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.

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.

CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice.

BACKGROUND: Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain's major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. RESULTS: Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. CONCLUSION: All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis.

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Differential cytokine profiling and microbial species involved in cecal microbiota modulations in SPF chicks immunized with a dual vaccine against Salmonella Typhimurium infection.

Salmonella Typhimurium (ST) infection in laying hens is a significant threat to public health and food safety. Host resistance against enteric pathogen invasion primarily relies on immunity and gut barrier integrity. This study applied the ST infection model and a dual live vaccine containing Salmonella Enteritidis (SE) strain Sm24/Rif12/Ssq and ST strain Nal2/Rif9/Rtt to investigate the cellular cytokine expression profiles and the differential community structure in the cecal microbiota of specific-pathogen-free (SPF) chicks and field-raised layers. The results showed that ST challenge significantly upregulated expressions of IL-1ß in SPF chicks. Vaccination, on the other hand, led to an elevation in IFNγ expression and restrained IL-1ß levels. In the group where vaccination preceded the ST challenge (S.STvc), heightened expressions of IL-1ß, IL-6, IL-10, and IL-12ß were observed, indicating active involvement of both humoral and cell-mediated immunity in the defense against ST. Regarding the cecal microbiota, the vaccine did not affect alpha diversity nor induce a significant shift in the microbial community. Conversely, ST infection significantly affected the alpha and beta diversity in the cecal microbiota, reducing beneficial commensal genera, such as Blautia and Subdoligranulum. MetagenomeSeq analysis reveals a significant increase in the relative abundance of Faecalibacterium prausnitzii in the groups (S.STvc and STvc) exhibiting protection against ST infection. LEfSe further demonstrated Faecalibacterium prausnitzii as the prominent biomarker within the cecal microbiota of SPF chicks and field layers demonstrating protection. Another biomarker identified in the S.STvc group, Eubacterium coprostanoligenes, displayed an antagonistic relationship with Faecalibacterium prausnitzii, suggesting the limited biological significance of the former in reducing cloacal shedding and tissue invasion. In conclusion, the application of AviPro Salmonella DUO vaccine stimulates host immunity and modulates cecal microbiota to defend against ST infection. Among the microbial modulations observed in SPF chicks and field layers with protection, Faecalibacterium prausnitzii emerges as a significant species in the ceca. Further research is warranted to elucidate its role in protecting layers against ST infection.

Oral Immunization with Attenuated Salmonella Choleraesuis Expressing the FedF Antigens Protects Mice against the Shiga-Toxin-Producing Escherichia coli Challenge.

Edema disease (ED) is a severe and lethal infectious ailment in swine, stemming from Shiga-toxin-producing Escherichia coli (STEC). An efficient, user-friendly, and safe vaccine against ED is urgently required to improve animal welfare and decrease antibiotic consumption. Recombinant attenuated Salmonella vaccines (RASV) administered orally induce both humoral and mucosal immune responses to the immunizing antigen. Their potential for inducing protective immunity against ED is significant through the delivery of STEC antigens. rSC0016 represents an enhanced recombinant attenuated vaccine vector designed for Salmonella enterica serotype Choleraesuis. It combines sopB mutations with a regulated delay system to strike a well-balanced equilibrium between host safety and immunogenicity. We generated recombinant vaccine strains, namely rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA), and assessed their safety and immunogenicity in vivo. The findings demonstrated that the mouse models immunized with rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA) generated substantial IgG antibody responses to FedF and rStx2eA, while also provoking robust mucosal and cellular immune responses against both FedF and rStx2eA. The protective impact of rSC0016 (pS-FedF) against Shiga-toxin-producing Escherichia coli surpassed that of rSC0016 (pS-rStx2eA), with percentages of 83.3%. These findings underscore that FedF has greater suitability for vaccine delivery via recombinant attenuated Salmonella vaccines (RASVs). Overall, this study provides a promising candidate vaccine for infection with STEC.

Salmonella Vaccine Study in Oxford (SALVO) trial: protocol for an observer-participant blind randomised placebo-controlled trial of the iNTS-GMMA vaccine within a European cohort.

