Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET).

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

O-antigen-deficient, live, attenuated Salmonella typhimurium confers efficient uptake, reduced cytotoxicity, and rapid clearance in chicken macrophages and lymphoid organs and induces significantly high protective immune responses that protect chickens against Salmonella infection.

In the present study, we developed an O-antigen-deficient, live, attenuated Salmonella Typhimurium (ST) strain (JOL2377) and assessed its safety, macrophage toxicity, invasion into lymphoid tissues, immunogenicity, and protection against Salmonella infection in chickens. The JOL2377 induced significantly lower cytotoxicity and higher level of cytokine response in IL-2, IL-10, IL-4, and IFN- γ than the WT strain upon macrophage uptake. It did not persist in macrophages or in chicken organs and rapidly cleared without systemic infection. None of the chicken were found to secrete Salmonella in feces into the environment exacerbating its attenuation. Interestingly JOL2377 successfully arrived in immunological hot-spots such as spleen, liver and bursa of Fabricius for an efficient antigen presentation and immune stimulation. Mucosal and parenteral immunization with JOL2377 significantly elicit antigen-specific humoral (IgY) and cell mediated responses marked by peripheral blood mononuclear cell proliferation, cytokine induction, increase in T-cell responses than non-immunized control. JOL2377 did not generate significant levels of LPS specific antibodies as compared to the WT strain due to the lack of immunogenic O-antigen component from its LPS structure. Upon virulent challenge, route dependent efficacy differences were leaving the intramuscular route is superior to the oral route on reducing splenic and liver colonization of the challenge ST. The least cytotoxicity, virulence, and superior immunogenicity of JL2377 that effectively engage both humoral and IFN- γ mediated CMI responses present an ideal scenario in host immune modulation to fight against intracellular pathogen Salmonella.

Salmonella-mediated therapy targeting indoleamine 2, 3-dioxygenase 1 (IDO) activates innate immunity and mitigates colorectal cancer growth.

Patients with colon cancer remain largely refractory to current immunotherapeutic strategies. This is, in part, due to the overexpression of the immune checkpoint protein indoleamine 2,3-dioxygenase 1 (IDO). IDO is an important enzyme contributing to tumor-mediated immunosuppression and also correlates with poor prognosis in colon cancer patients. The aim of this study was to assess the therapeutic efficacy of attenuated Salmonella typhimurium delivering an shRNA plasmid targeting IDO (shIDO-ST) in two mouse models of colorectal cancer. In vitro, the CT26 and MC38 murine colon cancer cell lines were shown to upregulate IDO expression following stimulation with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Transfection of both cell lines with shIDO plasmid reduced IDO protein expression and function. In vivo, shIDO-ST treatment significantly delayed CT26 and MC38 tumor progression compared to mice treated with scrambled shRNA control (shScr-ST) or the clinically tested IDO inhibitor epacadostat. Increased tumor infiltration of neutrophils was found to be the primary immune cell population associated with shIDO-ST treatment, suggesting robust activation of innate immunity. Although increased tumor expression of IDO is associated with resistance to antibody therapy against programed cell death-1 (anti-PD1), co-administration of anti-PD1 with shIDO-ST did not provide additional tumor growth control in either model of colorectal cancer. Altogether, we demonstrate that treatment with shIDO-ST markedly delays tumor growth in two immunocompetent colorectal mouse models and this appears to be a superior therapeutic strategy compared to epacadostat or blocking anti-PD1 antibody therapy in colon cancer.

Evaluation of the protective effect of immunization spf DBA/2J mice with selected bacterial, recombinant Hsp60 antigens during Salmonella Enteritidis challenge.

Salmonella Enteritidis is one of the most common causes of food poisoning in humans. Many attempts have been made to develop an effective vaccine against S. Enteritidis for use in poultry, but experiments aimed at the complete elimination of this pathogen from poultry farms have not provided satisfactory results. The development of new generation vaccines against salmonellosis, such as subunit vaccines based on heat shock proteins (HSPs), is strongly justified. The high immunogenicity of Hsp60 isolated from Procaryota, including Salmonella, has been suggested by the presence of IgG anti-Hsp60 antibodies in mice immunized with these proteins. The aim of the studies was to evaluate the protective effects of immunization with recombinant Hsp60 from selected gram-negative bacteria (S. Enteritidis, Escherichia coli, Pasteurella multocida, Histophilus somni) in spf DBA/2 J mice experimentally infected with S. Enteritidis. The study demonstrated that double subcutaneous immunization of mice with a dose of 10 µg rHsp60 induced a specific immune response of IgG antibodies in tested animals. The median lethal dose (LD50) for the murine model spf DBA/2 J orally infected with S. Enteritidis was estimated at 6.84 × 105 cfu/animal. Mice immunized with rHsp60 from gastrointestinal pathogens (S. Enteritidis and E. coli) showed better survival after experimental infection with a 3 × LD50 dose from S. Enteritidis, compared to animals immunized with proteins obtained from respiratory pathogens (P. multocida and H. somni). However, the log-rank analysis did not show significant differences in the survival rates between rHsp60-immunized mice and controls. S. Enteritidis was not isolated any less frequently from internal organs and faeces of rHsp60-immunized mice than from controls. Nevertheless, the level of haptoglobin (but not IL-6) was increased in all mice in which the presence of the pathogen was observed. Bacterial Hsp60 is an interesting candidate for a subunit vaccine, but its use in livestock animals must be further investigated.

A recombinant protein of Salmonella Typhi induces humoral and cell-mediated immune responses including memory responses.

Gram negative enteric bacteria, Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever is a major public health problem in developing countries. While a permanent solution to the problem would require improved sanitation, food and water hygiene, controlling the infection by vaccination is urgently required due to the emergence of multidrug resistant strains in multiple countries. The currently licensed vaccines are moderately efficacious with limited applicability, and no recommended vaccines exist for younger children. We had previously reported that a candidate vaccine based on recombinant outer membrane protein (rT2544) of S. Typhi is highly immunogenic and protective in mice. Here we show that rT2544-specific antiserum is capable of mediating bacterial lysis by the splenocytes through Antibody-Dependent Cellular Cytotoxicity (ADCC). Increased populations of rT2544-specific IgA and IgG secreting plasma cells are found in the spleen, mesenteric lymph nodes and peyer's patches. Cell-Mediated Immune Responses (CMIR) induced by rT2544 consist of Th1 cell differentiation and generation of cytotoxic T lymphocytes (CTL), which produce IFN-γ and are capable of destroying cells displaying T2544-derived antigens. rT2544 elicits pro-inflammatory cytokines (TNF-α, IL-6) from Bone Marrow-Derived Dendritic cells (BMDCs), while in vitro re-stimulation of rT2544-primed CD4+ T cells induces cell proliferation and generates higher amounts of Th1 cytokines, such as IFN-gamma, TNF-α and IL-2. Finally, the candidate vaccine induces immunological memory in the form of memory B and T lymphocytes. Taken together, the study further supports the potential of rT2544 as a novel and improved vaccine candidate against S. Typhi.

Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens.

Characterization and Evaluation of a Salmonella enterica Serotype Senftenberg Mutant Created by Deletion of Virulence-Related Genes for Use as a Live Attenuated Vaccine.

Natural infections of chickens with Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) are characterized by low-level intestinal invasiveness and insignificant production of antibodies. In this study, we investigated the potential effects of lon and cpxR gene deletions on the invasiveness of S Senftenberg into the intestinal epithelium of chickens and its ability to induce an immune response, conferring protection against S Senftenberg infection. With the allelic exchange method, we developed JOL1596 (Δlon), JOL1571 (ΔcpxR), and JOL1587 (Δlon ΔcpxR) deletion mutants from wild-type S Senftenberg. Deletion of the lon gene from S Senftenberg produced increased frequency of elongated cells, with significantly greater amounts of exopolysaccharide (EPS) than in the cpxR-deleted strain and the wild-type strain. The in vivo intestinal loop invasion assay showed a significant increase in epithelial invasiveness for JOL1596 (Δlon) and JOL1587 (Δlon ΔcpxR), compared to JOL1571 (ΔcpxR) and the wild-type strain. Furthermore, the S Senftenberg wild-type and mutant strains were internalized at high levels inside activated abdominal macrophages from chicken. The in vivo inoculation of JOL1587 (Δlon ΔcpxR) into chickens led to colonization of the liver, spleen, and cecum for a short time. Chickens inoculated with JOL1587 (Δlon ΔcpxR) showed significant increases in humoral, mucosal, and cellular immune responses specific to S Senftenberg antigens. Postchallenge, compared to the control group, the JOL1587 (Δlon ΔcpxR)-inoculated chickens showed not only lower persistence but also faster clearance of wild-type S Senftenberg from the cecum. We conclude that the increased intestinal invasiveness and colonization of internal organs exhibited by JOL1587 (Δlon ΔcpxR) led to the establishment of immunogenicity and conferred protective efficacy against S Senftenberg infections in chickens.

RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine.

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.

Dans la présente étude on évalua la capacité d'un vaccin vivant atténué génétiquement modifié de Salmonella Enteritidis (SE) à protéger contre une infection par Salmonella Typhimurium (ST) chez le poulet. Les poulets furent inoculés oralement avec le vaccin à leur premier jour de vie et reçurent un rappel à 5 semaines d'âge. Les oiseaux témoins reçurent par voie orale de la saline tamponnée. Les deux groupes furent challengés par voie orale avec une souche virulente de ST à 9 sem d'âge. Comparativement aux oiseaux témoins, les poulets vaccinés avaient des taux significativement plus élevés d'IgG systémiques et d'IgA locaux contre des antigènes spécifiques de ST et une réponse lympho-proliférative significativement plus importante aux antigènes de ST. L'excrétion de ST dans les fèces était significativement moindre dans le groupe vacciné comparativement au groupe témoin aux jours 9 et 13 après le challenge. De plus, après le challenge le groupe vacciné avait significativement moins de lésions macroscopiques marquées dans le foie et la rate et des dénombrements bactériens moindres dans les organes internes que le groupe témoin. Ces résultats indiquent que le vaccin SE vivant génétiquement modifié peut induire une réponse immunitaire humorale et cellulaire contre des antigènes de ST et peut conférer une protection contre un challenge avec une souche de ST virulente.(Traduit par Docteur Serge Messier).

