Conjugation of Meningococcal Lipooligosaccharides Through Their Non-Reducing Terminus Results in Improved Induction a Protective Immune Response.

The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-D-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

Re-classification within the serogroups O3 and O8 of Citrobacter strains.

BACKGROUND: Citrobacter strains are opportunistic pathogens often responsible for serious enteric as well as extra-intestinal diseases, and therefore the O-antigenic scheme, still in use in diagnostic identification, should be set for proper serotyping. The structures of more than 30 different Citrobacter O-antigens (O-polysaccharide chains of the lipopolysaccharides) of 43 Citrobacter O-serogroups have been elucidated so far. However, relationships between strains in several heterogeneous serogroups still need to be clarified by immunochemical studies. These include complex serogroups O3 and O8, represented by 20 and 7 strains, respectively, which are the subject of the present work. Earlier, the O-polysaccharide structures have been determined for Citrobacter O3 strain Be35/57 (PCM 1508) and Citrobacter O8 strain Be64/57 (PCM 1536). RESULTS: Serological studies (immunoblotting) carried out on Citrobacter lipopolysaccharides from different strains ascribed to serogroups O3 and O8 showed that each of these serogroups should be divided into non-cross-reacting subgroups. Based on the results of chemical analyses and 1H and 13C NMR spectroscopy the structure of Citrobacter O-antigens from strains PCM 1504 (O6) and PCM 1573 (O2) have been established. Chemical data combined with serological analyses showed that several Citrobacter strains should be reclassified into other serogroups. CONCLUSIONS: Immunochemical studies carried out on Citrobacter LPS, described in this paper, showed the expediency of reclassification of: 1) strains PCM 1504 and PCM 1573 from serogroups O6 and O2 to serogroups O3 and O8, respectively, 2) strains PCM 1503 and PCM 1505 from serogroups O3 and O8 to new serogroups O3a and O8a, respectively.

O-aminoacylation of bacterial glycoconjugates: from native structure to vaccine design.

The aminoacylation of bacterial polysaccharide antigens and its biological role are poorly understood, although it might be relevant in infection and immunity. Due to the lability of ester-linked substituents on glycoconjugate antigens, such groups usually escape detection during routine structural investigation. Of the few data available, those on the occurrence of glycine in the endotoxic lipopolysaccharides of Gram-negative bacteria are well documented. This article summarizes these data on glycine as an integral constituent of bacterial LPS and also some other amino-acid esters in the teichoic acids and phosphatidylglycerol of Gram-positive bacteria. The possible functions of such noncarbohydrate ester-linked substituents in bacterial antigens are discussed. Because glycine, an inherent component of bacterial lipopolysaccharides in the core region, is supposed to participate in epitope formation, such a structure may be considered for potential use in the construction of a vaccine with broad specificity.

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization.

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.

Structure of the O-polysaccharide of Citrobacter youngae O1 containing an alpha-D-ribofuranosyl group.

The lipopolysaccharide of Citrobacter youngae O1, strain PCM 1492 was degraded with acid or alkali under mild conditions, and the resultant polysaccharide was isolated by GPC and studied by sugar and methylation analyses and 1H and 13C NMR spectroscopies, including 2D COSY, TOCSY, NOESY and 1H, 13C HSQC experiments. The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [structure: see text] where substitution with the alpha-D-Ribf group is nonstoichiometric. This group occurs rarely in bacterial polysaccharides and is easily cleaved under mild acidic conditions. Studies with polyclonal rabbit antisera against whole cells of C. youngae PCM 1492 and PCM 1506 showed the serological identity of the lipopolysaccharides of C. youngae PCM 1492, PCM 1493 and PCM 1506, which are classified in serogroup O1.

The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strains.

Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: [structure: see text]. NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.

Conjugation of meningococcal lipooligosaccharides through their lipid A terminus conserves their inner epitopes and results in conjugate vaccines having improved immunological properties.

The importance of conserved inner saccharide epitopes to the immune performance of meningococcal lipooligosaccharide-protein conjugate vaccines was demonstrated in the following experiments. Two different oligosaccharides were obtained by chemical degradations of the same L7 lipooligosaccharide, and both were linked terminally to tetanus toxoid. One was a truncated oligosaccharide in which the inner epitopes were incomplete and was obtained by mild acid hydrolysis of the L7 lipooligosaccharide. This oligosaccharide was conjugated by direct reductive amination through its newly exposed terminal Kdo residue. The second, a full-length oligosaccharide, was obtained by O-deacylation of the L7 lipooligosaccharide, with subsequent removal of phosphate substituents from its lipid A moiety using alkaline phosphatase. This permitted the full-length oligosaccharide to be conjugated directly to tetanus toxoid by reductive amination through its newly exposed terminal 2-N-acyl-2-deoxy-D-glucopyranose residue. Comparison of the immune performance of the two conjugates in mice revealed, that while both were able to induce significant levels of L7-lipooligosaccharide-specific IgG antibody, the conjugate made with the full-length saccharide was able to induce antibodies with increased bactericidal activity against homologous meningococci.

[Glycine as an inherent component of bacterial lipopolysaccharides]. / Glicyna jako integralny skladnik bakteryjnych lipopolisacharydów.

The amino-acylation of bacterial polysaccharide antigens and the biological role of this phenomenon are poorly understood, although it might be relevant in the processes of the infection and immunity. Due to the lability of ester linked substituents on glyco-conjugate antigens, usually such groups escaped detection during routine structural investigation. Among the only few data available, these on the occurrence of glycine in endotoxic lipopolysaccharides of Gram-negative bacteria are well documented. This work summarises these data on glycine as an integral constituent of bacterial LPS and also on some other amino acid esters in teichoic acids and phosphatidylglycerol of Gram-positive bacteria. The possible functions are discussed of such noncarbohydrate ester linked substituents in bacterial antigens.