Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

Outer Membrane Vesicles Derived from Salmonella enterica Serotype Typhimurium Can Deliver Shigella flexneri 2a O-Polysaccharide Antigen To Prevent Shigella flexneri 2a Infection in Mice.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has been developed yet. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively biosynthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in the serum, with similar trends in IgA levels from vaginal secretions and fluid from bronchopulmonary lavage, both intranasally and intraperitoneally. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay, opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. IMPORTANCEShigella, the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. In addition, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella, from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

Effect of O-Antigen Chain Length Regulation on the Immunogenicity of Shigella and Salmonella Generalized Modules for Membrane Antigens (GMMA).

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.

Attenuated Salmonella Typhimurium expressing Salmonella Paratyphoid A O-antigen induces protective immune responses against two Salmonella strains.

Salmonella enterica serovar Paratyphi A is the main causative agent of paratyphoid fever in many Asian countries. As paratyphoid is spread by the fecal-oral route, the most effective means of controlling S. Paratyphi A infection is through the availability of clean water supplies and working sanitation services. Because sanitation facilities improve slowly in these poor areas and antibiotic resistance is severe, the development of a safe and effective vaccine remains a priority for controlling the spread of paratyphoid disease. In this study, we investigated the strategy of heterologous O-antigenic O2 serotype (S. Paratyphi A characterized) conversion in S. Typhimurium to prevent paratyphoid infections. A series of S. Typhimurium mutants were constructed with replacement of abe, wzxB1 and wbaVB1 genes with respective prt-tyvA1, wzxA1 and wbaVA1, and the results showed that only three genes including prt, wbaVA1 and wzxA1 from S. Paratyphi A presence enable S. Typhimurium to sufficiently express O2 antigen polysaccharide. We also constructed a series of live attenuated S. Typhimurium vaccine candidates expressing heterologous O2 O-antigens, and a mouse model was used to evaluate the immunogenicity of live vaccines. ELISA data showed that vaccine candidates could induce a comparatively high level of S. Paratyphi A and/or S. Typhimurium LPS-specific IgG and IgA responses in murine model, and IgG2a levels were consistently higher than IgG1 levels. Moreover, the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays. Our work highlights the potential for developing S. Typhimurium live vaccines against S. Paratyphi A.

[18F]FEPPA a TSPO Radioligand: Optimized Radiosynthesis and Evaluation as a PET Radiotracer for Brain Inflammation in a Peripheral LPS-Injected Mouse Model.

[18F]FEPPA is a specific ligand for the translocator protein of 18 kDa (TSPO) used as a positron emission tomography (PET) biomarker for glial activation and neuroinflammation. [18F]FEPPA radiosynthesis was optimized to assess in a mouse model the cerebral inflammation induced by an intraperitoneal injection of Salmonella enterica serovar Typhimurium lipopolysaccharides (LPS; 5 mg/kg) 24 h before PET imaging. [18F]FEPPA was synthesized by nucleophilic substitution (90 °C, 10 min) with tosylated precursor, followed by improved semi-preparative HPLC purification (retention time 14 min). [18F]FEPPA radiosynthesis were carried out in 55 min (from EOB). The non-decay corrected radiochemical yield were 34 ± 2% (n = 17), and the radiochemical purity greater than 99%, with a molar activity of 198 ± 125 GBq/µmol at the end of synthesis. Western blot analysis demonstrated a 2.2-fold increase in TSPO brain expression in the LPS treated mice compared to controls. This was consistent with the significant increase of [18F]FEPPA brain total volume of distribution (VT) estimated with pharmacokinetic modelling. In conclusion, [18F]FEPPA radiosynthesis was implemented with high yields. The new purification/formulation with only class 3 solvents is more suitable for in vivo studies.

First characterization of immunogenic conjugates of Vi negative Salmonella Typhi O-specific polysaccharides with rEPA protein for vaccine development.

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P=0.0001) and conjugate 2 (OSP-rEPA) (P≤0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSPADH-HSA) and conjugate 4 (OSPADH-rEPA) were insignificant (P=0.1684 and P=0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials.

A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice.

BACKGROUND: Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). METHODOLOGY: Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 µg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. PRINCIPLE FINDINGS: Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 µg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model. CONCLUSION: We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines.

Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

Evaluation of a LPS-based glycoconjugate vaccine against bovine Escherichia coli mastitis: Formation of LPS Abs in cows after immunization with E. coli core oligosaccharides conjugated to hemocyanine.

The immune response of cows against the core oligosaccharide of Escherichia coli rough mutants (core types R1-R4, K-12 and J-5) was investigated after immunization with a synthetic glycoconjugate composed of deacylated LPS conjugated to hemocyanine (22 animals). Ab formation was measured by ELISA using LPS or deacylated LPS conjugated to BSA as an Ag. The glycoconjugate immunogens were used to vaccinate cows (36 animals), which were then challenged intramammarily with E. coli O 157 (K1 negative, R1 core type). Compared with control groups no protection was observed, although high titers against the R1 core type were detected in vaccinated animals. Western blots using the immune sera showed that the Ab response was directed against the core region and not against the O-antigen, which may explain the failure of the vaccine.

Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2-5-year-old children.

