Protective immunity elicited by distinct polysaccharide antigens of classical and hypervirulent Klebsiella

项目摘要 该项目将增强我们对肺炎克雷伯氏菌（Kp）的体液免疫应答的理解， 其多糖抗原的长期目标是指导和优化广泛的疫苗开发。KP 包括肺炎、尿路感染和菌血症在内的感染在非洲国家急剧上升， 住院患者; CDC已宣布感染Kp和其他碳青霉烯类耐药肠杆菌 (CRE)威胁等级为紧急除了在美国常见的经典Kp外，新出现的高毒力Kp 能够在健康宿主中引起肝脓肿、菌血症和脑膜炎的菌株正在全球范围内传播。 这项工作建立在PI的细菌学背景，革兰氏阴性菌致病机理和建模 宿主适应性免疫应答，以研究靶向Kp多糖胶囊（K型）的抗体 和O抗原。我们已经发现，虽然小鼠能够产生针对Kp囊和O- 抗原，胶囊可能直接干扰O-抗体结合和Kp的杀伤。与我们的合作者在 Omniose公司已经开发并正在测试针对最流行的K-和 O型血生物接合是一种替代的生产工艺，使用重组E。大肠杆菌菌株， 同时产生所述胶囊或O-抗原和工程化载体蛋白，并将所述胶囊或O-抗原与所述工程化载体蛋白酶促连接， 两个.我们已经生产了多种K-和O-生物缀合物，这些缀合物在小鼠中表现出有希望的功效。 由于O-和K-疫苗都在开发中，我们将在小鼠中使用我们的新型生物缀合物疫苗， 保护实验，以确定O-抗原或K-抗原生物缀合物针对 经典和高毒力Kp分离株。此外，我们将用密切相关的菌株攻击O免疫小鼠。 迄今为止尚未包括在疫苗制剂中的相关O-抗原结构亚型， 商业开发。将通过小鼠血清测定胶囊对O抗原的潜在掩蔽作用 IgG ELISA。将开发血清杀菌试验（SBAs）和调理吞噬细胞杀伤试验（OPKAs 并与鼠保护相关。此外，利用细菌突变体、互补菌株和荚膜， 抑制剂，我们将利用多种技术确定胶囊对Kp O-抗体抑制的特异性 包括生物层干涉测量法、免疫荧光显微术和透射免疫电子显微术， 显微镜最后，我们将对Kp感染的人类患者进行首次纵向研究，分析 其血清中对Kp多糖特异性的抗体及其对激发Kp菌株的功能活性。 在这些研究结束时，K型和O型生物缀合物疫苗的相对效力将是 确定，O抗原亚型之间的交叉保护将得到解决，保护性免疫的相关性将 建立，并将定义O-抗原掩蔽的机制。我们的研究结果将有助于阐明人类抗体 Kp感染的免疫应答，并指导疫苗开发以靶向这种令人担忧的病原体。

PROJECT SUMMARY This project will enhance our understanding of the humoral immune response to Klebsiella pneumoniae (Kp) and its polysaccharide antigens with the long-term goal of guiding and optimizing broad vaccine development. Kp infections, including pneumonia, urinary tract infection, and bacteremia, are sharply on the rise among hospitalized patients; CDC has declared infections with Kp and other carbapenem-resistant Enterobacteriales (CRE) demand a threat level of urgent. Beyond classical Kp typically seen in the US, emerging hypervirulent Kp strains, capable of causing liver abscess, bacteremia, and meningitis in healthy hosts, are spreading globally. This work builds on the PI's background in bacteriology, Gram-negative bacterial pathogenesis, and modeling of host adaptive immune responses, to investigate antibodies targeting Kp's polysaccharide capsule (K-type) and O-antigen. We have found that, while mice are able to produce antibodies targeting Kp capsule and O- antigen, capsule may directly interfere with O-antibody binding and killing of Kp. With our collaborators at Omniose, we have developed and are testing novel bioconjugate vaccines targeting the most prevalent K- and O-types. Bioconjugation is an alternative manufacturing process that uses recombinant E. coli strains to concurrently produce the capsule or O-antigen and an engineered carrier protein, and to enzymatically link the two. We have produced multiple K- and O-bioconjugates that have demonstrated promising efficacy in mice. As both O- and K- vaccines are under development, we will use our novel bioconjugate vaccines in murine protection experiments to determine the relative effectiveness of O-antigen or K-antigen bioconjugates against classical and hypervirulent Kp isolates. Further, we will challenge O-immunized mice with strains of closely related O-antigen structural subtypes that have not been included to date in vaccine formulations being developed commercially. Potential masking of O-antigen by capsule will be determined through mouse serum IgG ELISAs. Serum bactericidal assays (SBAs) and opsonophagocytic killing assays (OPKAs) will be developed and correlated with murine protection. Further, with bacterial mutants, complemented strains, and capsule inhibitors, we will determine the specificity of Kp O-antibody inhibition by capsule, utilizing multiple techniques including biolayer interferometry, immunofluorescence microscopy, and transmission immunoelectron microscopy. Finally, we will perform a first-ever longitudinal study of human patients with Kp infection, analyzing their sera for antibodies specific to Kp polysaccharides and their functional activity against the inciting Kp strain. At the conclusion of these studies, the relative efficacy of both K- and O-type bioconjugate vaccines will be determined, cross-protection among O-antigen subtypes will be resolved, correlates of protective immunity will be established, and mechanisms of O-antigen masking will be defined. Our results will illuminate human antibody responses to Kp infection and guide vaccine development to target this worrisome pathogen.