Development of a Group B Streptococcus bioconjugate vaccine

项目摘要 无乳链球菌，通常称为B族链球菌（GBS），是新生儿腹泻的主要原因。 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6天之间的疾病，或迟发性侵袭性疾病，定义为发生于 7、89天在某些情况下，早发性疾病可以通过分娩期抗生素预防来预防; 然而，这一治疗战略对低收入和中等收入国家并不实用，因为这些国家占全球人口的95%， 所有新生儿GBS感染，也不能预防迟发性疾病。三种GBS血清型（Ia、Ib和III型） 占所有早发侵袭性GBS感染的>60%和所有晚发侵袭性GBS感染的>90%。因此，低成本 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。结合疫苗，由多糖组成 共价连接到载体蛋白，是用于预防多种细菌引起的疾病的救命疫苗。 病原体缀合疫苗常规地使用化学缀合来制造，化学缀合是 众所周知的复杂、劳动密集和昂贵，阻碍了新的结合疫苗的开发。意识 在这些缺点中，VaxNewMo已经提出了一种制造缀合物的替代方法， 在称为生物缀合的过程中利用原核糖基化系统的疫苗。VaxNewMo的 专有的生物缀合平台依赖于缀合酶将细菌多糖转移到 工程化载体蛋白使用E.大肠杆菌作为宿主。此外，由于生物缀合是酶促过程， 所产生的缀合物是非衍生的，并且多糖在结构上与所呈现的那些相同 感染到免疫细胞在第一阶段，我们开发了一种原型三价（血清型Ia，Ib和III） 生物缀合物疫苗，并证明它是免疫原性的，引起功能性抗体应答， 所有三种GBS血清型，并保护新生小鼠幼崽免受侵袭性血清III型GBS疾病。同相 II，我们将使用改进的二糖基化载体蛋白设计生产Ia-、Ib-、III-型生物缀合物， 模拟最终配方，建立生物处理能力和下游纯化工艺，以及 在小鼠中进行IND前使能研究。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分批补料生物反应器系统， 来自生物缀合物的组氨酸标签（其在市售人用疫苗中不可接受），以及 建立下游纯化工艺以及表征疫苗质量属性。在目标2中，我们将 使用GBS生物缀合物的单价和三价制剂在小鼠中进行剂量递增研究 在改进的二糖基化载体蛋白上生产的疫苗，以通过ELISA评估免疫原性， 使用调理吞噬杀伤测定（OPKA）的功能性抗体应答。此外，我们将评估 通过评价用GBS菌株（Ia，Ib， 或III）由三价生物缀合物接种的母亲所生。

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.