BACKGROUND: Escherichia coli O157:H7 causes severe enteritis and hemolytic-uremic syndrome, mostly in young children and older adults. Similar to the case with Shigella, serum IgG against the O-specific polysaccharide of E. coli O157:H7 may confer immunity by lysing the inoculum in the intestine. A phase 1 trial in adults showed that a vaccine of E. coli O157:H7 O-specific polysaccharide conjugated to recombinant exotoxin A of Pseudomonas aeruginosa (O157-rEPA) was safe and immunogenic. METHODS: A phase 2 trial of the O157-rEPA vaccine was conducted in 49 children 2-5 years old who were divided randomly into groups receiving 1 or 2 doses of vaccine. Adverse reactions were monitored. Serum IgG lipopolysaccharide (LPS) antibodies were determined. RESULTS: No significant adverse reactions were observed. At 1 week after the first dose was administered, most children (81%) responded with a >4-fold increase in serum IgG LPS antibodies. At 6 weeks after the first dose was administered, all children responded with a >8-fold increase; a second dose did not elicit a booster response. At 26 weeks after the first dose was administered, the geometric mean titer of serum IgG LPS antibodies was ~20-fold higher than was the prevaccination titer. These serum samples had high titers of bactericidal activity that were correlated roughly with serum IgG LPS antibody titers (r = .78). CONCLUSIONS: The O157-rEPA vaccine was safe and immunogenic in young children. A phase 3 trial of the administration of this conjugate vaccine concurrently with routine immunization in infants is planned.

Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4+ T cell help.

The relative lack of memory for IgG antipolysaccharide responses is believed to be secondary to the inability of polysaccharides to associate with MHC class II molecules and thus a failure to recruit cognate CD4+ T cell help. However, little is known concerning the role of T cells and the generation of memory for antipolysaccharide Ig responses to intact extracellular bacteria. We used heat-killed, intact Streptococcus pneumoniae, capsular type 14 (Pn14), to evaluate the IgM and IgG responses specific for the capsular polysaccharide (PPS14), the phosphorylcholine determinant of the cell wall C-polysaccharide, and the cell wall protein, pneumococcal surface protein A (PspA). We demonstrate that the IgG (but not IgM), anti-PPS14, and anti-PspA responses to Pn14 are CD4+ T cell dependent and TCR specific. Nevertheless, in contrast to the anti-PspA response, the IgG anti-PPS14 response shows no apparent memory, an accelerated kinetics of primary Ig induction, and a more rapid delivery of CD4+ T cell help. In contrast, the IgG anti-phosphorylcholine response, although also dependent on CD4+ T cells, is TCR nonspecific. We make similar observations using soluble conjugates of PPS14-PspA and C-polysaccharide-PspA. These data lead us to suggest that the central issue concerning the mechanisms underlying different functional outcomes for anti-bacterial IgG responses to capsular polysaccharide vs protein Ags is not necessarily based on the ability to recruit cognate CD4+ T cell help, but perhaps on the nature of the B cell Ag receptor signaling that occurs and/or on the responding B cell subpopulations.

Mice intradermally-inoculated with the intact lipopolysaccharide, but not the lipid A or O-chain, from Francisella tularensis LVS rapidly acquire varying degrees of enhanced resistance against systemic or aerogenic challenge with virulent strains of the pathogen.

The present study examines the relationship between the structure and important biological effects of the lipopolysaccharide (LPS) of the intracellular bacterial pathogen, Francisella tularensis LVS. It shows that treating mice with sub-immunogenic amounts of intact F. tularensis LPS rapidly induces an enhanced resistance to intradermal or aerogenic challenge with strains of the pathogen of varying virulence. However, neither the free Lipid A nor core-O-chain produced by mild acid hydrolysis of LPS appeared able to elicit this host defense mechanism.

Influence of the sequence of administration of beta-glucans and a Vibrio damsela vaccine on the immune response of turbot (Scophthalmus maximus L.).

Yeast (Saccharomyces cerevisae) beta-1,3 glucans were used as adjuvant in a Vibrio damsela vaccine for turbot (Scophthalmus maximus L.). Turbot were injected with the adjuvant prior, at the same time and after the vaccine. Several immune parameters (index and rate of phagocytosis, passive haemolytic plaque numbers, and agglutinating antibody titers) were determined at different times postinoculation. The highest activity of all the immune parameters was obtained when glucans were injected after the bacterin. It is concluded that the sequence of glucan administration is critical when used as a vaccine adjuvant.

Bacteremia versus endotoxemia in experimental mouse leukopenia--role of antibiotic chemotherapy.

Imipenem and ceftazidime have different specificities for penicillin-binding proteins and cause a differential release of lipopolysaccharide (LPS) from gram-negative bacteria in vitro. In studies in mice made leukopenic by the administration of cyclophosphamide, the innate relative resistance to the lethal effects of LPS was not significantly changed, but these animals became highly sensitized to bacterial infection. When leukopenic mice were challenged with graded doses of Escherichia coli O111:B4, an LD50 was achieved at a dose of approximately 10(6) cfu. Administration of either antibiotic resulted in a shift in the LD50 of approximately 500-fold, in contrast to D-galactosamine-treated LPS-sensitized mice, in which a < 10-fold increase in the LD50 was observed with antibiotic therapy. Further, if mice were made LPS-sensitive with D-galactosamine, no differences between leukopenic and normal mice were noted with antibiotic therapy.