Development of a prototype Klebsiella O polysaccharide conjugate vaccine

 描述（由申请方提供）：肺炎克雷伯菌（KP）是日益重要的革兰氏阴性细菌病原体，可引起肺炎、伤口和尿路感染、菌血症和脑膜炎。与其他肠杆菌科细菌一样，KP对超广谱β-内酰胺酶（ESBL）抗生素（包括第三代头孢菌素）具有耐药性。KP现在包含越来越多的产ESBL细菌，令人担忧的是，在过去十年中，KP对碳青霉烯的耐药性越来越强。碳青霉烯耐药的KP（KPC）现在占所有KP的8%，并且在向CDC报告的数据中，在过去十年中增加了6倍以上，导致住院时间延长，成本增加和死亡率增加。这些噩梦般的细菌需要使用有毒、效果较差的最后手段抗生素。基于正在开发的抗生素的管道越来越少，新抗生素在短期内上市的可能性很小。针对KP的疫苗可以引发抗体，所述抗体将为KP提供不受抗生素耐药性机制影响的连续治疗。我们以前开发了一种23价KP荚膜多糖疫苗，并进行了人体试验。由于相对较少的KP O血清型占KP感染的>70%，我们推断基于COPS的多价KP疫苗可以提供广泛的覆盖范围，并且生产难度和成本较低。我们建议开发一种原型结合疫苗，针对核心/O多糖从脂多糖（LPS）的毒性KP（O 1 K2）连接到一个既定的载体蛋白，CRM 197通过两种不同的共轭化学。在具体目标1中，我们将构建KP的重组减毒株，其可用作可用于安全地纯化大量KP COPS的试剂菌株。正如我们对其他GNB病原体（志贺氏菌、沙门氏菌、弗朗西斯菌）所做的那样，我们将在鸟苷生物合成途径酶中产生突变，并在小鼠攻毒研究中确认毒力丧失。我们将在编码KP胶囊的基因中产生第二个突变，这将允许更有效地纯化表面COPS抗原。在具体目标2中，我们将测试这样的假设：KP型O 1 COPS与既定蛋白载体CRM 197的缀合将诱导抗LPS抗体，这些抗体介导吞噬细胞对KP的调理吞噬作用，并将保护小鼠免受腹腔内和气管内小鼠中致命的同源KP攻击模型。我们将改变连接（随机偶联与末端连接）和接头的使用，以评估哪种构建体产生最佳抗体应答和保护。在这些研究结束时，我们计划获得一种KP COPS结合疫苗，该疫苗可作为多价KP COPS疫苗的原型，可用于预防和/或开发用于治疗KP感染的KP抗体富集IVIG，即使菌株对抗生素具有高度耐药性。我们还将拥有一种KP生产菌株，该菌株对于规模扩大是安全的，并允许更有效地纯化KP COPS。

 DESCRIPTION (provided by applicant): Klebsiella pneumoniae (KP) are increasingly important Gram-negative bacterial pathogens that cause pneumonia, wound and urinary tract infections, bacteremia and meningitis. Like other Enterobacteriaceae, KP have become resistant to extended spectrum of beta lactamases (ESBL) antibiotics, including third generation cephalosporins. KP now comprise an increasing percentage of these ESBL-producing bacteria and alarmingly, in the last decade KP have become increasingly resistant to carbapenem. Carbapenamse-resistant KP (KPC) now represent 8% of all KP and in data reported to the CDC have increased over 6 fold in the last decade, resulting in extended hospitalization, increased costs and mortality. These nightmare bugs necessitate the use of toxic, less effective last resort antibiotics. Based on the dwindling pipeline of antibiotics being developed, there is little likelihood that new antibiotics will be available in the near term. A vaccine against KP could elict antibodies that would provide adjunctive therapy for KP that would not be subject to antibiotic resistance mechanisms. We previously developed a 23-valent KP capsular polysaccharide vaccine that progressed to human trials. Since relatively few KP O serotypes account for >70% of KP infections, we reason that a multivalent COPS-based KP vaccine could provide wide coverage and be less difficult and expensive to produce. We propose to develop a prototype conjugate vaccine against the core/O polysaccharide from the lipopolysaccharide (LPS) of a virulent KP (O1K2) linked to an established carrier protein, CRM197 by two different conjugation chemistries. In Specific Aim 1 we will construct a recombinant attenuated strain of KP that can serve as a reagent strain that can be used to safely purify large amounts of KP COPS. As we have successfully done for other GNB pathogens (Shigella, Salmonella, Francisella) we will generate mutations in the guanosine biosynthesis pathway enzymes, and confirm loss of virulence in murine challenge studies. We will generate a second mutation in the genes encoding the KP capsule which will permit a more efficient purification of the surface COPS antigen. In Specific Aim 2 we will test the hypothesis that conjugation of KP type O1 COPS to an established protein carrier, CRM197 will induce anti-LPS antibodies that mediate opsonophagocytosis of KP by phagocytes and will protect mice from lethal homologous KP challenges in both intraperitoneal and intratracheal mouse challenge models. We will vary the linkage (random coupling versus end-linkage) and use of linkers to assess which construct elicits optimal antibody responses and protection. At the conclusion of these studies, we intend to have a KP COPS conjugate vaccine that may serve as a prototype for a multivalent KP COPS vaccine that can be used in the prevention and/or development of a KP-antibody-enriched IVIG for the treatment of KP infection, even if the strains are highly resistant to antibiotics. We also will have a production strain of KP that will be safe for scale-up and allow for more efficient purification of the KP COPS.