A method was developed for coupling carboxylic acid-containing oligosaccharides (OS) to proteins. The 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 (TT). The molar ratio of the OS to TT in three conjugates ranged from 11:1 to 19:1. The antigenicity of the OS was conserved in these conjugates as measured by ELISA and inhibition ELISA with polyclonal and monoclonal antibodies to A1 LOS. These conjugates induced IgG antibodies to A1 LOS in mice and rabbits. The immunogenicity of the conjugates in rabbits was enhanced by using 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 by ELISA and by Western blot which revealed a 3.6 KDa reactive band in these LOSs. The rabbit antisera showed bactericidal activity against the homologous strain A1, and the heterologous strains 44/76 and BB431. These results indicate that conjugates derived from A1 LOS can induce antibodies against many immunotype LOSs from different serogroup organisms including group B. OS- protein conjugates derived from meningococcal LOSs, therefore, may be a candidate vaccine to prevent meningitis caused by this pathogen.
