The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-D-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.
Keywords: Conjugate vaccines; Immunological properties; Meningococcal lipooligosaccharides.