In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

中文翻译：

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利用半乳糖氧化酶开发用于糖缀合物疫苗设计的化学酶平台。


在商业结合疫苗的制备中，荚膜多糖（CPS）必须经过化学修饰，以产生共价连接到蛋白质载体所需的反应基团。这种衍生化最常用的方法之一是对 CPS 结构中的邻位二醇进行高碘酸钠 (NaIO4) 氧化。该过程很大程度上是随机的并且具有结构破坏性，可能导致 CPS 结构及其抗原性发生显着变化。此外，CPS 的高碘酸盐活化通常会产生具有不同功效的异质结合疫苗产品。在这里，我们探索使用从镰刀菌中分离出的替代剂半乳糖氧化酶（GOase）。采用化学酶法产生针对肺炎链球菌的结合疫苗。使用比色测定和核磁共振波谱，我们发现 GOase 以位点特异性和可逆的方式在肺炎链球菌血清型 14 (Pn14p) 的 CPS 上生成醛基基序。直接比较 GOase 或 NaIO4 衍生的 Pn14p 说明化学氧化可能对 CPS 结构产生功能上的有害作用。与传统的高碘酸盐氧化组相比，使用 GOase 合成的缀合物进行免疫可显着改善体液反应。此外，通过测量调理吞噬杀伤作用在体外验证了功能性保护，并通过小鼠致死性挑战在体内验证了功能性保护。总体而言，这项工作引入了一种糖复合物开发策略，该策略克服了先前已知的在当前疫苗设计方法中发挥作用的限制。