Identification of novel immunogenic proteins from Bordetella pertussis

项目摘要 尽管疫苗接种覆盖率很高，但由革兰氏阴性专性人类病原体引起的百日咳爆发 百日咳杆菌（Bp）在许多国家都有发现。百日咳复发与免疫系统的转变相关 20世纪90年代，从引发长寿命Th 1/17免疫应答的全细胞疫苗（wPV）到无细胞疫苗 (aPV)其引起Th 1/2偏斜的免疫应答。此外，aPV不能防止鼻腔定植或 BP的传播目前的aPV由1-5种细菌蛋白组成，这些蛋白是根据它们在以下方面的作用而选择的： 致病性和引发抗体的能力。相比之下，wPV呈现不确定的大量抗原。 有限的抗原多样性和Th 2偏斜的免疫概况的组合是对免疫缺陷的可能解释。 aPV提供的不完整保护。最近的研究，包括我们实验室的研究也表明， 来自全球不同国家的循环Bp毒株（CBp）缺乏/降低了当前aPV的表达 抗原，这表明aPV对CBp株的有效性可能显著降低。人们日益 认识到CD 4 + T细胞应答对于清除免疫缺陷的长期保护性免疫应答至关重要。 整个呼吸道。然而，目前还不清楚目前的aPV抗原是最佳的CD 4 + T细胞靶。我们 将使用最先进的质谱，生物信息学，表型和功能分析，以确定 由人II类组织相容性抗原加工和呈递的CBp表达的蛋白质 和小鼠，并且刺激CD 4 + T细胞应答。这个基础数据集将与一个prime-pull相结合 疫苗接种策略和我们实验室开发的Th 1/17偏斜佐剂，以确定 新定义的抗原的免疫原性和保护功效以产生下一代aPV。 具体目标1：定义在MHC II上呈递的天然衍生的Bp肽的集合， 由CD 4 + T细胞识别。我们将鉴定来自循环Bp菌株（CBp）的Bp抗原， 在人和鼠II类细胞上表达，并使用增殖和流式细胞术测定来确定其中 抗原被wPV免疫的个体和恢复期小鼠的CD 4 + T细胞识别。 具体目的2：测试新抗原的免疫原性和保护效力，以对抗 循环Bp菌株及其在发病机制中的作用。我们将测试免疫原性和保护效力 使用Bp感染的小鼠模型的新蛋白质。我们将创造新蛋白质的缺失突变体 以确定它们在呼吸道发病机制和定植中的作用。 影响：我们鉴定、测试和利用新型Bp抗原的综合方法将允许合理设计 下一代aPV在呼吸道中引起持久保护，防止鼻咽携带， 从而减少百日咳疾病的传播。

PROJECT SUMMARY Despite high vaccination coverage, pertussis outbreaks caused by the gram-negative obligate human pathogen Bordetella pertussis (Bp) are observed in many countries. Pertussis resurgence correlates with the switch in the 1990s from whole cell vaccines (wPV) which elicit long-lived Th1/17 immune responses, to acellular vaccines (aPV) which elicit Th1/2 skewed immune responses. Furthermore, aPV do not prevent nasal colonization or transmission of Bp. Current aPV are comprised of 1-5 bacterial proteins that were selected for their roles in pathogenesis and ability to elicit antibodies. In contrast, wPV present an undefined large number of antigens. The combination of limited antigenic diversity and Th2 skewed immune profile is a likely explanation for the incomplete protection provided by aPV. Recent studies including that from our laboratory also show that circulating Bp strains (CBp) from globally diverse countries have absent/reduced expression of current aPV antigens, suggesting that aPV may be significantly less effective against CBp strains. There is increasing recognition that CD4+ T cell responses are critical for long-lived protective immune responses that clear the entire respiratory tract. However, it is not clear that current aPV antigens are optimal CD4+ T cell targets. We will use state-of-the-art mass spectrometry, bioinformatics, and phenotypic and functional assays to identify proteins expressed by CBp that are processed and presented on Class II histocompatibility antigens of humans and mice, and that stimulate CD4+ T cell responses. This foundational data set will be coupled with a prime-pull vaccination strategy and a Th1/17 skewing adjuvant developed in our laboratory, to determine the immunogenicity and protective efficacy of newly defined antigens to create a next-gen aPV. Specific Aim 1: Define the set of naturally derived Bp peptides presented on MHC II and recognized by CD4+ T cells. We will identify the Bp antigens from circulating Bp strains (CBp) that are expressed on human and murine Class II, and use proliferation and flow cytometry assays to determine which antigens are recognized by CD4+ T cells of wPV-immunized individuals and convalescent mice. Specific Aim 2: To test the immunogenicity and protective efficacy of novel antigens against circulating Bp strains and their role in pathogenesis. We will test the immunogenicity and protective efficacy of the novel proteins using a murine model of Bp infection. We will create deletion mutants of the novel proteins in CBp to determine their role in pathogenesis and colonization of the respiratory tract. IMPACT: Our integrated approach to identify, test, and leverage novel Bp antigens will permit the rational design of next-gen aPVs that elicit long-lasting protection in the respiratory tract, prevent nasopharyngeal carriage and thereby reduce the spread of the disease pertussis.