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

Bioconjug Chem. 2022 Jul 20;33(7):1350-1362. doi: 10.1021/acs.bioconjchem.2c00211. Epub 2022 Jun 10.


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 Qβ conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qβ glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Qβ conjugates was performed in mice. We found that the N-glycan-Qβ 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-Qβ immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Qβ 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-Qβ immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Qβ immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylglucosamine*
  • Allolevivirus / chemistry
  • Animals
  • Antibody Formation*
  • Antigens
  • Disaccharides
  • Glycoconjugates
  • Humans
  • Mammals
  • Mice
  • Polysaccharides / chemistry
  • Triazoles


  • Antigens
  • Disaccharides
  • Glycoconjugates
  • Polysaccharides
  • Triazoles
  • chitobiose
  • Acetylglucosamine