Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Probiotic Bacillus licheniformis ZW3 Alleviates DSS-Induced Colitis and Enhances Gut Homeostasis.

Despite Bacillus species having been extensively utilized in the food industry and biocontrol as part of probiotic preparations, limited knowledge exists regarding their impact on intestinal disorders. In this study, we investigated the effect of Bacillus licheniformis ZW3 (ZW3), a potential probiotic isolated from camel feces, on dextran sulfate sodium (DSS)-induced colitis. The results showed ZW3 partially mitigated body weight loss, disease activity index (DAI), colon shortening, and suppressed immune response in colitis mice, as evidenced by the reduction in the levels of the inflammatory markers IL-1ß, TNF-α, and IL-6 (p < 0.05). ZW3 was found to ameliorate DSS-induced dysfunction of the colonic barrier by enhancing mucin 2 (MUC2), zonula occluden-1 (ZO-1), and occludin. Furthermore, enriched beneficial bacteria Lachnospiraceae_NK4A136_group and decreased harmful bacteria Escherichia-Shigella revealed that ZW3 improved the imbalanced gut microbiota. Abnormally elevated uric acid levels in colitis were further normalized upon ZW3 supplementation. Overall, this study emphasized the protective effects of ZW3 in colitis mice as well as some potential applications in the management of inflammation-related diseases.

Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production Due to Aberrant Virion Morphogenesis and Enhances Inflammatory Cytokine Expression in Porcine Macrophages.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein by using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deleted from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with those of ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of noninfectious particles with a higher particle-to-infectious titer ratio in ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) than in those infected with ASFV-WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis, or egress but did affect ASFV assembly; noninfectious virions containing large aberrant tubular and bilobulate structures comprised nearly 98% of all virions observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virions as indicated by the H240R-deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in primary porcine alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.

Genome-wide identification of chicken bursae of Fabricius miRNAs in response to very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) can cause a highly contagious immunosuppressive disease in young chickens. MicroRNAs (miRNAs) are crucial regulators of gene expression and are involved in the pathogenesis of IBDV infection. To investigate the roles of miRNA in chicken bursae of Fabricius in response to very virulent IBDV (vvIBDV) infection, RNA sequencing was performed to compare the small RNA libraries from uninfected and vvIBDV-infected group which was infected for 3 days. A total of 77 differentially expressed (DE) miRNAs were identified in BF, of which 42 DE miRNAs were upregulated and 35 DE miRNAs were downregulated. A gene ontology analysis showed that genes associated with cellular processes, cells, and binding were enriched. Moreover, pathway analyses suggested that apoptosis, T cell receptor signaling pathways, and chemokine signaling pathways may be activated following vvIBDV infection. In addition, we predicted the target genes of DE miRNAs and constructed an miRNA-mRNA regulatory network. In total, 189 pairs of miRNA-target genes were identified, comprising 67 DE miRNAs and 73 mRNAs. In this network, gga-miR-1684b-3p was identified with the highest fold change, as well as gga-miR-1788-3p and gga-miR-3530-5p showed a high degree of change. The above three miRNAs were considered to play vital roles in vvIBDV-host interactions. This study was the first to perform a comprehensive analysis of DE miRNAs in the bursa of Fabricius in response to vvIBDV infection, and it provided new insights into molecular mechanisms underlying vvIBDV infection and pathogenesis.

Lactobacillus johnsonii activates porcine monocyte derived dendritic cells maturation to modulate Th cellular immune response.

Lactobacilli are abundant in the intestinal tract where they constantly regulate immune system via interacting with a great diversity of immune cells, such as dendritic cells (DCs). Notably, DCs are powerful antigen-presenting cells and they are capable of initiating primary immune responses. In this study, we studied the effects of Lactobacillus johnsonii (L. johnsonii) and Lactobacillus johnsonii cell-free supernatant (L. johnsonii-CFS) on the activation of porcine monocyte-derived dendritic cells (MoDCs) and their regulation of Th cellular immune responses in vitro. The MoDCs generated from porcine peripheral blood monocytes were stimulated by L. johnsonii and L. johnsonii-CFS, respectively. Pre-incubation with L. johnsonii increased expression of CD172a, CD80, major histocompatibility complex class II (MHCII) in MoDCs, and enhanced the ability of MoDCs to induce the proliferation of CD4+ T cell, while pre-incubation with L. johnsonii-CFS merely upregulated the expression of MHCII. Analysis of the cytokines showed that L. johnsonii stimulated up-regulation of Th1-type cytokines (IL-12p40, IFN-γ, TNF-α), pro-inflammatory cytokine IL-1ß, chemokine CCL20, and Treg-type / anti-inflammatory cytokines IL-10 in MoDCs. Notably, a high production of IL-10 was observed in the MoDCs treated with L. johnsonii-CFS, indicating L. johnsonii-CFS exerted anti-inflammatory effects. Furthermore, L. johnsonii induced up-regulation of TLR2 and TLR6, but L. johnsonii-CFS not. Moreover, MoDCs stimulated by L. johnsonii mainly promoted T cell differentiate into Th1/Th2/Treg cells and plays an important role in improving the balance between Th1/Th2/Treg-type cells, whereas MoDCs stimulated by L. johnsonii-CFS mainly directed T cell to Th2/Treg subset polarization. In conclusion, L. johnsonii and L. johnsonii-CFS exhibited the ability of modulating innate immunity by regulating immunological functions of MoDCs in vitro, suggesting their potential ability to use as microecological preparations and medicines.

Genome-wide analysis of differentially expressed mRNAs, lncRNAs, and circRNAs in chicken bursae of Fabricius during infection with very virulent infectious bursal disease virus.

BACKGROUND: Infectious bursal disease virus (IBDV) causes acute, highly contagious, immunosuppressive, and lethal infectious disease in young chickens and mainly infects the bursa of Fabricius (BF). To investigate interactions between IBDV and its host, RNA sequencing was applied to analyze the responses of the differentially expressed transcriptional profiles of BF infected by very virulent IBDV (vvIBDV). RESULTS: In total, 317 upregulated and 94 downregulated mRNAs were found to be significantly differentially expressed in infected chickens, compared to controls. Long non-coding RNA (lncRNA) and circular RNA (circRNA) alterations were identified in IBDV-infected chickens, and significantly different expression was observed in 272 lncRNAs and 143 circRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to assess the functions of significantly dysregulated genes, which showed that the JAK-STAT signaling pathway, the NOD-like receptor signaling pathway, and apoptosis may be activated by IBDV infection. We predicted interactions between differentially expressed genes and produced lncRNA-mRNA and circRNA-miRNA-mRNA regulator network. CONCLUSIONS: The present study identified the expression profiles of mRNAs, lncRNAs, and circRNAs during vvIBDV infection and provides new insights into the pathogenesis of IBDV and antiviral immunity of the host.

Lactobacillus johnsonii-activated chicken bone marrow-derived dendritic cells exhibit maturation and increased expression of cytokines and chemokines in vitro.

Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1ß and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.

Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. RESULTS: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. CONCLUSIONS: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

Porcine transmissible gastroenteritis virus inhibits NF-κB activity via nonstructural protein 3 to evade host immune system.

