Development of a Group B Streptococcus bioconjugate vaccine

项目总结 无乳链球菌，通常被称为B组链球菌(GBS)，是新生儿的主要病因 全世界的脑膜炎和败血症。GBS新生儿疾病表现为早发性侵袭性疾病，定义为 出生至出生6天之间的疾病，或晚发性侵袭性疾病，定义为在出生至出生6天之间发生的疾病 7天和89天。在某些情况下，早发性疾病可以通过产中预防性使用抗生素来预防； 然而，这种治疗策略对低收入和中等收入国家并不现实，这些国家占全球 所有新生儿的GBS感染，也不能预防迟发性疾病。三种GBS血清型(Ia、Ib和III型) 占所有早发性GBS感染的60%，占所有晚发型侵袭性GBS感染的90%。因此，一款低成本的 预防与这三种血清型相关的大多数侵袭性GBS新生儿疾病的疫苗策略 将是一个有价值的治疗干预选择。由多糖组成的结合疫苗 与载体蛋白共价连接的是用于预防多种细菌感染的疾病的救命疫苗 病原体。结合疫苗通常使用化学结合来制造，这是 臭名昭著的复杂、劳动密集型和昂贵，阻碍了新结合疫苗的开发。意识到 在这些缺点中，VaxNewMo一直在提出一种制造共轭化合物的替代方法 在称为生物偶联的过程中利用原核糖基化系统的疫苗。VaxNewMo‘s 专有的生物结合平台依靠结合酶将细菌多糖转移到 以大肠杆菌为宿主的工程载体蛋白。此外，由于生物偶联是一个酶过程，所以 产生的偶联物是非衍生化的，并且多糖的结构与所提供的相同 通过病原体本身传递给免疫细胞。在第一阶段，我们开发了一个三价原型(Ia、Ib和III型) 生物结合疫苗，并证明它是免疫原性的，引起功能性抗体反应 所有三种GBS血清型，并保护新生小鼠免受侵袭性血清III型GBS疾病的侵袭。同相 II，我们将使用改进的二糖化载体蛋白设计来生产Ia-，Ib-，III-型生物结合物 模仿最终配方，建立生物处理能力和下游提纯工艺 AS在小鼠身上进行前IND使能研究。在目标1中，我们将优化GBS Ia、Ib和III的生产 生物结合疫苗，从摇瓶转移到更工业化的补料分批生物反应器系统，移除 来自生物结合物的组氨酸标签(这在市场上的人类使用的疫苗中是不被接受的)，以及 建立下游纯化工艺，并确定疫苗质量属性。在目标2中，我们将 使用GBS生物结合物的单价和三价制剂在小鼠身上进行剂量递增研究 用改进的二糖化载体蛋白生产的疫苗通过ELISA法和 功能性抗体反应使用吞噬细胞杀伤试验(OPKA)。此外，我们还将评估 通过评估GBS毒株(Ia，Ib， 或三价生物结合疫苗母亲所生的孩子。

PROJECT SUMMARY Streptococcus agalactiae, commonly referred to as Group B Streptococcus (GBS), is a leading cause of neonatal meningitis and sepsis worldwide. GBS neonatal disease manifests as early onset invasive disease, defined as disease between birth and 6 days of life, or late onset invasive disease, defined as disease occurring between 7 and 89 days. In some instances, early onset disease is preventable with intrapartum antibiotic prophylaxis; however, this treatment strategy is not practical for low- and middle-income countries, which account for 95% of all neonatal GBS infections, nor does it prevent late onset disease. Three GBS serotypes (type Ia, Ib, and III) account for >60% of all early-onset and >90% of all late-onset invasive GBS infections. As such, a low-cost vaccine strategy to prevent the majority of invasive GBS neonatal disease associated with these three serotypes would be a valuable therapeutic intervention option. Conjugate vaccines, composed of polysaccharides covalently linked to carrier proteins, are life-saving vaccines used to prevent disease from multiple bacterial pathogens. Conjugate vaccines are conventionally manufactured using chemical conjugation, which is notoriously complex, labor intensive, and costly, hindering the development of new conjugate vaccines. 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 bacterial polysaccharides to engineered carrier proteins using E. coli as a host. Moreover, since bioconjugation is an enzymatic process, the conjugates produced are non-derivatized and the polysaccharides are structurally identical to those presented to immune cells by the pathogen itself. In Phase I, we developed a prototype trivalent (serotypes Ia, Ib, and III) bioconjugate vaccine and demonstrated that it was immunogenic, elicited functional antibody responses towards all three GBS serotypes, and protected newborn mouse pups from invasive serotype III GBS disease. In Phase II, we will produce type Ia-, Ib-, III-bioconjugates using an improved, di-glycosylated carrier protein design that mimics the final formulation, establish bioprocessing capabilities and downstream purification processes, as well as perform pre-IND enabling studies in mice. In Aim 1, we will optimize production of the GBS Ia, Ib and III bioconjugate vaccines, moving from shake flasks to more industrial fed-batch bioreactor systems, remove histidine tags from the bioconjugates (which are not acceptable in marketed vaccines for human use), and establish downstream purification processes as well as characterize vaccine quality attributes. In Aim 2, we will perform dose-escalation studies in mice using monovalent and trivalent formulations of GBS bioconjugate vaccines produced on the improved, di-glycosylated carrier protein to assess immunogenicity via ELISA and functional antibody responses using an opsonophagocytic killing assay (OPKA). Additionally, we will evaluate vaccine efficacy by evaluating survival of newborn mouse pups challenged with a GBS strain (either type Ia, Ib, or III) born to trivalent bioconjugate vaccinated mothers.