An attenuated Salmonella vaccine secreting Lawsonia intracellularis immunogenic antigens confers dual protection against porcine proliferative enteropathy and salmonellosis in a murine model

Porcine proliferative enteropathy (PPE) caused by Lawsonia intracellularis (LI) is a global cause for substantial economic losses in the swine industry. Here, we constructed live attenuated Salmonella typhimurium (ST) mutant strains expressing and secreting 4 selected immunogenic LI antigens, namely, optA, optB, Lawsonia flagellin (LfliC), and Lawsonia hemolysin (Lhly); the resultant recombinant strains were designated Sal-optA, Sal-optB, Sal-LfliC, or Sal-Lhly, respectively. Using the BALB/c mouse model, we demonstrate that mice vaccinated once orally, either with a mixture of all 4 recombinant strains or with an individual recombinant strain, show significant (p < 0.05) production of LI-specific systemic immunoglobulin (Ig) G and mucosal IgA responses compared to the Salmonella alone group. Upon restimulation of vaccinated splenocytes with the LI-specific antigens, significant (p < 0.05) and comparable production of interferon-γ responses are found in all vaccinated groups, except the Sal-Lhly group, which shows non-significant levels. Challenge studies were performed in C57BL/6 vaccinated mice. On challenge with the LI (10(6.9) 50% tissue culture infectious dose) 14 days post-vaccination, 20% (1/5) of mice in all vaccinated groups, except Sal-Lhly group, show the presence of the LI-specific genomic DNA (gDNA) in stool samples. In contrast, 40% (2/5) and 60% (3/5) of mice vaccinated with the Sal-Lhly strain and the attenuated Salmonella alone, respectively, were found positive for the LI-specific gDNA. Furthermore, 0% mortality was observed in mice vaccinated against the ST challenge compared to the 30% mortality observed in the unvaccinated control group. In conclusion, we demonstrate that the Salmonella-based LI-vaccines induce LI-specific humoral and cell-mediated immunities, and encompass the potential to offer dual protection against PPE and salmonellosis.

A Salmonella Typhi ghost induced by the E gene of phage φX174 stimulates dendritic cells and efficiently activates the adaptive immune response

Previously, we genetically engineered a Salmonella Typhi bacterial ghost (STG) as a novel inactivated vaccine candidate against typhoid fever. The underlying mechanism employed by the ghost in stimulating the adaptive immune response remains to be investigated. In this study, we aimed to evaluate the immunostimulatory effect of STG on mouse bone marrow-derived dendritic cells (BMDCs) and its activation of the adaptive immune response in vitro. Immature BMDCs were stimulated with STG, which efficiently stimulated maturation events in BMDCs, as indicated by upregulated expressions of CD40, CD80, and major histocompatibility complex class II molecules on CD11⁺ BMDCs. Immature BMDCs responded to STG stimulation by significantly increasing the expression of interleukin (IL)-6, which might indicate the induction of dendritic cell maturation in vivo (p < 0.05). In addition, ghost-stimulated murine BMDCs showed significant expressions of interferon gamma and IL-4, which can drive the development of Th1 and Th2 cells, respectively, in co-cultured CD4⁺ T cells in vitro. These results suggest that STG can effectively stimulate maturation of BMDCs and facilitate subsequent immune responses via potent immunomodulatory cytokine responses.

Effect of immunization routes and protective efficacy of Brucella antigens delivered via Salmonella vector vaccine

An anti-Brucella vaccine candidate comprised of purified Brucella lipopolysaccharide (LPS) and a cocktail of four Salmonella Typhimurium (ST)-Brucella vectors was reported previously. Each vector constitutively expressed highly conserved Brucella antigens (rB), viz., lumazine synthase (BLS), proline racemase subunit A, outer membrane protein-19 (Omp19), and Cu-Zn superoxide dismutase (SOD). The present study determined a relative level of protection conferred by each single strain. Upon virulent challenge, the challenge strain was recovered most abundantly in non-immunized control mice, with the ST-Omp19-, ST-BLS-, LPS-, and ST-SOD-immunized mice showing much less burden. Indirect enzyme-linked immunosorbent assay-based assay also confirmed the induction of antigen-specific immunoglobulin G for each antigen delivered. In a route-wise comparison of the combined vaccine candidate, intraperitoneal (IP), intramuscular (IM), and subcutaneous immunizations revealed an indication of highly efficient routes of protection. Splenocytes of mice immunized via IM and IP routes showed significant relative expression of IL-17 upon antigenic pulsing. Taken together, each of the Brucella antigens delivered by ST successfully induced an antigen-specific immune response, and it was also evident that an individual antigen strain can confer a considerable degree of protection. More effective protection was observed when the candidate was inoculated via IP and IM routes.

Construction of a live attenuated Salmonella strain expressing FanC protein to prevent bovine enterotoxigenic Escherichia coli and evaluation of its immunogenicity in mice

To construct a novel vaccine candidate against bovine enterotoxigenic Escherichia coli (ETEC), FanC, the major subunit of K99 fimbriae adhesion, was inserted into secretion plasmid pYA3560 containing a β-lactamase secretion system. This was then transformed into Δasd Δcrp Salmonella (S.) Typhimurium and designated as JOL950. Secretion of recombinant fanC fimbrial antigens was confirmed by immunoblot analysis. Groups of mice were inoculated with single or double doses of JOL950. Another group was used as a negative control. Compared to control mice, all immunized mice had significantly higher levels (p < 0.05) of serum immunoglobulin (Ig)G, and secretory IgA against FanC. The IgG2a and IgG1 titer assays revealed that immunization highly induced IgG2a compared to that of IgG1, indicating that T helper-1- related cell-mediated immune responses may be elicited by JOL950. The results show that both systemic and mucosal immunities against selected fimbrial antigens of bovine ETEC expressed by a live attenuated S. Typhimurium strain are prominently produced in mice immunized with JOL950 via an oral route.

Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate

PURPOSE: Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid. MATERIALS AND METHODS: The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization. RESULTS: Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4+ and CD8+ T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4+ and CD8+ T-cell population and IL-6 cytokine in mRNA levels. CONCLUSION: Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.

Hens immunized with live attenuated Salmonella strains expressing virulence-associated genes in avian pathogenic Escherichia coli passively transfer maternal antibodies to chicks

We investigated whether maternal antibodies (mAbs) elicited by dams immunized with recombinant vaccine candidates against avian pathogenic Escherichia coli (APEC) can passively confer protective immunity to chicks. In the present study, pBP244 plasmids carrying selected antigens of APEC were transformed into Salmonella Typhimurium JOL912, which was used as a vaccine candidate against APEC. The hens were immunized with the vaccine candidates using prime or booster doses. The levels of IgG and sIgA specific to the selected antigens increased significantly following prime immunization. To evaluate the persistence of passively transferred mAbs, the levels of IgY and IgA were determined in egg yolks and whites, respectively. The eggs from the immunized group showed consistently increased levels of IgY and IgA until week 16 post-laying (PL) and week 8 PL, respectively, relative to the control group. The presence of mAbs was observed in chicks that hatched from the hens, and titers of plasma IgY were consistently raised in those from the immunized hens by day 14 post-hatching. Further, chicks from the immunized hens were protected from challenge with a virulent APEC strain, whereas those from non-immunized hens showed acute mortality.