Genetic and structural characterization of L11 lipooligosaccharide from Neisseria meningitidis serogroup A strains.

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for alpha-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates.

A method, using high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD), has been developed to determine the concentrations of Streptococcus pneumoniae capsular polysaccharides and polysaccharide conjugates used in formulating a conjugate vaccine for the prevention of pneumococcal infections. In an effort to determine optimum hydrolysis conditions for the analysis, pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TFA) hydrolysis, methanolysis followed by TFA hydrolysis, or hydrofluoric acid (HF) hydrolysis followed by TFA hydrolysis. For quantification purposes, best results were obtained by methanolysis followed by TFA hydrolysis for uronic acid-containing polysaccharides, and by TFA hydrolysis for all the others. For the quantification of all the polysaccharides (from native to conjugated forms), a monosaccharide reference mixture (Rha, Gal and GlcA) hydrolyzed along with the samples can be used as standards for routine analysis. This is much more convenient than to hydrolyze a well-characterized reference polysaccharide (necessary standard only for type 1 capsular polysaccharide). This method is rapid, very sensitive (less than 10 microg of polysaccharide is required), and may replace advantageously the currently used colorimetric assays used to determine polysaccharides content. Moreover, it can be readily adapted for use with other bacterial polysaccharide preparations as well.

Intranasal immunization with pneumococcal conjugate vaccines with LT-K63, a nontoxic mutant of heat-Labile enterotoxin, as adjuvant rapidly induces protective immunity against lethal pneumococcal infections in neonatal mice.

Immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus toxoid (TT) (Pnc-TT) elicits protective immunity in an adult murine pneumococcal infection model. To assess immunogenicity and protective immunity in early life, neonatal (1 week old) and infant (3 weeks old) mice were immunized intranasally (i.n.) or subcutaneously (s.c.) with Pnc-TT of serotype 1 (Pnc1-TT). Anti-PPS-1 and anti-TT immunoglobulin G (IgG) and IgM antibodies were measured in serum and saliva, and vaccine-induced protection was evaluated by i.n. challenge with serotype 1 pneumococci. Pnc1-TT was immunogenic in neonatal and infant mice when administered s.c. without adjuvant: a majority of the young mice were protected from bacteremia and a reduction of pneumococcal density in the lungs was observed, although antibody responses and protective efficacy remained lower than in adults. The addition of LT-K63, a nontoxic mutant of heat-labile enterotoxin, as adjuvant significantly enhanced PPS-1-specific IgG responses and protective efficacy following either s.c. or i.n. Pnc1-TT immunization. Mucosal immunization was particularly efficient in neonates, as a single i.n. dose of Pnc1-TT and LT-K63 induced significantly higher PPS-1-specific IgG responses than s.c. immunization and was sufficient to protect neonatal mice against pneumococcal infections, whereas two s.c. doses were required to induce complete protection. In addition, i.n. immunization with Pnc1-TT and LT-K63 induced a vigorous salivary IgA response. This suggests that mucosal immunization with pneumococcal conjugate vaccines and LT-K63 may be able to circumvent some of the limitations of neonatal antibody responses, which are required for protective immunity in early life.