Plantarum NC8 Research Articles

Immune Responses Induced by a Recombinant Lactiplantibacillus plantarum Surface-Displaying the gD Protein of Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

Synergistic effects of oral inoculation with a recombinant Lactobacillus plantarum NC8 strain co-expressing interleukin-2 and interleukin-17B on the efficacy of the infectious bronchitis vaccine in chickens

Mucosal vaccination strategies are easier to implement than others in large-scale poultry farming. However, the adjuvants that are approved for veterinary use, which are predominantly aluminum- and oil-emulsion-based adjuvants, are not suitable for mucosal vaccination and carry a risk of adverse reactions. In this study, we engineered a novel Lactobacillus plantarum NC8 strain that co-expresses chicken interleukin-2 (IL-2) and IL-17B, which we designated NC8-ChIL2-17B, and evaluated its potential as an oral immunoadjuvant. The immunomodulatory properties of NC8-ChIL2-17B were evidenced by its ability to activate macrophages and inhibit the proliferation of infectious bronchitis virus (IBV) in vitro. We then confirmed its immunoadjuvant activity in vivo by orally administering NC8-ChIL2-17B along with a commercial IBV vaccine to chicks. The results indicated that NC8-ChIL2-17B enhanced the immune response elicited by the IBV vaccine and increased the levels of IBV-specific IgG and sIgA antibodies produced in response to IBV infection. Additionally, administration of NC8-ChIL2-17B promoted weight gain and beneficially modulated the gut microbiota, resulting in improved chicken performance. These findings suggest that oral administration of NC8-ChIL2-17B is a promising strategy to enhance the immune efficacy of the IBV vaccine in chickens, offering an efficacious alternative adjuvant.

Lactobacillus Plantarum NC8 and its metabolite acetate alleviate type 1 diabetes via inhibiting NLRP3

A healthy organism is the result of host-microbiome co-evolution. Microbial metabolites can also stimulate immune cells to reduce intestinal inflammation and permeability. Gut dysbiosis will lead to a variety of autoimmune diseases, such as Type 1 diabetes (T1D). Most of probiotics, such as Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium bifidium, and Streptococcus thermophiles, can improve the intestinal flora structure of the host, reduce intestinal permeability, and relieve symptoms of T1D patients if ingested above probiotics in sufficient amounts. Lactobacillus Plantarum NC8, a kind of Lactobacillus, whether it has an effect on T1D, and the mechanism of it regulating T1D is still unclear. As a member of the inflammatory family, NLRP3 inflammasome can enhance inflammatory responses by promoting the production and secretion of proinflammatory cytokines. Many previous studies had shown that NLRP3 also plays an important role in the development of T1D. When the NLRP3 gene is deleted, the disease progression of T1D will be delayed. Therefore, this study investigated whether Lactobacillus Plantarum NC8 can alleviate T1D by regulating NLRP3. The results demonstrated that Lactobacillus Plantarum NC8 and its metabolites acetate play a role in T1D by co-modulating NLRP3. Lactobacillus Plantarum NC8 and acetate can reduce the damage of T1D in the model mice, even if orally administered them in the early stage of T1D. The number of Th1/Th17 cells in the spleen and pancreatic lymph nodes (PLNs) of T1D mice were significantly reduced by oral Lactobacillus Plantarum NC8 or acetate. The expression of NLRP3 in the pancreas of T1D mice or murine macrophages of inflammatory model were significantly inhibited by treatment with Lactobacillus Plantarum NC8 or acetate. In addition, the number of macrophages in the pancreas were significantly reduced by the treatment with Lactobacillus Plantarum NC8 or acetate. In summary, this study indicated that the regulatory mechanism of Lactobacillus Plantarum NC8 and its metabolite acetate to T1D maybe via inhibiting NLRP3 and provides a novel insights into the mechanism of the alleviated role of probiotics to T1D.

Antitumor effect of invasive Lactobacillus plantarum delivering associated antigen gene sHSP between Trichinella spiralis and Lewis lung cancer cells.

Cancer is a frequent disease that seriously harms human health, but there are no ideal therapies for it. Currently, some food-grade microorganisms such as Lactobacillus plantarum have shown better anti-tumor effects. Here, recombinant Lactobacillus plantarum lives vector vaccine NC8-sHSP was generated by using the invasive Lactobacillus plantarum NC8 expressing FnBPA to deliver the associated antigen gene sHSP between trichinella spiralis and Lewis lung cancer cells (LLC) to host cells. NC8-sHSP colonized the mouse intestine to deliver plasmids to intestinal epithelial cells and controlled the growth of LLC by inducing humoral, cellular, and mucosal immunity. The tumor inhibition rates were 62.36% and 68.37% in the prophylactic assay and 40.76% and 44.22% in the treatment assay, respectively. Recombination of Lactobacillus plantarum did not cause significant damage. In conclusion, the recombinant invasive Lactobacillus plantarum constructed in this study has better anti-Lewis lung cancer effects in mice, which will provide new ideas for the application of food-grade microorganisms in anti-tumor and the development of oral tumor vaccines.

