Formation and immunological evaluation of Moraxella catarrhalis glycoconjugates based on synthetic oligosaccharides.

Published work has shown that glycoconjugate vaccines, based on truncated detoxified lipopolysaccharides from Moraxella catarrhalis attached through their reducing end to a carrier protein, gave good protection for all three serotypes A, B, and C in mice immunisation experiments. The (from the non-reducing end) truncated LPS structures were obtained from bacterial glycosyl transferase knock-out mutants and contained the de-esterified Lipid A, two Kdo residues and five glucose moieties. This work describes the chemical synthesis of the same outer Moraxella LPS structures, spacer-equipped and further truncated from the reducing end, i.e., without the Lipid A part and containing four or five glucose moieties or four glucose moieties and one Kdo residue, and their subsequent conjugation to a carrier protein via a five­carbon bifunctional spacer to form glycoconjugates. Immunisation experiments both in mice and rabbits of these gave a good antibody response, being 2-7 times that of pre-immune sera. However, the sera produced only recognized the immunizing glycan immunogens and failed to bind to native LPS or whole bacterial cells. Comparative molecular modelling of three alternative antigens shows that an additional (2 â†’ 4)-linked Kdo residue, not present in the synthetic structures, has a significant impact on the shape and volume of the molecule, with implications for antigen binding and cross-reactivity.

Immune responses against group B Streptococcus monovalent and pentavalent capsular polysaccharide tetanus toxoid conjugate vaccines in Balb/c mice.

Immunization of pregnant women with Group B Streptococcus (GBS) capsular polysaccharide (CPS) conjugate vaccine (CV) could protect young infants against invasive GBS disease. We evaluated the immunogenicity of investigational five GBS monovalent (serotypes Ia, Ib, II, III, and V) CPS-tetanus toxoid (TT)-CV with adjuvant and GBS pentavalent CPS-TT-CV with adjuvant (GBS5-CV-adj) and without adjuvant (GBS5-CV-no-adj), in Balb/c mice. Aluminum phosphate was the adjuvant in the formulations, where included. The homotypic immunoglobulin G (IgG) geometric mean concentration (GMC) and opsonophagocytic activity (OPA) geometric mean titer (GMT) did not differ after the third dose of the GBS5-CV-adj vaccine compared with the monovalent counterparts for all five serotypes. The GBS5-CV-adj induced higher post-vaccination serotype-specific IgG GMCs and OPA GMTs compared to GBS5-CV-no_adj. The GBS5-CV with and without adjuvant should be considered for further development as a potential vaccine for pregnant women to protect their infants against invasive GBS disease.

A detailed picture of a protein-carbohydrate hydrogen-bonding network revealed by NMR and MD simulations.

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents.

Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen Bonds with a Protein.

Hydroxyl proton resonances of uniformly 13C-labeled Manα(1-2)Manα(1-2)ManαOMe (Man3) bound to cyanovirin-N (CV-N) were detected at ambient temperature in aqueous solution by NMR spectroscopy. The directions of the hydroxyl groups were determined on the basis of NOEs, and a previously unknown hydrogen-bonding network between Man3 and CV-N was discovered. This is the first report on detecting hydroxyl protons of a protein-bound carbohydrate in aqueous solution by NMR. Approaches such as those presented here may open the door for accurately determining intermolecular hydrogen bonds in carbohydrate-protein complexes.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

NMR of a uniformly 13C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13C spectral dispersion of 13C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.