Fungal sensing enhances neutrophil metabolic fitness by regulating antifungal Glut1 activity.

Combating fungal pathogens poses metabolic challenges for neutrophils, key innate cells in anti-Candida albicans immunity, yet how host-pathogen interactions cause remodeling of the neutrophil metabolism is unclear. We show that neutrophils mediate renal immunity to disseminated candidiasis by upregulating glucose uptake via selective expression of glucose transporter 1 (Glut1). Mechanistically, dectin-1-mediated recognition of ß-glucan leads to activation of PKCδ, which triggers phosphorylation, localization, and early glucose transport by a pool of pre-formed Glut1 in neutrophils. These events are followed by increased Glut1 gene transcription, leading to more sustained Glut1 accumulation, which is also dependent on the ß-glucan/dectin-1/CARD9 axis. Card9-deficient neutrophils show diminished glucose incorporation in candidiasis. Neutrophil-specific Glut1-ablated mice exhibit increased mortality in candidiasis caused by compromised neutrophil phagocytosis, reactive oxygen species (ROS), and neutrophil extracellular trap (NET) formation. In human neutrophils, ß-glucan triggers metabolic remodeling and enhances candidacidal function. Our data show that the host-pathogen interface increases glycolytic activity in neutrophils by regulating Glut1 expression, localization, and function.

RTEC-intrinsic IL-17-driven inflammatory circuit amplifies antibody-induced glomerulonephritis and is constrained by Regnase-1.

Antibody-mediated glomerulonephritis (AGN) is a clinical manifestation of many autoimmune kidney diseases for which few effective treatments exist. Chronic inflammatory circuits in renal glomerular and tubular cells lead to tissue damage in AGN. These cells are targeted by the cytokine IL-17, which has recently been shown to be a central driver of the pathogenesis of AGN. However, surprisingly little is known about the regulation of pathogenic IL-17 signaling in the kidney. Here, using a well-characterized mouse model of AGN, we show that IL-17 signaling in renal tubular epithelial cells (RTECs) is necessary for AGN development. We also show that Regnase-1, an RNA binding protein with endoribonuclease activity, is a negative regulator of IL-17 signaling in RTECs. Accordingly, mice with a selective Regnase-1 deficiency in RTECs exhibited exacerbated kidney dysfunction in AGN. Mechanistically, Regnase-1 inhibits IL-17-driven expression of the transcription factor IκBξ and, consequently, its downstream gene targets, including Il6 and Lcn2. Moreover, deletion of Regnase-1 in human RTECs reduced inflammatory gene expression in a IκBξ-dependent manner. Overall, these data identify an IL-17-driven inflammatory circuit in RTECs during AGN that is constrained by Regnase-1.

The m6A reader IMP2 directs autoimmune inflammation through an IL-17- and TNFα-dependent C/EBP transcription factor axis.

Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBPß and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPß/δ-encoding transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification. Induction of C/EBPs is enhanced by an m6A methylase "writer" and suppressed by a demethylase "eraser." The only m6A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPß/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPß/δ. Imp2-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.

Local antifungal immunity in the kidney in disseminated candidiasis.

Disseminated candidiasis is a hospital-acquired infection that results in high degree of mortality despite antifungal treatment. Autopsy studies revealed that kidneys are the major target organs in disseminated candidiasis and death due to kidney damage is a frequent outcome in these patients. Thus, the need for effective therapeutic strategies to mitigate kidney damage in disseminated candidiasis is compelling. Recent studies have highlighted the essential contribution of kidney-specific immune response in host defense against systemic infection. Crosstalk between kidney-resident and infiltrating immune cells aid in the clearance of fungi and prevent tissue damage in disseminated candidiasis. In this review, we provide our recent understanding on antifungal immunity in the kidney with an emphasis on IL-17-mediated renal defense in disseminated candidiasis.

Uremia Coupled with Mucosal Damage Predisposes Mice with Kidney Disease to Systemic Infection by Commensal Candida albicans.

