Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

Potent O-antigen-deficient (rough) mutants of Salmonella Typhimurium secreting Lawsonia intracellularis antigens enhance immunogenicity and provide single-immunization protection against proliferative enteropathy and salmonellosis in a murine model.

The obligate intracellular pathogen Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), poses a substantial economic loss in the swine industry worldwide. In this study, we genetically engineered an O-antigen-deficient (rough) Salmonella strain secreting four selected immunogenic LI antigens, namely OptA, OptB, LfliC, and Lhly. The genes encoding these antigens were individually inserted in the expression vector plasmid pJHL65, and the resultant plasmids were transformed into the ∆asd ∆lon ∆cpxR ∆rfaL Salmonella Typhimurium (ST) strain JOL1800. The individual expression of the selected LI antigens in JOL1800 was validated by an immunoblotting assay. We observed significant (P < 0.05) induction of systemic IgG and mucosal IgA responses against each LI antigen or Salmonella outer membrane protein in mice immunized once orally with a mixture of four JOL1800-derived strains. Further, mRNA of IL-4 and IFN-γ were highly upregulated in splenic T cells re-stimulated in vitro with individual purified antigens. Subsequently, immunized mice showed significant protection against challenge with 106.9 TCID50 LI or 2 × 109 CFU of a virulent ST strain. At day 8 post-challenge, no mice in the immunized groups showed the presence of LI-specific genomic DNA (gDNA) in stool samples, while 50% of non-immunized mice were positive for LI-specific gDNA. Further, all the immunized mice survived the virulent ST challenge, compared to a 20% mortality rate observed in the control mice. Collectively, the constructed rough ST-based LI vaccine candidate efficiently elicited LI and ST-specific humoral and cell-mediated immunity and conferred proper dual protection against PE and salmonellosis.

Salmonella Typhimurium, the major causative agent of foodborne illness inactivated by a phage lysis system provides effective protection against lethal challenge by induction of robust cell-mediated immune responses and activation of dendritic cells.

Salmonella Typhimurium infection via foodborne transmission remains a major public health threat even in developed countries. Vaccines have been developed to reduce the disease burden at the pre-harvest stage, but the cell-mediated immune response against intracellular invasion of the pathogen is not sufficiently elicited by conventional killed Salmonella vaccines, which are safer than live vaccines. In this study, we developed a genetically inactivated vaccine candidate by introducing lysis plasmid pJHL454 harboring the λ phage holin-endolysin system into S. Typhimurium; we designated this vaccine JOL1950. In vitro expression of endolysin was validated by immunoblotting, and complete inactivation of JOL1950 cells was observed following 36 h of the lysis. Electron microscopic examinations by scanning electron microscopy and immunogold labeling transmission EM revealed conserved surface antigenic traits of the JOL1950 cells after lysis. An in vivo immunogenicity study in mice immunized with lysed cells showed significantly increased serum IgG, IgG1, and IgG2a levels. Further, we observed markedly increased in vitro cell proliferation and upregulation of Th1, Th2, and Th17 cytokines in the repulsed splenic T-cells of immunized mice. In dendritic cells (DCs) treated with lysed JOL1950, we observed a significant increase in dendritic cell activation, co-stimulatory molecule production, and levels of immunomodulatory cytokines. In addition, Th1 and Th17 cytokines were also released by naïve CD4+ T-cells pulsed with primed DCs. Lysed JOL1950 also protected against lethal challenge in immunized mice. Together, these results indicate that our vaccine candidate has great potential to induce cell-mediated immunity against S. Typhimurium by facilitating the activation of DCs.

Bacterial ghosts as adjuvants: mechanisms and potential.

Bacterial ghosts (BG) are empty cell envelopes derived from Gram-negative bacteria. They contain many innate immunostimulatory agonists, and are potent activators of a broad range of cell types involved in innate and adaptive immunity. Several considerable studies have demonstrated the effectiveness of BG as adjuvants as well as their ability to induce proinflammatory cytokine production by a range of immune and non-immune cell types. These proinflammatory cytokines trigger a generalized recruitment of T and B lymphocytes to lymph nodes that maximize the chances of encounter with their cognate antigen, and subsequent elicitation of potent immune responses. The plasticity of BG has allowed for the generation of envelope-bound foreign antigens in immunologically active forms that have proven to be effective vaccines in animal models. Besides their adjuvant property, BG also effectively deliver DNA-encoded antigens to dendritic cells, thereby leading to high transfection efficiencies, which subsequently result in higher gene expressions and improved immunogenicity of DNA-based vaccines. In this review, we summarize our understanding of BG interactions with the host immune system, their exploitation as an adjuvant and a delivery system, and address important areas of future research interest.

A novel Salmonella strain inactivated by a regulated autolysis system and expressing the B subunit of Shiga toxin 2e efficiently elicits immune responses and confers protection against virulent Stx2e-producing Escherichia coli.

BACKGROUND: Salmonella Typhimurium (S. Typhimurium) inactivated by a regulated autolysis system was genetically engineered to express the homo-pentameric B subunit of Shiga toxin 2e (Stx2eB) on its surface. To prepare a strain able to yield autolyzed Salmonella bearing Stx2eB, the plasmid pJHL184 harboring stx 2eB gene was transformed into the attenuated S. Typhimurium strain, JOL1454. Stx2eB subcloned into the antigen delivery cassette of the plasmid was expressed as fusion protein with the outer membrane protein RESULTS: The expression of Stx2eB fused to the signal peptide in JOL1454 was validated by immunoblot analysis. To determine the immunogenicity of JOL1454, female BALB/c mice were intramuscularly injected with 1 × 108 CFU of the inactivated cells at weeks 0 and 2. Significantly elevated levels of IgG and IgA specific to Stx2eB was observed at weeks 4 and 6 post-immunization (PI) (P <0.05). Proportion of CD3+CD4+ T lymphocyte subpopulation was also significantly augmented in in vivo stimulated splenocytes relative to that in the control group. The increased titers of IgG1 and IgG2a, and of immunomodulatory cytokines indicated that the immunization elicited Th1 and Th2 immune responses. Further, immunomodulatory cytokine genes (IL-6, IL-17A, IL21 and JOL1454) efficiently upregulated in naïve porcine peripheral blood mononuclear cells (PBMCs) pulsed with JOL1454. At week 6 PI, following the challenge with a virulent Stx2e-producing Escherichia coli in the mice, all immunized mice survived whereas approximately 30% of the mice in the control group died. CONCLUSIONS: JOL1454 provided superior immunogenicity and effective protection against challenge with a sublethal dose, which demonstrates its potential as a candidate vaccine against edema disease.

Prevalence and risk factors of seafood-borne Vibrio vulnificus in Asia: a systematic review with meta-analysis and meta-regression.

Vibrio vulnificus is a free-living marine bacterium associated with the contamination of fish and shellfish-the most consumed seafood in Asia. Owing to its potentially lethal clinical consequences, the consumption of seafood contaminated with V. vulnificus has become a growing public health concern. This systematic review with meta-analysis and meta-regression aimed to integrate data on the prevalence of seafood-borne V. vulnificus specifically in Asia and assess the potential risk factors that can influence the outcomes. A comprehensive literature search of four electronic databases yielded 279 relevant studies, among which 38 fulfilled the inclusion criteria. These selected studies were subjected to risk-of-bias assessment and data extraction by three independent researchers. A meta-analysis of the eligible studies estimated the overall prevalence of seafood-borne V. vulnificus in Asia to be 10.47% [95% confidence interval (CI): 6.8-15.8%], with bivalve shellfish, such as oysters, mussels, clams, and cockles being the most contaminated seafood. The highest prevalence was reported in Japan, where 47.6% of the seafood samples tested positive for V. vulnificus. The subgroup and meta-regression analyses identified three potential covariates-detection method, publication year, and country-associated with between-study heterogeneity. Furthermore, data visualization displayed the variations in V. vulnificus prevalence across the studies, associated with differences in sample type, sample size, and sampling stage. This study provides valuable insights into the prevalence of V. vulnificus in fish and shellfish across the entire Asian continent and highlights the potential factors that cause variation in the prevalence rates among the studies. These findings underscore the importance of enhancing hygiene measures throughout the seafood supply chain to mitigate V. vulnificus infection risks and ensure the safety of consumers.

Integrating meta-analysis with a quantitative microbial risk assessment model to investigate Campylobacter contamination of broiler carcasses.

This study investigated the prevalence and associated risk factors of Campylobacter in South Korean broilers using a random-effects meta-analysis. Subsequently, to facilitate the design of preventive measures, the prevalence estimate from the meta-analysis was incorporated into a stochastic risk assessment model to quantify the Campylobacter contamination levels on broiler carcasses. The baseline model was developed based on the most common practices along the South Korean broiler processing line, with no interventions. Meta-analysis results revealed Campylobacter prevalence across the chicken supply chain in the following order: farms (60.6 % [57.3-63.4]), retail markets (43.90 % [24.81-64.99]), slaughterhouses (27.71 % [18.56-39.21]), and processing plants (14.50 % [3.96-41.09]). The model estimated a 52 % (36.1-70.8) Campylobacter prevalence at the end of chilling, with an average contamination level of 4.62 (2.50-6.74) log CFU/carcass. Sensitivity analysis indicated that Campylobacter fecal shedding (r = 0.95) and the amount of feces on bird exteriors (r = 0.17) at pre-harvest were the main factors for carcass contamination, while soft scalding (r = -0.22) and air chilling (r = -0.12) can serve as critical control points (CCPs) at harvest. Scenario analysis indicated that a combination of hard scalding, inside-outside bird washing, spray washing, and chlorinated water immersion chilling can offer a 30.9 % reduction in prevalence and a reduction of 2.23 log CFU/carcass in contamination levels compared to the baseline model. Apart from disinfection and sanitation interventions carried out during meat processing, the implementation of robust control measures is indispensable to mitigate Campylobacter prevalence and concentration at broiler farms, thereby enhancing meat safety and public health. Furthermore, given the high Campylobacter prevalence in the retail markets, future studies should explore the potential risk of cross-contamination at post-harvest stage.

Microbial quality and bacteria pathogens in private wells used for drinking water in northeastern Ohio.

In agricultural intensive areas, drinking contaminated water from private wells is considered an important cause of acute gastroenteric illnesses (AGI), particularly among high-risk populations. In the summer of 2009, the microbial water quality of 180 randomly selected private wells in two northeastern Ohio counties, a region with a high concentration of dairy farms, was assessed. Forty-five percent (82/180) of water samples were contaminated with total coliforms. Generic Escherichia coli were present in 9% (16/180) of samples. Using real-time polymerase chain reaction, E. coli O157:H7 was identified in 4% (7/180) of specimens. Campylobacter spp. DNA could not be amplified from 70 of the samples tested for this organism. The frequency of generic E. coli contamination varied among townships (P < 0.001). Well structure (i.e. age and depth) or other common measures of pollution potential (depth of water, hydrology, topography, net recharge soil media) was not correlated with coliforms and E. coli contamination. Importantly, the presence of the pathogen E. coli O157:H7 was not associated with the presence of fecal indicators in the water samples: Only one of the seven E. coli O157-positive samples was also positive for generic E. coli. Appropriate risk management and communication processes are needed to reduce the potential waterborne disease outbreaks in agricultural intensive areas.

Identification of risk profiles for Salmonella prevalence in pig supply chains in South Korea using meta-analysis and a quantitative microbial risk assessment model.

International travel and the globalization of food supplies have increased the risk of epidemic foodborne infections. Salmonella strains, particularly non-typhoidal Salmonella (NTS), are major zoonotic pathogens responsible for gastrointestinal diseases worldwide. In this study, the prevalence and Salmonella contamination in pigs/carcasses throughout the South Korean pig supply chain and the associated risk factors were evaluated using Systematic reviews and meta-analyses (SRMA), and quantitative microbial risk assessment (QMRA). The prevalence of Salmonella in finishing pigs, which is one of the major starting inputs of the QMRA model was calculated through SRMA of studies conducted in south Korea in order to complement and enhance the robustness of the model. Our findings revealed that the pooled Salmonella prevalence in pigs was 4.15% with a 95% confidence interval (CI) of 2.56 to 6.66%. Considering the pig supply chain, the highest prevalence was detected in slaughterhouses (6.27% [95% CI: 3.36; 11.37]), followed by farms (4.16% [95% CI: 2.32; 7.35]) and meat stores (1.21% [95% CI: 0.42; 3.46]). The QMRA model predicted a 3.9% likelihood of Salmonella-free carcasses and a 96.1% probability of Salmonella-positive carcasses at the end of slaughter, with an average Salmonella concentration of 6.38 log CFU/carcass (95% CI: 5.17; 7.28). This corresponds to an average contamination of 1.23 log CFU/g (95% CI: 0.37; 2.48) of pork meat. Across the pig supply chain, the highest Salmonella contamination was predicted after transport and lairage, with an average concentration of 8 log CFU/pig (95% CI: 7.15; 8.42). Sensitivity analysis indicated that Salmonella fecal shedding (r = 0.68) and Salmonella prevalence in finishing pigs (r = 0.39) at pre-harvest were the most significant factors associated with Salmonella contamination in pork carcasses. Although disinfection and sanitation interventions along the slaughter line can reduce contamination levels to some extent, effective measures should be taken to reduce Salmonella prevalence at the farm level to improve the safety of pork consumption.

Prevalence, Seroprevalence and Risk Factors of Avian Influenza in Wild Bird Populations in Korea: A Systematic Review and Meta-Analysis.

Since the first recorded outbreak of the highly pathogenic avian influenza (HPAI) virus (H5N1) in South Korea in 2003, numerous sporadic outbreaks have occurred in South Korean duck and chicken farms, all of which have been attributed to avian influenza transmission from migratory wild birds. A thorough investigation of the prevalence and seroprevalence of avian influenza viruses (AIVs) in wild birds is critical for assessing the exposure risk and for directing strong and effective regulatory measures to counteract the spread of AIVs among wild birds, poultry, and humans. In this study, we performed a systematic review and meta-analysis, following the PRISMA guidelines, to generate a quantitative estimate of the prevalence and seroprevalence of AIVs in wild birds in South Korea. An extensive search of eligible studies was performed through electronic databases and 853 records were identified, of which, 49 fulfilled the inclusion criteria. The pooled prevalence and seroprevalence were estimated to be 1.57% (95% CI: 0.98, 2.51) and 15.91% (95% CI: 5.89, 36.38), respectively. The highest prevalence and seroprevalence rates were detected in the Anseriformes species, highlighting the critical role of this bird species in the dissemination of AIVs in South Korea. Furthermore, the results of the subgroup analysis also revealed that the AIV seroprevalence in wild birds varies depending on the detection rate, sample size, and sampling season. The findings of this study demonstrate the necessity of strengthening the surveillance for AIV in wild birds and implementing strong measures to curb the spread of AIV from wild birds to the poultry population.

Systematic review and network meta-analysis to compare vaccine effectiveness against porcine edema disease caused by Shiga toxin-producing Escherichia coli.

