Preparation, characterization, and immunogenicity of meningococcal lipooligosaccharide-derived oligosaccharide-protein conjugates

Abstract

A method was developed for coupling carboxylic acid-containing oligosaccharides (OS) to proteins. An OS was isolated from Neisseria meningitidis group A strain A1 lipooligosaccharide (LOS). This LOS has no human glycolipid-like lacto-N-neotetraose structure and contains multiple immunotypes, including L8, found in group B and C strains. The carboxylic acid at 2-keto-3-deoxyoctulosonic acid of the OS was linked through adipic acid dihydrazide to tetanus toxoid. The molar ratio of the OS to tetanus toxoid in three conjugates ranged from 11:1 to 19:1. The antigenicity of the OS was conserved in these conjugates, as measured by an enzyme-linked immunosorbent assay (ELISA) and an inhibition ELISA with polyclonal and monoclonal antibodies to A1 LOS. These conjugates induced immunoglobulin G antibodies to A1 LOS in mice and rabbits. The immunogenicity of the conjugates in rabbits was enhanced by use of monophosphoryl lipid A plus trehalose dimycolate as an adjuvant. The resulting rabbit antisera cross- reacted with most of 12 prototype LOSs and with LOSs from two group B disease strains, 44/76 and BB431, in an ELISA and in Western blotting (immunoblotting), which revealed a 3.6-kDa reactive band in these LOSs. The rabbit antisera showed bactericidal activity against homologous strain A1 and heterologous strains 44/76 and BB431. These results indicate that conjugates derived from A1 LOS can induce antibodies against many LOS immunotypes from different organism serogroups, including group B. OS-protein conjugates derived from meningococcal LOSs may therefore be candidate vaccines to prevent meningitis caused by meningococci.