INTRODUCTION: Invasive non-typhoidal Salmonellosis (iNTS) is mainly caused by Salmonella enterica serovars Typhimurium and Enteritidis and is estimated to result in 77 500 deaths per year, disproportionately affecting children under 5 years of age in sub-Saharan Africa. Invasive non-typhoidal Salmonellae serovars are increasingly acquiring resistance to first-line antibiotics, thus an effective vaccine would be a valuable tool in reducing morbidity and mortality from infection. While NTS livestock vaccines are in wide use, no licensed vaccines exist for use in humans. Here, a first-in-human study of a novel vaccine (iNTS-GMMA) containing S. Typhimurium and S. Enteritidis Generalised Modules for Membrane Antigens (GMMA) outer membrane vesicles is presented. METHOD AND ANALYSIS: The Salmonella Vaccine Study in Oxford is a randomised placebo-controlled participant-observer blind phase I study of the iNTS-GMMA vaccine. Healthy adult volunteers will be randomised to receive three intramuscular injections of the iNTS-GMMA vaccine, containing equal quantities of S. Typhimurium and S. Enteritidis GMMA particles adsorbed on Alhydrogel, or an Alhydrogel placebo at 0, 2 and 6 months. Participants will be sequentially enrolled into three groups: group 1, 1:1 randomisation to low dose iNTS-GMMA vaccine or placebo; group 2, 1:1 randomisation to full dose iNTS-GMMA vaccine or placebo; group 3, 2:1 randomisation to full dose or lower dose (dependant on DSMC reviews of groups 1 and 2) iNTS-GMMA vaccine or placebo.The primary objective is safety and tolerability of the vaccine. The secondary objective is immunogenicity as measured by O-antigen based ELISA. Further exploratory objectives will characterise the expanded human immune profile. ETHICS AND DISSEMINATION: Ethical approval for this study has been obtained from the South Central-Oxford A Research Ethics Committee (Ethics REF:22/SC/0059). Appropriate documentation and regulatory approvals have been acquired. Results will be disseminated via peer-reviewed articles and conferences. TRIAL REGISTRATION NUMBER: EudraCT Number: 2020-000510-14.

Vaccine value profile for Salmonella enterica serovar Paratyphi A.

In Asia, there are an estimated 12 million annual cases of enteric fever, a potentially fatal systemic bacterial infection caused by Salmonella enterica serovars Typhi (STy) and Paratyphi A (SPA). The recent availability of typhoid conjugate vaccines (TCV), an increasing incidence of disease caused by SPA and growing antimicrobial resistance (AMR) across the genus Salmonella makes a bivalent STy/SPA vaccine a useful public health proposition. The uptake of a stand-alone paratyphoid vaccine is likely low thus, there is a pipeline of bivalent STy/SPA candidate vaccines. Several candidates are close to entering clinical trials, which if successful should facilitate a more comprehensive approach for enteric fever control. Additionally, the World Health Organization (WHO) has made advancing the development of vaccines that protect young children and working aged adults against both agents of enteric fever a priority objective. This "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by SPA prevalent in Asia. Information is included on stand-alone SPA candidate vaccines and candidate vaccines targeting SPA combined with STy. Out of scope for the first version of this VVP is a wider discussion on the development of a universal Salmonella combination candidate vaccine, addressing both enteric fever and invasive non-typhoidal Salmonella disease, for use globally. This VVP is a detailed, high-level assessment of existing, publicly available information to inform and contextualize the public health, economic, and societal potential of pipeline vaccines and vaccine-like products for SPA. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and "Full Vaccine Value Assessment" that will inform the value proposition of an SPA vaccine. This VVP was developed by an expert working group from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations as well as in collaboration with stakeholders from the WHO South-East Asian Region. All contributors have extensive expertise on various elements of the VVP for SPA and collectively aimed to identify current research and knowledge gaps.

Development of toxin-antitoxin self-destructive bacteria, aimed for salmonella vaccination.

The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria.

"EvoVax" - A rationally designed inactivated Salmonella Typhimurium vaccine induces strong and long-lasting immune responses in pigs.

Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm) poses a considerable threat to public health due to its zoonotic potential. Human infections are mostly foodborne, and pork and pork products are ranked among the top culprits for transmission. In addition, the high percentage of antibiotic resistance, especially in monophasic S.Tm, limits treatment options when needed. Better S.Tm control would therefore be of benefit both for farm animals and for safety of the human food chain. A promising pre-harvest intervention is vaccination. In this study we tested safety and immunogenicity of an oral inactivated S.Tm vaccine, which has been recently shown to generate an "evolutionary trap" and to massively reduce S.Tm colonization and transmission in mice. We show that this vaccine is highly immunogenic and safe in post-weaning pigs and that administration of a single oral dose results in a strong and long-lasting serum IgG response. This has several advantages over existing - mainly live - vaccines against S.Tm, both in improved seroconversion and reduced risk of vaccine-strain persistence and reversion to virulence.

Salmonella Typhimurium-based inactivated vaccine containing a wide spectrum of bacterial antigens which mimics protein expression changes during different stages of an infection process.

