Immune recognition of Klebsiella pneumoniae O2v1 and O2v2 O-antigen subtypes

项目总结 这项研究项目将加深我们对猪瘟O抗原诱导的免疫反应的了解。 肺炎克雷伯氏菌(Kp)，是疫苗开发的重要靶点。这样做的长期目标是 建议是为了加深我们对先天和获得性免疫反应的了解，这些反应是由两种亚型的 Kp、O2v1和O2v2的常见血清群O2。我们试图理解单曲的加入是如何 与O2v1相比，O2v2的支链半乳糖改变了其细菌适应性和宿主免疫敏感性。 此外，我们将确定这两种亚型之间的交叉反应，以及感染或接种 一种亚型提供对另一种亚型的交叉保护，这些信息对疫苗的设计至关重要，可以 广泛瞄准这种抗药性日益增强的微生物。金黄色葡萄球菌感染，包括肺炎、尿路 住院患者的感染和血液感染急剧上升；疾控中心已经宣布 感染KP和其他耐碳青霉烯类肠杆菌需要达到紧急威胁级别。 这个项目建立在PI细菌学、小鼠感染模型和发病机制研究的背景之上 通过特别关注KP毒力因子O-抗原和宿主 免疫原性反应。我们以前已经证明，针对KP的生物结合疫苗的使用是 有益于预防疾病，并寻求增加我们对未来的了解和覆盖范围 疫苗。Kp历史上有11个已知的O-抗原血清群。最近，更多的O抗原亚型 在这些血清群中，已经确定了。然而，在致病适合性、免疫原性、功能性 抗体反应和相关亚型之间的交叉保护还不是很清楚。 我们构建了一株表达O-抗原O2v2亚型的经典KP菌株的突变体，以产生 另外，表达O2v1的同源菌株。我们将测试这些细菌是否表现出类似的表型表达 通过测定被膜、高粘滞度、菌毛和生物膜来测定毒力因子。一只健壮的小鼠 肺炎模型将被用来确定肺健康和描绘宿主的先天免疫 对每种病原体的反应。中性粒细胞和补体介导的细菌杀灭试验将用于 进一步探索细菌对先天免疫攻击的抵抗力，文献表明，这可能会在 O2v2菌株。接下来，我们将进行一系列实验，测试O2v1和O2v2是否具有交叉保护作用。vbl.使用 经典的免疫学技术，包括酶联免疫吸附试验和流式细胞仪，我们将确定免疫球蛋白亚类 以及这些病原体呼吸道感染所激发的效应细胞因子。最后，使用新的O2v1或 O2v2生物结合疫苗之后与O2v1或O2v2毒株的挑战，我们将评估交叉保护 防止体重减轻和肺炎死亡率。我们将使用血清进一步表征抗体的功能 杀菌试验和吞噬细胞试验。这些研究将极大地促进我们对 对KPO抗原的免疫反应，并协助疫苗设计以对抗这种病原体。

PROJECT SUMMARY This research project will enhance our understanding of the immune responses elicited by the O-antigen of Klebsiella pneumoniae (Kp), an important target in putative vaccine development. The long-term goal of this proposal is to further our knowledge of the innate and adaptive immune responses elicited by two subtypes of the common serogroup O2 of Kp, O2v1 and O2v2. We seek to understand how the addition of the single branched galactose of O2v2 changes its bacterial fitness and host immune susceptibility relative to O2v1. Further, we will determine cross-reactivity between these two subtypes and whether infection or vaccination with one subtype confers cross-protection against the other, information critical to the design of vaccines that can broadly target this increasingly antibiotic-resistant organism. Kp infections, including pneumonia, urinary tract infection, and bloodstream infection, are sharply on the rise among hospitalized patients; CDC has declared that infections with Kp and other carbapenem-resistant Enterobacteriales demand a threat level of urgent. This project builds on the PI's background in bacteriology, murine models of infection, and pathogenesis studies of Gram-negative bacteria, by focusing specifically on the Kp virulence factor O-antigen and the host immunogenic response. We have previously demonstrated that use of bioconjugate vaccines targeting Kp are beneficial in preventing disease and seek to increase our understanding and breadth of coverage of future vaccines. Kp historically has eleven known serogroups of O-antigen. Recently, additional O-antigen subtypes within these serogroups have been identified. Yet, differences in pathogenic fitness, immunogenicity, functional antibody response, and cross-protection between related subtypes are not well understood. We have constructed a mutant of a classical Kp strain expressing O-antigen subtype O2v2 to generate an otherwise isogenic strain expressing O2v1. We will test if these bacteria exhibit similar phenotypic expression of virulence factors by measuring capsule, hypermucoviscosity, fimbriae and biofilm. A well-established murine model of pneumonia will be leveraged to determine pulmonary fitness and delineate host innate immune response to each pathogen. Neutrophil and complement-mediated bacterial killing assays will be employed to further explore bacterial resistance to innate immune attack, which the literature indicates may be enhanced in O2v2 strains. Next, we will perform a range of experiments testing if O2v1 and O2v2 are cross-protective. Using classically immunological techniques including ELISA and flow cytometry, we will determine the IgG subclasses and effector cytokines elicited by respiratory tract infection with these pathogens. Lastly, using novel O2v1 or O2v2 bioconjugate vaccines followed by challenge with O2v1 or O2v2 strains, we will assess cross-protection against weight loss and mortality from pneumonia. We will further characterize antibody functionality using serum bactericidal and opsonophagocytic killing assays. These studies will significantly advance our understanding of immune response to Kp O-antigen and aid in vaccine design to combat this pathogen.