Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA; Center for Molecular Medicine, University of Georgia, Athens, Georgia, USA.
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2022 Jan;298(1):101453. doi: 10.1016/j.jbc.2021.101453. Epub 2021 Nov 25.
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 (NaIO) 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 NaIO 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.
在制备商业结合疫苗时，荚膜多糖 (CPS) 必须经过化学修饰，以生成与蛋白质载体共价连接所需的反应基团。为此，最常用的方法之一是 CPS 结构中存在的邻二醇的高碘酸钠 (NaIO) 氧化。该过程在很大程度上是随机的，对结构具有破坏性，可能导致 CPS 结构及其抗原性发生重大变化。此外，CPS 的高碘酸盐活化通常会导致具有可变功效的异质结合疫苗产品。在这里，我们探索了使用替代试剂——来自镰孢菌属的半乳糖氧化酶 (GOase)，在化学酶方法中生成针对肺炎链球菌的结合疫苗。使用比色测定法和 NMR 光谱法，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 上生成醛基模体。GOase 或 NaIO 衍生的 Pn14p 的直接比较说明了化学氧化对 CPS 结构可能具有功能上的有害作用。与传统的高碘酸盐氧化组相比，用 GOase 合成的缀合物进行免疫接种可显著改善体液反应。此外，通过体外测定调理吞噬杀伤作用和体内通过在小鼠中的致死性挑战来验证功能保护。总的来说，这项工作引入了一种糖缀合物开发策略，克服了在当前疫苗设计方法中起作用的限制。