Salmonella infections are still considered a persistent problem in veterinary medicine. Vaccination is one of the tools for decreasing the burden of many pathogens on animals. However, the efficiency of available commercial or experimental vaccines against non-typhoid Salmonella strains is not yet sufficient. We followed the path of an inactivated vaccine that is safe and well accepted, but whose presented antigen spectrum is limited. We improved this issue by using diverse cultivation conditions mimicking bacterial protein expression during the natural infection process. The cultivation process was set up to simulate the host environment to enhance the expression of SPI-1 (Salmonella pathogenicity island) proteins, SPI-2 proteins, siderophore-related proteins, and flagellar proteins. Three different cultivation media were used and subsequent cultures were mixed together, inactivated, and used for the immunization of post-weaned piglets. A mixture of recombinant Salmonella proteins was also used as a recombinant vaccine for comparison. The clinical symptoms during the subsequent experimental infection, antibody response, and organ bacterial loads were examined. One day after the infection, we observed an increased rectal temperature in the group of unvaccinated animals and the animals vaccinated with the recombinant vaccine. The increase in the temperature of the pigs vaccinated with the inactivated Salmonella mixture was significantly lower. In the same group, we also found lower bacterial loads in the ileum content and the colon wall. The IgG response to several Salmonella antigens was enhanced in this group, but it did not reach the titers of the group vaccinated with the recombinant vaccine. To summarize, the pigs vaccinated with an inactivated mixture of Salmonella cultures mimicking protein expression changes during the natural infection exhibited less serious clinical symptoms and lower bacterial load in the body after the experimental infection compared to the unvaccinated pigs and the pigs vaccinated with a mixture of recombinant Salmonella proteins.

Reduced immunogenicity of a live Salmonella enterica serovar Typhimurium vaccine in aged mice.

Introduction: Non-typhoidal Salmonella (NTS) is responsible for a high burden of foodborne infections and deaths worldwide. In the United States, NTS infections are the leading cause of hospitalizations and deaths due to foodborne illnesses, and older adults (≥65 years) are disproportionately affected by Salmonella infections. Due to this public health concern, we have developed a live attenuated vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), against Salmonella enterica serovar Typhimurium, a common serovar of NTS. Little is known about the effect of age on oral vaccine responses, and due to the decline in immune function with age, it is critical to evaluate vaccine candidates in older age groups during early product development. Methods: In this study, adult (six-to-eight-week-old) and aged (18-month-old) C57BL/6 mice received two doses of CVD 1926 (109 CFU/dose) or PBS perorally, and animals were evaluated for antibody and cell-mediated immune responses. A separate set of mice were immunized and then pre-treated with streptomycin and challenged orally with 108 CFU of wild-type S. Typhimurium SL1344 at 4 weeks postimmunization. Results: Compared to PBS-immunized mice, adult mice immunized with CVD 1926 had significantly lower S. Typhimurium counts in the spleen, liver, and small intestine upon challenge. In contrast, there were no differences in bacterial loads in the tissues of vaccinated versus PBS aged mice. Aged mice exhibited reduced Salmonella-specific antibody titers in the serum and feces following immunization with CVD 1926 compared to adult mice. In terms of T cell responses (T-CMI), immunized adult mice showed an increase in the frequency of IFN-γ- and IL-2-producing splenic CD4 T cells, IFN-γ- and TNF-α-producing Peyer's Patch (PP)-derived CD4 T cells, and IFN-γ- and TNF-α-producing splenic CD8 T cells compared to adult mice administered PBS. In contrast, in aged mice, T-CMI responses were similar in vaccinated versus PBS mice. CVD 1926 elicited significantly more PP-derived multifunctional T cells in adult compared to aged mice. Conclusion: These data suggest that our candidate live attenuated S. Typhimurium vaccine, CVD 1926, may not be sufficiently protective or immunogenic in older humans and that mucosal responses to live-attenuated vaccines decrease with increasing age.

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.

Screening of lipid-A related genes and development of low-endotoxicity live-attenuated Salmonella gallinarum by arnT deletion that elicits immune responses and protection against fowl typhoid in chickens.

