The use of controlled human infection models to identify correlates of protection for invasive Salmonella vaccines.

Controlled human infection model (CHIM) studies, which involve deliberate exposure of healthy human volunteers to an infectious agent, are recognised as important tools to advance vaccine development. These studies not only facilitate estimates of vaccine efficacy, but also offer an experimental approach to study disease pathogenesis and profile vaccine immunogenicity in a controlled environment, allowing correlation with clinical outcomes. Consequently, the data from CHIMs can be used to identify immunological correlates of protection (CoP), which can help accelerate vaccine development. In the case of invasive Salmonella infections, vaccination offers a potential instrument to prevent disease. Invasive Salmonella disease, caused by the enteric fever pathogens Salmonella enterica serovar Typhi (S. Typhi) and S. Paratyphi A, B and C, and nontyphoidal Salmonella (iNTS), remains a significant cause of mortality and morbidity in low- and middle-income countries, resulting in over 200,000 deaths and the loss of 15 million DALYs annually. CHIM studies have contributed to the understanding of S. Typhi infection and provided invaluable insight into the development of vaccines and CoP following vaccination against S. Typhi. However, CoP are less well understood for S. Paratyphi A and iNTS. This brief review focuses on the contribution of vaccine-CHIM trials to our understanding of the immune mechanisms associated with protection following vaccines against invasive Salmonella pathogens, particularly in relation to CoP.

Generation and evaluation of Salmonella entericaserovar Choleraesuis mutant strains as a potential live-attenuated vaccine.

BACKGROUND: Salmonella entericaserovar Choleraesuis (S.C) is a swine enteric pathogen causing paratyphoid fever, enterocolitis, and septicemia in piglets. S. C is mainly transmitted through the fecal-oral route. Vaccination is an effective strategy for preventing and controlling Salmonella infection. RESULTS: Herein, we used CRISPR-Cas9 technology to knockout the virulence regulatory genes, rpoS, and slyA of S. C and constructed the ∆rpoS, ∆slyA, and ∆rpoS ∆slyA strains. The phenotypic characteristics of the mutant strains remained unchanged compared with the parental wild-type strain. In vivo study, unlike the wild-type strain, the ∆slyA and ∆rpoS ∆slyA strains alleviated splenomegaly, colon atrophy, and lower bacterial loads in the spleen, liver, ileum, and colon. These mutant strains survived in Peyer's patches (PPs) and mesenteric lymph nodes (MLN) for up to 15 days post-infection. Furthermore, the immunization of the ∆rpoS ∆slyA strain induced robust humoral and cellular immune responses. CONCLUSIONS: Consequently, vaccination with ∆rpoS ∆slyA conferred a high percentage of protection against lethal invasive Salmonella, specifically S. C, in mice. This study provided novel insights into the development of live-attenuated vaccines against the infection of S. C.

An integrated comparative genomics, subtractive proteomics and immunoinformatics framework for the rational design of a Pan-Salmonella multi-epitope vaccine.

Salmonella infections pose a significant global public health concern due to the substantial expenses associated with monitoring, preventing, and treating the infection. In this study, we explored the core proteome of Salmonella to design a multi-epitope vaccine through Subtractive Proteomics and immunoinformatics approaches. A total of 2395 core proteins were curated from 30 different isolates of Salmonella (strain NZ CP014051 was taken as reference). Utilizing the subtractive proteomics approach on the Salmonella core proteome, Curlin major subunit A (CsgA) was selected as the vaccine candidate. csgA is a conserved gene that is related to biofilm formation. Immunodominant B and T cell epitopes from CsgA were predicted using numerous immunoinformatics tools. T lymphocyte epitopes had adequate population coverage and their corresponding MHC alleles showed significant binding scores after peptide-protein based molecular docking. Afterward, a multi-epitope vaccine was constructed with peptide linkers and Human Beta Defensin-2 (as an adjuvant). The vaccine could be highly antigenic, non-toxic, non-allergic, and have suitable physicochemical properties. Additionally, Molecular Dynamics Simulation and Immune Simulation demonstrated that the vaccine can bind with Toll Like Receptor 4 and elicit a robust immune response. Using in vitro, in vivo, and clinical trials, our findings could yield a Pan-Salmonella vaccine that might provide protection against various Salmonella species.

Vaccine value profile for invasive non-typhoidal Salmonella disease.

Invasive non-typhoidal Salmonella (iNTS) disease is an under-recognized high-burden disease causing major health and socioeconomic issues in sub-Saharan Africa (sSA), predominantly among immune-naïve infants and young children, including those with recognized comorbidities such as HIV infection. iNTS disease is primarily caused by Salmonella enterica serovar Typhimurium sequence type (ST) 313 and 'African-restricted clades' of Salmonella Enteritidis ST11 that have emerged across the African continent as a series of epidemics associated with acquisition of new antimicrobial resistance. Due to genotypes with a high prevalence of antimicrobial resistance and scarcity of therapeutic options, these NTS serovars are designated by the World Health Organization as a priority pathogen for research and development of interventions, including vaccines, to address and reduce NTS associated bacteremia and meningitis in sSA. Novel and traditional vaccine technologies are being applied to develop vaccines against iNTS disease, and the results of the first clinical trials in the infant target population should become available in the near future. The "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by Salmonella Enteritidis and Salmonella Typhimurium prevalent in sSA. Information is included on stand-alone iNTS disease candidate vaccines and candidate vaccines targeting iNTS disease combined with another invasive serotype, Salmonella Typhi, that is also common across sSA. Out of scope for the first version of this VVP is a wider discussion on either diarrheagenic NTS disease (dNTS) also associated with Salmonella Enteritidis and Salmonella Typhimurium or the development of a multivalent Salmonella vaccines targeting key serovars for use globally. This VVP for vaccines to prevent iNTS disease is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic, and societal value of pipeline vaccines and vaccine-like products. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and a "Full Vaccine Value Assessment" that will inform the value proposition of an iNTS disease vaccine. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the World Health Organization African Region. All contributors have extensive expertise on various elements of the iNTS disease VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

Deletion of an immune evasion gene, steD, from a live Salmonella enterica serovar Typhimurium vaccine improves vaccine responses in aged mice.

Introduction: Non-typhoidal Salmonella (NTS) generally causes self-limiting gastroenteritis. However, older adults (≥65 years) can experience more severe outcomes from NTS infection. We have previously shown that a live attenuated S. Typhimurium vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), was immunogenic in adult but not aged mice. Here we describe modification of CVD 1926 through deletion of steD, a Salmonella effector responsible for host immune escape, which we hypothesized would increase immunogenicity in aged mice. Methods: Mel Juso and/or mutuDC cells were infected with S. Typhimurium I77, CVD 1926, and their respective steD mutants, and the MHC-II levels were evaluated. Aged (18-month-old) C57BL/6 mice received two doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and the number of FliC-specific CD4+ T cells were determined. Lastly, aged C57BL/6 mice received three doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and then were challenged perorally with wild-type S. Typhimurium SL1344 (108 CFU). These animals were also evaluated for antibody responses. Results: MHC-II induction was higher in cells treated with steD mutants, compared to their respective parental strains. Compared to PBS-vaccinated mice, CVD 1926 ΔsteD elicited significantly more FliC-specific CD4+ T cells in the Peyer's Patches. There were no significant differences in FliC-specific CD4+ T cells in the Peyer's patches or spleen of CVD 1926- versus PBS-immunized mice. CVD 1926 and CVD 1926 ΔsteD induced similar serum and fecal anti-core and O polysaccharide antibody titers after three doses. After two immunizations, the proportion of seroconverters for CVD 1926 ΔsteD was 83% (10/12) compared to 42% (5/12) for CVD 1926. Compared to PBS-immunized mice, mice immunized with CVD 1926 ΔsteD had significantly lower S. Typhimurium counts in the spleen, cecum, and small intestine upon challenge. In contrast, there were no differences in bacterial loads in the tissues of PBS-vaccinated and CVD 1926-immunized animals. Conclusion: These data suggest that the steD deletion enhanced the immunogenicity of our live attenuated S. Typhimurium vaccine. Deletion of immune evasion genes could be a potential strategy to improve the immunogenicity of live attenuated vaccines in older adults.

Evaluation of immunogenicity and protective efficacy of outer membrane vesicles from Salmonella Typhimurium and Salmonella Choleraesuis.

