Nitin Machindra Kamble

Interaction of a live attenuated Salmonella Gallinarum vaccine candidate with chicken bone marrow-derived dendritic cells

ABSTRACT Salmonella enterica serovar Gallinarum (SG) is a Gram-negative intracellular host-adapted pathogen that causes fowl typhoid. Attenuated strains of SG are proven and widely used vaccine candidates because of advantages like induction of strong humoral and cell-mediated immune responses. In the present study, we investigated the interaction of chicken bone marrow-derived dendritic cells (chBM-DCs) with an attenuated SG (JOL1355) strain that secretes a heat-labile enterotoxin B subunit protein previously shown to successfully vaccinate chickens. ChBM-DCs were isolated and cultured in the presence of recombinant chicken GM-CSF and IL-4 cytokines. The chBM-DCs were infected with JOL1355 at an multiplicity of infection of 10. JOL1355 was able to invade dendritic cells (DCs); however, the survival of JOL1355 in DCs decreased over time. At 24 h post infection, IL-6, IL-10 and IFN-γ transcript levels were significantly increased in JOL1355-infected DCs compared to non-stimulated DCs. Flow cytometry analysis showed an increased proportion of cells producing CD40, CD80, and MHC class II in the JOL1355-infected cultures compared to the non-stimulated control. In addition, JOL1355-stimulated chBM-DCs could induce significant expression of IL-2 in co-culture with autologous CD4+ T cells. Based on these results, we conclude that chBM-DCs are capable of internalizing the live attenuated SG vaccine candidate and the infected chBM-DCs show signs of maturation as evidenced by the upregulated expression of costimulatory molecules and cytokines.

Orally administered live attenuated Salmonella Typhimurium protects mice against lethal infection with H1N1 influenza virus

Pre-stimulation of toll-like receptors (TLRs) by agonists has been shown to increase protection against influenza virus infection. In this study, we evaluated the protective response generated against influenza A/Puerto Rico/8/1934 (PR8; H1N1) virus by oral and nasal administration of live attenuated Salmonella enterica serovar Typhimurium, JOL911 strain, in mice. Oral and nasal inoculation of JOL911 significantly increased the mRNA copy number of TLR-2, TLR4 and TLR5, and downstream type I interferon (IFN) molecules, IFN-α and IFN-β, both in peripheral blood mononuclear cells (PBMCs) and in lung tissue. Similarly, the mRNA copy number of interferon-inducible genes (ISGs), Mx and ISG15, were significantly increased in both the orally and the nasally inoculated mice. Post PR8 virus lethal challenge, the nasal JOL911 and the PBS control group mice showed significant loss of body weight with 70% and 100% mortality, respectively, compared to only 30% mortality in the oral JOL911 group mice. Post sub-lethal challenge, the significant reduction in PR8 virus copy number in lung tissue was observed in oral [on day 4 and 6 post-challenge (dpc)] and nasal (on 4dpc) than the PBS control group mice. The lethal and sub-lethal challenge showed that the generated stimulated innate resistance (StIR) in JOL911 inoculated mice conferred resistance to acute and initial influenza infection but might not be sufficient to prevent the PR8 virus invasion and replication in the lung. Overall, the present study indicates that oral administration of attenuated S. Typhimurium can pre-stimulate multiple TLR pathways in mice to provide immediate early StIR against a lethal H1N1 virus challenge.

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

ABSTRACT 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.

Inhibition of Salmonella-induced apoptosis as a marker of the protective efficacy of virulence gene-deleted live attenuated vaccine.

Vaccination is one of the best protection strategies against Salmonella infection in humans and chickens. Salmonella bacteria must induce apoptosis prior to initiating infection, pathogenesis and evasion of host immune responses. In this study, we evaluated the efficacy of vaccinating chickens against Salmonella Enteritidis (SE) using a vaccine candidate strain (JOL919), constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. In present study day old chickens were inoculated with 1×10(7)cfu (colony forming unit) of JOL919 per os. We measured cell-mediated immunity, protective efficacy and extent of apoptosis induction in splenocytes. Seven days post-immunization, the number of CD3+CD4+ and CD3+ CD8+ T cells was significantly higher in the immunized group compared to the control group, indicating a significant augmentation of systemic immune response. The internal organs of chickens immunized with JOL919 had a significantly lower challenge-strain recovery, indicating effective protection and clearance of the challenge strain. Post-challenge, the number of apoptotic cells in the immunized group was significantly lower than in the control group. Additionally, AV/PI (Annexin V/propidium iodide) staining was performed to differentiate between apoptotic cells and necrotic cells, which corroborated TUNEL-assay (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) results. The proportions of AV+/PI- and AV+/PI+ cells, which represent the proportions of early apoptotic and late apoptotic/early necrotic cells present, respectively, were significantly lower in the immunized group. Our findings suggest that the apoptotic splenocytes in immunized chickens significantly decreased in number, which occurred concomitantly with a significant rise in systemic immune response and bacterial clearance. This suggests that inhibition of apoptosis may be a marker of protection efficacy in immunized chickens.