In the present study, lipid-A gene mutants of Salmonella gallinarum (SG) were screened, and the arnT mutant exhibited optimal acidic and oxidative-stress and macrophage-survival. Modifying lipid-A by arnT-deletion resulted in significantly reduced endotoxicity, virulence, and mortality. Therefore, the arnT-deleted vaccine-candidate strain JOL2841 was constructed and demonstrated to be safe due to appropriate clearance by the chicken immune system. The reduced-endotoxicity of JOL2841 was evident from the downregulation of TNFα and IL-1ß inflammatory cytokines, no inflammatory signs in organ gross-examination, and histopathological analysis. The IgY and IgA antibody titres, CD4, and CD8 T-cell population improvements, and IL-4, IL-2, and INFγ expression decipher the profound Th2 and Th1 immunogenicity. Consequently, JOL2841 exhibited prominent protection against wild-type SG challenge, as revealed by organ pathogen-load determination, organ gross-examination, and histopathological examination. Overall, the study represented the first report of arnT deficient SG resulted in negligible endotoxicity, low-virulence, safety and coordinated elicitation of humoral and cell-mediated immune response in chickens.

Investigation of a gel-based delivery method for the administration of a live, attenuated Salmonella Typhimurium vaccine.

Poultry vaccines are often administered using water as a suspension media and applied using an oral or coarse spray method. Gel-based vaccine diluents have been developed as an alternative vaccine delivery method. Gels are more viscous, and droplets adhere more effectively to feathers giving the vaccine a longer time to be ingested (through preening). Application of gel diluents with live bacterial vaccines, however, is limited. The present study tested a gel diluent prepared in various media, using a live, attenuated Salmonella Typhimurium vaccine, Vaxsafe ST. Reconstitution in gel diluent did not negatively affect vaccine viability or motility. The invasive capacity of vaccine suspended in gel diluent into cultured intestinal epithelial cells was also tested. Results demonstrated that vaccine suspended in gel diluent retained invasiveness. Day old chicks were orally administered with Vaxsafe ST suspended in gel diluent to characterize in vivo colonization capacity of the vaccine. The results revealed that the VaxSafe ST suspended in gel diluent could efficiently colonize the caeca of chicks, which is needed for the development of effective immunity.

Self-administration of a Salmonella vaccine by domestic pigs.

Hand vaccinating is time consuming and inefficient. Oral vaccines delivered by drenching are less likely to be used due to a lack of labor on farms. Current environmental enrichment (EE) technologies do not allow pigs to express certain natural behaviors such as rooting and getting a reward. We developed a sprayer so that domestic pigs can self-apply any liquid. By adding an attractant (pig maternal pheromone), the use of EE devices by individual pigs can be increased. In this study, we used a Salmonella oral vaccine to evaluate efficacy of three delivery methods: (1) Control, no vaccine, (2) hand drenching as labeled, and (3) self-administration by this EE rooting device. All pigs sprayed themselves within 80 min of exposure to the EE device. While control pigs had little or no Salmonella serum and oral fluid IgG or IgA, hand-drenched and self-vaccinated pigs built similar levels of both serum and oral fluid IgA and IgG. We conclude we were able to significantly reduce human labor needed and achieved 100% efficacy in eliciting a serologic response when pigs self-administered a Salmonella vaccine. This technology could benefit commercial pig production while providing an enriched behavioral environment. Self-vaccination could also assist in control or immunization of feral swine and improve domestic pig health and food safety.

Effect of dietary ß-glucan on intestinal microbial diversity and Salmonella vaccine immunogenicity and efficacy in pigs.

Alternatives to antibiotics to improve animal performance, limit the negative impact of infectious disease, and/or reduce colonization with foodborne pathogens is a major focus of animal agricultural research. ß-glucans, a generally-recognized-as-safe (GRAS) product derived from various sources, are used in swine and can serve as both a prebiotic and/or stimulant of the immune system given the expression of ß-glucan receptors on immune cells. When supplied in the diet of nursery pigs, it is unclear how dietary additives, particularly those known to modulate immune status, impact immunogenicity and efficacy of mucosal-delivered vaccines. Salmonellosis is one of the most common bacterial foodborne infections in the United States, and consumption of contaminated pork is a major source of human infection. Reduction of foodborne Salmonella in pigs via vaccination is one strategy to reduce contamination risk and subsequently reduce human disease. We examined the ability of dietary ß-glucan to modulate fecal microbial diversity, and immunogenicity and efficacy of a mucosally-delivered, live-attenuated Salmonella vaccine during the nursery period. While dietaryß-glucan did modulate fecal alpha diversity, it did not alter the induction of peripheral Salmonella-specific IFN-γ secreting Tcells or Salmonella-specific IgA in oral fluids. In addition, vaccination reduced Salmonella enterica serovar Typhimurium fecal shedding and tissue colonization. Overall, addition of ß-glucan to the nursery diet of pigs impacted the microbiota but did not alter mucosal vaccine immunogenicity and efficacy.