Infections are the second major cause of mortality in patients with kidney disease and accompanying uremia. Both vascular access and non-access-related infections contribute equally to the infection-related deaths in patients with kidney disease. Dialysis is the most common cause of systemic infection by Candida albicans in these patients. C albicans also reside in the gastrointestinal tract as a commensal fungus. However, the contribution of gut-derived C albicans in non-access-related infections in kidney disease is unknown. Using a mouse model of kidney disease, we demonstrate that uremic animals showed increased gut barrier permeability, impaired mucosal defense, and dysbiosis. The disturbance in gut homeostasis is sufficient to drive the translocation of microbiota and intestinal pathogen Citrobacter rodentium to extraintestinal sites but not C albicans Interestingly, a majority of uremic animals showed fungal translocation only when the gut barrier integrity is disrupted. Our data demonstrate that uremia coupled with gut mucosal damage may aid in the translocation of C. albicans and cause systemic infection in kidney disease. Because most of the individuals with kidney disease suffer from some form of gut mucosal damage, these results have important implications in the risk stratification and control of non-access-related opportunistic fungal infections in these patients.

Restoring glucose uptake rescues neutrophil dysfunction and protects against systemic fungal infection in mouse models of kidney disease.

Disseminated candidiasis caused by the fungus Candida albicans is a major clinical problem in individuals with kidney disease and accompanying uremia; disseminated candidiasis fatality is twice as common in patients with uremia as those with normal kidney function. Many antifungal drugs are nephrotoxic, making treatment of these patients particularly challenging. The underlying basis for this impaired capacity to control infections in uremic individuals is poorly understood. Here, we show in multiple models that uremic mice exhibit an increased susceptibility to systemic fungal infection. Uremia inhibits Glut1-mediated uptake of glucose in neutrophils by causing aberrant activation of GSK3ß, resulting in reduced ROS generation and hence impaired killing of C. albicans in mice. Consequently, pharmacological inhibition of GSK3ß restored glucose uptake and rescued ROS production and candidacidal function of neutrophils in uremic mice. Similarly, neutrophils isolated from patients with kidney disease and undergoing hemodialysis showed similar defect in the fungal killing activity, a phenotype rescued in the presence of a GSK3ß inhibitor. These findings reveal a mechanism of neutrophil dysfunction during uremia and suggest a potentially translatable therapeutic avenue for treatment of disseminated candidiasis.

IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival.

Lymph-node (LN) stromal cell populations expand during the inflammation that accompanies T cell activation. Interleukin-17 (IL-17)-producing helper T cells (TH17 cells) promote inflammation through the induction of cytokines and chemokines in peripheral tissues. We demonstrate a critical requirement for IL-17 in the proliferation of LN and splenic stromal cells, particularly fibroblastic reticular cells (FRCs), during experimental autoimmune encephalomyelitis and colitis. Without signaling via the IL-17 receptor, activated FRCs underwent cell cycle arrest and apoptosis, accompanied by signs of nutrient stress in vivo. IL-17 signaling in FRCs was not required for the development of TH17 cells, but failed FRC proliferation impaired germinal center formation and antigen-specific antibody production. Induction of the transcriptional co-activator IκBζ via IL-17 signaling mediated increased glucose uptake and expression of the gene Cpt1a, encoding CPT1A, a rate-limiting enzyme of mitochondrial fatty acid oxidation. Hence, IL-17 produced by locally differentiating TH17 cells is an important driver of the activation of inflamed LN stromal cells, through metabolic reprogramming required to support proliferation and survival.

IL-17 Receptor Signaling Negatively Regulates the Development of Tubulointerstitial Fibrosis in the Kidney.

Chronic inflammation has an important role in the development and progression of most fibrotic diseases, for which no effective treatments exist. Tubulointerstitial fibrosis (TF) is characterized by irreversible deposition of fibrous tissue in chronic kidney diseases. Prolonged injurious stimuli and chronic inflammation regulate downstream events that lead to TF. In recent years, interleukin-17 (IL-17) has been strongly linked to organ fibrosis. However, the role of IL-17 receptor signaling in TF is an active area of debate. Using the unilateral ureteral obstruction (UUO) mouse model of TF, we show that IL-17 receptor A-deficient mice (Il17ra-/- ) exhibit increased TF in the obstructed kidney. Consequently, overexpression of IL-17 restored protection in mice with UUO. Reduced renal expression of matrix-degrading enzymes results in failure to degrade ECM proteins, thus contributing to the exaggerated TF phenotype in Il17ra -/- mice. We demonstrate that the antifibrotic kallikrein-kinin system (KKS) is activated in the obstructed kidney in an IL-17-dependent manner. Accordingly, Il17ra-/- mice receiving bradykinin, the major end-product of KKS activation, prevents TF development by upregulating the expression of matrix-degrading enzymes. Finally, we show that treatment with specific agonists for bradykinin receptor 1 or 2 confers renal protection against TF. Overall, our results highlight an intriguing link between IL-17 and activation of KKS in protection against TF, the common final outcome of chronic kidney conditions leading to devastating end-stage renal diseases.

Unexpected kidney-restricted role for IL-17 receptor signaling in defense against systemic Candida albicans infection.

Kidney injury is a frequent outcome in patients with disseminated Candida albicans fungal infections. IL-17 receptor (IL-17R) signaling is critical for renal protection against disseminated candidiasis, but the identity and function of IL-17-responsive cells in mediating renal defense remains an active area of debate. Using BM chimeras, we found that IL-17R signaling is required only in nonhematopoietic cells for immunity to systemic C. albicans infection. Since renal tubular epithelial cells (RTEC) are highly responsive to IL-17 in vitro, we hypothesized that RTEC might be the dominant target of IL-17 activity in the infected kidney. We generated mice with a conditional deletion of IL-17 receptor A (Il17ra) in RTEC (Il17raΔRTEC). Strikingly, Il17raΔRTEC mice showed enhanced kidney damage and early mortality following systemic infection, very similar to Il17ra-/- animals. Increased susceptibility to candidiasis in Il17raΔRTEC mice was associated with diminished activation of the renal protective Kallikrein-kinin system (KKS), resulting in reduced apoptosis of kidney-resident cells during hyphal invasion. Moreover, protection was restored by treatment with bradykinin, the major end-product of KKS activation, which was mediated dominantly via bradykinin receptor b1. These data show that IL-17R signaling in RTEC is necessary and likely sufficient for IL-17-mediated renal defense against fatal systemic C. albicans infection.

The Kallikrein-Kinin System: A Novel Mediator of IL-17-Driven Anti-Candida Immunity in the Kidney.

The incidence of life-threatening disseminated Candida albicans infections is increasing in hospitalized patients, with fatalities as high as 60%. Death from disseminated candidiasis in a significant percentage of cases is due to fungal invasion of the kidney, leading to renal failure. Treatment of candidiasis is hampered by drug toxicity, the emergence of antifungal drug resistance and lack of vaccines against fungal pathogens. IL-17 is a key mediator of defense against candidiasis. The underlying mechanisms of IL-17-mediated renal immunity have so far been assumed to occur solely through the regulation of antimicrobial mechanisms, particularly activation of neutrophils. Here, we identify an unexpected role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in C. albicans-infected kidney, and we show that the KKS provides significant renal protection in candidiasis. Microarray data indicated that Klk1 was upregulated in infected kidney in an IL-17-dependent manner. Overexpression of Klk1 or treatment with bradykinin rescued IL-17RA-/- mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following C. albicans infection. Furthermore, combining a minimally effective dose of fluconazole with bradykinin markedly improved survival compared to either drug alone. These results indicate that IL-17 not only limits fungal growth in the kidney, but also prevents renal tissue damage and preserves kidney function during disseminated candidiasis through the KKS. Since drugs targeting the KKS are approved clinically, these findings offer potential avenues for the treatment of this fatal nosocomial infection.

Evaluation of immunogenicity and protective efficacy of adjuvanted Salmonella Typhimurium ghost vaccine against salmonellosis in chickens.

