Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Despite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immuniza-tion programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently docu-mented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface pro-tein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugationmethodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive ami-nation, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0 % of PS6B was in the conjugate fraction, whereas 24%was observed in the conjugate fraction with reduc-tive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46 % and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) in-volved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens