 诱导10E8样bNAbs的关键挑战是：(A)艾滋病毒多肽和 蛋白质，(B)凹陷对bNab接近角度的严格限制， 膜-近端表位环境和(C)大多数可溶性的10E8表位缺失， 类似原生的三聚体(例如SOSIP或NFL或UFO)。 启动类似10E8的bNab诱导的一个有前途的策略是生殖系靶向，在这种策略中，合适的 10E8类前体使用工程免疫原专门激活，从而选择BCR 有可能在缺乏自身反应性的情况下产生广泛的中和作用。这种方法 也将有助于绕过与表位凹陷位置相关的空间问题，通过 引发具有已知遗传和结构潜力的前体成熟为与之兼容的bNAbs 更多的空间限制。在这项提案中，我们将设计出表位支架免疫原， 利用计算设计和定向进化，激活类似10E8的前体。我们最初会 用人天然B细胞分选法进行体外免疫原试验。我们将进一步产生敲入鼠 这会过度表达10E8样前体，我们将用这些小鼠来测试B细胞的启动和 体内增强，首先过继将敲打B细胞转移到野生型小鼠，以便 模拟人类10E8bNab前体细胞和竞争对手B细胞的频率。 众所周知，bNAbs是高度突变的，在种系初始意愿之后疫苗诱导bNAbs 可能需要用其他免疫原进行顺序免疫，以增强牧羊人的亲和力 B细胞受体的成熟。我们将开发不同类别的增强免疫原， 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变异体稳定在10E8强烈结合的构象中。我们会 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、 跟踪和优化亲和力的单个B细胞分选、测序和中和试验 成熟。 总而言之，这些研究寻求开发新的艾滋病毒候选疫苗，并还 将艾滋病毒疫苗研究转向基于战略的简化论方法 鉴定bNAb前体，发展最先进的蛋白质工程 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 体内成熟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 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.