A multivalent O-antigen bioconjugate vaccine for the prevention of Klebsiella pneumoniae infections

项目总结 肺炎克雷伯菌是医疗保健和社区相关感染的主要原因。此外， 肺炎克雷伯菌经常对第三代头孢菌素和 碳青霉烯类。事实上，对碳青霉烯类耐药克雷伯氏菌被疾控中心认为是一个紧急威胁，要求 咄咄逼人，立即采取行动。美国疾病控制与预防中心提出的对抗抗生素耐药性的五项核心行动之一 是为了继续投资和开发预防肺炎克雷伯菌和其他药物的疫苗 具有抗药性的细菌感染。因此，VaxNewMo开发了一种针对 肺炎克雷伯菌临床分离株居多。结合疫苗，由共价连接的多糖组成 对于一种载体蛋白，是用于预防多种细菌病原体疾病的救命疫苗。 传统上，结合疫苗是使用化学结合来制造的，这是出了名的复杂， 劳动强度大，而且不精确，阻碍了针对现有和 新出现的细菌威胁，如肺炎克雷伯菌。清楚地意识到这些缺点，VaxNewMo一直在推进 一种利用原核糖基化系统制造结合疫苗的替代方法 这一过程称为生物偶联。VaxNewMo的专利生物结合平台依赖于结合酶 将细菌多糖转移到载体蛋白上，所有这些都在实验室安全的细菌E.Coli内。此外，由于 生物偶联是一个酶驱动的过程，产生的偶联物是非衍生化的，因此 在结构上与病原体本身呈现给免疫细胞的那些相同。生物偶联可以用来 只需引入编码不同基因的新遗传信息即可快速产生许多连接物 将多糖血清型转化为具有生物结合能力的大肠杆菌菌株。作为一个例子，我们开发了 一种针对临床上80%肺炎克雷伯菌分离株的多价O抗原生物结合疫苗。 在这个快速通道应用中，我们将验证疫苗的免疫原性，并随后确定 在小鼠和兔子中优化剂量，通过以下方法评估功能性抗体反应和疫苗效力 进行挑战研究。在第一阶段，我们将评估单价和多价O-的免疫原性 在小鼠身上进行剂量递增研究的抗原生物偶联制剂。免疫原性将是 用酶联免疫吸附试验检测治疗前后血清特异性总免疫球蛋白及其亚型抗体浓度 对配制成疫苗的每个O-抗原进行免疫。一旦确认具有免疫原性，我们将 进入第二阶段。在第二阶段，我们将使用可扩展的微生物批量生产疫苗 生物反应器系统。随后，我们将通过血清杀菌试验来评估功能性抗体反应。 (SBA)和吞噬细胞杀伤试验(OPKA)，以及在接种疫苗的小鼠中进行挑战研究 用小鼠优化剂量的多价O抗原生物结合疫苗。最后，我们将确认 单价和多价O-抗原的免疫原性和功能性抗体应答(SBA和OPKA) 兔体内的生物结合制剂，广泛用于结合疫苗开发的动物模型。

PROJECT SUMMARY Klebsiella pneumoniae is a leading cause of healthcare- and community-associated infections. Moreover, K. pneumoniae is frequently resistant to last line antibiotics like third generation cephalosporins and carbapenems. In fact, carbapenem-resistant Klebsiella is considered an Urgent Threat by the CDC requiring aggressive, immediate action. One of the five core actions proposed by the CDC to combat antibiotic resistance is for continued investment and development of vaccines to prevent K. pneumoniae as well as other drug resistant bacterial infections. As such, VaxNewMo developed a multivalent conjugate vaccine targeting the majority of K. pneumoniae clinical isolates. Conjugate vaccines, composed of a polysaccharide covalently linked to a carrier protein, are life-saving vaccines used to prevent disease from multiple bacterial pathogens. Conventionally, conjugate vaccines are manufactured using chemical conjugation, which is notoriously complex, labor intensive, and imprecise, hindering the development of new conjugate vaccines against existing and emerging bacterial threats, like K. pneumoniae. Well aware of these drawbacks, VaxNewMo has been advancing an alternative method for manufacturing conjugate vaccines that utilizes prokaryotic glycosylation systems in a process termed bioconjugation. VaxNewMo’s proprietary bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to a carrier protein all within the lab safe bacterium E. coli. Moreover, since bioconjugation is an enzyme driven process, the conjugates produced are non-derivatized and are therefore structurally identical to those presented to immune cells by the pathogen itself. Bioconjugation can be used to rapidly produce many conjugates simply by introducing new genetic information encoding for a different polysaccharide serotype into a bioconjugation competent strain of E. coli. As an example of this, we developed a multivalent O-antigen bioconjugate vaccine targeting >80% of K. pneumoniae isolates encountered in the clinic. In this Fast-Track application, we will validate the vaccine for immunogenicity and subsequently determine optimized doses in mice and rabbits, assess functional antibody responses as well as vaccine efficacy by performing challenge studies. In Phase I, we will assess immunogenicity of monovalent and multivalent O- antigen bioconjugate formulations by performing dose-escalation studies in mice. Immunogenicity will be assessed by ELISA for serotype-specific total IgG and IgG subtype antibody concentrations pre- and post- immunizations to each O-antigen formulated into the vaccine. Once validated for immunogenicity, we will proceed to Phase II. In Phase II, we will produce the vaccine in larger batches using a scalable microbial bioreactor system. Subsequently, we will assess functional antibody responses via a serum bactericidal assay (SBA) and an opsonophagocytic killing assay (OPKA) as well as perform challenge studies in mice vaccinated with a mouse optimized dose of the multivalent O-antigen bioconjugate vaccine. Finally, we will confirm immunogenicity and functional antibody responses (SBA and OPKA) of monovalent and multivalent O-antigen bioconjugate formulations in rabbits, a widely utilized animal model for conjugate vaccine development.