Construction of a Lactobacillus plantarum-based claudin-3 targeting delivery system for the development of vaccines against Eimeria tenella.

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection.

Construction of constitutive expression of Eimeria tenella eukaryotic initiation factor U6L5H2 on the surface of Lactobacillus plantarum and evaluation of its immunoprotective efficiency against chicken coccidiosis

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens.

Recombinant invasive Lactobacillus plantarum expressing the J subgroup avian leukosis virus Gp85 protein induces protection against avian leukosis in chickens.

Avian leukosis, caused by avian leukosis virus (ALV), is an infectious tumor disease and severely hinders the development of the poultry industry. The use of Lactobacillus plantarum (L. plantarum) could effectively alleviate viremia in the early period of J subgroup ALV (ALV-J) infection. In this study, an invasive L. plantarum NC8 expressing Gp85 protein of ALV-J was constructed. After chickens were orally administered the recombinant invasive NC8, the levels of expression of CD4+ and CD8+ T lymphocytes in peripheral blood and spleen by flow cytometry and the proliferation ability of splenocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were examined, and the contents of cytokines, the anti-ALV-J antibody in serum, and mucosal antibody sIgA in intestinal lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The immunoprotective efficiency was evaluated by monitoring the infection rate, the percent of cloacal swabs and survival, body weight gain, the organ indexes, and relative virus loads after challenge with ALV-J. The results showed that the recombinant invasive strain (FnBPA-gp85) could promote the expression levels of the CD8+T cells in peripheral blood and spleen, the proliferation of splenocytes, the secretions of cytokines interleukin 2 (IL-2) and γ-interferon (IFN-γ), and the production of IgG and sIgA compared with the PBS and FnBPA control groups in chickens. The FnBPA-gp85 group was exhibited the highest immune protection against ALV-J infection. The above results indicated that the recombinant invasive NC8 could promote the cellular immunity, humoral immunity, and mucosal immunity responses in chicken and provide a new method for exploring the live vaccine against ALV-J.Key points• The FnBPA-gp85 strain could enhance cellular immunity response.• The FnBPA-gp85 strain could improve the immune protection against ALV-J infection.

Lactobacillus plantarum surface-displayed ASFV (p54) with porcine IL-21 generally stimulates protective immune responses in mice

African classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

Oral immunization with recombinant Lactobacillus plantarum expressing Nudix hydrolase and 43 kDa proteins confers protection against Trichinella spiralis in BALB/c mice

Trichinellosis is a significant food-borne zoonotic parasitic disease caused by parasite Trichinella. Given the side effects of anti-Trichinella drugs (e.g., Mebendazole) aroused in the course of treatments, an effective vaccine against the parasite is called for. The therapies available to date are in most instances targeting a single stage of Trichinella, resulting in an incomplete protective immunity against the parasite in terms of the complexity of its developmental stages. In this study, a recombinant dual-expression double anchor vector NC8-pLp-TsNd-S-pgsA′-gp43 was constructed carrying two antigen genes from Trichinella spiralis (T. spiralis), encoding the gp43 and T. spiralis Nudix hydrolase (TsNd) proteins which were mainly expressed in muscle larva (ML) and intestinal infective larva stages of the parasite respectively. These two proteins were to be expressed by Lactobacillus plantarum NC8 (L. plantarum NC8) which was designed to express the two anchored peptides, a truncated poly-γ-glutamic acid synthetase A (pgsA′) and the surface layer protein of Lactobacillus acidophilus (SlpA), on its surface for attaching expressed foreign proteins. Oral immunization with the above recombinant vaccine induced higher levels of specific serum IgG and mucosal secretory IgA (SIgA) in BALB/c mice. In addition, cytokines, interferon-γ (IFN- γ), interleukin-4 (IL-4) and IL-17 released by lymphocytes, and CD4+ levels displayed on the surfaces of splenic and mesenteric lymph cells were significantly enhanced by the vaccination. Moreover, after larval challenges, a 75.67 % reduction of adult worms (AW) at 7 days post-infection (dpi) and 57.14 % reduction of ML at 42 dpi were observed in mice immunized with the recombinant vaccine. Furthermore, this oral vaccination reduced the counts of encysted larvae presented in tongue and masseter muscles after infected with T. spiralis in mice. The overall results demonstrated that the recombinant vaccine developed in this study could induce specific humoral, mucosal, and cellular immune responses, and provides protections against different stages (adult worms and muscle larva) of T. spiralis infections in BALB/c mice, which could make it a promising oral vaccine candidate against trichinellosis.

