Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Enzymic properties of an N-acetylglucosaminide 3-alpha-L-fucosyltransferase of a wheat-germ agglutinin-resistant melanoma clone.

A fucosyltransferase was solubilized by extraction with Triton CF-54 from a wheat-germ agglutinin-resistant variant of mouse B16 melanoma. Through affinity chromatography on GDP hexanolamine--Sepharose a 44-fold enrichment of its specific activity was obtained. Analysis of its specificity indicated that the enzyme is an N-acetylglucosaminide 3-alpha-L-fucosyltransferase, which is able to transfer fucose to oligosaccharides containing Gal(beta 1-4)GlcNAc and Gal(beta 1-4)Glc structures. The enzyme is activated by divalent cations and has a maximum of activity at pH 5. It is unable to transfer fucose to sialylated glycoproteins, 6-alpha-sialyllactose or 3-alpha-sialyllactose. As suggested by its precipitation in the presence of antibodies raised in rabbit against a soluble human milk N-acetylglucosaminide 3-alpha-L-fucosyltransferase, these two enzymes seem to be structurally related.

Co-purification of the Lewis blood group N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase and an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase from human milk.

The Lewis blood group-specified N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase and an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase have been copurified over 500,000-fold from human milk by affinity chromatography on GDP-hexanolamine agarose. The purified enzyme preparation migrates as two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr = 53,000 and 51,000. Analysis of the acceptor substrate specificity of the transferase(s) and structural characterization of the reaction products indicate that the enzyme(s) forms the Fuc alpha 1 goes to 4GlcNAc, Fuc alpha 1 goes to 3GlcNAc, and Fuc alpha 1 goes to 3Glc linkages with oligosaccharide acceptors containing the nonreducing terminal sequences Gal beta 1 goes to 3GlcNAc, Gal beta 1 goes to 4GlcNAc, and Gal beta 1 goes to 4Glc, respectively. The two fucosyltransferase activities are activated to the same extent by a variety of divalent metal ions, inactivated at identical rates by thermal denaturation or reaction with N-ethylmaleimide, and inhibited to the same extent by rabbit antiserum prepared against the purified fucosyltransferase(s). In addition, kinetic analysis of the initial rate data obtained using acceptors for one of the fucosyltransferase activities as an inhibitor of the second suggests that acceptors for both fucosyltransferase activities bind at a single active site.