Impact of Immunoglobulin Isotype and Epitope on the Functional Properties of Vibrio cholerae O-Specific Polysaccharide-Specific Monoclonal Antibodies.

Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

Immunodiagnostic of Vibrio cholerae O1 using localized surface plasmon resonance (LSPR) biosensor.

V. cholerae O1 is a gram-negative bacilli that causes an acute gastrointestinal disease called cholera. V. cholerae can enter into the biofilm phase in a period of life; hence, it is challenging to recognize these bacteria. Accordingly, using localized surface plasmon resonance (LSPR) features of the nanoparticles, an accurate detection method based on the antigen-antibody reaction was used. Ordinarily, immobilization of plasmonic nanoparticles by monoclonal antibodies was performed and UV-visible spectroscopy, dynamic light scattering (DLS), and zeta potential (Zp) measurements verified the conjugation process. In the vicinity of several concentrations of V. cholerae O1, the consistency of the engineered nanobioprobe was then investigated using LSPR monitoring and colorimetric assay. Finally, the ELISA and PCR techniques contrasted the sensitivity of nanobiosensors. The results showed that by applying monoclonal antibodies as a sensor feature, the nanobioprobe showed high sensitivity to target bacterial analysis. Thus, the limit of detection in this immunoassay-based biosensor was calculated to be a sharp reduction in the absorption of 10 CFU/mL of V. cholerae O1 with approximately 5 nm of redshift, while the shift of light refraction in the LSPR band was extended to approximately 18 nm by raising the antigen concentration to 104 CFU/mL. This LSPR biosensor can therefore be used for V. cholerae O1 (Inaba strain) detection as a simple, sensitive, and reliable diagnostic tool. In conclusion, the built biosensor will facilitate and speed up V. cholerae O1 (Inaba strain) classification by controlling the specific antigen to prevent the unintended spread of cholera disease.

Synthesis of glycocluster-containing conjugates for a vaccine against cholera.

Glycoclusters displaying synthetic fragments of the O-specific polysaccharide (OSP) of Vibrio cholerae O1 serotype Inaba on a carbohydrate platform were prepared by Cu(i)-catalysed azide alkyne cycloaddition (CuAAC, click chemistry). The clusters were subsequently conjugated to BSA via squaric acid chemistry. Their immunoreactivity was compared with those of similar conventional conjugates, i.e. made from single oligosaccharides presented in non cluster form, using plasma of patients recovering from cholera. The results showed that the conjugates were displayed in immunologically relevant manners and that the immunoreactivity of hexasaccharide-cluster conjugates was similar to that of a conjugate displaying OSP isolated from wild type V. cholerae, further supporting the immunologic relevance of antigens made from synthetic oligosaccharides.

Development of lipopolysaccharide-mimicking peptides and their immunoprotectivity against Vibrio cholerae serogroup O1.

Vibrio cholerae serogroup O1 is the main causative agent of cholera diseases defined by life threatening rice watery diarrhea. Cholera routine vaccination has failed in controlling epidemics in developing countries because of their hard and expensive production. In this study, our aim was to investigate phage displayed mimotopes that could mimic V. cholerae lipopolysaccharide (LPS). Although LPS of Vibrio, as an endotoxin, can stimulate the immune system, thereby making it a suitable candidate for cholera vaccine, its toxicity remains as a main problem. Phage particles displaying 12 amino acid peptides were selected from phage library mimicking the antigenic epitopes of LPS from vibrio. The screening was carried out using single-domain antibody fragment VHH against LPS as target through three rounds of selection. Three clones with highest affinity to VHH were selected. To find out a new and efficient vaccine against cholera, these three phage particles containing high-affinity peptides were administered to mice to investigate the active and passive immunity. Out of 20 particles, three showed the highest affinity toward VHH. ELISA was carried out with immunized mice sera using LPS and three selected phages particles individually. ETEC, Shigella sonnei, and clinical isolates were used as bacterial targets. These three selected phages (individually or in combination) could stimulate mice immune system producing active and passive immunity. The mice immunized with phage particles could protect about 14 LD50 of V. cholerae. In conclusion, these peptides are mimicking LPS and can potentially act as vaccine candidates against V. cholerae. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

O-Specific Polysaccharide-Specific Memory B Cell Responses in Young Children, Older Children, and Adults Infected with Vibrio cholerae O1 Ogawa in Bangladesh.

