Abstract
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.
Highlights
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
To assess the difference between a traditional chemical strategy harnessing sodium periodate (NaIO4) oxidation to derivatize CPS as compared with the enzymatically directed oxidation through galactose oxidase, a representative pneumococcal polysaccharide found in the current pneumococcal conjugate vaccine (Prevnar 20; Pfizer, Inc) was selected
All conjugate vaccine administered mice showed minimal symptoms relative to adjuvant mice, with a 100% survival rate over the course of study. In both functional protection studies, the Pneumococcal Type 14 capsular polysaccharide (Pn14p) group did show activity, which can be attributed to the IgM response elicited through immunization with CPS alone—an effect that is used clinically for short-term protection against encapsulated pneumococcus currently [27]. These results show that a novel chemoenzymatic approach for reversible activation of CPS can be harnessed to form glycoconjugate vaccines that are site-specific and nondestructive for the competent polysaccharide antigen and is capable of providing a more robust humoral response with equal protection as the type found in commercial preparations
Summary
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. Current pneumococcal vaccine formulations are designed to provide protection against individual serotypes of Spn by eliciting an immune response specific to the capsular polysaccharide (CPS) coating the surface of the bacterium.
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