Role of neuronal and inducible nitric oxide synthases in the guinea pig ileum myenteric plexus during in vitro ischemia and reperfusion

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Westernimmunoblotting and real time RT-PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOSexpressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS-immunoreactive neurons, protein and mRNA levels were up-regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by Nx-propyl-L-arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis.(VLID)266645

Generalized Modules for Membrane Antigens (GMMA), an outer membrane vesicle-based vaccine platform, for efficient viral antigen delivery.

Vaccine platforms enable fast development, testing, and manufacture of more affordable vaccines. Here, we evaluated Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles (OMVs) generated by genetically modified Gram-negative bacteria, as a vaccine platform for viral pathogens. Influenza A virus hemagglutinin (HA), either physically mixed with GMMA (HA+STmGMMA mix), or covalently linked to GMMA surface (HA-STmGMMA conjugate), significantly increased antigen-specific humoral and cellular responses, with HA-STmGMMA conjugate inducing further enhancement than HA+STmGMMA mix. HA-STmGMMA conjugate protected mice from lethal challenge. The versatility for this platform was confirmed by conjugation of rabies glycoprotein (RABVG) onto GMMA through the same method. RABVG+STmGMMA mix and RABVG-STmGMMA conjugate exhibited similar humoral and cellular response patterns and protection efficacy as the HA formulations, indicating relatively consistent responses for different vaccines based on the GMMA platform. Comparing to soluble protein, GMMA was more efficiently taken up in vivo and exhibited a B-cell preferential uptake in the draining lymph nodes (LNs). Together, GMMA enhances immunity against viral antigens, and the platform works well with different antigens while retaining similar immunomodulatory patterns. The findings of our study imply the great potential of GMMA-based vaccine platform also against viral infectious diseases

Impact of O-Acetylation on <i>S. flexneri</i> 1b and 2a O-Antigen Immunogenicity in Mice

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity

Combined Chemical Synthesis and Tailored Enzymatic Elongation Provide Fully Synthetic and Conjugation-Ready <i>Neisseria meningitidis</i> Serogroup X Vaccine Antigens

Studies on the polymerization mode of <i>Neisseria meningitidis</i> serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total <i>in vitro</i> construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates