Identification of novel immunogenic proteins from Bordetella pertussis

项目总结 尽管疫苗接种率很高，但由革兰氏阴性人类特有病原体引起的百日咳暴发 百日咳杆菌(BP)在许多国家都有发现。百日咳的复苏与疾病的转折有关 20世纪90年代，从诱导长寿Th1/17免疫反应的全细胞疫苗(WPV)到无细胞疫苗 (APV)引起Th1/2扭曲的免疫反应。此外，APV不能防止鼻腔定植或 BP的传播。目前的APV由1-5个细菌蛋白组成，这些蛋白被选为在 发病机制和诱导抗体的能力。相比之下，WPV呈现大量未定义的抗原。 有限的抗原多样性和Th2歪曲的免疫图谱的结合可能是对 APV提供的保护不完整。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/减少了当前APV的表达 抗原，这表明APV对CBP毒株的效果可能显著降低。有越来越多的 认识到CD4T细胞反应对于长寿的保护性免疫反应至关重要，这种免疫反应可以清除 整个呼吸道。然而，目前尚不清楚目前的APV抗原是否是最佳的CD4T细胞靶点。我们 将使用最先进的质谱学、生物信息学以及表型和功能分析来识别 CBP在人类II类组织相容性抗原上加工和呈递的表达蛋白 和小鼠，刺激CD4T细胞反应。这一基础数据集将与素数拉动相结合 疫苗接种策略和我们实验室开发的Th1/17偏斜佐剂，以确定 新定义的抗原的免疫原性和保护效力，以创造下一代APV。 具体目标1：确定MHC II和MHC II上呈现的一组自然衍生的BP多肽 被CD4T细胞识别。我们将从循环中的BP菌株(CBP)中鉴定BP抗原 在人和小鼠的第II类分子上表达，并用增殖和流式细胞术检测 WPV免疫个体和恢复期小鼠的CD4T细胞识别抗原。 特异性目标2：检测新抗原的免疫原性和保护效果 循环BP菌株及其在发病机制中的作用。我们将测试其免疫原性和保护效果 利用BP感染的小鼠模型对新的蛋白质进行了研究。我们将创造新蛋白质的缺失突变体 以确定它们在呼吸道的发病和定植中的作用。 影响：我们识别、测试和利用新BP抗原的集成方法将允许合理设计 下一代APV在呼吸道中产生长期保护，防止鼻咽携带和 从而减少百日咳的传播。

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.