Outer membrane vesicles (OMVs) are membranous structures frequently observed in Gram-negative bacteria that contain bioactive substances. These vesicles are rich in bacterial antigens that can activate the host's immune system, making them a promising candidate vaccine to prevent and manage bacterial infections. The aim of this study was to assess the immunogenicity and protective efficacy of OMVs derived from Salmonella enterica serovar Typhimurium and S. Choleraesuis, while also focusing on enhancing OMV production. Initial experiments showed that OMVs from wild-type strains did not provide complete protection against homologous Salmonella challenge, possible due to the presence of flagella in the purified OMVs samples, which may elicit an unnecessary immune response. To address this, flagellin-deficient mutants of S. Typhimurium and S. Choleraesuis were constructed, designated rSC0196 and rSC0199, respectively. These mutants exhibited reduced cell motility and their OMVs were found to be flagellin-free. Immunization with non-flagellin OMVs derived from rSC0196 induced robust antibody responses and improved survival rates in mice, as compared to the OMVs derived from the wild-type UK-1. In order to enhance OMV production, deletions of ompA or tolR were introduced into rSC0196. The deletion of tolR not only increase the yield of OMVs, but also conferred complete protection against homologous S. Typhimurium challenge in mice. Collectively, these findings indicate that the flagellin-deficient OMVs with a tolR mutation have the potential to serve as a versatile vaccine platform, capable of inducing broad-spectrum protection against significant pathogens.

Research Note: Validation of a new differentiation approach using the commercial ASAPTM media to detect the Salmonella 441/014 vaccine strain.

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAPTM). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods.

Reversal of diabetes by an oral Salmonella-based vaccine in acute and progressive diabetes in NOD mice.

Type 1 diabetes (T1D)-associated hyperglycemia develops, in part, from loss of insulin-secreting beta cells. The degree of glycemic dysregulation and the age at onset of disease can serve as indicators of the aggressiveness of the disease. Tracking blood glucose levels in prediabetic mice may demonstrate the onset of diabetes and, along with animal age, also presage disease severity. In this study, an analysis of blood glucose levels obtained from female NOD mice starting at 4 weeks until diabetes onset was undertaken. New onset diabetic mice were orally vaccinated with a Salmonella-based vaccine towards T1D-associated preproinsulin combined with TGFß and IL10 along with anti-CD3 antibody. Blood glucose levels were obtained before and after development of disease and vaccination. Animals were classified as acute disease if hyperglycemia was confirmed at a young age, while other animals were classified as progressive disease. The effectiveness of the oral T1D vaccine was greater in mice with progressive disease that had less glucose excursion compared to acute disease mice. Overall, the Salmonella-based vaccine reversed disease in 60% of the diabetic mice due, in part, to lessening of islet inflammation, improving residual beta cell health, and promoting tolerance. In summary, the age of disease onset and severity of glucose dysregulation in NOD mice predicted response to vaccine therapy. This suggests a similar disease categorization in the clinic may predict therapeutic response.

Persistence of vaccine origin Salmonella Typhimurium through the poultry production continuum, and development of a rapid typing scheme for their differentiation from wild type field isolates.

Salmonella enterica serovar Typhimurium is one of the top Salmonella serovars annually linked to poultry production and corresponding human illnesses. Because of this, vaccination of commercial poultry against Salmonella Typhimurium has been a focal point in recent years. There are several commercially available Salmonella Typhimurium vaccines available for use in poultry production. Among these are modified live vaccines, including Poulvac ST (Zoetis), Megan Egg (AviPro), and Megan Vac 1 (AviPro). In this study, analyses of 27 field isolates of Salmonella Typhimurium from poultry sources indicated evidence for the persistence of some vaccine-origin strains through the commercial production cycle. Further analyses of 26,812 database isolates indicated vaccine-origin isolates are persisting frequently through processing, are present on retail meat products, and are even occasionally found in human patients. A novel polymerase chain reaction (PCR) was created and validated which enables simultaneous identification of Salmonella enterica sp., the Salmonella Typhimurium serovar, and differentiation of wild type Salmonella Typhimurium from live attenuated vaccines involving mutations in the cya/crp or aroA genes. The PCR was developed considering whole genome differences between the vaccines and wild type field isolates and was validated using different field isolates and recovered vaccine strains. This method enables poultry producers to rapidly determine if recovered field isolates have a vaccine origin.

