Mechanistic Studies of Combination Adjuvants to Target B Cells in Vaccines

为了充分响应RFA-AI-20 - 004，我们建议研究NKT细胞与 TLR佐剂。我们以前已经证明，我们可以使用NKT细胞和TLR7佐剂的组合， 产生高亲和力保护性抗细菌聚糖疫苗。我们现在的目标是了解 这两种佐剂之间的协同作用使其优化并产生疫苗制剂， 一次用药后的保护。此外，我们认为，机械研究也将允许 限制佐剂的潜在副作用。为此，我们的建议是建立在两个独立的， 互补的具体目的：目的1：评价内体TLR配体增强NKT细胞增殖的能力。 应答及其B细胞辅助活性。我们已经通过以下方法产生了纳摩尔亲和力的抗聚糖抗体： 联合NKT细胞和TLR7激动剂。这种反应依赖于NKT和CD4 T细胞的帮助。我们 假设每种内体TLR配体在组合使用中可能具有不同的增强作用。 我们将通过RNA的单细胞技术研究NKT，T滤泡辅助细胞，树突状细胞和B细胞的反应 以及我们将要研究的每个TLR/NKT细胞对的疫苗接种动物模型中的蛋白质表达。B细胞 将使用受体测序和病原体攻击来测量每种组合的功效。 目的2：评估两种佐剂共同递送至特定细胞类型的重要性， 内体/溶酶体区室。我们假设，与尊重的物理方面的佐剂 抗原摄取和递送到处理区室是关键的。通过共同递送抗原和佐剂 特定的细胞和在选定的隔室，使用合成化学来控制它们的配对， 分离后，将检查佐剂活性的机制。这种分子的生物学后果 基于佐剂组合的设计将在剂量、毒性和功效方面进行评价。我们预计 以开发在单次疫苗施用后诱导保护的稳健制剂。 我们的整个建议完全集中在旨在发展保护性B细胞免疫的疫苗上， 模型系统是针对肺炎链球菌的缀合物疫苗。

In full response to RFA-AI-20-004, we propose to study the mechanisms of cooperativity between NKT cell and TLR adjuvants. We have previously shown that we could use a combination of NKT cell and TLR7 adjuvants to produce high affinity protective anti-bacterial glycan vaccines. Our goal is now to understand the mechanisms of this cooperative effects between the two adjuvants to optimize it and produce a vaccine formulation that promotes protection after a single administration. In addition, we contend that mechanistic studies will also allow the limitation of the potential side effects of adjuvants. To this end, our proposal is built on two separate and complementary specific aims: Aim 1: Evaluate endosomal TLR ligands for their ability to enhance NKT cell responses and their B cell helper activities. We have produced nanomolar affinity anti-glycan antibodies by combining NKT cell and TLR7 agonists. This response was dependent on NKT and CD4 T cell help. We hypothesize that each endosomal TLR ligand may have a different enhancing effect in a combinatorial usage. We will investigate NKT, T follicular helper, dendritic, and B cell responses by single cell technologies for RNA and protein expression in animal models of vaccination for each TLR/NKT cell pair that we will study. B cell receptor sequencing and pathogen challenges will be used to measure the efficacy of each combination. Aim 2: Evaluate the importance of the co-delivery of the two adjuvants to particular cell types and endosomal/lysosomal compartments. We hypothesize that the physical aspects of adjuvanticity with respect to antigen uptake and delivery to processing compartments are critical. By co-delivering antigen and adjuvants to particular cells and within chosen compartments, using synthetic chemistry to control their pairing and separation, mechanisms of adjuvant activity will be examined. The biological consequences of this molecular based design of adjuvant combinations will be evaluated with respect to dosage, toxicity, and efficacy. We expect to develop robust formulations that induce protection after a single vaccine administration. Our entire proposal is focused exclusively on vaccines aimed at developing protective B cell immunity, and our model system is a conjugate vaccine against Streptococcus pneumoniae.