Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or highmolecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28-to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169-to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.

We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.