Recombinant Lactobacillus plantarum NC8 strain expressing porcine rotavirus VP7 induces specific antibodies in BALB/c mice.

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.

Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production.

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino-acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting the growth of the most susceptible bacterial strains at picomolar concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here, we report the development of pRV610gcc, an easily modifiable 11.2-kbp plasmid that enables the production of GccF in L. plantarum NC8. gcc gene expression relies on native promoters in the cloned cluster, and NC8(pRV610gcc) produces mature GccF at levels similar to KW30. Key findings are that the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence but glycosylation of the loop serine is both sequence and spatially specific, that glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation, that neither of the putative thioredoxin proteins is essential for peptide maturation, and that removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity.IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

Phenotypic and genetic characterization of differential galacto-oligosaccharide utilization in Lactobacillus plantarum

Several Lactobacillus plantarum strains are marketed as probiotics for their potential health benefits. Prebiotics, e.g., galacto-oligosaccharides (GOS), have the potential to selectively stimulate the growth of L. plantarum probiotic strains based on their phenotypic diversity in carbohydrate utilization, and thereby enhance their health promoting effects in the host in a strain-specific manner. Previously, we have shown that GOS variably promotes the strain-specific growth of L. plantarum. In this study we investigated this variation by molecular analysis of GOS utilization by L. plantarum. HPAEC-PAD analysis revealed two distinct GOS utilization phenotypes in L. plantarum. Linking these phenotypes to the strain-specific genotypes led to the identification of a lac operon encoding a β-galactosidase (lacA), a permease (lacS), and a divergently oriented regulator (lacR), that are predicted to be involved in the utilization of higher degree of polymerization (DP) constituents present in GOS (specifically DP of 3–4). Mutation of lacA and lacS in L. plantarum NC8 resulted in reduced growth on GOS, and HPAEC analysis confirmed the role of these genes in the import and utilization of higher-DP GOS constituents. Overall, the results enable the design of highly-selective synbiotic combinations of L. plantarum strain-specific probiotics and specific GOS-prebiotic fractions.

Construction and evaluation of recombinant Lactobacillus plantarum NC8 delivering one single or two copies of G protein fused with a DC-targeting peptide (DCpep) as novel oral rabies vaccine

Rabies remains an important public health threat in most developing countries. To develop a more effective and safe oral vaccine against rabies, we constructed recombinant Lactobacillus plantarum NC8 carrying one or two copies of the G gene with a dendritic cell-targeting peptide (DCpep) fused at the C-terminal designated NC8-pSIP409-sRVG or NC8-pSIP409-dRVG, respectively. The immunogenicity and protective efficacy of these recombinant Lactobacillus plantarum against RABV were evaluated by oral administration in a mouse model. The results showed that recombinant NC8-pSIP409-dRVG possessed more G protein, resulting in more functional maturation of DCs. After three cycle of oral immunization, NC8-pSIP409-dRVG induced significantly higher levels of specific IgG antibody and mixed Th1/Th2 with a strong Th1-biasd immune response in mice. Most importantly, although the titers of RABV neutralizing antibody (VNA) were below the threshold of 0.5 IU/mL, the NC8-pSIP409-dRVG could protect 60 % of inoculated mice against lethal RABV challenge. These data reveal that recombinant NC8-pSIP409-dRVG may be a novel and promising oral vaccine candidate to prevent and control of animal rabies.

A potential vaccine candidate towards chicken coccidiosis mediated by recombinant Lactobacillus plantarum with surface displayed EtMIC2 protein

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.

The Construction and Immunoadjuvant Activities of the Oral Interleukin-17B Expressed by Lactobacillus plantarum NC8 Strain in the Infectious Bronchitis Virus Vaccination of Chickens.

Interleukin-17B (IL-17B) is a protective cytokine of the IL-17 family and plays an essential role in the regulation of mucosal inflammation. However, little is known about the role of IL-17B in the control of viral infections. In this study, a recombinant Lactobacillus plantarum, designated as NC8-ChIL17B, was constructed to express the chicken IL-17B (ChIL-17B) gene. The recombinant ChIL17B (rChIL17B) protein was about 14 kDa and was anchored to the surface of NC8 cells. In vitro, it was found that the rChIL17B protein inhibited the proliferation of the infectious bronchitis virus (IBV) through activation of nuclear factor kappa B (NF-κB) and the JAK (Janus kinase)-STAT (signal transducers and activators of transcription) signaling. Moreover, to evaluate the immunoadjuvant activities of NC8-ChIL17B, 40 three-day-old specific pathogen-free (SPF) chickens were divided into four groups. Three groups were orally vaccinated with fresh NC8, NC8-ChIL17B, and phosphate buffered saline (PBS), along with the infectious bronchitis virus vaccine, and the other group was the PBS-negative control. The results of the IBV-specific antibody titer and the concentration of the cytokines IL-2, IL-4, IL-6, and interferon gamma (IFN-γ) in sera, as well as the concentration of secretory immunoglobulin A (sIgA) in the tracheal and small intestinal mucosa, the number of cluster of differentiation 4 positive (CD4+) and cluster of differentiation 8 positive (CD8+) T cells in the blood, and the expression of immune-related genes all indicated that NC8-ChIL17B efficiently enhanced the humoral and cellular immune responses to IBV vaccine. Moreover, the viral loads in the NC8-ChIL17B- and IBV-vaccinated group were significantly lower than in the control groups, suggesting a significant promotion of the immunoprotection of IBV vaccination against the virulent IBV strain. Therefore, ChIL-17B is a promising, effective adjuvant candidate for chicken virus vaccines.

Protection against Trichinella spiralis in BALB/c mice via oral administration of recombinant Lactobacillus plantarum expressing murine interleukin-4

Interleukin-4 (IL-4), an immunomodulatory cytokine derived from activated T lymphocytes were shown to regulate Th2-type immune responses. It plays an important role in anti-parasitic infections. In this study, a recombinant plasmid was designed using murine IL-4 co-expressed with pgsA anchor system of Lactobacillus plantarum NC8 and was delivered by live Lactobacillus plantarum NC8, which exhibited an enhanced immunogenicity in protection of BALB/c mice from infection with Trichinella spiralis. The results showed that the levels of serum IgG1 and mucosal secretory IgA (sIgA) were both increased significantly in mice orally inoculated with NC8-pSIP409-pgsA-mIL-4, and the Th2 phenotype immune response was up-regulated. A 29.9 % reduction in adult worm burden at 7 days post-infection (dpi) and 83.3 % reduction in muscle larvae burden at 28 dpi were observed in immune-stimulated mice with NC8-pSIP409-pgsA-mIL-4. Moreover, weight loss and pathological changes were also improved in mice of NC8-pSIP409-pgsA-mIL-4 group. Taken together, it suggests that NC8-pSIP409-pgsA-mIL-4 could improve the intestinal mucosal immunity and promoted the elimination of the adult worm in Trichinella-infected mice. This study laid the foundation for the development of a novel vaccines against Trichinellosis.

Genome Analysis of Lactobacillus plantarum Isolated From Some Indian Fermented Foods for Bacteriocin Production and Probiotic Marker Genes.

In this study, Lactobacillus plantarum strain DHCU70 isolated from dahi, a fermented milk product and L. plantarum strain DKP1 isolated from kinema, a fermented soybean food of India, respectively were evaluated for their bacteriocin production and probiotic properties. Both strains of L. plantarum (DHCU70 and DKP1) were found to have potent antimicrobial activity against Kocuria rhizophila ATCC 9341. Bacteriocin produced by L. plantarum strains DHCU70 and DKP1 did not exhibit inhibition of cell wall, DNA and fatty acids biosynthesis mechanisms as evaluated by whole cell reporter assays. We characterized the bacteriocin encoding genes in L. plantarum strains DHCU70 and DKP1 by whole genome sequence which consisted of a single and circular chromosome with genome size of 3.38 Mb (GC content of 44.3%) and 3.39 Mb, respectively and a GC content of 44.3%. L. plantarum DHCU70 has 3252 number of protein encoding genes comprising 89 number of RNA genes (69tRNA, 16rRNA, 4nc RNA) whereas L. plantarum DKP1 has total of 3277 number of protein encoding genes with 89 number. of RNA genes (69tRNA, 16S rRNA, 4nc RNA). Analysis revealed the presence of 20.5 kb long and 23 numbers of plantaricin encoding locus (pln locus) for production of antimicrobial compound. BAGEL analysis has shown that the pln locus of both the strains of L. plantarum showed maximum sequence similarity with plantaricin NC8 of L. plantarum NC8, originally isolated from grass silage. Annotated whole genome sequence of both strains DHCU70 and DKP1 was analyzed for the presence of probiotic marker genes. The probiotic properties of these strains of were also evaluated in vitro. Due to the presence of genes responsible for antimicrobial activity and probiotic properties, both strains of L. plantarum may be considered as a suitable probiotic candidate in food industry.