Cholera caused by Vibrio cholerae O1 confers at least 3 to 10 years of protection against subsequent disease regardless of age, despite a relatively rapid fall in antibody levels in peripheral blood, suggesting that memory B cell responses may play an important role in protection. The V. cholerae O1-specific polysaccharide (OSP) component of lipopolysaccharide (LPS) is responsible for serogroup specificity, and it is unclear if young children are capable of developing memory B cell responses against OSP, a T cell-independent antigen, following cholera. To address this, we assessed OSP-specific memory B cell responses in young children (2 to 5 years, n = 11), older children (6 to 17 years, n = 21), and adults (18 to 55 years, n = 28) with cholera caused by V. cholerae O1 in Dhaka, Bangladesh. We also assessed memory B cell responses against LPS and vibriocidal responses, and plasma antibody responses against OSP, LPS, and cholera toxin B subunit (CtxB; a T cell-dependent antigen) on days 2 and 7, as well as days 30, 90, and 180 after convalescence. In all age cohorts, vibriocidal responses and plasma OSP, LPS, and CtxB-specific responses peaked on day 7 and fell toward baseline over the follow-up period. In comparison, we were able to detect OSP memory B cell responses in all age cohorts of patients with detectable responses over baseline for 90 to 180 days. Our results suggest that OSP-specific memory B cell responses can occur following cholera, even in the youngest children, and may explain in part the age-independent induction of long-term immunity following naturally acquired disease.

Synthesis and molecular structure of the 5-methoxycarbonylpentyl α-glycoside of the upstream, terminal moiety of the O-specific polysaccharide of Vibrio cholerae O1, serotype Inaba.

The trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed reaction of methyl 6-hydroxyhexanoate with 3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-levulinoyl-α-d-mannopyranosyl trichloroacetimidate followed by a two-step deprotection (hydrogenolysis over Pd/C catalyst and Zemplén deacylation, to simultaneously remove the acetyl and levulinoyl groups) gave 5-(methoxycarbonyl)pentyl 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranoside. The structure of the latter, for which crystals were obtained in the analytically pure state for the first time, followed from its NMR and high-resolution mass spectra and was confirmed by X-ray crystallography. The molecule has two approximately linear components; a line through the aglycon intersects a line through the mannosyl and tetronylamido groups at 120°. The crystal packing separates the aglycon groups from the tetronylamido and mannosyl groups, with only C-H...O hydrogen bonding among the aglycon groups and N-H...O, O-H...O and C-H...O links among the tetronylamido and mannosyl groups. A carbonyl oxygen atom accepts the strongest O-H...O hydrogen bond and two strong C-H...O hydrogen bonds. The geometric properties were compared with those of related molecules.

Structural characterization of the extracellular polysaccharide from Vibrio cholerae O1 El-Tor.

The ability to form biofilms is important for environmental survival, transmission, and infectivity of Vibrio cholerae, the causative agent of cholera in humans. To form biofilms, V. cholerae produces an extracellular matrix composed of proteins, nucleic acids and a glycoconjugate, termed Vibrio exopolysaccharide (VPS). Here, we present the data on isolation and characterization of the polysaccharide part of the VPS (VPS-PS), which has the following structure: -4)-α-GulpNAcAGly3OAc-(1-4)-ß-D-Glcp-(1-4)-α-Glcp-(1-4)-α-D-Galp-(1- where α-D-Glc is partially (∼20%) replaced with α-D-GlcNAc. α-GulNAcAGly is an amide between 2-acetamido-2-deoxy-α-guluronic acid and glycine. Apparently, the polysaccharide is bound to a yet unidentified component, which gives it high viscosity and completely suppresses any NMR signals belonging to the sugar chains of the VPS. The only reliable method to remove this component at present is a treatment of the whole glycoconjugate with concentrated hydrochloric acid.

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C-fragment carrier.

We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT-Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate-protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT-Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein.

Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum.

Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.

Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC-ESI-QqTOF-MS/MS.

In this manuscript, we present the determination of glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.

Molecular architecture and assembly principles of Vibrio cholerae biofilms.

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion; Bap1 allowed the developing biofilm to adhere to surfaces; and heterogeneous mixtures of Vibrio polysaccharide, RbmC, and Bap1 formed dynamic, flexible, and ordered envelopes that encased the cell clusters.

Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines.

Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.

[Evaluation of toxin producing abilities of non-O1/non-O139 serogroup Vibrio cholerae isolated from humans].