In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Detección in silico y por PCR de una cepa vacunal viva atenuada de Salmonella Typhimurium. La aplicación de vacunas vivas atenuadas contra Salmonella Typhimurium ha ayudado significativamente a controlar Salmonella en productos avícolas. Debido a que el Servicio de Inspección de Seguridad Alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA-FSIS) califica todas las Salmonella como positivas, independientemente del serovar. Las cepas atenuadas de la vacuna que se identifican en el procesamiento contribuyen negativamente a los estándares de desempeño de Salmonella. Este estudio fue diseñado para determinar la incidencia de una vacuna viva atenuada de Salmonella serovar Typhimurium identificada en productos de pollo de engorde por el FSIS y para desarrollar un ensayo de PCR para la detección de aislados. Se recolectaron y ensamblaron secuencias de lectura corta de Salmonella Typhimurium de muestras de pollos de engorde introducidas en la plataforma de detección de patógenos del Centro Nacional de Información Biotecnológica (NCBI) por el USDA-FSIS entre los años 2016 al 2022. Estos se analizaron utilizando la herramienta de búsqueda de alineación local básica con una secuencia exclusiva para las cepas de campo, seguida de una secuencia exclusiva para la cepa vacunal. Los ensayos de PCR se desarrollaron contra cepas de campo y vacunales centrándose en eventos de transposición en los genes crp y cya y se validaron mediante la detección de aislados de Salmonella serovar Typhimurium. Entre 2016 y 2022, se encontraron 1708 aislados de Salmonella Typhimurium de origen avícola en el sistema de detección de patógenos del NCBI, lo que corresponde al 7.99 % de todas las Salmonellas identificadas. De ellas, 104 (5.97%) fueron identificadas como cepa vacunal. El ensayo de PCR diferenció las cepas de campo de la cepa de la vacuna cuando se aplicó a los aislados y también fue capaz de detectar la cepa de la vacuna a partir del ADN aislado de cultivos de enriquecimiento por toda la noche de Salmonella con serovares mixtos. Las vacunas vivas atenuadas contra Salmonella son una herramienta fundamental para el control de Salmonella y se utilizan ampliamente en la industria. Con las próximas regulaciones que probablemente se centrarán en Salmonella Typhimurium, junto con otros serovares, es necesario distinguir entre los aislados que pertenecen a la cepa vacunal y los que son responsables de causar enfermedades humanas.

Chitosan-nanoparticle-based oral Salmonella enteritidis subunit vaccine elicits cross-protection against Salmonella typhimurium in broilers.

Non-typhoidal Salmonella infection is a significant health and economic burden in poultry industry. Developing an oral vaccine to induce robust mucosal immunity in the intestines of birds, especially cross protection against different Salmonella serotypes is challenging. Therefore, a potent oral vaccine platform that can mitigate different serotypes of Salmonella is warranted for the poultry industry. We reported earlier that the Salmonella enteritidis (SE) immunogenic outer membrane proteins (OMPs) and flagellin (FLA) entrapped in mannose chitosan nanoparticles (OMPs-FLA-mCS NPs) administered prime-boost (d-3 and 3-wk later) by oral inoculation elicits mucosal immunity and reduces challenge SE colonization by over 1 log10 CFU in birds. In this study, we sought to evaluate whether the SE antigens containing OMPs-FLA-mCS NPs vaccine induces cross-protection against Salmonella typhimurium (ST) in broilers. Our data indicated that the OMPs-FLA-mCS NPs vaccine induced higher cross-protective antibody responses compared to commercial Poulvac ST vaccine (contains a modified-live ST bacterium). Particularly, OMPs-FLA-mCS-NP vaccine elicited OMPs and FLA antigens specific increased production of secretory IgA and IgY antibodies in samples collected at both post-vaccination and post-challenge timepoints compared to commercial vaccine group. Notably, the vaccine reduced the challenge ST bacterial load by 0.8 log10 CFU in the cecal content, which was comparable to the outcome of Poulvac ST vaccination. In conclusion, our data suggested that orally administered OMPs-FLA-mCS-NP SE vaccine elicited cross protective mucosal immune responses against ST colonization in broilers. Thus, this candidate vaccine could be a viable option replacing the existing both live and killed Salmonella vaccines for birds.

The impact of lipid A modification on biofilm and related pathophysiological phenotypes, endotoxicity, immunogenicity, and protection of Salmonella Typhimurium.

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Immunogenicity and Efficacy of Live-Attenuated Salmonella Typhimurium Vaccine Candidate CVD 1926 in a Rhesus Macaque Model of Gastroenteritis.

