Synthesis and Immunological Study of N-Glycan-Bacteriophage Qβ Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core

N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Q beta conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Q beta glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Q beta conjugates was performed in mice. We found that the N-glycan-Q beta conjugates raised significant IgG antibodies that recognize N-glycans, but, surprisingly, most of the glycan-dependent antibodies were directed to the shared chitobiose core and were nonspecific for respective N-glycan structures. The linker chemistry was found to affect antibody specificity with adipic acid-linked N-glycan-Q beta immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Q beta immunogens with a triazole linker preferentially recognized the innermost N-acetylglucosamine moiety at the reducing end. We also found that sialylation of the N-glycans significantly suppressed the immune response. Furthermore, the N-glycan-Q beta immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Q beta immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.

Automated summary

N-Glycosylation plays an important role in biological recognition, but there are very few specific antibodies available for research and treatment. In this study, phage Q beta conjugates with representative N-glycans were synthesized to raise N-glycan-specific antibodies. However, most of the antibodies recognized the shared chitobiose core instead of specific N-glycan structures. The linker chemistry and sialylation of N-glycans were found to affect antibody specificity. Adipic acid-linked N-glycan-Q beta immunogens raised antibodies capable of recognizing both the N-acetylglucosamine moieties of the chitobiose core, while triazole-linked immunogens preferentially recognized the innermost N-acetylglucosamine moiety. Furthermore, the presence of sialylation significantly suppressed the immune response. These findings highlight the challenges in eliciting mammalian N-glycan-specific antibodies through conventional vaccine design and immunization.