BACKGROUND: Salmonella Typhimurium, a non-host-adapted Gram-negative intracellular pathogen, is capable of infecting a variety of animal hosts and humans. OBJECTIVE: This study utilized the prime-booster immunization strategy using Salmonella Typhimurium-LTB (S. Typhimurium-LTB) ghost with the aim of inducing a robust immune response for the prevention of avian salmonellosis. In addition, the effect of Montanide(TM) ISA 70VG adjuvant on S. Typhimurium-LTB ghost vaccination was investigated. ANIMALS AND METHODS: A total of 75 chickens were divided into three groups (n=25) for intramuscular immunization: group A (non-immunized control injected with sterile PBS), group B (immunized with S. Typhimurium-LTB ghost), and group C (immunized with S. Typhimurium-LTB ghost plus Montanide(TM) ISA70VG adjuvant). RESULTS: Compared with group A, the immunized chickens (groups B and C) exhibited increased titers of antigen specific plasma IgG and intestinal secretory IgA antibodies. In addition, group C showed enhanced induction of the humoral immune response compared to group B. The populations of splenic CD3+CD4+ and CD3+CD8+ T-cells increased significantly in both immunized groups. In addition, increased mRNA expression of the Th1 cytokines, IFN-γ, and IL-2 were observed in S. Typhimurium antigen-stimulated peripheral blood mononuclear cells from groups B and C chickens. Chickens from both vaccinated groups showed significant protection against virulent S. Typhimurium oral challenge compared to non-vaccinated chickens and a lower challenge strain count was recovered from the internal organs of group C. CONCLUSIONS: Injection of S. Typhimurium-LTB ghost with or without Montanide(TM) ISA70VG adjuvant is capable of inducing protective immunity against the virulent S. Typhimurium infection in chickens.

Utilization of a Modified Phage E Protein Lysis System Accounts for Increased Biomass in Salmonella Gallinarum Ghosts.

A major limiting issue of bacterial ghost technology involves the stable maintenance of Phix174 lysis gene E expression. Unwanted leaky expression of gene E in the absence of induction temperature results in reduced biomass production of host bacterium, consequently leading to the lower yield of bacterial ghost. To mitigate the leaky expression status of lysis gene E, we utilized a novel E-lysis system in which gene E is located between sense λpR promoter with a CI857 regulator and antisense ParaBAD promoter with the AraC regulator. In the presence of L-arabinose at 28 C, unwanted transcription of lysis gene E from λpR promoter is repressed by a simultaneous transcription event from ParaBAD promoter by means of anti-sense RNA-mediated inhibition. Tight repression of lysis gene E in the absence of induction temperature resulted in higher bacterial cell number in culture suspension and, consequently, higher production of Salmonella Gallinarum (SG) ghost biomass. The safety and protective efficacy of the SG ghost vaccine were further examined in chickens. All of the immunized chickens showed significantly higher mucosal and systemic antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response. Vaccination of chickens with SG ghost preparation offered efficient protection against wild-type SG challenge.

Immune responses to oral vaccination with Salmonella-delivered avian pathogenic Escherichia coli antigens and protective efficacy against colibacillosis.

In this study, the immune responses to and protective efficacy of a live attenuated Salmonella-delivered vaccine candidate secreting the papA, papG, iutA, and clpG antigens of Escherichia coli were evaluated against infection with avian pathogenic E. coli (APEC) in layer chickens. Primary vaccination was done at age 7 d and booster vaccination at age 5 wk. The levels of intestinal secretory immunoglobulin A specific to the 4 antigens were significantly higher in the vaccinated group than in the control group. A potent lymphocyte-proliferation response and increased levels of interferon-γ, interleukin-2, and interleukin-6 in the plasma and in culture supernatants of antigen-stimulated lymphocytes from the vaccinated group suggested significant induction of the cell-mediated immune response in this group compared with the control group. Upon challenge with a virulent APEC strain at 8 wk of age, the vaccinated group had no deaths, whereas the control group had a 15% mortality rate. In addition, the morbidity rate was significantly higher in the control group (55%) than in the vaccinated group (15%). Thus, giving primary and booster vaccination with the Salmonella-delivered APEC vaccine candidate significantly elevated both mucosal and cellular immune responses, which protected the chickens against colibacillosis.

Dans la présente étude, les réponses immunitaires et l'efficacité de protection d'un vaccin candidat administré via un Salmonella vivant atténué secrétant les antigènes papA, papG, iutA, et clpG d'Escherichia coli furent évaluées lors d'une infection chez des poules pondeuses avec un isolat d'E. coli pathogène pour les oiseaux (APEC). Une vaccination initiale fut faite à 7 jours d'âge et un rappel à l'âge de 5 semaines. Les quantités d'immunoglobulines A intestinales spécifiques aux quatre antigènes étaient significativement plus élevées dans le groupe vacciné comparativement au groupe témoin. Une réponse proliférative des lymphocytes et des niveaux augmentés d'interféron-γ, d'interleukine-2, et d'interleukine-6 dans le plasma et le surnageant de cultures de lymphocytes stimulés par des antigènes provenant des animaux du groupe vacciné suggère une induction significative de la réponse à médiation cellulaire dans ce groupe comparativement au groupe témoin. Lors d'une infection défi à 8 semaines d'âge avec une souche virulente d'APEC, aucune mortalité ne fut notée dans le groupe vacciné, alors que dans le groupe témoin on nota un taux de mortalité de 15 %. De plus, le taux de morbidité était significativement plus élevé dans le groupe témoins (55 %) comparativement au groupe vacciné (15 %). Ainsi, en donnant une primo-vaccination et un rappel avec le vaccin candidat APEC livré par Salmonella on induit des réponses significativement élevées de l'immunité humorale et de l'immunité cellulaire qui protégèrent les poulets contre la colibacillose.(Traduit par Docteur Serge Messier).

A novel approach for the generation of Salmonella Gallinarum ghosts and evaluation of their vaccine potential using a prime-booster immunization strategy.

A novel, regulatory E-lysis cassette was used in this study to avoid the untimely expression of lysis gene E and to achieve stable and improved production of Salmonella Gallinarum (SG) ghosts. A prime-booster immunization strategy using these ghosts was subsequently utilized with the aim of inducing a robust immune response for the prevention of acute fowl typhoid infection. In the first animal experiment, a total of 54 chickens were equally divided into three groups (n=18): group A (non-immunized control), group B (prime-boost immunized), and group C (singly immunized). Chickens from both immunized groups demonstrated significant increases in plasma IgG, intestinal secretory IgA, and antigen-specific lymphocyte proliferative responses. After virulent SG challenge, group B chickens immunized with the prime-boost regimen showed optimized protection. In the second animal experiment, total 20 chickens were equally divided into two groups (n=10): group A (non-immunized control), group B (prime-boost immunized) and the immunogenicity of the ghosts was further evaluated after a booster dose of the immunization. In the second animal experiment, the population of CD3+CD4+ positive T cells in the immunized chickens was significantly higher after booster immunization. In addition, increased gene expression levels of Th1 cytokines, IFN-γ, and IL-2 were observed in SG-specific antigen stimulated peripheral blood mononuclear cells of prime-boost immunized chickens compared to non-immunized chickens. In summary, the current study describes a novel approach for stable production of a safety-enhanced SG ghost preparation, and demonstrates that utilization of a prime-boost immunization strategy has an advantage over single immunization because it induces a robust immune response for optimum protection against fowl typhoid.

Characterization of a Salmonella Typhimurium ghost carrying an adjuvant protein as a vaccine candidate for the protection of chickens against virulent challenge.

In this study we describe the generation of a safe, immunogenic, genetically inactivated Salmonella Typhimurium ghost vaccine candidate carrying the Escherichia coli heat-labile enterotoxin B subunit (LTB) protein as an adjuvant molecule. An asd(+) p15A ori(-) plasmid pJHL187-LTB harbouring the E lysis gene cassette and a foreign antigen delivery cassette containing the eltB gene was used to transform a Δasd Salmonella Typhimurium (JOL1311) strain to construct the ghost strain, JOL1499. Incubation of mid-logarithmic phase JOL1499 cultures at 42°C resulted in co-expression of the eltB and E lysis genes, leading to the generation of Salmonella Typhimurium ghost cells carrying the LTB protein (Salmonella Typhimurium-LTB ghost). The production of LTB in Salmonella Typhimurium-LTB ghost preparations was confirmed by western blot analysis, and functional activity of the LTB protein to bind with GM1 receptors was determined by means of GM1 enzyme-linked immunosorbent assay. Efficacy of the Salmonella Typhimurium-LTB ghost as a vaccine candidate was evaluated in a chicken model using 56 chickens at 5 weeks old, which were divided into four groups (n = 14): group A was designated the non-vaccinated control group, whereas the birds in groups B, C, and D were immunized intramuscularly with 10(9), 10(8), and 10(7) ghost cells, respectively. Compared with the non-immunized chickens (group A), immunized chickens (groups B, C and D) exhibited increased titres of plasma IgG and intestinal secretory IgA antibodies. After oral challenge with 10(9) colony-forming units of a virulent Salmonella Typhimurium strain, the vaccinated group B birds showed a decrease in internal organ colonization with the challenge strain.

An immunogenic Salmonella ghost confers protection against internal organ colonization and egg contamination.

The tightly regulated expression of the PhiX174 lysis gene E from a multi-copy plasmid led to the stable production of an Salmonella Enteritidis bacterial ghost. The present study was conducted to evaluate induction of the humoral and cell-mediated immune responses induced after single or double intramuscular immunization with the S. Enteritidis ghost and to assess its protective effect on colonization of the intestinal tract, visceral and reproductive organs, internal egg contamination, and egg production of laying chickens. A total of 60 chickens were equally divided into three groups (n=20); group A (non-immunized control), group B (immunized at 8 and 16 weeks of age) and group C (immunized at 16th week of age). Chickens from both immunized groups B and C demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative responses. The population of CD3+CD4+ positive T cells in the immunized chickens was also significantly increased after immunization and virulent challenge. In addition, the immunized groups B and C showed significantly higher egg production and a lower percentage of S. Enteritidis contaminated eggs after challenge compared to those of group A. A comparison of challenge strain isolation from the immunized-challenged and non-immunized-challenged layer hens showed that the double immunization group induced excellent protection against intestinal, liver, splenic, and ovarian Salmonella colonization; however, the single immunized chickens showed lower counts only in the splenic and ovarian organs. Overall, the data give compelling evidence that vaccination with the S. Enteritidis ghost induced robust protective immunity against experimental avian salmonellosis and may contribute to the reduce incidence of egg contamination.

Comparative evaluation of Salmonella Enteritidis ghost vaccines with a commercial vaccine for protection against internal egg contamination with Salmonella.

The study was conducted for the comparative evaluation of the vaccine potential of Salmonella Enteritidis (S. Enteritidis, SE) ghost, SE ghost carrying Escherichia coli heat labile enterotoxin B subunit (LTB) protein, and a commercial vaccine. Group A chickens were used as a non-vaccinated control, group B chickens were immunized with the ghost carrying LTB protein, group C chickens were immunized with the ghost and, group D chickens were immunized with a commercial vaccine. Group D chickens showed the swelling at the injection site, while no adverse reactions were observed at injection sites of the group B and C chickens. Chickens from the immunized groups B, C, and D demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative responses. After challenge with a virulent SE strain via intravenous route, groups B, C, and D showed significantly higher egg production and lower internal egg contamination and lower recovery of the challenge strain from internal organs compared to non-immunized-challenged control group A. In conclusion, these data indicate that immunization of chickens with the ghost and ghost carrying LTB is safe, without causing any adverse reaction, and is effective as the commercial vaccine in terms of reduction in internal egg contamination and internal organ colonization of Salmonella.

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine.

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.

Salmonella enterica serovar enteritidis ghosts carrying the Escherichia coli heat-labile enterotoxin B subunit are capable of inducing enhanced protective immune responses.

The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3(+) CD4(+) subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.

The B subunits of cholera and Escherichia coli heat-labile toxins enhance the immune responses in mice orally immunised with a recombinant live P-fimbrial vaccine for avian pathogenic E. coli.

This study aimed to investigate the adjuvant effect of recombinant attenuated Salmonella expressing cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) for the P-fimbriae subunit-based vaccine of avian pathogenic E. coli (APEC) in a murine model. The PapA-specific sIgA and IgG responses were significantly enhanced after immunisation with the Salmonella-PapA vaccine in the presence of CTB or LTB. The group immunised with the Salmonella-LTB strain promoted Th1-type immunity, whereas that immunised with the Salmonella-CTB strain produced Th2-type immunity. We concluded that both Salmonella-CTB and -LTB strains can enhance the immune response to PapA, and that the LTB strain may be a more effective adjuvant for APEC vaccination, which requires higher Th1-type immunity for protection. Thus, our findings provide evidence that immunisation with an adjuvant, LTB, is one of the strategies of developing effective vaccines against P-fimbriated APEC.