Salmonella Paratyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever.

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers.

Dual role of splenic mononuclear and polymorphonuclear cells in the outcome of ciprofloxacin treatment of Salmonella enterica infections.

OBJECTIVES: To determine the immune cell populations associated with Salmonella enterica serovar Typhimurium before and after ciprofloxacin treatment using a murine model of systemic infection. The effect of depletion of immune cells associating with Salmonella on treatment outcome was also determined. METHODS: We infected mice with a Salmonella enterica serovar Typhimurium strain expressing GFP and used multicolour flow cytometry to identify splenic immune cell populations associating with GFP-positive Salmonella before and after treatment with ciprofloxacin. This was followed by depletion of different immune cell populations using antibodies and liposomes. RESULTS: Our results identified CD11b+CD11chi/lo (dendritic cells/macrophages) and Ly6G+CD11b+ (neutrophils) leucocytes as the main host cell populations that are associated with Salmonella after ciprofloxacin treatment. We therefore proceeded to test the effects of depletion of such populations during treatment. We show that depletion of Ly6G+CD11b+ populations resulted in an increase in the number of viable bacterial cells in the spleen at the end of ciprofloxacin treatment. Conversely, treatment with clodronate liposomes during antimicrobial treatment, which depleted the CD11b+CD11chi/lo populations, resulted in lower numbers of viable bacteria in the tissues. CONCLUSIONS: Our study identified host cells where Salmonella bacteria persist during ciprofloxacin treatment and revealed a dual and opposing effect of removal of Ly6G+CD11b+ and CD11b+CD11chi/lo host cells on the efficacy of antimicrobial treatment. This suggests a dichotomy in the role of these populations in clearance/persistence of Salmonella during antimicrobial treatment.

Within-host spatiotemporal dynamics of systemic Salmonella infection during and after antimicrobial treatment.

OBJECTIVES: We determined the interactions between efficacy of antibiotic treatment, pathogen growth rates and between-organ spread during systemic Salmonella infections. METHODS: We infected mice with isogenic molecularly tagged subpopulations of either a fast-growing WT or a slow-growing ΔaroC Salmonella strain. We monitored viable bacterial numbers and fluctuations in the proportions of each bacterial subpopulation in spleen, liver, blood and mesenteric lymph nodes (MLNs) before, during and after the cessation of treatment with ampicillin and ciprofloxacin. RESULTS: Both antimicrobials induced a reduction in viable bacterial numbers in the spleen, liver and blood. This reduction was biphasic in infections with fast-growing bacteria, with a rapid initial reduction followed by a phase of lower effect. Conversely, a slow and gradual reduction of the bacterial load was seen in infections with the slow-growing strain, indicating a positive correlation between bacterial net growth rates and the efficacy of ampicillin and ciprofloxacin. The viable numbers of either bacterial strain remained constant in MLNs throughout the treatment with a relapse of the infection with WT bacteria occurring after cessation of the treatment. The frequency of each tagged bacterial subpopulation was similar in the spleen and liver, but different from that of the MLNs before, during and after treatment. CONCLUSIONS: In Salmonella infections, bacterial growth rates correlate with treatment efficacy. MLNs are a site with a bacterial population structure different to those of the spleen and liver and where the total viable bacterial load remains largely unaffected by antimicrobials, but can resume growth after cessation of treatment.

Inferring within-host bottleneck size: A Bayesian approach.

Recent technical developments in microbiology have led to new discoveries on the within-host dynamics of bacterial infections in laboratory animals. In particular, they have highlighted the importance of stochastic bottlenecks at the onset of invasive disease. A number of approaches exist for bottleneck-size estimation with respect to within-host bacterial infections; however, some are more appropriate than others under certain circumstances. A Bayesian comparison of several approaches is made in terms of the availability of isogenic multitype bacteria (e.g., WITS), knowledge of post-bottleneck dynamics, and the suitability of dilution with monotype bacteria. A sampling approach to bottleneck-size estimation is also introduced. The results are summarised by a guiding flowchart, which we hope will promote the use of quantitative models in microbiology to refine the analysis of animal experiment data.

Design of glycoconjugate vaccines against invasive African Salmonella enterica serovar Typhimurium.

Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S. Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM(197)). The OAg-CRM(197) conjugates varied in (i) OAg source, with three S. Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM(197) ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S. Typhimurium glycoconjugate by challenge with live Salmonella. We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM(197) ratio. In addition, we found that bactericidal activity can be influenced by S. Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S. Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S. Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.

Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection. Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

LuxS affects flagellar phase variation independently of quorum sensing in Salmonella enterica serovar typhimurium.

