Publications by authors named "Sang-Kee Kang"

71 Publications

Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route.

Vaccine 2021 Jul 12;39(30):4072-4081. Epub 2021 Jun 12.

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun 25354, Republic of Korea; Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun 25354, Republic of Korea; Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.
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http://dx.doi.org/10.1016/j.vaccine.2021.06.010DOI Listing
July 2021

Additive anti-inflammation by a combination of conjugated linoleic acid and α-lipoic acid through molecular interaction between both compounds.

Food Sci Biotechnol 2020 Mar 15;29(3):419-429. Epub 2019 Oct 15.

1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 08826 Republic of Korea.

Alpha lipoic acid (LA) and conjugated linoleic acid (CLA) have been well-documented on a variety of functional effects in health foods. The main purpose of this study was focused on the additive anti-inflammatory activity of the combination of LA and CLA in vitro. Raw 264.7 cells induced by lipopolysaccharide were treated with LA and CLA individually or in combination at a variety of concentration ranges. Co-treating 25 μM of LA and 25 μM of CLA significantly inhibited pro-inflammatory cytokines compared to the same concentration of single LA- or CLA-treated group. The molecular mechanism of anti-inflammation by a combination of these compounds was attributed to extracellular signal-regulated kinase-1 (ERK1) and peroxisome proliferator-activated receptor gamma (PPARγ). Also, the molecular interaction between both compounds was confirmed by NMR. Our findings suggested that the combination of CLA and LA showed potential additive effect on anti-inflammation through the molecular interaction of both compounds.
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http://dx.doi.org/10.1007/s10068-019-00677-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105553PMC
March 2020

Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Vet Microbiol 2020 Mar 4;242:108604. Epub 2020 Feb 4.

Viral Disease Division, Animal and Plant Quarantine Agency (APQA), Gimcheon, Gyeongbuk, 39660, Republic of Korea. Electronic address:

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.
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http://dx.doi.org/10.1016/j.vetmic.2020.108604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117268PMC
March 2020

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

Tissue Eng Regen Med 2020 02 9;17(1):33-44. Epub 2019 Dec 9.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

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.
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http://dx.doi.org/10.1007/s13770-019-00228-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992806PMC
February 2020

Erratum to: Study on the fatty acid profile of phospholipid and neutral lipid in Hanwoo beef and their relationship to genetic variation.

J Anim Sci Technol 2019 Nov 30;61(6):388. Epub 2019 Nov 30.

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea.

[This corrects the article DOI: 10.5187/jast.2019.61.2.69.].
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http://dx.doi.org/10.5187/jast.2019.61.6.388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906128PMC
November 2019

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

J Vet Sci 2019 Nov;20(6):e70

Institute of Green-Bio Science & Technology, Seoul National University Graduate School of International Agricultural Technology, Pyeongchang 25354, Korea.

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 , 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, 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 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 and studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.
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http://dx.doi.org/10.4142/jvs.2019.20.e70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883195PMC
November 2019

Novel production of natural bacteriocin via internalization of dextran nanoparticles into probiotics.

Biomaterials 2019 10 15;218:119360. Epub 2019 Jul 15.

Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea; Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Recently, probiotics has drawn much attention as an alternative of antibiotics because overuse use of antibiotics has caused widespread bacterial resistance. Given that prebiotics enhance the activity of probiotics, we prepared phthalyl dextran nanoparticles (PDNs) by conjugation of phthalic anhydride with dextran to form a prebiotic and checked its effects on the cellular and antimicrobial properties of the probiotics. First, we found that the internalization of PDNs by probiotics was dependent on temperature, time, and glucose transporters. Internalization of PDNs enhanced the production of antimicrobial peptides by probiotics through self-defense mechanism and resulted in higher antimicrobial activities against Gram-positive and -negative pathogens compared to probiotics themselves. Moreover, pediocin produced by PDN-internalized probiotics was able to suppress pathogenic gut infections and alter the population of gut microbiome in vivo. The enhanced antimicrobial property of Pediococcus acidilactidi internalized with PDNs could decrease the number of pathogens and increase beneficial bacteria species in mice. Furthermore, the composition of gut microbiome was changed, and resulted in preventing reduction of the diversity of the microflora. Our results indicate that PDNs as a new type of prebiotic can regulate probiotic bacterial metabolism, suggesting a new avenue for probiotic modulation and their use in addressing the challenge of bacterial resistance.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119360DOI Listing
October 2019

Study on the fatty acid profile of phospholipid and neutral lipid in Hanwoo beef and their relationship to genetic variation.

