Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate.

BACKGROUND: Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. RESULTS AND CONCLUSION: We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

Influence of Flaxseed Oil on Fecal Microbiota, Egg Quality and Fatty Acid Composition of Egg Yolks in Laying Hens.

Although there have been many attempts to produce ω-3 fatty acid-rich eggs using alpha-linolenic acid (ALA) that is a popular fatty acid in the poultry feed industry, only limited knowledge about the effects of ALA-enriched diets on chicken fecal microbiota is currently available. Herein we examined the changes in the fecal microbiota composition, egg quality traits and fatty acid composition of the egg yolks of laying hens fed ALA-rich flaxseed oil for 8 weeks. The animals fed the experimental diets that contained 0 % (group C), 0.5 % (group T1), and 1.0 % (group T2) of flaxseed oil, respectively, and eggs and feces were obtained for the analyses. ω-3 fatty acids, including ALA, were increased in T1 and T2 compared with C. Furthermore, the freshness of eggs was improved with no side effects on the eggs. The diet also changed the fecal microbiota; Firmicutes was increased in T1 and T2 (48.6 to 83 and 79.6 %) and Bacteroidetes was decreased (40.2 to 8.8 and 4.2 %). Principal coordinate analysis revealed that Lactobacillus, among the 56 examined genera, was the most influenced bacterial group in terms of the fecal microbial community shifts. These results indicate that ALA-rich diets influenced both the egg and fecal microbiota in beneficial manners in laying hens although the association between the fatty acid composition of the egg yolk and the fecal microbiota was not clear. This study is a first step to understand the effect of flaxseed oil as well as intestinal microbiota of laying hens.

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant.

BACKGROUND: To initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer's patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant. RESULTS: For efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB. CONCLUSIONS: Our results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.

Determination of C-Terminal δ-Catenin Responsible for Inducing Dendritic Morphogenesis.

δ-Catenin induces dendritic morphogenesis in several cells and it was reported that deletion of C-terminal 207 amino acid of δ-catenin completely abolished the dendritic morphogenesis. However, exact domain responsible for inducing dendritic morphogenesis in C-terminus of δ-catenin was not mapped. Here, we report that expression of ΔC47 (lacking 47 amino acid of C-terminus: 1-1200), ΔC77 (lacking 77 amino acid of C-terminus: 1-1170) deletion mutants of δ-catenin induced the dendritic morphogenesis of HEK293T and NIH3T3 cells as full-length δ-catenin did. In agreement with previous report, ΔC207 deletion mutant did not show the dendritic morphogenesis of the cells. Interestingly, introducing 107 amino acid deletion of C-terminus (ΔC107 mutant: 1-1140) and 177 amino acid deletion of C-terminus (ΔC177 mutant: 1-1070) showed limited primary and secondary dendritic process and notable spine-like process formation. These results suggest that 1140-1170 amino acid of C-terminal δ-catenin is required for primary and secondary dendrite-like process formation.

Formic acid as an alternative reducing agent for the catalytic nitrate reduction in aqueous media.

Formic acid was used for the nitrate reduction as a reductant in the presence of Pd:Cu/gamma-alumina catalysts. The surface characteristics of the bimetallic catalyst synthesized by wet impregnation were investigated by SEM, TEM-EDS. The metals were not distributed homogeneously on the surface of catalyst, although the total contents of both metals in particles agreed well with the theoretical values. Formic acid decomposition on the catalyst surface, its influence on solution pH and nitrate removal efficacy was investigated. The best removal of nitrate (50 ppm) was obtained under the condition of 0.75 g/L catalyst with Pd:Cu ratio (4:1) and two fold excess of formic acid. Formic acid decay patterns resembled those of nitrate removal, showing a linear relationship between k(f) (formic acid decay) and k (nitrate removal). Negligible amount of ammonia was detected, and no nitrite was detected, possibly due to buffering effect of bicarbonate that is in situ produced by the decomposition of formic acid, and due to the sustained release of H2 gas.

Enhanced Efficacy of Immunization with a Foot-and-Mouth Disease Multi-Epitope Subunit Vaccine Using Mannan-Decorated Inulin Microparticles.

BACKGROUND: Despite the many advantages of recombinant subunit vaccines, they have critical weaknesses that include a low efficacy for promoting cellular and humoral immune responses against antigens because of their poor immunogenicity, and a rapidly cleared properties as a result of proteolytic enzymes in the body. To circumvent these problems, we developed mannan-decorated inulin acetate microparticles (M-IA MPs) that functioned as carriers and adjuvants for immunization with the recombinant foot-and-mouth disease multi-epitope subunit vaccine (M5BT). METHODS: The M5BT-loaded M-IA MPs were obtained by a double-emulsion solvent-evaporation method. Their properties including morphology, size and release ability were determined by field emission scanning electron microscope, dynamic light-scattering spectrophotometer and spectrophotometer. To assess the immunization efficacy of the MPs, mice were immunized with MPs and their sera were analyzed by ELISA. RESULTS: The M-IA MPs obtained by a double-emulsion solvent-evaporation method were spherical and approximately 2-3 µm, and M5BT was encapsulated in the M-IA MPs. The M5BT-loaded M-IA MPs showed higher antigen-specific IgG, IgG1, IgG2a and anti-FMDV antibodies than the M5BT-loaded IA MPs and the Freund's adjuvant as a control. CONCLUSION: The M-IA MPs showed a powerful and multifunctional polymeric system that combined two toll-like receptor agonists compared to the conventional adjuvant.

Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0.

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles.

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

Lichen secondary metabolites in Flavocetraria cucullata exhibit anti-cancer effects on human cancer cells through the induction of apoptosis and suppression of tumorigenic potentials.

Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, F. cucullata exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of F. cucullata contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited in vitro and in vivo tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, F. cucullata and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.