Salmonella Typhimurium is a common cause of foodborne gastroenteritis and a less frequent but important cause of invasive disease, especially in developing countries. In our previous work, we showed that a live-attenuated S. Typhimurium vaccine (CVD 1921) was safe and immunogenic in rhesus macaques, although shed for an unacceptably long period (10 days) postimmunization. Consequently, we engineered a new strain, CVD 1926, which was shown to be safe and immunogenic in mice, as well as less reactogenic in mice and human cell-derived organoids than CVD 1921. In this study, we assessed the reactogenicity and efficacy of CVD 1926 in rhesus macaques. Animals were given two doses of either CVD 1926 or saline perorally. The vaccine was well-tolerated, with shedding in stool limited to a mean of 5 days. All CVD 1926-immunized animals had both a serological and a T cell response to vaccination. At 4 weeks postimmunization, animals were challenged with wild-type S. Typhimurium I77. Unvaccinated (saline) animals had severe diarrhea, with two animals succumbing to infection. Animals receiving CVD 1926 were largely protected, with only one animal having moderate diarrhea. Vaccine efficacy in this gastroenteritis model was 80%. S. Typhimurium vaccine strain CVD 1926 was safe and effective in rhesus macaques and shed for a shorter period than other previously tested live-attenuated vaccine strains. This strain could be combined with other live-attenuated Salmonella vaccine strains to create a pan-Salmonella vaccine.

Salmonella reduces tumor metastasis by downregulation C-X-C chemokine receptor type 4.

Tumor metastasis is the main reason for the death of most cancer patients. C-X-C chemokine receptor type 4 (CXCR4) has been demonstrated to be overexpressed in numerous types of cancer. CXCR4 selectively binds with stromal cell-derived factor 1 (SDF1), also known as C-X-C family chemokine ligand 12 (CXCL12) (CXCL12/SDF-1), which induced tumor proliferation and metastasis. Recently, the use of conventional cancer treatments had some limitation; bacteria treatment for cancer becomes a trend that overcomes these limitations. Plenty of studies show that Salmonella has anti-tumor and anti-metastatic activity. The current study aimed to investigate Salmonella suppresses CXCR4 protein expression and tumor cell migration ability in B16F10 melanoma and LL2 lung carcinoma cells. Salmonella reduced CXCR4 protein expression through downregulating Protein Kinase-B (Akt)/Mammalian Target of Rapamycin (mTOR) signaling pathway. In cells transfected with constitutively active Akt plasmids, a reverse effect of Salmonella-induced inhibition of CXCR4 was observed. Tumor cells have chemotactic response to CXCL12 in migration assay, and we found that Salmonella reduced tumor chemotactic response after CXCL12 treatment. The C57BL/6 mice were intravenously injected with B16F10 and LL2 cells pre-incubated with or without Salmonella, the tumor size and lung weight of Salmonella group had obviously decreased, indicating anti-metastatic effect that confirmed the findings from the in vitro experiments.

Salmonella alters heparanase expression and reduces tumor metastasis.

Salmonella causes salmonellosis, is a facultative anaerobe and is one of the common Gram-negative bacteria. Salmonella has anti-tumor potential and tumor-targeting activity. The heparin sulfate on cell surfaces can be cleaved by heparanase that is an endo-ß-D-glucuronidase. Heparanase can destroy the extracellular matrix and is involved in tumor metastasis and angiogenic activity. Previously, Salmonella was demonstrated to inhibit tumor metastasis. It remains unclear whether Salmonella inhibits metastasis by regulating heparanase. The expression of heparanase in Salmonella-treated tumor cells was found to be decreased. Transwell and wound-healing assays demonstrated the inhibition of cell migration after Salmonella treatment. Salmonella was found to influence the levels of phosphate-protein kinase B (P-AKT) and phosphate-extracellular regulated protein kinases (P-ERK), which are involved in heparanase expression. Salmonella reduced the heparanase expression induced upregulating PERK and PAKT signaling pathways. The mice bearing an experimental metastasis tumor model was used to evaluate the anti-tumor metastatic effects of Salmonella. Compared with the control group, Salmonella significantly reduced the number of metastatic nodules and enhanced survival. The results of our study indicate that Salmonella plays a vital role in the inhibition of tumor metastasis through the downregulation of heparanase.

Robust immunity induced by multi-epitope DnaK peptides, potential vaccine candidates against Salmonella: An in vitro study.