The comprehensive effect size of several commercial vaccines and vaccine candidates against edema disease (ED) has not been evaluated to date. To integrate the effectiveness of ED vaccines reported so far and to compare and evaluate the posterior-effect estimates of each vaccine type with network models, we identified eligible studies (n = 12) from the electronic databases using specified search strings. Data for dichotomous outcomes (i.e., mortality and clinical symptoms) and continuous outcomes (i.e., fecal shedding and average daily gain) were extracted and analyzed. Conventional meta-analysis shows that, compared with that in non-vaccinated pigs, vaccinated animals are likely to show reduced mortality (OR = 0.07) and clinical signs of ED (OR = 0.11), and increased productivity (SMD = 0.73). Although reduced fecal shedding (SMD = - 1.29) was observed in vaccinated pigs, this could not be fully determined on insufficient grounds. In contrast to mortality and clinical symptoms, fecal shedding (I2 = 88%) and average daily gain (I2 = 85%) showed immense heterogeneity, which was attributed to the small sample size and vaccination route, respectively. According to the Bayesian network meta-analysis, the plasmid-based DNA vaccine demonstrated a better effect for all outcomes compared to other types of vaccines. However, these findings should be carefully interpreted with consideration to potential mediators, insufficient data, and inconsistent network models.

Risk factors influencing swine influenza A virus infection in South Korea: A systematic review and meta-analysis of prevalence and seroprevalence.

The past and current burden of swine influenza A viruses (swIAV) must be estimated since pigs act as mixing vessels and are considered a potential source of newly emerging IAV variants. The objective of this systematic review and meta-analysis was to integrate data on the prevalence and seroprevalence of swIAV in South Korean domestic pigs and evaluate important risk factors that influence these outcomes. Eight databases were searched for studies that evaluated the prevalence and seroprevalence of swIAV in South Korean pigs using a specified search string; twenty-seven eligible studies were identified after application of a set of pre-determined inclusion criteria by three authors. The reported prevalence and seroprevalence were pooled separately in proportions between 0 and 1, using a random-effect meta-analysis. To identify and quantify potential sources of heterogeneity, subgroup, and meta-regression analyses were conducted using covariates (publication type, swIAV subtype, growth stage of pigs, sampling region, publication year, sampling season, facility, detection method, sample type, and sample size). The overall prevalence and seroprevalence in domestic pigs were 0.05 [95% confidence intervals (CIs): 0.05-0.12] and 0.35 (95% CIs: 0.14-0.63), respectively. To identify the impact of covariates on effect size, a suitable meta-regression model was determined using predictor importance estimates with corrected Akaike information criterion values. Consequently, the best-fit model included two covariates, publication year and sample size, which were significantly associated with high heterogeneity in the subgroup analysis. Furthermore, data visualization depicted a significant non-linear association between swIAV prevalence and seroprevalence and specific growth stages of pigs. These findings suggest that the periodic monitoring of pigs at different growth stages in large farms may help to establish the status of swIAV-spread across species in the region, and thereby minimize pandemic risk.

Evidence-Based Approaches for Determining Effective Target Antigens to Develop Vaccines against Post-Weaning Diarrhea Caused by Enterotoxigenic Escherichia coli in Pigs: A Systematic Review and Network Meta-Analysis.

In this study, we conducted a meta-analysis (MA) and systematic review to evaluate the effectiveness of vaccines against post-weaning diarrhea (PWD), caused by enterotoxigenic Escherichia coli (ETEC), in piglets. A Bayesian network meta-analysis (NMA) was also performed to compare the effects of combining different target antigens on vaccine efficacy. Relevant electronic databases were searched using pre-specified search terms, and 17 studies were selected based on three outcomes: diarrhea, mortality, and average daily weight gain (ADWG). In pairwise MA, the vaccinated group showed a significant decrease in diarrhea (OR = 0.124 [0.056, 0.275]) and mortality (OR = 0.273 [0.165, 0.451]), and a significant increase in ADWG (SMD = 0.699 [0.107, 1.290]) compared with those in controls. Furthermore, NMA results showed that all vaccine groups, except for group D (LT enterotoxin), were effective against PWD. Rank probabilities indicated that the F4 + F18 + LT combination was the best regimen for preventing diarrhea (SUCRA score = 0.92) and mortality (SUCRA score = 0.89). NMA also demonstrated that, among the vaccine groups, those inducing simultaneous anti-adhesion and antitoxin immunity had the highest efficacy. Our results provide evidence-based information on the efficacy of vaccines in reducing PWD incidence in pigs and may serve as guidelines for antigen selection for commercial vaccine development in the future.

