Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→.

Hypocrellin A (HA) is an excellent perylenequinone photosensitizer from Shiraia fruiting bodies. A dominant bacterium Pseudomonas fulva SB1 in the fruiting body was found to promote HA biosynthesis. The bacterial LPS were purified and the O-specific polysaccharide (OPS) consisted of rhamnose (Rha), galactose (Gal) and N-acetyl-galactosamine (GalNAc) with an average molecular weight of 282.8 kDa. Although the OPS composing of Rhap and Galp backbone showed elicitation capability on fungal HA accumulation, the highest HA production (303.76 mg/L) was achieved by LPS treatment at 20 μg/mL on day 3 of the mycelium culture. The generation of nitric oxide (NO) in Shiraia mycelia was triggered by LPS, which was partially blocked by inhibitors of nitric oxide synthase (NOS) and nitrate reductase (NR), leading to the depressed HA production. Transcriptome analysis revealed that NO mediated LPS-induced HA production via upregulating the expressions of critical genes associated with central carbon metabolism and downstream HA biosynthesis genes. This is the first report of LPS-induced NO to regulate fungal secondary metabolite production, which provides new insights on the role of bacterial LPS in bacterium-fungus interactions and an effective strategy to enhance hypocrellin production.

Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Pseudomonas sp. Strain L1, the endophytic bacteria of Lolium perenne (ryegrass) plants growing in soil in an industrial area in the Silesia region (Zabrze, Southern Poland). The high-molecular-weight O-PS fraction liberated from Pseudomonas sp. L1 lipopolysaccharide by mild acid hydrolysis was studied using chemical methods, MALDI-TOF mass spectrometry, and 1D and 2D NMR spectroscopy techniques. It was found that the O-specific polysaccharide was built of tetrasaccharide repeating units composed of d-FucpN, d-Fucp4N, and two d-QuipN residues. The following structure of the O-PS of Pseudomonas sp. Strain L1 was established: [Formula: see text].

O-specific polysaccharides (O-PSs) isolated from lipopolysaccharides of Serratia spp., strains 10.1WK and 1XS, which are endophytic bacteria of Oenothera biennis (common evening-primrose) and Lotus corniculatus (bird\'s-foot trefoil), plants growing on a petroleum hydrocarbon polluted site in the Silesia region, were investigated. The high-molecular-weight O-PS fractions liberated from lipopolysaccharides by mild acid hydrolysis were studied using chemical methods, MALDI-TOF mass spectrometry, and a set of 1D and 2D NMR spectroscopy techniques. It was found that both O-specific polysaccharides were built of an identical trisaccharide repeating unit composed of d-Rhap and d-Manp residues. The following structure of the O-PSs of Serratia spp. strains 10.1WK and 1XS was established: →4)-α-d-Rhap-(1 → 3)-β-d-Manp-(1 → 4)-β-d-Rhap-(1→.

Utilizing sugar, methylation, and absolute configurations analyses as well as NMR spectroscopy, the chemical repeating unit of the O-specific polysaccharide of Pectobacteriumversatile CFBP6051T was identified as: The polymer contains residues of an unusual, higher-branched monosaccharide, named erwiniose (3,6,8-trideoxy-4-C-(R-1-hydroxyethyl)-d-gulo-octose). Comparison of the P. versatile CFBP6051T O-polysaccharide with those isolated from strains of other Pectobacterium species indicated high differentiation in their structures within this genus.

The structure of the O-specific polysaccharide (OPS) from Aeromonas encheleia strain A4 lipopolysaccharide was investigated. A. encheleia strain A4, classified into the new provisional serogroup PGO1 predominating among aeromonads in Polish aquaculture, was isolated from common carp tissues during an outbreak of MAI/MAS disease on a fish farm. The high-molecular-weight OPS fraction liberated from the lipopolysaccharide after mild acid hydrolysis followed by gel-permeation chromatography was studied with chemical methods, mass spectrometry, and one- and two-dimensional 1H and 13C NMR spectroscopy techniques. Inter-residue correlations were identified in 1H,13C-heteronuclear multiple-bond correlation (HMBC) and 1H,1H NOESY experiments. It was found that the O-specific polysaccharide of A. encheleia strain A4 consists of branched pentasaccharide repeating units with the following structure:→2)[α-d-Fucp3NRHb-(1→3)]-α-l-Rhap-(1→3)-β-l-Rhap-(1→4)-α-l-Rhap-(1→3)-β-d-QuipNAc-(1→.

Vibrio cholerae, a noninvasive mucosal pathogen, is endemic in more than 50 countries. Oral cholera vaccines, based on killed whole-cell strains of Vibrio cholerae, can provide significant protection in adults and children for 2-5 years. However, they have relatively limited direct protection in young children. To overcome current challenges, in this study, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from V. cholerae O1 El Tor Inaba strain PIC018 with Qβ virus-like particles efficiently via squarate chemistry. The Qβ-OSP conjugate was characterized with mass photometry (MP) on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qβ-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae O1 Inaba. This was the first time that Qβ was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier.

There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.