A capsule-based bioconjugate vaccine to prevent Klebsiella pneumoniae infections

项目总结 肺炎克雷伯氏菌是一种被包裹的人类病原体，能够引起无数的人类感染。 最近，肺炎克雷伯菌也成为继发性细菌性肺炎最常见的原因之一。 在新冠肺炎患者中。在过去的40年里，肺炎克雷伯菌已经进化成两种不同的致病类型，即已知的 如经典肺炎克雷伯菌(CKP)和超强毒力肺炎克雷伯菌(HvKp)。CKP通常充当一个 在住院或免疫功能受损的个体中引起疾病的机会性病原体。事实上，CKP是 每年占所有医疗保健相关感染的5%，是医院感染的主要原因 肺炎在美国。此外，CKP分离株往往是碳青霉烯类耐药(CR)，限制了治疗选择。 在美国，肺炎克雷伯菌多位点序列类型258(ST258)菌株约占所有碳青霉烯类抗生素的70%。 耐药肺炎克雷伯菌感染。相反，hvkp通常会在健康人群中引起社区获得性感染。 经常表现为社区获得性肺炎的宿主。像ST258感染一样，hvKp感染也有 高死亡率接近40%-60%。目前，还没有获得许可的疫苗来预防K。 肺炎感染，临床试验中无一例。然而，初步数据显示，ckp和hvkp都是 感染可以通过针对其胶囊多糖(CPS)的疫苗来预防。结合疫苗 由共价连接到免疫原性载体蛋白上的CPS组成。而结合物的临床益处 疫苗是有充分记录的，针对肺炎克雷伯菌的新结合疫苗的开发是滞后的， 可能是由于高技术进入壁垒和与结合疫苗生产相关的高成本。 此外，大多数结合疫苗是多价的，进一步增加了制造的复杂性。为了 为了简化结合疫苗的生产，我们开发了一种名为生物结合的体内结合平台。 生物偶联允许同时生产CPS、载体蛋白及其随后的 所有的共价键都在大肠杆菌内。我们生物结合平台的关键是我们的专利结合酶PGLS， 它几乎将任何多糖连接到融合到载体蛋白的独特的氨基酸序列上。 此外，生物结合是模块化的，允许快速生产多种不同的CPS-蛋白质 共轭关系。利用我们的生物结合平台，我们正在开发一种基于多价CPS的生物结合疫苗 预防大多数肺炎克雷伯菌感染。在这个第一阶段的STTR计划中，最初有四种血清型 选择(K1，K2，KL106，KL107)作为这些血清型与所有hvKp(K1和K2)分离株的80%相关 世界范围内，70%的ST258(KL106和KL107)分离株在美国。在目标1中，我们将生产一种四价化合物 (K1，K2，KL106，KL107)修饰载体蛋白内部糖基化的生物结合疫苗，其 有望改善稳定性和免疫原性等结合特性。在目标2中，我们将测试 四价生物结合疫苗在剂量递增研究中，以确定最佳剂量。最后，在目标3中，我们 将用ST258毒株(KL106和KL107)挑战安慰剂或生物结合疫苗接种组的小鼠 或hvKp毒株(K1和K2)，并评估作为疫苗效力替代品的存活率。

PROJECT SUMMARY Klebsiella pneumoniae is an encapsulated human pathogen capable of causing a myriad of human infections. Recently, K. pneumoniae has also emerged as one the most common causes of secondary bacterial pneumonia in COVID-19 patients. Over the last 40 years, K. pneumoniae has evolved into two distinct pathotypes, known as classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). cKp commonly acts as an opportunistic pathogen causing disease in hospitalized or immunocompromised individuals. In fact, cKp is annually responsible for 5% of all healthcare-associated infections and is the leading cause of nosocomial pneumonia in the US. Furthermore, cKp isolates are often carbapenem-resistant (CR), limiting treatment options. In the US, K. pneumoniae multilocus sequence type 258 (ST258) strains account for ~70% of all carbapenem- resistant K. pneumoniae infections. Conversely, hvKp usually cause community-acquired infections in healthy hosts that frequently manifest as community-acquired pneumonia. Like ST258 infections, hvKp infections have high mortality rates approaching 40-60%. Currently, there are no licensed vaccines available to prevent K. pneumoniae infections and none in clinical trials. Nevertheless, preliminary data demonstrate both cKp and hvKp infections can be prevented by vaccines that target their capsular polysaccharide (CPS). Conjugate vaccines consist of a CPS covalently attached to an immunogenic carrier protein. While the clinical benefits of conjugate vaccines are well documented, the development of new conjugate vaccines targeting K. pneumoniae is lagging, likely due to the high technological barriers to entry and high costs associated with conjugate vaccine production. In addition, most conjugate vaccines are multivalent, further increasing manufacturing complexities. In order to simplify conjugate vaccine production, we have developed an in vivo conjugation platform termed bioconjugation. Bioconjugation allows for the simultaneous production of the CPS, the carrier protein and their subsequent covalent linkage all within E. coli. Key to our bioconjugation platform is our patented conjugating enzyme, PglS, which attaches virtually any polysaccharide to a unique amino acid sequence fused to the carrier protein. Furthermore, bioconjugation is modular, allowing for rapid production of multiple, different CPS-protein conjugates. Using our bioconjugation platform, we are developing a multivalent CPS-based bioconjugate vaccine to prevent the majority of K. pneumoniae infections. In this Phase I STTR program, four serotypes were initially selected (K1, K2, KL106, KL107) as these serotypes are associated with >80% of all hvKp (K1 and K2) isolates worldwide and >70% of ST258 (KL106 and KL107) isolates in the US. In Aim 1, we will produce a tetravalent (K1, K2, KL106, KL107) bioconjugate vaccine on a modified carrier protein glycosylated at an internal site, which is expected to improve conjugate characteristics such as stability and immunogenicity. In Aim 2, we will test the tetravalent bioconjugate vaccine in a dose-escalation study to determine an optimal dose. Finally, in Aim 3, we will challenge groups of placebo- or bioconjugate-vaccinated mice with either a ST258 strain (KL106 and KL107) or a hvKp strain (K1 and K2) and assess survival as a surrogate for vaccine efficacy.