J Anim Sci Technol 2019 Mar 31;61(2):69-76. Epub 2019 Mar 31.

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea.

Maize which has very high omega-6 fatty acid content has been used as a main feed grain for Hanwoo beef production to increase marbling, and thus omega-6 to omega-3 fatty acids ratio in Hanwoo beef is expected to be biased. To elucidate the current status of omega fatty acids ratio in Hanwoo beef, fatty acid profiles of neutral lipid and phospholipid fraction were analyzed separately using 55 Hanwoo steers' muscle slaughtered at Pyeongchang, Korea from Oct. to Nov. 2015. In addition, an association study was conducted to evaluate associations between single nucleotide polymorphism (SNP) markers from references and omega fatty acid profiles in phospholipid of Hanwoo beef samples using analysis of variance (ANOVA). In neutral lipid fraction, composition of saturated and monounsaturated fatty acids was higher and polyunsaturated fatty acids was lower compared to those in phospholipid fraction. The mean n-6/n-3 ratios of Hanwoo were 56.059 ± 16.180 and 26.811 ± 6.668 in phospholipid and neutral lipid, respectively. There were three SNPs showing statistically significant associations with omega fatty acid content. GA type of rs41919985 in fatty acid synthase (FASN) was significantly associated with the highest amount of C20:5 n-3 ( = 0.031). CC type of rs41729173 in fatty acid-binding protein 4 (FABP4) was significantly associated with the lowest amount of C22:2n-6 ( = 0.047). AG type of rs42187261 in FADS1 was significantly linked to the lowest concentration of C20:4 n-6 ( = 0.044). The total n-6/n-3 ratio of the steer which has all four SNP types in above loci (27.905) was much lower than the mean value of the total n-6/n-3 ratio in phospholipid of the 55 Hanwoo steers (56.059 ± 16.180). It was found that phospholipid and neutral lipid of Hanwoo have very high n-6/n-3 ratios compared to the reported data from different cow breeds. Four SNPs in genes related with fatty acid metabolism showed significant associations with the fatty acid profile of phospholipid and may have potential as SNP markers to select Hanwoo steers in terms of n-6/n-3 balance in the future.
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http://dx.doi.org/10.5187/jast.2019.61.2.69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582934PMC
March 2019

Pullulan Nanoparticles as Prebiotics Enhance the Antibacterial Properties of Through the Induction of Mild Stress in Probiotics.

Front Microbiol 2019 6;10:142. Epub 2019 Feb 6.

Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea.

Synbiotics, which are the combination of probiotics and prebiotics, have recently attracted attention because of their synergistic net health benefits. Probiotics have been used as alternatives to antibiotics. Among the probiotics, (LP) has shown strong antimicrobial activity against , a major livestock pathogen. In this study, we aimed to investigate the antimicrobial activity of phthalyl pullulan nanoparticle (PPN)-treated LP. Interestingly, when PPNs were added to LP, the PPNs were internalized into the LP through an energy-dependent and galactose transporter-dependent mechanism. Additionally, more plantaricin, a natural antibacterial peptide, was secreted from PPN-treated LP than from untreated or pullulan-treated LP. Furthermore, antimicrobial activity against Gram-negative and Gram-positive by PPN-treated LP was higher than those of untreated or pullulan-treated LP. It is thought that the enhanced antimicrobial properties of the PPN-treated LP are due to intracellular stimulation. Overall, this research provides a new method of producing plantaricin in LP through intracellular stimulation by internalized PPNs.
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http://dx.doi.org/10.3389/fmicb.2019.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372531PMC
February 2019

Oral Delivery of Probiotics Using pH-Sensitive Phthalyl Inulin Tablets.

J Microbiol Biotechnol 2019 Feb;29(2):200-208

Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun 25354, Republic of Korea.

