Immunogenicity of a Bivalent Adjuvanted Glycoconjugate Vaccine against Salmonella Typhimurium and Salmonella Enteritidis

Salmonella enterica serovars Typhimurium and Enteritidis are the predominant causes of invasive non-typhoidal Salmonella (iNTS) disease. Considering the co-endemicity of S. Typhimurium and S. Enteritidis, a bivalent vaccine formulation against both pathogens is necessary for protection against iNTS disease, thus investigation of glycoconjugate combination is required. In the present work, we investigated the immune responses induced by S. Typhimurium and S. Enteritidis monovalent and bivalent glycoconjugate vaccines adjuvanted with aluminum hydroxide (alum) only or in combination with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG). Humoral and cellular, systemic and local, immune responses were characterized in two different mouse strains. All conjugate vaccines elicited high levels of serum IgG against the respective O-antigens (OAg) with bactericidal activity. The bivalent conjugate vaccine induced systemic production of antibodies against both S. Typhimurium and S. Enteritidis OAg. The presence of alum or alum + CpG adjuvants in vaccine formulations significantly increased the serum antigen-specific antibody production. The alum + CpG bivalent vaccine formulation triggered the highest systemic anti-OAg antibodies and also a significant increase of anti-OAg IgG in intestinal washes and fecal samples, with a positive correlation with serum levels. These data demonstrate the ability of monovalent and bivalent conjugate vaccines against S. Typhimurium and S. Enteritidis to induce systemic and local immune responses in different mouse strains, and highlight the suitability of a bivalent glycoconjugate formulation, especially when adjuvanted with alum + CpG, as a promising candidate vaccine against iNTS disease

A Computational Approach in the Diagnostic Process of COVID-19: The Missing Link between the Laboratory and Emergency Department

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Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella.

Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197 Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with SalmonellaS Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance

Relationship between Antibody Susceptibility and Lipopolysaccharide O-Antigen Characteristics of Invasive and Gastrointestinal Nontyphoidal Salmonellae Isolates from Kenya

Background: Nontyphoidal Salmonellae (NTS) cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal. Methodology/Principal findings: We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis) from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible and expressed more O-antigen than Typhimurium (p\u3c0.0001 for both comparisons). For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r = 0.29, 95% CI = 0.10-0.45, p = 0.002). Both serovars expressed O-antigen populations ranging 21-33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20%-50% glucosylated. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features. Conclusion/Significance: Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. No clear differences were found in the phenotype of isolates from blood and stool, suggesting that the same isolates can cause invasive disease and gastroenteritis. Genome studies are required to understand whether invasive and gastrointestinal isolates differ at the genotypic level

O:2-CRM(197) conjugates against Salmonella Paratyphi A.

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A

Evaluation of the Immunogenicity and Biological Activity of the Citrobacter freundii Vi-CRM197 Conjugate as a Vaccine for Salmonella enterica Serovar Typhi▿ †

Typhoid fever remains a major health problem in developing countries. Young children are at high risk, and a vaccine effective for this age group is urgently needed. Purified capsular polysaccharide from Salmonella enterica serovar Typhi (Vi) is licensed as a vaccine, providing 50 to 70% protection in individuals older than 5 years. However, this vaccine is ineffective in infants. Vi conjugated to a carrier protein (i.e., an exoprotein A mutant from Pseudomonas aeruginosa [rEPA]) is highly immunogenic, provides long-term protection, and shows more than 90% protective efficacy in children 2 to 5 years old. Here, we describe an alternative glycoconjugate vaccine for S. Typhi, Vi-CRM197, where Vi was obtained from Citrobacter freundii WR7011 and CRM197, the mutant diphtheria toxin protein, was used as the carrier. We investigated the optimization of growth conditions for Vi production from C. freundii WR7011 and the immunogenicity of Vi-CRM197 conjugates in mice. The optimal saccharide/protein ratio of the glycoconjugates was identified for the best antibody production. We also demonstrated the ability of this new vaccine to protect mice against challenge with Vi-positive Salmonella enterica serovar Typhimurium

Characterization of Citrobacter sp. line 328 as a source of Vi for a Vi-CRM197 glycoconjugate vaccine against Salmonella Typhi

Introduction: Salmonella enterica serovar Typhi is the causative agent of typhoid fever with over 22 million cases and over 200,000 deaths reported annually. A vaccine is much needed for use in young children and the Novartis Vaccines Institute for Global Health (NVGH) is developing a conjugate vaccine which targets S. Typhi Vi capsular polysaccharide. Methodology: Here we describe a method suitable for industrial scale production of the Vi antigen based on expression by a Citrobacter line. We optimized the production of Vi by selecting a suitable Citrobacter strain (Citrobacter 328) that yields high and stable expression of Vi in chemically defined medium under industrial-scale fermentation conditions. Results: Vi-CRM197 made using Vi from Citrobacter 328 elicited high anti-Vi antibody levels in mice and rabbits. Conclusions: Citrobacter 328 is a suitable strain for production of Vi for conjugate anti-Typhi vaccines. Being a BSL-1 organism, which grows in defined medium and stably produces high yields of Vi, it offers excellent potential for safe production of inexpensive vaccines for populations at risk of typhoid fever

O:2-CRM197 conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM197, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM197 as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A