A Live Salmonella Gallinarum Vaccine Candidate Secreting an Adjuvant Protein Confers Enhanced Safety and Protection Against Fowl Typhoid

SUMMARY Live attenuated vaccines are used for effective protection against fowl typhoid (FT) in domestic poultry. In this study, a lon/cpxR/asd deletion mutant of Salmonella Gallinarum expressing the B subunit of a heat labile toxin (LTB) from Escherichia coli, a known adjuvant, was cloned in a recombinant p15A ori plasmid, JOL1355, and evaluated as a vaccine candidate in chickens. The plasmid was shown to be stable inside the attenuated Salmonella Gallinarum cell after three successive generations. Moreover, from an environmental safety point of view, apart from day 1 the JOL1355 strain was not detected in feces through day 21 postinoculation. For the efficacy of JOL1355, a total of 100 chickens were equally divided into two groups. Group A (control) chickens were intramuscularly inoculated with phosphate-buffered saline at 4 and 8 wk of age. Group B chickens were primed and boosted via the intramuscular route with 200 &mgr;L of a bacterial suspension of JOL1355 containing 1 × 108 colony forming units. All the chickens in Group A and B were challenged at 3 wk postbooster by oral inoculation with a wild-type Salmonella Gallinarum strain, JOL420. The JOL1355-immunized group showed significant protection and survival against the virulent challenge compared to the nonimmunized group. In addition, Group B exhibited a significantly higher humoral immune response, and the chickens remained healthy without any symptoms of anorexia, diarrhea, or depression. Group B also exhibited a significantly lower mortality rate of 4% compared to the 46% of the control group, which can be attributed to higher immunogenicity and better protection. The Group B chickens had significantly lower lesion scores for affected organs, such as the liver and spleen, compared to those of the control chickens (P < 0.01). These findings suggest that JOL1355 is a promising candidate for a safe and highly immunogenic vaccine against FT.

Intracellular delivery of HA1 subunit antigen through attenuated Salmonella Gallinarum act as a bivalent vaccine against fowl typhoid and low pathogenic H5N3 virus

Introduction of novel inactivated oil-emulsion vaccines against different strains of prevailing and emerging low pathogenic avian influenza (LPAI) viruses is not an economically viable option for poultry. Engineering attenuated Salmonella Gallinarum (S. Gallinarum) vaccine delivering H5 LPAI antigens can be employed as a bivalent vaccine against fowl typhoid and LPAI viruses, while still offering economic viability and sero-surveillance capacity. In this study, we developed a JOL1814 bivalent vaccine candidate against LPAI virus infection and fowl typhoid by engineering the attenuated S. Gallinarum to deliver the globular head (HA1) domain of hemagglutinin protein from H5 LPAI virus through pMMP65 constitutive expression plasmid. The important feature of the developed JOL1814 was the delivery of the HA1 antigen to cytosol of peritoneal macrophages. Immunization of chickens with JOL1814 produced significant level of humoral, mucosal, cellular and IL-2, IL-4, IL-17 and IFN-γ cytokine immune response against H5 HA1 and S. Gallinarum antigens in the immunized chickens. Post-challenge, only the JOL1814 immunized chicken showed significantly faster clearance of H5N3 virus in oropharyngeal and cloacal swabs, and 90% survival rate against lethal challenge with a wild type S. Gallinarum. Furthermore, the JOL1814 immunized were differentiated from the H5N3 LPAI virus infected chickens by matrix (M2) gene-specific real-time PCR. In conclusion, the data from the present showed that the JOL1814 can be an effective bivalent vaccine candidate against H5N3 LPAI and fowl typhoid infection in poultry while still offering sero-surveillance property against H5 avian influenza virus.

Live-attenuated auxotrophic mutant of Salmonella Typhimurium expressing immunogenic HA1 protein enhances immunity and protective efficacy against H1N1 influenza virus infection

AIM To evaluate the efficacy of attenuated Salmonella Typhimurium (JOL912) as a live bacterial vaccine vector. MATERIALS & METHODS The JOL912 engineered to deliver HA1 protein from influenza A/Puerto Rico/8/1934 (H1N1; PR8) virus was coined as JOL1635 and further evaluated for immunogenicity and protective efficacy. RESULTS The JOL1635 stably harbored the HA1 gene within pMMP65 plasmid with periplasmic expression and effective delivery of HA1 protein to RAW264.7 cells. The JOL1635 immunized chickens showed the significant increase in HA1-specific IgG, sIgA antibody, IFN-γ, IL-6 cytokine and cellular immune responses. The postoral challenge, the JOL1635-immunized chickens showed a faster clearance of PR8 virus cloacal shedding than the control group. CONCLUSION Generated JOL1635 can establish specific immunogenicity and protection against the PR8 virus in chickens.

Homologous prime-boost immunization with live attenuated Salmonella enterica serovar Senftenberg and its preventive efficacy against experimental challenge with various strains of S. Senftenberg.

BackgroundThe heterogeneity observed regarding persistence, and subsequent fecal shedding pattern of the Salmonella Senftenberg (S. Senftenberg) serovar in chicken’s calls for development of the optimized immunization strategy which can provide protection against various S. Senftenberg isolated. Optimization of an immunization strategy with a live attenuated S. Senftenberg (Δlon and ΔcpxR) vaccine candidate (JOL1587) was undertaken in this study to evaluate the ability of a homologous prime-boost immunization strategy (using JOL1587) to confer protection against four different S. Senftenberg isolates in chickens.ResultsAfter oral immunization with JOL1587, the humoral, mucosal and cell-mediated immune responses were significantly higher in double immunized chickens than in single immunized and control group chickens. A significant increase in the multifunctional cytokine IL-6 and in helper and cytotoxic T cell populations after a booster immunization also indicated the advantage of double over single immunization. The four different S. Senftenberg field isolates were characterized by their persistence levels in chickens, and were subsequently used for challenge experiments to evaluate the differences in protective efficacy conferred by single and double immunization. Chickens from the doubleimmunized group exhibited significant reduction in the shedding of all four wild-type S. Senftenberg challenge strains below the detection limit in the fecal samples. Single immunized chickens showed a decrease in fecal shedding, but failed to exhibit complete protection against all the challenge strains.ConclusionAlthough single immunization with JOL1587 showed a reduction in the fecal shedding of challenge strains, only the homologous prime-boost immunization strategy provided an adequate immune response for increased protection against all four challenge strains of S. Senftenberg from the feces of chickens.