Innate Cytokine Responses and Toll-Like Receptor Induced by Recombinant Porcine Rotavirus VP6 and VP7 Proteins Expressing in <i>Lactobacillus plantarum</i> NC8 Strain Colonization in Mice

The significant function of Toll-like receptors (TLR) is the detection of microbes by host guard cells that guide to the innate immune responses and to the successive adaptive. The current study patterns of TLR2, TLR3 and TLR9 expressing antigen presenting cells (APCs) in blood of mice after colonization with L. plantarum NC8 strain were assessed. The power of L. plantarum on serum innate cytokine and TLR responses stimulated by recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep and NC8-pSIP409-pgsA were also assessed. We confirmed that L. plantarum NC8 stimulated powerful TLR2 expressing APC responses in blood Recombinant strain stimulated a TLR3 response in spleen, and TLR9 responses were stimulated in blood or in spleen. Recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep on TLR2 and TLR9 expressing APC responses has a preservative outcome, reliable with the DCpep adjuvant outcome. In serum the recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep has increased the IL-4 and IFN-γ responses, except that on the TLR3 and TLR9 expressing CD14 APC responses it had an oppressive consequence in spleen and the IFN-α response in serum-stimulated by PRV. Our results give details that following PRV infection after immunization with NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep, the systemic TLR2, TLR3, and TLR9 expressing cDC and macrophage/monocyte responses.

Immune responses of mice inoculated with recombinant Lactobacillus plantarum NC8 expressing the fusion gene HA2 and 3M2e of the influenza virus and protection against different subtypes of influenza virus

To evaluate the efficiency of preventing pathogenic avian influenza by vaccination with recombinant Lactobacillus plantarum (L. plantarum) that expresses conserved antigens, the mucosal and systemic immune responses in animals vaccinated with recombinant L. plantarum NC8-409-1 (NC8-pSIP409-pgsA'-HA2) and NC8-409-2 (NC8-pSIP409-pgsA'-3M2e-HA2) were evaluated. Our results showed that recombinant L. plantarum NC8-409-1 and NC8-409-2 could substantially stimulate the specific IgA titer in the intestine and the specific IgG antibody titer in the serum. We also found that recombinant L. plantarum induced increases in the number of B220+ IgA+ cells in Peyer’s patches (PPs), in lymphocyte proliferation and in the number of IFN-γ+ producing CD8+ T cells after immunization in mice. Most importantly, the mice that were vaccinated with recombinant L. plantarum NC8-409-2 and NC8-409-1 were to some extent protected against infection challenge with the H9N2 and H1N1 viruses. In particular, NC8-409-2 provided up to 80% protection against the H9N2 virus, and NC8-409-1 provided up to 60% protection. Lung tissue pathology was also reduced. Therefore, recombinant L. plantarum NC8-409-2 and NC8-409-1 may be candidate oral vaccines against bird flu.

Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide

Porcine epidemic diarrhea virus (PEDV) is one of the most important causative pathogens of swine diarrhea, which is widely prevalent throughout the world and is responsible for significant economic losses in the commercial pig industry, both domestic and abroad. The spike (S) protein in the PEDV capsid structure can carry the major B lymphocyte epitope, which induces production of neutralizing antibodies and provides immunoprotective effects. Moreover, the conserved region encoded by the S gene can be considered a target for establishing a new diagnostic method and is a new candidate for vaccine design. In this study, use of anchorin pgsA' allowed the fusion protein of S-DCpep to express on the surface of recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA'-S-DCpep) NC8 strain. Mice were immunized by lavage administration of the recombinant NC8-pSIP409-pgsA'-S-DCpep, which was observed to induce DC activation and high production of sIgA and IgG antibodies in experimental animals, while also eliciting production of significantly more IgA+B220+ B cells. More importantly, secretion of cytokines IFN-γ, IL-4 and IL-17 in mice that were vaccinated with NC8-pSIP409-pgsA'-S-DCpep was remarkably increased. The results of our study suggest that NC8-pSIP409-pgsA'-S-DCpep potently triggers cellular and humoral immune responses. The obtained experimental results can provide a theoretical basis that lays the foundation for production of a novel oral vaccine against PED.