The Effectiveness of Commercial Vaccination against Lawsonia intracellularis in Mitigating the Reduction in ADWG, the Increased Mortality and Fecal Shedding of the Vaccinated Pigs: A Systematic Review and Meta-Analysis.

In this study, a systematic review and meta-analysis was conducted to assess the efficacy of commercial vaccines against PPE in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. Of the 373 articles reviewed, 16 fulfilled the pre-specified inclusion criteria. Three independent reviewers extracted the data, and vaccine effectiveness was assessed using the outcomes of interest. The majority of studies had a low or unclear risk of bias as assessed using the ARRIVE guidelines. The results of the meta-analysis indicated that the vaccination resulted in statistically significant reductions in bacterial fecal shedding (odds ratio, OR = 0.122, 95% confidence interval, CI 0.054−0.278) and mortality rate (risk ratio, RR = 0.199; 95% CI, 0.066−0.605). Furthermore, ADWG was significantly increased in the vaccinated pigs compared to the unvaccinated controls (standardized mean difference (SMD) = 0.606, 95% CI 0.243−0.969). In the subgroup analysis, the production phase and study type significantly influenced the effect size (p < 0.1). The Egger's regression test showed no evidence of publication bias (p > 0.1). The effectiveness of commercially available vaccines against PPE-related weight loss, fecal shedding, and mortality suggests that the vaccines may help control PPE on affected swine farms.

Fucoxanthin Exerts Anti-Tumor Activity on Canine Mammary Tumor Cells via Tumor Cell Apoptosis Induction and Angiogenesis Inhibition.

Fucoxanthin is a carotenoid derived from brown algae. It is known to exhibit anticancer activity, including the promotion of apoptosis and cell cycle arrest in several tumors. However, it remains unclear whether fucoxanthin exhibits anticancer activity against mammary gland tumors. In this study, we evaluated fucoxanthin activity against canine mammary tumor cells (CMT-U27) and human umbilical vein endothelial cells (HUVECs) to investigate its effect on cell viability, migration, tube formation, and angiopoietin 2 (Ang2) expression. Our results showed that fucoxanthin induced apoptosis via caspase activation in CMT-U27 cells. In rat aortic ring assay, fucoxanthin suppressed endothelial cell sprouting. Furthermore, fucoxanthin inhibited tube formation and migration in HUVECs. The number of migrated cells was assessed using CMT-U27 cells. The results demonstrated that fucoxanthin exerted anti-angiogenic activity on HUVECs and CMT-U27 cells by promoting Ang2 expression. In conclusion, our results demonstrated that fucoxanthin induced tumor cell death and inhibited angiogenesis, suggesting that fucoxanthin could be considered as a promising therapeutic agent for canine mammary gland tumors.

Salmonella Enteritidis ghost vaccine carrying the hemagglutinin globular head (HA1) domain from H1N1 virus protects against salmonellosis and influenza in chickens.