LuxS catalyzes the synthesis of the quorum-sensing signaling molecule autoinducer 2. We show that in Salmonella enterica serovar Typhimurium, deletion of the luxS gene polarizes flagellar phase variation toward the more immunogenic phase 1 flagellin. This phenotype is complementable by luxS in trans but is independent of quorum-sensing signals.

Plant lectins ConBr and CFL modulate expression toll-like receptors, pro-inflammatory cytokines and reduce the bacterial burden in macrophages infected with Salmonella enterica serovar Typhimurium.

BACKGROUND: Plant lectins have long been used in biomedical research as immunomodulators against tumor cells and microbial infections. PURPOSE: To test the ability of plant lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) to activate antimicrobial and immunomodulatory activities of murine peritoneal macrophages (pMØ) infected with a virulent strain of Salmonella enterica serovar Typhimurium (STm). METHODS: We incubated pMØ with non-toxic amounts of ConBr and CFL either before (preventive schedule) or after (curative schedule) exposure to STm. RESULTS: In uninfected pMØ, ConBr and CFL greatly increased levels of mRNA transcripts for IL-1ß, TNF-α and IL-6 and the inducible nitric oxide synthase (iNOs), but not IL-10 and IL-12. Exposure to naïve splenocytes of culture supernatants of pMØ previously stimulated with CFL resulted in expression of IL-12 and IFN-γ. Both preventive and curative treatment schedules significantly reduced the intracellular load of Salmonella. Experiments in infected macrophages exposed to lectins in the preventive schedule showed that mRNA transcripts for IL-6 and TNF-α were increased by CFL, whereas ConBr enhanced IL-12 (subunit p40). In the curative schedule, CFL induced significant expression of IL-12 (p40) whereas ConBr enhanced expression IL-1ß and TNF-α genes. The lectin treatments did not influence on iNOs expression in pMØ infected with STm C5 regardless of the treatment schedule. Curative treatments with CFL increased approximately 130-fold expression of TLR-4 whist expression of TLR-9 was increased by treatments with ConBr. CONCLUSION: We conclude that lectins ConBr and CFL have immunomodulatory properties that are beneficial on control of cells infected by Salmonella.

Effects of tetanus toxin after intracerebral microinjection are antagonized by drugs enhancing GABAergic transmission in adult fowls.

In adult fowls (Gallus domesticus), the behavioural effects of small doses of tetanus toxin, after unilateral microinjection into the nucleus mesencephalicus profundus (NMP), homologous to the mammalian substantia nigra, were studied. A rich pattern of stereotyped movements, vocalization, ipsilateral head-neck rotation, wing abduction, occasional ipsilateral circling and escape responses from the box were observed; the intensity of such symptomatology was dose-related and fully reversible within approx. 4 hr. Pretreatment with sodium valproate (100 and 200 mg/kg, i.m.) or with ethanolamine-sulphate (EOS, 1.6 mumol into the NMP) completely prevented the effects of tetanus toxin. The present findings show that fowls can represent an ideal species for studying acute central effects of tetanus toxin and more interestingly that drugs enhancing GABAergic mechanisms are able to prevent the effects of tetanus toxin.

Comparative convulsant potencies of two carbapenem derivatives in C57 and DBA/2 mice.

The behavioural and convulsant effects of imipenem and meropenem were studied after intraperitoneal administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizure, and in C57 mice, a strain not prone to seizure. DBA/2 mice were more susceptible than C57 mice to seizures induced by imipenem-cilastatin or meropenem. Imipenem was also 1.9 times more potent than meropenem in inducing clonus in DBA/2 mice. To investigate the possibility that the seizure-inducing activity of imipenem might be due to a probenecid-like effect of cilastatin, animals were treated with imipenem alone. No significant differences were observed between imipenem-cilastatin and imipenem-treated animals. Thus, it is reasonable to exclude a probenecid-like effect of cilastatin. Although the main mechanism for seizure-like activity of imipenem cannot be easily determined, we believe that several mechanisms may be involved. An increased excitation of the central nervous system (CNS) by inhibition of GABA binding to receptors and a slow clearance of imipenem from the CNS may be postulated. Cilastatin did not induce seizures. In addition, meropenem, a compound structurally related to imipenem, showed weak or no convulsant effects.

Effects of rufloxacin in Salmonella typhimurium infection in mice.

This study was undertaken to investigate the efficacy of rufloxacin, a new quinolone which is interesting due to its pharmacokinetics characterized by a long plasma half-life, in the treatment of systemic salmonella infections in the mouse typhoid model. Innately susceptible BALB/c and resistant CBA mice were used to investigate the efficacy of rufloxacin in controlling systemic salmonella infections when given for brief or prolonged periods. The present study shows that rufloxacin is not only very effective on both mouse strains, but can completely eradicate the salmonellae from livers and spleens when given early in the infection of CBA resistant mice.

Salmonella: immune responses and vaccines.

Salmonella infections are a serious medical and veterinary problem world-wide and cause concern in the food industry. Vaccination is an effective tool for the prevention of Salmonella infections. Host resistance to Salmonella relies initially on the production of inflammatory cytokines leading to the infiltration of activated inflammatory cells in the tissues. Thereafter T- and B-cell dependent specific immunity develops allowing the clearance of Salmonella microorganisms from the tissues and the establishment of long-lasting acquired immunity to re-infection. The increased resistance that develops after primary infection/ vaccination requires T-cells cytokines such as IFNgamma TNFalpha and IL12 in addition to opsonising antibody. However for reasons that are not fully understood seroconversion and/or the presence of detectable T-cell memory do not always correlate with the development of acquired resistance to infection.Whole-cell killed vaccines and subunit vaccines are used in the prevention of Salmonella infection in animals and in humans with variable results. A number of early live Salmonella vaccines derived empirically by chemical or u.v. mutagenesis proved to be immunogenic and protective and are still in use despite the need for repeated parenteral administration. Recent progress in the knowledge of the genetics of Salmonella virulence and modern recombinant DNA technology offers the possibility to introduce multiple defined attenuating and irreversible mutations into the bacterial genome. This has recently allowed the development of Salmonella strains devoid of significant side effects but still capable of inducing solid immunity after single oral administration. Live attenuated Salmonella vaccines have been used for the expression of heterologous antigens/proteins that can be successfully delivered to the immune system. Furthermore Salmonella can transfer plasmids encoding foreign antigens under the control of eukaryotic promoters (DNA vaccines) to antigen-presenting cells resulting in targeted delivery of DNA vaccines to these cells. Despite the great recent advances in the development of Salmonella vaccines a large proportion of the work has been conducted in laboratory rodents and more research in other animal species is required.

Comparative effect of gentamicin and pefloxacin treatment on the late stages of mouse typhoid.

The present study compares the ability of gentamicin and pefloxacin to eradicate a Salmonella infection in BALB/c mice when the treatment is instituted in the late stages of the infection. The results indicate that pefloxacin is highly effective in the treatment of mouse typhoid even when the therapy is instituted after the suppression of bacterial growth in the reticuloendothelial system (RES). Conversely, gentamicin treatment only reduced the bacterial load in the RES of infected mice, but neither induced the clearance of the organisms from the RES, nor prevented the resurgence of bacterial growth. Even when using gentamicin at a high dosage, bacterial clearance could not be accomplished.

Modulatory effect of HHV-6 on MCP-1 production by human monocytes.

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells.

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.

Treatment of PBMC with killed Helicobacter pylori subverts the environment of inflammatory cytokines.

It is well known that inflammation induced by Helicobacter pylori is characterized by the local production of cytokines and chemokines. In the present study, we analyse the kinetics of MCP-1, IL-12 and IL-4 induction during the interaction of peripheral blood mononuclear cells with killed and/or live H. pylori. Our results demonstrate that live H. pylori does not induce IL-4 release whereas it stimulates MCP-1 and IL-12 production. In addition, the neutralization of IL-12 with monoclonal antibodies determines a lower MCP-1 release. These data demonstrate that MCP-1 production is in part supported by IL-12 induced by live H. pylori. On the contrary, killed H. pylori stimulates the IL-4 but not MCP-1 and IL-12 production. The combined treatment with killed and live H. pylori upregulates the IL-4 production and at the same time downregulates IL-12 and MCP-1 production.

Differential induction of TNFalpha and IL-18 in human peripheral blood mononuclear cells infected with Leishmania major or Leishmania donovani.

Several cytokines are involved in the host response to Leishmania. However, the role played by cytokines during infection with different species of Leishmania is not univocal. In this work, the production of tumor necrosis factor alpha (TNFalpha) and interleukin 18 (IL-18) during interaction of human phagocytes with Leishmania major or L. donovani was comparatively investigated. Peripheral blood mononuclear cells (PBMC) and monocytes from healthy donors were used. The release of TNFalpha and IL-18 during infection of cells with different species of Leishmania "in vitro" was evaluated. L. donovani induced in both PBMC and monocytes significantly more TNFalpha and IL-18 with respect to L. major. The amounts of TNFalpha released by PBMC were always significantly higher than those released by monocytes of the same donors.