AIM: Determination of non-O1/non-O139 Vibrio cholerae toxin (CT) gene expression by using EIA, and biological effect of non-O1/non-O139 V. cholerae supernatant on cell cultures evaluation. MATERIALS AND METHODS: 39 V. cholerae strains from various serological groups were studied. Hemolytic activity of strains was determined by using Greig test, and cholera toxin production--in GM1-EIA and in continuous cell lines by registering cytotonic, cytotoxic and proteolitic effect. RESULTS: GM1-EIA method does not detect CT production in 29 museum strains of non-O1/non-O139 V. cholerae in vitro. CT was detected only in 1 non-O1/non-O139 V. cholerae strain supernatant with OD = 0.577 that is substantially lower than in O1 V. cholerae strains (OD = 2.176). In cell cultures non-O1/non-O139 V. cholerae supernatants diluted to 1:100 caused elongation only in single cells. CONCLUSION: Cytological model is a more sensitive technique to evaluate toxin producing abilities of non-O1/non-O139 V. cholerae strains and is appropriate for use.

Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model.

BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry.

We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

Enhanced stereoselectivity of alpha-mannosylation under thermodynamic control using trichloroacetimidates.

O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of alpha-(1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-D-mannose). The blockwise synthesis of larger fragments of such O-PSs involves oligosaccharide glycosyl donors that contain a nonparticipating 2-O-glycosyl group at the position vicinal to the anomeric center where the new glycosidic linkage is formed. Such glycosyl donors may bear at C-4 either a latent acylamino (e.g., azido) or the 3-deoxy-L-glycero-tetronamido group. While monosaccharide glycosyl donors, even those bearing a nonparticipating group at O-2 (e.g., methyl), and the 4-N-(3-deoxy-L-glycero-tetronyl) side chain form alpha-linked oligosaccharides with excellent stereoselectivity, alpha-mannosylation with analogous oligosaccharide donors in this series is adversely affected by the presence of the side chain. Consequently, the unwanted beta-product is formed in a considerable amount. Conducting the reaction at elevated temperature under thermodynamic control substantially enhances formation of the alpha-linked oligosaccharide. This effect is much more pronounced when glycosyl trichloroacetimidates, rather than thioglycosides or glycosyl chlorides, are used as glycosyl donors.

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba.

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.

Negative matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry fragmentation of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1 in the presence of anionic dopants.

Oligosaccharides (tri- to hexamers) that represent terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, have been studied by negative matrix-assisted laser desorption/ionization time of flight/time-of-flight mass spectrometry (MALDI ToF/ToF MS). The [M - H(+)](-) ions are formed after expulsion of a proton from molecules studied under condition of MALDI MS analysis in the negative mode. Several ammonium salts (chloride, nitrate, hydrogencarbonate and hydrogensulfate) were used as additives to increase the formation of negative ions from saccharides. The most efficient was the addition of ammonium hydrogencarbonate, which increased the amount of [M - H(+)](-) ions more than six times. Between three fragmentation pathways, the new conjugated transfer of electrons within the second downstream unit of oligosaccharides was discovered. Production of these ions, which has not been observed in any other kinds of measurement, distinguishes substances belonging to Ogawa and Inaba serotypes. The negative MALDI ToF/ToF mass spectra are simpler and, at the same time, more informative, as compared with positive and negative electrospray ionization ion trap as well as with positive MALDI ToF/ToF analysis.

Immunogenicity of synthetic saccharide fragments of Vibrio cholerae O1 (Ogawa and Inaba) bound to Exotoxin A.

Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS. These studies demonstrate that the 'functional' B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.

Positive-ion fragmentation in matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1.

Saccharides (mono- to hexamers) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba have been studied by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI ToF/ToF) mass spectrometry (MS). Cationized adducts are the characteristic ions formed through the capture of sodium or potassium cations under MALDI MS conditions. Three characteristic pathways dominate in the fragmentation of model substances under MALDI ToF/ToF collision-induced dissociation MS/MS conditions of measurement. The first is the elimination which shortens the length of the oligosaccharide. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. In the second, the conjugated transfer of electrons in the upstream unit of oligosaccharides takes place. The third route brings about the elimination of one alpha-hydroxy-gamma-butyrolactone molecule from the 4-(3-deoxy-L-glycero-tetronamido) group. The MALDI ToF/ToF MS/MS provided sufficient information about molecular mass, the number of saccharide residues, the structure of saccharides, the C(4)- amide of 3-deoxy-L-glycero-tetronic acid (DGT) of the compounds investigated, and allows to distinguish between Ogawa and Inaba serotypes.