This study evaluated the attenuated Salmonella Enteritidis (SE) ghost strain JOL2114 (Δlon ΔcpxR Δasd), which displays on the bacterial surface the H1N1 hemagglutinin globular head portion (HA1; amino acid residues 63-286) on the bacterial surface for protective efficacy against Salmonella and H1N1 challenge in the chicken model, as the birds are the predominant reservoirs for both diseases. The ghost system enhanced the lysis process by converging two lysis processes found in bacteriophages: bacteriophage PhiX174 lysis gene E and holin-endolysin genes found in bacteriophage λ, complemented with accessory lysis-related proteins Rz/Rz1. The present lysis machinery resulted in complete lysis of host-attenuated SE strains in about 24 hrs of incubation under a non-permissible temperature of 42 °C in the absence of L-arabinose, an antisense inducer that blocks lysis gene expression during the growth phase. SE ghost JOL2114 surface display of HA1 was confirmed by Western blot analysis resulting in an immune-reactive band of 31 kDa in size. Chicken immunization via intramuscular and oral routes yielded both SE and HA1 antigen-specific immune responses. Protective humoral and cell-mediated immune responses were effectively elicited against both Salmonella and influenza challenge. This efficient strategy of ghost generation employs a dual system of phage lysis for biological generation of SE ghosts that preserves the surface antigenic architecture, offering a rapid and effective way to generate vaccines that could be deployed in urgent circumstances to protect against both Salmonella and influenza infection.

Construction of a novel tetravalent dengue vaccine with a Salmonella Typhimurium bacterial ghost and evaluation of its immunogenicity and protective efficacy using a murine model.

Efforts to develop a safe, effective, and affordable dengue vaccine have focused on providing simultaneous immunity against all four serotypes of the dengue virus (DENV). In the current study, Salmonella Typhimurium (ST) lysed by gene E activation was genetically constructed to deliver the envelope protein domain III (EDIII) of all four serotypes of DENV using a foreign antigen delivery and expression vector, pJHL184. Each DENV-EDIII protein expressed in the constructed strain was validated by immunoblot analysis. To assess the immunogenicity and protective efficacy of the constructs against dengue infection, BALB/c mice were injected once orally with either the individual ST-EDIII constructs or a mix of all four ST-EDIII constructs followed by intramuscular administration of the purified EDIII protein. Significantly elevated titers of EDIII-specific IgG, IgG1, and IgG2a were observed in the immunized mice (P < 0.01). Furthermore, lymphocyte proliferative activity and CD3+CD4+ T-cell subpopulations increased significantly in vitro in re-pulsed splenic T cells compared with those from non-immunized mice. In addition, a lower viral load was detected in the BG-EDIII vaccinated group after challenge with DENV-infected K562 cells. Collectively, the results demonstrate that DENV-EDIII expressed in the inactivated ST strain could induce robust humoral and cell-mediated immunity specific to the target antigen and could provide significant protective potential.

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 (106.9 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.

F18+Escherichia coli flagellin expression in Salmonella has immunoadjuvant effects in a ghost vaccine candidate containing E. coli Stx2eB, FedF and FedA against porcine edema disease.

Flagellin, a major structural component of bacteria flagella, is responsible for facilitating motility and invading host cells. The binding capacity of flagellin to toll-like receptor 5 (TLR5) and its trigger action for innate immunity make it an attractive vaccine adjuvant. In this work, we explored the efficacy of F18+STEC flagellin as an adjuvant in a ghost vaccine candidate against porcine edema disease (ED). TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling following ligand exposure was detected in HEK-Blue™-hTLR5 cells stimulated with flagellin expressed in the Salmonella Typhimurium (ST) ghost, JOL1485. Mice immunized with JOL1485 mixed with the vaccine candidate showed significantly increased secretory IgA specific to flagellin during the study period. JOL1485 efficiently induced maturation of naïve dendritic cells, resulting in production of immunomodulatory cytokines associated with Th1 and Th2 immunity. Particularly, marked increases in IL-6 inducing B-cell differentiation and IL-12 associated with Th1 immunity were observed in CD4 + T-cells stimulated with JOL1485 primed DCs. Furthermore, all mice immunized with JOL1485 mixed with the vaccine candidate showed higher protection efficacy against challenge and significantly enhanced clearance of the challenge strain, compared to mice immunized with only the vaccine candidate. Collectively, these results indicated that flagellin expression in JOL1485 may have immunostimulatory properties that serve as a potent adjuvant for the vaccines against ED.

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.