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

项目摘要 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，委员会认为， K.肺炎通常对最后一线抗生素如第三代头孢菌素具有耐药性， 碳青霉烯类。事实上，碳青霉烯类耐药克雷伯氏菌被CDC认为是一种紧急威胁， 积极的立即行动CDC提出的对抗抗生素耐药性的五项核心行动之一 是继续投资和开发疫苗来预防K。肺炎以及其他药物 耐药细菌感染。因此，VaxNewMo开发了一种多价缀合物疫苗， 大多数K。肺炎临床分离株。结合疫苗，由共价连接的多糖组成 到载体蛋白，是用于预防多种细菌病原体引起的疾病的救命疫苗。 常规地，缀合物疫苗使用化学缀合来制造，化学缀合众所周知是复杂的， 劳动密集型和不精确，阻碍了针对现有和 新出现的细菌威胁，如K。肺炎。VaxNewMo深知这些缺点， 一种用于制备缀合物疫苗的替代方法， 这一过程被称为生物共轭。VaxNewMo的专有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，这一切都在实验室安全的细菌E.杆菌而且由于 生物缀合是酶驱动的过程，所产生的缀合物是非衍生化的，因此 在结构上与病原体本身呈递给免疫细胞的那些相同。生物缀合可用于 通过简单地引入新的编码不同的基因的遗传信息， 多糖血清型转化为具有生物接合能力的E.杆菌作为一个例子，我们开发了 靶向>80%的K.肺炎分离株在临床上遇到的。 在本快速通道申请中，我们将验证疫苗的免疫原性，并随后确定 在小鼠和兔中优化剂量，通过以下方法评估功能性抗体应答以及疫苗效力： 进行挑战研究。在I期，我们将评估单价和多价O- 通过在小鼠中进行剂量递增研究来制备抗原生物缀合物制剂。免疫原性将是 通过ELISA评估接种前和接种后的IgG型特异性总IgG和IgG亚型抗体浓度。 对配制成疫苗的每种0抗原进行免疫。一旦免疫原性得到验证，我们将 进入第二阶段。在第二阶段，我们将使用一种可扩展的微生物， 生物反应器系统随后，我们将通过血清杀菌试验评估功能性抗体应答 (SBA)和调理吞噬细胞杀伤试验（OPKA），并在接种疫苗的小鼠中进行攻毒研究 用小鼠优化剂量的多价O-抗原生物缀合物疫苗接种。最后，我们将确认 单价和多价O-抗原免疫原性和功能性抗体应答（SBA和OPKA） 兔中的生物缀合物制剂，一种广泛用于缀合物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.