Boosting efficacy of oral vaccine candidates by enabling spore display of nitrated antigens

项目摘要 许多疾病可以通过让免疫系统识别特定抗原来预防或治疗。 细菌疾病值得高度关注，因为许多细菌病原体缺乏有效的疫苗和 显示出抗生素耐药率的上升。这些病原体经常找到许多逃避免疫系统的方法。 检测，包括改变它们最具免疫原性的抗原。虽然许多与毒力相关的蛋白质可以 它们在病原体血清型之间高度保守，通常表现出弱的免疫原性，不足以 得出免疫系统的反应。在这个项目中，我们问：有没有策略来照亮LIVE 提高免疫细胞识别能力的细菌抗原？此外，我们能否将这些战略结合起来 货架稳定的递送载体，对世界各地的患者来说管理起来很简单？ 我们提出了一种变革性的方法来扩大用于活细菌疫苗的候选抗原的列表。 通过教导枯草芽孢杆菌产生和利用一种免疫原性氨基酸来传播媒介。这种氨基酸有 已被证明当被替换到自体蛋白表面时可以终止免疫自我耐受 在老鼠身上。蛋白质中硝化残基的定点导入导致了新表位的呈现 这是被辅助T细胞识别的，随后激活产生多克隆抗体的B细胞。它 理所当然的是，许多弱免疫原性外来抗原的免疫原性可以提高。 使用这一策略，尽管这还没有经过测试。一个挑战是，之前的研究也建立了一个 MHC第二类基因在对硝化抗原的免疫应答中的关键作用，但知之甚少。 我们的项目将研究孢子展示的硝化抗原作为变革性疫苗接种的可能性。 细菌疾病平台，以志贺氏菌侵袭蛋白抗原为模型系统。我们将首先表演 具有弱免疫原性但跨病原体高度保守的志贺氏菌抗原的动物研究 以确定硝化作用是否能提高其免疫原性。为了更好地了解硝化在哪里 应放置残留物以供免疫细胞机器进行最佳识别，我们将开发一种高通量 微生物展示平台筛选MHC-II对非天然多肽配体的偏好。同时，我们将 开发工具，使免疫原性氨基酸能够在融合到 枯草杆菌的孢子衣。这种非致病生物的重组孢子可以口服和 暴露在恶劣条件下后保持免疫效力。基于孢子的平台承诺 克服疫苗制造、运输和管理方面的限制；然而，该平台具有 免疫原性低。我们使用这个平台形成硝化残基的策略可以克服这一限制。 从这个项目中，我们将深入了解硝化作用对增强免疫原性的要求， 我们将朝着以货架稳定性和口服给药为特征的免疫平台技术前进。

Project Summary Many diseases could be prevented or treated by enlisting the immune system to recognize a specific antigen. Bacterial diseases warrant heightened attention as many bacterial pathogens lack efficacious vaccines and exhibit rising rates of antibiotic resistance. These pathogens often find many ways of evading immune system detection, including varying their most immunogenic antigens. While many virulence-related proteins can be strongly conserved across pathogen serotypes, they often exhibit weak immunogenicity that is insufficient to draw the response of the immune system. In this project, we ask: Are there strategies to shine a light on live bacterial antigens for increased recognition by immune cells? Furthermore, can we couple these strategies to shelf-stable delivery vectors that are simple to administer to patients across the world? We propose a transformational approach to expand the list of candidate antigens for use in live bacterial vaccine vectors by teaching Bacillus subtilis to produce and harness an immunogenic amino acid. This amino acid has been demonstrated to terminate immune self-tolerance when substituted on the surface of autologous proteins in mice. Site-specific introduction of nitrated residues within proteins has resulted in presentation of a neoepitope that is recognized by helper T cells for subsequent activation of B cells that produce polyclonal antibodies. It stands to reason that the immunogenicity of many weakly immunogenic foreign antigens could be increased using this strategy, though this has not yet been tested. One challenge is that prior studies also established a critical but poorly understood role of the MHC Class II locus in enabling immune response to nitrated antigens. Our project will investigate the potential of spore-displayed nitrated antigens as a transformational vaccination platform for bacterial disease, with the Shigella invasion protein antigens as a model system. We will first perform animal studies with Shigella antigens that are weakly immunogenic but strongly conserved across pathogen serotypes to determine if nitration can increase their immunogenicity. To better understand where nitrated residues should be placed for optimal recognition by immune cell machinery, we will develop a high-throughput microbial display platform to screen MHC-II preference towards unnatural peptide ligands. In parallel, we will develop tools to enable site-specific incorporation of the immunogenic amino acid within proteins fused to the spore coat of B. subtilis. Recombinant spores of this non-pathogenic organism can be orally administered and maintain immunization efficacy after exposure to harsh conditions. The spore-based platform has promise to overcome limitations in the manufacture, transport, and administration of vaccines; however, the platform has low immunogenicity. Our strategy to form nitrated residues using this platform could overcome that limitation. From this project, we will gain insights about the requirements for enhanced immunogenicity due to nitration, and we will advance towards a platform technology for immunization that features shelf-stability and oral delivery.