Genomic Analysis of Glycosyltransferases Responsible for Galactose-α-1,3-Galactose Epitopes in Streptococcus pneumoniae: Implications for Broadly Protective Vaccination Strategy

Background: The origin of natural anti-galactose-α-1,3-galactose (anti-Gal) antibodies in humans is only partially understood. The gut microbiome has been proposed as an important source of galactose-α-1,3-galactose (αGal) epitopes that drive the maturation of anti-Gal–reactive B cells. Certain bacteria expressing αGal epitopes, notably Escherichia coli O86:B7, have been shown to elicit anti-Gal antibody responses in α1,3-galactosyltransferase knockout (α3GalT1 KO) mice. In this study, we investigated the interaction between currently widely used bacteria polysaccharide vaccine, the 23-valent pneumococcal polysaccharide vaccine (PPV23), which contains capsular polysaccharides (CPS) from multiple Streptococcus pneumoniae serotypes, and host anti-Gal antibodies. Methods: We conducted a genomic analysis to identify α1,3-galactosyltransferase (α3GalT1) genes in S. pneumoniae strains. Binding of PPV23 to anti-Gal monoclonal antibodies was evaluated by ELISA, and αGal epitope content in PPV23 was estimated using a four-parameter logistic (4PL) model fitted to the ELISA calibration data. To assess in vivo immunogenicity, we immunized α3GalT1 KO mice with PPV23 and measured serum anti-Gal IgG and IgM titers before and after vaccination. Results: Genomic analysis revealed the presence of α3GalT1 genes in S. pneumoniae strains. PPV23 showed specific binding to anti-Gal monoclonal antibodies as detected by ELISA. Quantitative modeling indicated that αGal epitopes are present at low abundance within PPV23, consistent with limited expression of αGal among a minority of included serotypes. Immunization of α3GalT1 KO mice with PPV23 induced a significant rise in anti-Gal IgG titers (mean value from 124 to 384), whereas anti-Gal IgM titers remained relatively unchanged (mean value at the range of 6500–7500). High baseline anti-Gal IgM levels observed in α3GalT1 KO mice are consistent with age-dependent induction by the gut microbiota. Conclusions: These results provide genetic and immunological evidence that αGal epitopes derived from S. pneumoniae are present in PPV23 and can engage pre-existing anti-Gal antibodies. Our findings underscore a complex interplay among bacterial glycosyltransferase genes, vaccine polysaccharide composition, and host anti-Gal antibody repertoires, which may influence vaccine immunogenicity. Consideration of host natural antibody profiles may therefore be important for interpreting responses to carbohydrate-based vaccines and for guiding vaccine design.