SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 µg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 µg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 µg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 µg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

Intracellular Survival and Pathogenicity Modulation of Salmonella Lon, CpxR, and RfaL Mutants Used as Live Bacterial Vectors under Abiotic Stress, Unveiling the Link between Stress Response and Virulence in Epithelial Cells.

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Bacteria-enabled oral delivery of a replicon-based mRNA vaccine candidate protects against ancestral and delta variant SARS-CoV-2.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolution has resulted in many variants, contributing to the striking drop in vaccine efficacy and necessitating the development of next-generation vaccines to tackle antigenic diversity. Herein we developed a multivalent Semliki Forest virus replicon-based mRNA vaccine targeting the receptor binding domain (RBD), heptad repeat domain (HR), membrane protein (M), and epitopes of non-structural protein 13 (nsp13) of SARS-CoV-2. The bacteria-mediated gene delivery offers the rapid production of large quantities of vaccine at a highly economical scale and notably allows needle-free mass vaccination. Favorable T-helper (Th) 1-dominated potent antibody and cellular immune responses were detected in the immunized mice. Further, immunization induced strong cross-protective neutralizing antibodies (NAbs) against the B.1.617.2 delta variant (clade G). We recorded a difference in induction of immunoglobulin (Ig) A response by the immunization route, with the oral route eliciting a strong mucosal secretory IgA (sIgA) response, which possibly has contributed to the enhanced protection conferred by oral immunization. Hamsters immunized orally were completely protected against viral replication in the lungs and the nasal cavity. Importantly, the vaccine protected the hamsters against SARS-CoV-2-induced pneumonia. The study provides proof-of-principle findings for the development of a feasible and efficacious oral mRNA vaccine against SARS-CoV-2 and its variants.

Structure, Substrate Specificity and Role of Lon Protease in Bacterial Pathogenesis and Survival.

Proteases are the group of enzymes that carry out proteolysis in all forms of life and play an essential role in cell survival. By acting on specific functional proteins, proteases affect the transcriptional and post-translational pathways in a cell. Lon, FtsH, HslVU and the Clp family are among the ATP-dependent proteases responsible for intracellular proteolysis in bacteria. In bacteria, Lon protease acts as a global regulator, governs an array of important functions such as DNA replication and repair, virulence factors, stress response and biofilm formation, among others. Moreover, Lon is involved in the regulation of bacterial metabolism and toxin-antitoxin systems. Hence, understanding the contribution and mechanisms of Lon as a global regulator in bacterial pathogenesis is crucial. In this review, we discuss the structure and substrate specificity of the bacterial Lon protease, as well as its ability to regulate bacterial pathogenesis.

Oral immunization with an attenuated Salmonella Gallinarum encoding the H9N2 haemagglutinin and M2 ectodomain induces protective immune responses against H9N2 infection in chickens.

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated Salmonella Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens (n = 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly (P < 0.05) greater antigen-specific humoral and cell-mediated immune responses in chickens compared with those vaccinated with SG alone. A conventional H9N2 vaccine induced significantly (P < 0.05) greater HA1 and HA2 antibody responses than SG-based H9N2 vaccine strains, but significantly (P < 0.05) less robust M2e-specific responses. Upon challenge with the virulent H9N2 virus on day 28 post-vaccination, chickens vaccinated with either the SG-based H9N2 or conventional H9N2 vaccines exhibited comparable lung inflammation and viral loads, although both were significantly lower (P < 0.05) than in the group vaccinated with SG alone. In conclusion, our results showed that SG-based vaccination stimulated efficient immune responses against virulent H9N2. Further studies are needed to fully develop this approach as a preventive strategy for low pathogenic avian influenza viruses affecting poultry. RESEARCH HIGHLIGHTS S. gallinarum expressing HA1, HA2 and M2e antigens are immunogenic and safe. Salmonella has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.

Non-Structural Protein-W61 as a Novel Target in Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV): An In-Vitro and In-Silico Study on Protein-Protein Interactions with Nucleoprotein and Viral Replication.

The non-structural protein (NSs) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NSs and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at positions C6S (Cystein at 6th position to Serine), W61Y (Tryptophan 61st to Tyrosine), and S207T (Serine 207th to Threonine), three mutants of NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NSs. Over-expression of wild-type NSs in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NSs-NP interaction and viral replication. Thus, the present study identified a critical NSs site, which could be targeted for development of therapeutic regimens against SFTSV.

Expression kinetics of cytokines and the humoral antibody response concerning short-term protection induced by radiation-attenuated Trypanosoma evansi in bovine calves.

'Surra', an economically important disease of livestock, is caused by the parasitic blood protozoon Trypanosoma evansi. Both innate and adaptive immunity contribute to the protection against this infection. T-helper cells play a crucial role in the antibody-mediated clearance of T. evansi. We present here the data on the kinetics of expression of important Th1, Th2 and Th17 cytokines, vis-a-vis the dynamics of humoral response in bovine calves following immunization with γ-radiation-attenuated live T. evansi and later challenged with homologous virulent T. evansi. Significant upregulation of the pro-inflammatory Th1 and Th17 cytokines was correlated with the IgG2-mediated protection in the immunized bovine calves post-challenge. The calves were immunized with 5 × 106 500 Gy γ-radiation-attenuated live T. evansi (horse isolate) thrice at 15 days intervals through the subcutaneous route and subsequently, challenged with 1 × 103 virulent T. evansi on day 50. Significantly high serum IgG (1:1600) and IgM (1:800) titres were recorded on week 2 PC, whereas the peak serum IgG2 titre (1:800) was recorded on week 6 PC. Significant upregulation of IFN-γ, TNF, IL-1ß, and IL-2 was recorded between days 1 to 3 PC, while the same for IL-17 was recorded on day 14 PC. The immunized calves were free from parasitemia post-challenge and were clinically healthy till the end of the experiment. Significant upregulation of IL-10 and IL-4 transcripts and a corresponding increase of serum IgG1 titre in the placebo group helped patency of the parasite in an anti-inflammatory environment and clinical exacerbation of the disease. The expression of the important Th1 cytokines was crucial for antibody-mediated short-term protection against a lethal challenge of T. evansi in cattle.

Harnessing transcriptionally driven chromosomal instability adaptation to target therapy-refractory lethal prostate cancer.

Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.

Targeting primary and metastatic tumor growth in an aggressive breast cancer by engineered tryptophan auxotrophic Salmonella Typhimurium.

The global cancer burden is growing and accounted for 10 million deaths in 2020. The resurgence of chemo- and radiation resistance have contributed to the treatment failures in many cancer types. Therefore, alternative strategies are desired for the effective cancer therapy. Bacteria-mediated cancer therapy presents an attarctive alternative option for the treatment and diagnosis of cancers. Herein, we describe an engineered Salmonella Typhimurium (ST) auxotrophic for tryptophan as a cancer therapeutic. The tryptophan auxotrophy was sufficient to render ST avirulent and highly safe to mice. The auxotroph recovered from the infected tumors had improved ability to target and colonize the tumors. We show that tryptophan auxotrophy reduced the fitness of ST in healthy tissues, but not in the tumors. We evaluated the auxotroph in highly aggressive metastatic 4T1 breast cancer model to inhibit primary tumor growth and lung metastases. The therapy greatly suppressed the primary growth with tumor-free survival of 40% mice. Importantly, therapy abolished the metastatic dissemination of tumor to lungs. Further, therapy markedly diminished the macrophage population in the tumors that may have contributed to the therapeutic benefit recorded. Collectively, results highlight the therapeutic efficacy of the tryptophan auxotrophic ST in an aggressive metastatic cancer model.

Oral mRNA Vaccines Against Infectious Diseases- A Bacterial Perspective [Invited].

The mRNA vaccines from Pfizer/BioNTech and Moderna were granted emergency approval in record time in the history of vaccinology and played an instrumental role in limiting the pandemic caused by SARS-CoV-2. The success of these vaccines resulted from over 3 decades of research from many scientists. However, the development of orally administrable mRNA vaccine development is surprisingly underexplored. Our group specializing in Salmonella-based vaccines explored the possibility of oral mRNA vaccine development. Oral delivery was made possible by the exploitation of the Semliki Forest viral replicon and Salmonella vehicle for transgene amplification and gene delivery, respectively. Herein we highlight the prospect of developing oral replicon-based mRNA vaccines against infectious diseases based on our recent primary studies on SARS-CoV-2. Further, we discuss the potential advantages and limitations of bacterial gene delivery.

Coordinated interaction between Lon protease and catalase-peroxidase regulates virulence and oxidative stress management during Salmonellosis.

This study investigates the interplay between Lon protease and catalase-peroxidase (KatG) in relation to virulence modulation and the response to oxidative stress in Salmonella Typhimurium (ST). Proteomic comparison of ST wild-type and lon deletion mutant led to the recognition of a highly expressed KatG protein product among five other protein candidates that were significantly affected by lon deletion. By employing a bacterium two-hybrid assay (B2H), we demonstrated that the catalytic domain of Lon protease potentially interacts with the KatG protein that leads to proteolytic cleavage. Assessment of virulence gene expression in single and double lon and katG mutants revealed katG to be a potential positive modulator of both Salmonella pathogenicity Island-1 (SPI-1) and -2, while lon significantly affected SPI-1 genes. ST double deletion mutant, ∆lon∆katG was more susceptible to survival defects within macrophage-like cells and exhibited meager colonization of the mouse spleen compared to the single deletion mutants. The findings reveal a previously unknown function of Lon and KatG interaction in Salmonella virulence. Taken together, our experiments demonstrate the importance of Lon and KatG to cope with oxidative stress, for intracellular survival and in vivo virulence of Salmonella.

Evaluation of host and bacterial gene modulation during Lawsonia intracellularis infection in immunocompetent C57BL/6 mouse model.

BACKGROUND: Proliferative enteritis caused by Lawsonia intracellularis undermines the economic stability of the swine industry worldwide. The development of cost-effective animal models to study the pathophysiology of the disease will help develop strategies to counter this bacterium. OBJECTIVES: This study focused on establishing a model of gastrointestinal (GI) infection of L. intracellularis in C57BL/6 mice to evaluate the disease progression and lesions of proliferative enteropathy (PE) in murine GI tissue. METHODS: We assessed the murine mucosal and cell-mediated immune responses generated in response to inoculation with L. intracellularis. RESULTS: The mice developed characteristic lesions of the disease and shed L. intracellularis in the feces following oral inoculation with 5 × l07 bacteria. An increase in L. intracellularis 16s rRNA and groEL copies in the intestine of infected mice indicated intestinal dissemination of the bacteria. The C57BL/6 mice appeared capable of modulating humoral and cell-mediated immune responses to L. intracellularis infection. Notably, the expression of genes for the vitamin B12 receptor and for secreted and membrane-bound mucins were downregulated in L. intracellularis -infected mice. Furthermore, L. intracellularis colonization of the mouse intestine was confirmed by the immunohistochemistry and western blot analyses. CONCLUSIONS: This is the first study demonstrating the contributions of bacterial chaperonin and host nutrient genes to PE using an immunocompetent mouse model. This mouse infection model may serve as a platform from which to study L. intracellularis infection and develop potential vaccination and therapeutic strategies to treat PE.

Highly feasible immunoprotective multicistronic SARS-CoV-2 vaccine candidate blending novel eukaryotic expression and Salmonella bactofection.

Introduction: The emergence of SARS-CoV-2 variants has raised concerns on future vaccine efficacy as most vaccines target only the spike protein. Hence, vaccines targeting multiple SARS-CoV-2 proteins will offer broader protection and improve our preparedness to combat the pandemic. Objectives: The study aimed to develop a novel vaccine strategy by combining a eukaryotic vector expressing multiple SARS-CoV-2 genes and Salmonella-mediated in vivo DNA delivery. Methods: The eukaryotic vector was designed to function as a DNA-launched RNA replicon in a self-replicating and self-amplifying mRNA mechanism. By exploiting the self-cleaving peptide, P2A, we fused four SARS-CoV-2 targets, including receptor-binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13, in a single open reading frame. Western blot and immunofluorescence assays were used to determine protein expression. In mice, the vaccine's safety and immunogenicity were investigated. Results: Western blot analysis revealed co-expression all four proteins from the vaccine construct, confirming the efficiency of Salmonella-mediated gene delivery and protein expression. The vaccine candidate was safe and elicited robust antigen-specific antibody titers in mice, and a recall response from splenocytes revealed induction of strong cell-mediated immunity. Flow cytometry demonstrated an increase in sub-populations of CD4+ and CD8+ T cells with the highest CD4+ and CD8+ T cells recorded for HR and RBD, respectively. Overall, humoral and cellular immune response data suggested the induction of both Th1 and Th2 immunity with polarization towards an antiviral Th1 response. We recorded a potent SARS-CoV-2 neutralizing antibody titers in the immunized mice sera. Conclusions: The Salmonella bactofection ensured optimum in vivo gene delivery, and through a P2A-enabled efficient multicistronic expression, the vaccine candidate elicited potent anti-SARS-CoV-2 immune responses. These findings provide important insight into development of an effective multivalent vaccine to combat SARS-CoV-2 and its variants.

A Simplified SARS-CoV-2 Mouse Model Demonstrates Protection by an Oral Replicon-Based mRNA Vaccine.

A mouse model of SARS-CoV-2 that can be developed in any molecular biology lab with standard facilities will be valuable in evaluating drugs and vaccines. Here we present a simplified SARS-CoV-2 mouse model exploiting the rapid adenoviral purification method. Mice that are sensitive to SARS-CoV-2 infection were generated by transducing human angiotensin-converting enzyme 2 (hACE2) by an adenovirus. The expression kinetics of the hACE2 in transduced mice were assessed by immunohistochemistry, RT-PCR, and qPCR. Further, the ability of the hACE2 to support viral replication was determined in vitro and in vivo. The hACE2 expression in the lungs of mice was observed for at least nine days after transduction. The murine macrophages expressing hACE2 supported viral replication with detection of high viral titers. Next, in vivo studies were carried out to determine viral replication and lung disease following SARS-CoV-2 challenge. The model supported viral replication, and the challenged mouse developed lung disease characteristic of moderate interstitial pneumonia. Further, we illustrated the utility of the system by demonstrating protection using an oral mRNA vaccine. The multicistronic vaccine design enabled by the viral self-cleaving peptides targets receptor binding domain (RBD), heptad repeat domain (HR), membrane glycoprotein (M) and epitopes of nsp13 of parental SARS-CoV-2. Further, Salmonella and Semliki Forest virus replicon were exploited, respectively, for gene delivery and mRNA expression. We recorded potent cross-protective neutralizing antibodies in immunized mice against the SARS-CoV-2 delta variant. The vaccine protected the mice against viral replication and SARS-CoV-2-induced weight loss and lung pathology. The findings support the suitability of the model for preclinical evaluation of anti-SARS-CoV-2 therapies and vaccines. In addition, the findings provide novel insights into mRNA vaccine design against infectious diseases not limiting to SARS-CoV-2.

Salmonella delivered Lawsonia intracellularis novel epitope-fusion vaccines enhance immunogenicity and confers protection against Lawsonia intracellularis in mice.

Attenuated Salmonella-mediated vaccine constructs were designed by employing selected discontinuous immunodominant epitopes of LatA, FliC, and PAL antigens of Lawsonia intracellularis to create vaccines against porcine proliferative enteropathy (PPE). Whole protein sequences were subjected to in silico prediction of dominant epitopes, the stability of fusions, and hydropathicity and to ensure that the fused epitopes were feasible for expression in a Salmonella system. Two fusion constructs, one comprising LatA epitopes and the other FliC-PAL-FliC epitopes, were built into a prokaryotic constitutive expression system and transformed into the auxotrophic Salmonella host strain JOL1800. Epitope selection eliminated the majority of less immunodominant regions of target proteins and resulted in an efficient secretion platform that induced significant protective responses. Overall, our results demonstrated that the Salmonella-mediated LI- multi-epitope vaccines elicited significant humoral and cellular immune responses. Additionally, the challenge study suggested that the vaccinated mice were protected against experimental Lawsonia intracellularis infection. Based on the outcomes of the study, Salmonella-mediated LI- multi-epitope vaccines have the potential to prevent PPE.

Deletion of the lon gene augments expression of Salmonella Pathogenicity Island (SPI)-1 and metal ion uptake genes leading to the accumulation of bactericidal hydroxyl radicals and host pro-inflammatory cytokine-mediated rapid intracellular clearance.

In the present study, we characterized the involvement of Lon protease in bacterial virulence and intracellular survival in Salmonella under abiotic stress conditions resembling the conditions of a natural infection. Wild type (JOL401) and the lon mutant (JOL909) Salmonella Typhimurium were exposed to low temperature, pH, osmotic, and oxidative stress conditions and changes in gene expression profiles related to virulence and metal ion uptake were investigated. Expression of candidate genes invF and hilC of Salmonella Pathogenicity Island (SPI)-1 and sifA and sseJ of SPI-2 revealed that Lon protease controls SPI-1 genes and not SPI-2 genes under all stress conditions tested. The lon mutant exhibited increased accumulation of hydroxyl (OH·) ions that lead to cell damage due to oxidative stress. This oxidative damage can also be linked to an unregulated influx of iron due to the upregulation of ion channel genes such as fepA in the lon mutant. The deletion of lon from the Salmonella genome causes oxidative damage and increased expression of virulence genes. It also prompts the secretion of host pro-inflammatory cytokines leading to early clearance of the bacteria from host cells. We conclude that poor bacterial recovery from mice infected with the lon mutant is a result of disrupted bacterial intracellular equilibrium and rapid activation of cytokine expression leading to bacterial lysis.

O-antigen-deficient, live, attenuated Salmonella typhimurium confers efficient uptake, reduced cytotoxicity, and rapid clearance in chicken macrophages and lymphoid organs and induces significantly high protective immune responses that protect chickens against Salmonella infection.

In the present study, we developed an O-antigen-deficient, live, attenuated Salmonella Typhimurium (ST) strain (JOL2377) and assessed its safety, macrophage toxicity, invasion into lymphoid tissues, immunogenicity, and protection against Salmonella infection in chickens. The JOL2377 induced significantly lower cytotoxicity and higher level of cytokine response in IL-2, IL-10, IL-4, and IFN- γ than the WT strain upon macrophage uptake. It did not persist in macrophages or in chicken organs and rapidly cleared without systemic infection. None of the chicken were found to secrete Salmonella in feces into the environment exacerbating its attenuation. Interestingly JOL2377 successfully arrived in immunological hot-spots such as spleen, liver and bursa of Fabricius for an efficient antigen presentation and immune stimulation. Mucosal and parenteral immunization with JOL2377 significantly elicit antigen-specific humoral (IgY) and cell mediated responses marked by peripheral blood mononuclear cell proliferation, cytokine induction, increase in T-cell responses than non-immunized control. JOL2377 did not generate significant levels of LPS specific antibodies as compared to the WT strain due to the lack of immunogenic O-antigen component from its LPS structure. Upon virulent challenge, route dependent efficacy differences were leaving the intramuscular route is superior to the oral route on reducing splenic and liver colonization of the challenge ST. The least cytotoxicity, virulence, and superior immunogenicity of JL2377 that effectively engage both humoral and IFN- γ mediated CMI responses present an ideal scenario in host immune modulation to fight against intracellular pathogen Salmonella.

Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India.

To incorporate immune competence traits in swine breeding programs, association between immune responsiveness and susceptibility to specific infectious diseases must be established. In order to understand the differences in immune competence between indigenous (Zovawk) and exotic (Large White Yorkshire: LWY) pigs reared in India, we carried out a time course expression analysis of immune-regulating key cytokine genes (interleukin (IL)-2, interferon (IFN)-γ, IL-4 and IL-10) in the phytohemagglutinin-P stimulated peripheral blood mononuclear cells (PBMCs). The IL-2 transcript levels in PBMCs increased several thousand-fold when compared to unstimulated cells in both the breeds, albeit the response in that of Zovawk was remarkably higher. Higher and earlier IFN-γ and IL-4 expression levels in Zovawk pigs suggest that both TH 1 and TH 2 immune responsiveness of this indigenous breed affords better preparedness for danger signals. Moreover, the low expression levels of IL-10 depict a regulated adaptive immune responsiveness. Remarkable difference between the two breeds of the pigs is evident showing a clear advantage of the Zovawk over LWY in terms of a shorter lag period of adaptive immune response. These findings provide a lead for understanding the genetic differences with respect to immune competence levels of indigenous pigs compared to exotic counterparts.