Serogroup 19 capsule maleability leading to vaccine failure

肺炎链球菌(简称肺炎球菌)是一种重要的人类共生致病菌。一把钥匙 肺炎球菌适合性和毒力的决定因素是其产生保护性多糖(PS)的能力。 胶囊的形式可以是生化上截然不同的“血清型”。肺炎球菌胶囊PS 结合疫苗(PCV)诱导保护性抗体，介导吞噬细胞杀伤(OPK) 肺炎球菌，并有效地减少了由下列血清型引起的全球疾病负担 疫苗。尽管取得了这一成功，但接种疫苗的人偶尔会经历PCV的突破性感染 血清型，这些“疫苗失败”病例的原因尚不清楚。关于疫苗失效的调查 主要集中在宿主因素和无效的抗体反应上，而微生物学方面 很大程度上没有得到解决。此外，与19A和19F密切相关的血清型是最常见的血清型 与疫苗失败病例有关，但很少有研究评估它们在这一现象中的作用。 为了评价胶囊延展性的广度及其对临床结果的影响，我们正在研究 19A/19F型包膜变异与逃避抗包膜免疫反应之间的潜在联系 疫苗失败了。初步分析确定了多个候选机制，通过这些机制 在19A/19F胶囊中，聚合酶Wzy可以调节相当大的胶囊变异性，同时保留大部分 太空舱功能。我们还发现了一种用传统方法进行血清分型为“19F”的菌株，但实际上 产生了一种新的胶囊PS结构，这里称为19X。因此，19A/19F类胶囊类型的完全多样性 还没有被定义。因为即使是胶囊结构的微小变化也会消除交叉保护 免疫，可能是一些变种，它们与19A/19F肺炎球菌无法区分 传统的血清分型方法仍然可以通过抗19A/19F胶囊抗体逃避OPK 因此，导致这些血清型的疫苗失败。 在这个R21提案中，我们将执行定向突变来测试Wzy多态对 19A/19F胶囊结构，并测试这些假定胶囊的变化对逃避反胶囊的影响 抗体介导的OPK体外实验(AIM 1)。我们还将从结构上/基因/抗原学上描述 新的19x胶囊类型并对扩展的基因组数据库进行生物信息学分析，目标是 确定在免疫人群中发现的其他假定的胶囊变异(目标2)。重要的是，工具和 这里提出的概念将推动未来对胶囊PS延展性的影响的研究 肺炎球菌血清型和其他使用类似途径的医学上重要的细菌病原体 多聚糖合成。独立于糖生物学进展，阐明19A/19F Wzy的分子基础 酶的特异性将立即提高分子分型策略的精确度。

Streptococcus pneumoniae (the “pneumococcus”) is an important human commensal pathogen. A key determinant of pneumococcal fitness and virulence is its ability to produce a protective polysaccharide (PS) capsule which can take the form of >100 biochemically distinct “serotypes”. Pneumococcal capsule PS conjugate vaccines (PCVs) induce protective antibodies that mediate opsonophagocytic killing (OPK) of pneumococci and have effectively reduced the global burden of disease caused by serotypes included in vaccines. Despite this success, immunized people occasionally experience breakthrough infections by PCV serotypes, and the cause for these cases of “vaccine failure” remains unclear. Investigation of vaccine failure has largely focused on host factors and ineffective antibody response, while microbiological aspects have gone largely unaddressed. Furthermore, closely-related serotypes 19A and 19F are the serotypes most commonly implicated in vaccine failure cases, but few studies have evaluated their role in this phenomenon. Appreciating the breadth of capsule malleability and its impact on clinical outcomes, we are examining a potential link between serotype 19A/19F capsule variants, evasion of anti-capsule immune responses, and vaccine failure. Preliminary analyses identified multiple candidate mechanisms through which polymorphisms in the 19A/19F capsule polymerase Wzy can mediate considerable capsule variability while preserving most capsule features. We also found a strain that was serotyped as “19F” by conventional methods, but in fact produces a novel capsule PS structure, herein called 19x. Thus, the full diversity of 19A/19F-like capsule types is yet to be defined. As even small changes in capsule structure can abrogate cross-protective immunity, it is possible that some variants, which are indistinguishable from 19A/19F pneumococci in conventional serotyping methods, can nonetheless evade OPK by anti-19A/19F capsule antibodies in vaccinated individuals and, thus, spur the vaccine failure attributed to these serotypes. In this R21 proposal, we will perform directed mutagenesis to test the impact of Wzy polymorphisms on 19A/19F capsules structure and test the effect of these putative capsule changes on evading anti-capsule antibody-mediated OPK in vitro (Aim 1). We will also structurally/genetically/antigenically characterize the novel 19x capsule type and perform bioinformatics analysis of expansive genomic databases with the goal of identifying other putative capsule variants found among immunized populations (Aim 2). Importantly, tools and concepts developed here will fuel future investigation of the impact capsule PS malleability has in additional pneumococcal serotypes and other medically important bacterial pathogens that employ similar pathways for glycan synthesis. Independent of glycobiological advances, elucidation of the molecular basis of 19A/19F Wzy enzymatic specificity would immediately improve the precision of the molecular typing strategies.