BACKGROUND: Transmissible gastroenteritis virus (TGEV), a member of the family Coronaviridae, causes lethal watery diarrhea in piglets. Previous studies have revealed that the coronaviruses develop various strategies to evade the host innate immunity through the inhibition of nuclear factor kappa B (NF-κB) signaling pathway. However, the ability of TGEV to inhibit the host innate immune response by modulating the NF-κB signaling pathway is not clear. METHODS: In this study, a dual luciferase reporter assay was used to confirm the inhibition of NF-κB by TGEV infection and to identify the major viral proteins involved in the inhibition of NF-κB signaling. Real-time quantitative PCR was used to quantify the mRNA expression of inflammatory factors. The deubiquitination of Nsp3 domains and its effect on IκBα and p65 were analyzed by western blotting. The ubiquitination level of IκBα was analyzed by immunoprecipitation. RESULTS: In ST and IPEC-J2 cells, TGEV exhibited a dose-dependent inhibition of NF-κB activity. Individual TGEV protein screening revealed the high potential of non-structural protein 3 (Nsp3) to inhibit NF-κB signaling, and leading to the downregulation of the NF-κB-induced cytokine production. We demonstrated that the inhibitory effect of Nsp3 was mainly mediated through the suppression of IκBα degradation as well as the inhibition of p65 phosphorylation and nuclear translocation. Furthermore, the amino acid residues at positions 590-1,215 in Nsp3 were demonstrated to inhibit the degradation of IκBα by inhibiting the IκBα ubiquitination. CONCLUSION: TGEV infection can inhibit the activation of the NF-κB signaling pathway, which is mainly mediated by Nsp3 through the canonical pathway. The amino acid residues at positions 590-1,215 in Nsp3 compose the critical domain that mediates NF-κB inhibition. We speculate that this inhibitory effect is likely to be related to the structure of PLP2 with deubiquitinating enzyme activity of the amino acid residues at positions 590-1,215 in Nsp3. Our study provides a better understanding of the TGEV-mediated innate immune modulation and lays the basis for studies on the pathogenesis of coronavirus.

Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea.

Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40-65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3'- or 5'-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/µL, 1.92 × 102 copies/µL, 1.74 × 102 copies/µL, and 1.76 × 102 copies/µL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

N-linked glycosylation sites in G protein of infectious hematopoietic necrosis virus (IHNV) affect its virulence and immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis in salmonid fish, resulting in substantial economic losses to the aquaculture industry worldwide. The G protein, which harbors the major antigenic determinants of IHNV, is an envelope glycoprotein that plays an important role in both pathogenicity and immunogenicity of IHNV. Previous studies have demonstrated that changes to viral glycosylation sites may affect replication and immunogenicity, but little is known about the specific contributions of G protein glycosylation to IHNV replication and pathogenicity. In this study, we predicted four N-linked glycosylation sites at position 56, 379, 401, and 438 Asp (N) in G protein, and using a reverse genetics system developed in our laboratory, constructed nine recombinant viruses with single, triple, or quadruple glycosylation site disruptions using alanine substitutions in the following combinations: rIHNV-N56A, rIHNV-N379A, rIHNV-N401A, rIHNV-N438A, rIHNV-N56A-N379A-N401A, rIHNV-N56A-N379A-N438A, rIHNV-N56A-N401A-N438A, rIHNV-N379A-N401A-N438A, and rIHNV-N56A-N379A-N401A-N438A. Our results confirmed that all four asparagines are sites of N-linked glycosylation, and Western blot confirmed that mutation of each predicted N-glycosylation sited impaired glycosylation. Among the nine recombinant IHNVs, replication levels decreased significantly in vitro and in vivo in the triple and quadruple mutants that combined mutation of asparagines 401 and 438, indicating the importance of glycosylation at these sites for efficient replication. Moreover, juvenile rainbow trout mortality after challenge by each of the nine mutants showed that, while eight mutants suffered almost 100% cumulative mortality over 30 days, the mutant with a single alanine substitution at position 438 resulted in cumulative mortality of less than 50% over 30 days. This mutant also elicited specific anti-IHNV IgM production earlier than other mutants, suggesting that glycosylation of asparagine 438 may be important for viral immune escape. In conclusion, our study reveals the effect of G protein glycosylation on the pathogenicity and immunogenicity of IHNV and provides a foundation for developing a live-attenuated vaccine.

Oral delivery of a Lactococcus lactis strain secreting bovine lactoferricin-lactoferrampin alleviates the development of acute colitis in mice.

Ulcerative colitis (UC) is a chronic relapsing disease. Treatment of UC would benefit from specific targeting of therapeutics to the intestine. Previous studies have demonstrated that bovine lactoferricin and lactoferrampin have bactericidal, anti-inflammatory, and immunomodulatory effects. Here, we investigated whether oral administration of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactococcus lactis (LL-LFCA) strain could alleviate experimental colitis. LFCA derived from LL-LFCA inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. In mice, administration of LL-LFCA decreased the disease activity index and attenuated dextran sulfate sodium (DSS)-induced body weight loss and colon shortening. LL-LFCA treatment also ameliorated DSS-induced colon damage, inhibited inflammatory cell infiltration, significantly decreased myeloperoxidase activity, and ameliorated DSS-induced disruption of intestinal permeability and tight junctions. In addition, 16S rDNA sequencing showed that LL-LFCA reversed DSS-induced gut dysbiosis. The production of proinflammatory mediators in serum and the colon was also reduced by administration of LL-LFCA. In vitro, LFCA derived from LL-LFCA decreased the messenger RNA expression of proinflammatory factors. The underlying mechanisms may involve inhibition of the nuclear factor kappa B (NF-κB) pathway. The results demonstrate that LL-LFCA ameliorates DSS-induced intestinal injury in mice, suggesting that LL-LFCA might be an effective drug for the treatment of inflammatory bowel diseases.

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)-GN438A expresses VP2 gene-encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross-protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV-N438A-ΔNV-EGFP and rIHNV-N438A-ΔNV-VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild-type (wt) IHNV HLJ-09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ-09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV-N438A-ΔNV-VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.

Construction of Lactobacillus casei ghosts by Holin-mediated inactivation and the potential as a safe and effective vehicle for the delivery of DNA vaccines.

BACKGROUND: Bacterial ghosts (BGs) are empty bacterial cell envelopes generated by releasing the cellular contents. In this study, a phage infecting Lactobacillus casei ATCC 393 (L. casei 393) was isolated and designated Lcb. We aimed at using L. casei 393 as an antigen delivery system to express phage-derived holin for development of BGs. RESULTS: A gene fragment encoding holin of Lcb (hocb) was amplified by polymerase chain reaction (PCR). We used L. casei 393 as an antigen delivery system to construct the recombinant strain pPG-2-hocb/L. casei 393. Then the recombinants were induced to express hocb. The immunoreactive band corresponding to hocb was observed by western-blotting, demonstrating the efficiency and specificity of hocb expression in recombinants. The measurements of optical density at 600 nm (OD600) after induction showed that expression of hocb can be used to convert L. casei cells into BGs. TEM showed that the cytomembrane and cell walls of hocb expressing cells were partially disrupted, accompanied by the loss of cellular contents, whereas control cells did not show any morphological changes. SEM showed that lysis pores were distributed in the middle or at the poles of the cells. To examine where the plasmid DNA was associated, we analyzed the L. casei ghosts loading SYBR Green I labeled pCI-EGFP by confocal microscopy. The result demonstrated that the DNA interacted with the inside rather than with the outside surface of the BGs. To further analyze where the DNA were loaded, we stained BGs with MitoTracker Green FM and the loaded plasmids were detected using EGFP-specific Cy-3-labeled probes. Z-scan sections through the BGs revealed that pCI-EGFP (red) was located within the BGs (green), but not on the outside. Flow cytometry and qPCR showed that the DNA was loaded onto BGs effectively and stably. CONCLUSIONS: Our study constructed L. casei BGs by a novel method, which may be a promising technology for promoting the further application of DNA vaccine, providing experimental data to aid the development of other Gram-positive BGs.

Expression of the alpha toxin of Clostridium perfringens in Lactobacillus casei genome and evaluation of its immune effects in mice.

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine.

Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus.

BACKGROUND: Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) has led to serious economic losses to the swine industry worldwide. In this study, an oral recombinant Lactobacillus casei vaccine against PEDV infection targeting the intestinal microfold (M) cells and dendritic cells (DCs) for delivering the core neutralizing epitope (COE) of PEDV spike protein was developed with M cell-targeting peptide (Col) and dendritic cell-targeting peptide (DCpep). The immunogenicity of the orally administered recombinant strains was evaluated. RESULTS: After immunization, significantly higher levels of anti-PEDV specific IgG antibodies with PEDV neutralizing activity in the sera and mucosal sIgA antibodies in the tractus genitalis, intestinal mucus, and stools were detected in mice orally administered with the recombinant strain pPG-COE-Col-DCpep/L393, which expressed DCpep and Col targeting ligands fused with the PEDV COE antigen, compared to mice orally immunized with the recombinant strain pPG-COE/L393 without the DCpep and Col targeting ligands. Moreover, in response to restimulation with the PEDV COE antigen in vitro, a significant difference in splenocyte proliferation response and Th2-associated cytokine IL-4 level was observed in the group of mice orally immunized with pPG-COE-Col-DCpep/L393 (p < 0.05) compared to the groups of mice that received pPG-COE-Col/L393 and pPG-COE-DCpep/L393, respectively. CONCLUSIONS: The intestinal M cells- and DCs-targeting oral delivery of genetically engineered Lactobacillus expressing the COE antigen of PEDV can efficiently induce anti-PEDV mucosal, humoral, and cellular immune responses via oral administration, suggesting a promising vaccine strategy against PEDV infection.

EvaGreen-based real-time PCR assay for sensitive detection of salmonid alphavirus.

Salmonid alphaviruses (SAVs), which include the etiological agents of salmon pancreas disease (PD) and sleeping disease (SD), are significant viral pathogens of European salmonid aquaculture, resulting in substantial economic losses to the salmonid-farming industry. Even though many countries including China have not reported the presence of SAV infections, these countries may be seriously threatened by these diseases as the salmon fish import trade increases. Thus, it is indeed necessary to develop efficient detection methods for the diagnosis and prevention of SAV infection. Real-time PCR assays have been increasingly used in viral detection, and in many cases scientists prefer dye-based real-time PCR assays for their high sensitivity and low cost. In this study, we developed a novel, sensitive, low-cost detection method, EvaGreen-based real-time PCR assay for the detection of SAV. This assay exhibited high specificity for SAV1, SAV2, and SAV5 and was able to detect SAV at concentrations as low as 1.5 × 101 copies, making them more sensitive than the approved conventional RT-PCR method (detection limit, 1.5 × 106 copies). Assessment of infected fish samples showed that the sensitivity of EvaGreen-based assay was higher than previously developed SYBR Green assay (227 assay). Thus, we report that the EvaGreen real-time PCR assays is an economical alternative diagnostic method for the rapid detection of SAV1, SAV2, and SAV5 infection, providing improved technical support for the clinical diagnosis and epidemiological investigation of SAV.

Oral immunization with a recombinant Lactobacillus expressing CK6 fused with VP2 protein against IPNV in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonid fish causing high mortality with serious economic losses to salmonid aquaculture. Ideally, the method of oral immunization should prevent the infection of rainbow trout juveniles with IPNV. In the present study, genetically engineered Lactobacillus casei 393 pPG-612-VP2/L. casei 393 and pPG-612-CK6-VP2/L. casei 393 constitutively expressing VP2 protein of IPNV were constructed. The recombinant strains pPG-612-CK6-VP2/L. casei 393 and pPG-612-VP2/L. casei 393 were orally administrated to juvenile rainbow trouts, and significant titers of IgM and IgT of pPG-612-CK6-VP2/L. casei 393 were observed. The results demonstrate that the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in trouts immunized with pPG-612-CK6-VP2/L. casei 393 showed that the recombinant strain could induce a strong cellular immune response. The IL-1ß, IL-8, CK6, MHC-II, Mx, ß-defensin, and TNF-1α levels in the spleen and gut suggest that the target molecular chemokine has the ability to attract relevant immune cells to participate in the inflammatory response and enhance the function of the innate immune response. Additionally, the pPG-612-CK6-VP2/L. casei 393 induced the expression of cytokines, which have the effect of promoting inflammation to drive the differentiation of macrophages and clear target cells. After challenging with IPNV, the reduction in viral load caused by pPG-612-CK6-VP2/L. casei 393 was significantly higher than that of the other groups. Thus, the recombinant pPG-612-CK6-VP2/L. casei 393 is a promising candidate for the development of an oral vaccine against IPNV.