Enteric fever is a common yet serious issue, most troublesome in underdeveloped and developing nations affecting all age group primarily children. Pitfalls of existing vaccines along with rapidly rising Multi-Drug-Resistant Salmonella strains necessitate the need for the development of new vaccine candidates having potential to provide complete protection. Several vaccine strategies are being pursued to stimulate protective immunity against typhoid, including conjugate vaccines for the elicitation of cellular and humoral responses as both arms of immunity are essential for complete protection. Bacterial HSPs are highly immunogenic to produce humoral and cellular immune responses. In this study, we are reporting in vitro immunostimulatory activity of immunodominant multi-epitope protective antigenic DnaK peptides identified earlier by immunoinformatics approach. Remarkable increase in antibody titer, lymphocyte proliferation, cytokines and NO level with individual /mixture of DnaK peptides as compared to control demonstrate immunogenic potential of these peptides that effectively augments both humoral and cellular immune responses. None of the peptides cause any hemolysis in human RBCs. Overall; our findings strongly elucidate the immune-stimulatory potential of DnaK peptides to be explored as potent vaccine candidates against multiple pathogens.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

Efficacy of a nanoparticle vaccine administered in-ovo against Salmonella in broilers.

Salmonella is a zoonotic pathogen that persists in poultry. Salmonella vaccines that can be delivered in-ovo can be cost-effective and can decrease Salmonella load in poultry. This study evaluates the efficacy of a Salmonella chitosan-nanoparticle (CNP) vaccine, administered in-ovo, in broilers. CNP vaccine was synthesized with Salmonella Enteritidis (SE) outer-membrane-proteins (OMPs) and flagellin proteins. At embryonic-d18, one-hundred-thirty-six eggs were injected with 200µl PBS or 1000µg CNP into the amniotic cavity. At d1-of-age, 132 chicks were allocated in 6 pens/treatment with 11 chicks/pen. At d7, birds were orally challenged with 1×109 CFU/bird SE. At d1, 8h-post-challenge, d14, and d21, serum anti-SE-OMPs IgY were analyzed. At d14 and d21, cloacal swabs and bile anti-SE-OMPs IgA, CD4+/CD8+-T-cell ratios, and ceca SE loads were analyzed. At d21, cecal tonsil IL-1ß, IL-10, and iNOS mRNA were analyzed. Body-weight-gain (BWG) and feed-conversion-ratio (FCR) were recorded weekly. Data were analyzed by Student's t-test at P<0.05. There were no significant differences in BWG or FCR between vaccinated birds compared to control. At d1, CNP-vaccinated birds had 5.62% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 8h-post-challenge, CNP-vaccinated birds had 6.39% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 2wk-post-challenge, CNP-vaccinated birds had 7.34% lower levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 1wk-post-challenge, CNP-vaccinated birds had 15.30% greater levels (P<0.05) of bile anti-SE-OMPs IgA, compared to control. At d14 and d21, CNP-vaccinated birds had 0.62 and 0.85 Log10 CFU/g, decreased SE ceca load (P<0.05), respectively, compared to control. There were no significant differences in CD4+/CD8+-T-cell ratios between vaccinated birds compared to control. There were no significant differences in IL-1ß, IL-10, iNOS mRNA between vaccinated birds compared to control. Findings demonstrate that the in-ovo administration of CNP vaccine can induce an antigen-specific immune response against SE and can decrease SE cecal load in broilers.

Assessment of microbiological correlates and immunostimulatory potential of electron beam inactivated metabolically active yet non culturable (MAyNC) Salmonella Typhimurium.

This study investigates the microbiological and immunological basis underlying the efficacy of electron beam-inactivated immune modulators. The underlying hypothesis is that exposure to eBeam-based ionization reactions inactivate microorganisms without modifying their antigenic properties and thereby creating immune modulators. The immunological correlates of protection induced by such eBeam based Salmonella Typhimurium (EBST) immune modulators in dendritic cell (DC) (in vitro) and mice (in vivo) models were assessed. The EBST stimulated innate pro inflammatory response (TNFα) and maturation (MHC-II, CD40, CD80 and CD86) of DC. Immuno-stimulatory potential of EBST was on par with both a commercial Salmonella vaccine, and live Salmonella cells. The EBST cells did not multiply under permissive in vitro and in vivo conditions. However, EBST cells remained metabolically active. EBST immunized mice developed Salmonella-specific CD4+ T-cells that produced the Th1 cytokine IFNγ at a level similar to that induced by the live attenuated vaccine (AroA- ST) formulation. The EBST retained stable immunogenic properties for several months at room temperature, 4°C, and -20°C as well as after lyophilization. Therefore, such eBeam-based immune modulators have potential as vaccine candidates since they offer the safety of a "killed" vaccine, while retaining the immunogenicity of an "attenuated" vaccine. The ability to store eBeam based immune modulators at room temperature without loss of potency is also noteworthy.