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.

Identification of salmonella pathogenicity island-2 type III secretion system effectors involved in intramacrophage replication of S. enterica serovar typhimurium: implications for rational vaccine design.

UNLABELLED: Salmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells. IMPORTANCE: Live attenuated strains of Salmonella enterica serotype Typhi have generated much interest in the search for improved vaccines against typhoid fever and as vaccine vectors for the delivery of heterologous antigens. A promising vaccine candidate is the ΔaroC ΔssaV S. Typhi strain, which owes its attenuation mainly to lack of a type III secretion system (SPI-2 T3SS). The SPI-2 T3SS is important for bacterial proliferation inside macrophages, but not all of the effectors involved in this process have been identified. Here, we show that 10 effectors of the related strain S. Typhimurium contribute to intracellular replication in macrophages. Moreover, we establish that a poly-effector mutant strain of S. Typhimurium can have a severe replication defect and maintain a functional SPI-2 T3SS, which can be exploited for delivery of heterologous antigens.

Identification of an outer membrane protein of Salmonella enterica serovar Typhimurium as a potential vaccine candidate for Salmonellosis in mice.

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine.

BACKGROUND: Salmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine. RESULTS: The protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Naïve mice receiving either cells or sera from immunized mice were partially protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-γ, and IL-10 cytokines in splenocytes induced with STM cell lysate. CONCLUSIONS: Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.

Flagellin from recombinant attenuated Salmonella enterica serovar Typhimurium reveals a fundamental role in chicken innate immunity.

Recombinant attenuated Salmonella vaccines have been extensively studied, with a focus on eliciting specific immune responses against foreign antigens. However, very little is known about the innate immune responses, particularly the role of flagellin, in the induction of innate immunity triggered by recombinant attenuated Salmonella in chickens. In the present report, we describe two Salmonella enterica serovar Typhimurium vaccine strains, wild-type (WT) or flagellin-deficient (flhD) Salmonella, both expressing the fusion protein (F) gene of Newcastle disease virus. We examined the bacterial load and spatiotemporal kinetics of expression of inflammatory cytokine, chemokine, and Toll-like receptor 5 (TLR5) genes in the cecum, spleen, liver, and heterophils following oral immunization of chickens with the two Salmonella strains. The flhD mutant exhibited an enhanced ability to establish systemic infection compared to the WT. In contrast, the WT strain induced higher levels of interleukin-1ß (IL-1ß), CXCLi2, and TLR5 mRNAs in cecum, the spleen, and the heterophils than the flhD mutant at different times postinfection. Collectively, the present data reveal a fundamental role of flagellin in the innate immune responses induced by recombinant attenuated Salmonella vaccines in chickens that should be considered for the rational design of novel vaccines for poultry.

Suppression of murine melanoma growth by a vaccine of attenuated Salmonella carrying heat shock protein 70 and Herpes simplex virus-thymidine kinase genes.

Attenuated Salmonella can invade tumor cells and acts as a eukaryotic expression vector for gene propagation. We constructed a bi-gene, eukaryotic co-expression DNA vaccine of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and Herpes simplex virus-thymidine kinase (HSV-tk) and used attenuated Salmonella as a vector to treat murine melanoma. In vitro, recombinant Salmonella can carry plasmid stably and can invade into the cytoplasm of B16 tumor cells expressing the protein of the mtHSP70/HSV-tk gene by Western blot assay. In vivo, after the recombinant Salmonella was injected into tumors, the HSV-tk precursor drug ganciclovir (GCV) was administered to start the HSV-tk killing of tumor cells. We found that the mtHSP70/HSV-tk recombinant bacteria can raise CD8+ T lymphocytes in peripheral blood by flow cytometry and in tumor tissues by immunofluorescence detection, increase IFN­Î³ contents in tumor tissue by ELISA and significantly suppress tumor growth.

Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity.

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its LPS. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg(2+) culture conditions, which induce lipid A modifications. LpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA Abs and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 compared with vector-only-immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an Ag-delivery system.

Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid.

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry.

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium DeltaaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4log(10) colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium DeltaaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to DeltaspaS or DeltassaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the DeltaaroA mutant. Expression in the DeltaaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry.

Adjuvant effects for oral immunization provided by recombinant Lactobacillus casei secreting biologically active murine interleukin-1{beta}.

Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1beta (IL-1beta), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1beta as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1beta gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinant L. casei secreting IL-1beta, expression of IL-6 was detected in vivo using a ligated-intestinal-loop assay. The release of IL-6 from Peyer's patch cells was also detected in vitro. Intragastric immunization with heat-killed Salmonella enterica serovar Enteritidis (SE) in combination with IL-1beta-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinant L. casei secreting bioactive murine IL-1beta provided adjuvant effects for intragastric immunization.