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

Abstract

bDespite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, 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 protein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, 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 reductive 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) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens.

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

“…1A). Recently, DMTMM has been applied to many challenging synthetic problems, for example, in peptide synthesis and bioconjugation chemistry (24)(25)(26). Hayama et al have shown its use in the coupling of the fluorescent derivatization agent, 4-(1-pyrene)butanoic acid hydrazide, to carboxyl groups of microcystins (27).…”

Conjugation of PspA4Pro with Capsular Streptococcus pneumoniae Polysaccharide Serotype 14 Does Not Reduce the Induction of Cross-Reactive Antibodies

“…The use of pneumococcal surface proteins as an alternative to heterologous proteins, such as CRM 197 or tetanus toxoid as a carrier for CPS, would be a way to improve pneumococcal vaccine coverage. PspA has been shown to be an efficient carrier for capsular polysaccharide, as well as an antigen able to induce protective antibodies when conjugated (11)(12)(13)30). PspA is one of the pneumococcal surface proteins described as a protein vaccine candidate, and the main reason is its capacity to induce protective antibodies (25,28,31).…”

“…However, the introduction of PCVs has been followed by an increase in disease rates caused by serotypes not included in their formulation, also known as serotype replacement (9,10). In order to circumvent this limitation, we have been evaluating pneumococcal surface protein A (PspA) as a carrier protein in a pneumococcal conjugate in order to broaden the vaccine coverage and possibly reduce serotype replacement (11)(12)(13).…”

Vaccine Potential of Pneumococcal Proteins

“…Furthermore, as demonstrated for pneumolysoids, PspA can also be an effective carrier of CPS when tested in mouse models [66,67]. Indeed, available comparative immunogenicity and protection studies from humans, mice, and rabbits support this notion [5À7,51,53,63À65].…”

“…We have also summarized evidence supporting development of multi-component protein vaccines, which have significantly enhanced protective efficacy relative to individual antigens. The success of pneumolysoid-CPS [33,35,36] and PspA-CPS [66,67] conjugate formulations in animal models provides excellent "proof of concept" for this strategy. They also provide an opportunity to include antigens that elicit improved protection against diseases such as meningitis and otitis media, against which the existing conjugate vaccines have uncertain efficacy.…”