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Background: Invasive non-typhoidal Salmonella (iNTS) serovars S. Typhimurium and S. Enteritidis are major etiologic agents of invasive bacterial disease among infants and young children in sub-Saharan Africa, including in Mali. Early studies of iNTS serovars in several countries indicated that S. Typhimurium was more prevalent than S. Enteritidis, including in Mali before 2008. We investigated genomic and associated phenotypic changes associated with an increase in the relative proportion of iNTS caused by S. Enteritidis versus S. Typhimurium in Bamako, Mali, during the period 2002–2012. Methodology/Principal findings: Comparative genomics studies identified homologs of tetracycline resistance and arsenic utilization genes that were associated with the temporal shift of serovars causing iNTS shift, along with several hypothetical proteins. These findings, validated through PCR screening and phenotypic assays, provide initial steps towards characterizing the genomic changes consequent to unknown evolutionary pressures associated with the shift in serovar prevalence. Conclusions/Significance: This work identified a shift to S. Enteritidis from the more classic S. Typhimurium, associated with iNTS in Bamako, Mali, during the period 2002–2012. This type of shift in underlying iNTS pathogens are of great importance to pediatric public health in endemic regions of sub-Saharan Africa. Additionally, this work demonstrates the utility of combining epidemiologic data, whole genome sequencing, and functional characterization in the laboratory to identify and characterize genomic changes in the isolates that may be involved with the observed shift in circulating iNTS agents. [ABSTRACT FROM AUTHOR]

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea. [ABSTRACT FROM AUTHOR]

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30–43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63–74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, systematic surveillance of young children with suspected invasive bacterial disease (e.g., septicemia, meningitis) has revealed non-typhoidal Salmonella (NTS) to be a major pathogen exhibiting high case fatality (~20%). Where infant vaccination against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae has been introduced to prevent invasive disease caused by these pathogens, as in Bamako, Mali, their burden has decreased markedly. In parallel, NTS has become the predominant invasive bacterial pathogen in children aged <5 years. While NTS is believed to be acquired orally via contaminated food/water, epidemiologic studies have failed to identify the reservoir of infection or vehicles of transmission. This has precluded targeting food chain interventions to diminish disease transmission but conversely has fostered the development of vaccines to prevent invasive NTS (iNTS) disease. We developed a mathematical model to estimate the potential impact of NTS vaccination programs in Bamako. Methodology/Principal Findings: A Markov chain transmission model was developed utilizing age-specific Bamako demographic data and hospital surveillance data for iNTS disease in children aged <5 years and assuming vaccine coverage and efficacy similar to the existing, successfully implemented, Hib vaccine. Annual iNTS hospitalizations and deaths in children <5 years, with and without a Salmonella Enteritidis/Salmonella Typhimurium vaccine, were the model’s outcomes of interest. Per the model, high coverage/high efficacy iNTS vaccination programs would drastically diminish iNTS disease except among infants age <8 weeks. Conclusions/Significance: The public health impact of NTS vaccination shifts as disease burden, vaccine coverage, and serovar distribution vary. Our model shows that implementing an iNTS vaccine through an analogous strategy to the Hib vaccination program in Bamako would markedly reduce cases and deaths due to iNTS among the pediatric population. The model can be adjusted for use elsewhere in Africa where NTS epidemiologic patterns, serovar prevalence, and immunization schedules differ from Bamako. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. [ABSTRACT FROM AUTHOR]

Background. In 2002, following establishment of a clinical microbiology laboratory in the government hospital that admits children with severe illnesses in Bamako, Mali, surveillance to identify pathogens causing invasive bacterial infections (septicemia, bacteremia, meningitis, etc) was initiated. Methods. Parents/guardians of children aged <16 years admitted to l’Hôpital Gabriel Touré with high fever or clinical syndromes compatible with focal invasive bacterial disease were asked for consent to culture their child’s blood/body fluid. Standard bacteriologic techniques speciated isolates; Salmonella serovars were determined. Results. From July 2002 through June 2014, 687 nontyphoidal Salmonella (NTS) isolates were obtained from 667 children; 667 yielded a single serovar and 20 grew 2 Salmonella serovars, 1 being NTS. Four serovars accounted for 87% of the 687 NTS isolates, including Salmonella Enteritidis (n = 244 [35.5%]), Salmonella Typhimurium (n = 221 [32.2%]), I:4,[5],12:i:- (n = 42 [6.1%]), and Salmonella Dublin (n = 89 [13.0%]). Of 553 patients with invasive NTS from whom 1 of the 4 predominant serovars was isolated in pure culture, 448 (81.0%) were aged <5 years and case fatality was 20.3%; Salmonella Enteritidis case fatality (27.8%) was higher than for other serovars (P = .0009). NTS disease showed a seasonal peak following the rainy season and into the cool, dry season. Since 2010, Salmonella Enteritidis cases have risen and Salmonella Typhimurium fallen. Conclusions. NTS has become the predominant invasive pathogen as Haemophilus influenzae type b and pneumococcal vaccine use in Mali has diminished invasive disease due to those pathogens. The age distribution and limited serovars involved make control of NTS disease by vaccines epidemiologically feasible, if products under development prove safe and efficacious. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella (NTS) serovars S. Enteritidis and S. Typhimurium are a major cause of invasive bacterial disease (e.g., bacteremia, meningitis) in infants and young children in sub-Saharan Africa and also occasionally cause invasive disease in highly susceptible hosts (young infants, the elderly, and immunocompromised subjects) in industrialized countries. No licensed vaccines exist against human NTS infections. NTS core and O polysaccharide (COPS) and FliC (Phase 1 flagellin subunits) each constitute protective antigens in murine models. S. Enteritidis COPS conjugated to FliC represents a promising vaccine approach that elicits binding and opsonophagocytic antibodies and protects mice against lethal challenge with virulent S. Enteritidis. We examined the protective efficacy of fractional dosages of S. Enteritidis COPS:FliC conjugate vaccines in mice, and also established that protection can be passively transferred to naïve mice by administering sera from mice immunized with conjugate. Mice were immunized with three doses of either 10 µg, 2.5 µg (full dose), 0.25 µg, or 0.025 µg S. Enteritidis COPS:FliC conjugate at 28 day intervals. Antibody titers to COPS and FliC measured by ELISA fell consonant with progressively smaller vaccine dosage levels; anti-FliC IgG responses remained robust at fractional dosages for which anti-COPS serum IgG titers were decreased. Nevertheless, >90% protection against intraperitoneal challenge was observed in mice immunized with fractional dosages of conjugate that elicited diminished titers to both FliC and COPS. Passive transfer of immune sera from mice immunized with the highest dose of COPS:FliC to naïve mice was also protective, demonstrating the role of antibodies in mediating protection. These results provide important insights regarding the potency of Salmonella glycoconjugate vaccines that use flagellin as a carrier protein. [ABSTRACT FROM AUTHOR]