Probiotics show low cell viability after oral administration because they have difficulty surviving in the stomach due to low pH and enzymes. For the oral delivery of probiotics, developing a formula that protects the probiotic bacteria from gastric acidity while providing living cells is mandatory. In this study, we developed tablets using a new pH-sensitive phthalyl inulin (PI) to protect probiotics from gastric conditions and investigated the effects of different compression forces on cell survival. We made three different tablets under different compression forces and measured survivability, disintegration time, and kinetics in simulated gastric-intestinal fluid. During tableting, there were no significant differences in probiotic viability among the different compression forces although disintegration time was affected by the compression force. A higher compression force resulted in higher viability in simulated gastric fluid. The swelling degree of the PI tablets in simulated intestinal fluid was higher than that of the tablets in simulated gastric fluid due to the pH sensitivity of the PI. The probiotic viability formulated in the tablets was also higher in acidic gastric conditions than that for probiotics in solution. Rapid release of the probiotics from the tablet occurred in the simulated intestinal fluid due to the pH sensitivity. After 6 months of refrigeration, the viability of the PI probiotics was kept. Overall, this is the first study to show the pH-sensitive properties of PI and one that may be useful for oral delivery of the probiotics.
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http://dx.doi.org/10.4014/jmb.1811.11021DOI Listing
February 2019

Tracing of the fecal microbiota of commercial pigs at five growth stages from birth to shipment.

Sci Rep 2018 04 16;8(1):6012. Epub 2018 Apr 16.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea.

The intestinal microbiota affect various physiological traits of host animals such as brain development, obesity, age, and the immune system. In the swine industry, understanding the relationship between intestinal microbiota and growth stage is essential because growth stage is directly related to the feeding system of pigs, thus we studied the intestinal microbiota of 32 healthy pigs across five sows at 10, 21, 63, 93, and 147 d of ages. The intestinal microbiota were altered with growth of pigs and were separated into three distinct clusters. The relative abundance of several phyla and genera were significantly different between growth stages. We observed co-occurrence pattern of the intestinal microbiota at each growth stage. In addition, we predicted the functions of the intestinal microbiota and confirmed that several KEGG pathways were significantly different between growth stages. We also explored the relationship between the intestinal microbiota and innate factors such as the maternal effect and gender. When pigs were young, innate factors affected on construction of intestinal microbiota, however this tendency was disappeared with growth. Our findings broaden the understanding of microbial ecology, and the results will be used as a reference for investigating host-microbe interactions in the swine industry.
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http://dx.doi.org/10.1038/s41598-018-24508-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902624PMC
April 2018

A new way of producing pediocin in Pediococcus acidilactici through intracellular stimulation by internalized inulin nanoparticles.

Sci Rep 2018 04 12;8(1):5878. Epub 2018 Apr 12.

Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea.

One of the most challenging aspects of probiotics as a replacement for antibiotics is to enhance their antimicrobial activity against pathogens. Given that prebiotics stimulate the growth and/or activity of probiotics, we developed phthalyl inulin nanoparticles (PINs) as prebiotics and observed their effects on the cellular and antimicrobial activities of Pediococcus acidilactici (PA). First, we assessed the internalization of PINs into PA. The internalization of PINs was largely regulated by glucose transporters in PA, and the process was energy-dependent. Once internalized, PINs induced PA to produce substantial amounts of antimicrobial peptide (pediocin), which is effective against both Gram-positive (Salmonella Gallinarum) and Gram-negative (Listeria monocytogenes) pathogens. When treated with small-sized PINs, PA witnessed a nine-fold increase in antimicrobial activity. The rise in pediocin activity in PA treated with PINs was accompanied by enhanced expression of stress response genes (groEL, groES, dnaK) and pediocin biosynthesis genes (pedA, pedD). Although the mechanism is not clear, it appears that the internalization of PINs by PA causes mild stress to activate the PA defense system, leading to increased production of pediocin. Overall, we identified a prebiotic in nanoparticle form for intracellular stimulation of probiotics, demonstrating a new avenue for the biological production of antimicrobial peptides.
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http://dx.doi.org/10.1038/s41598-018-24227-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897564PMC
April 2018

A New Method of Producing a Natural Antibacterial Peptide by Encapsulated Probiotics Internalized with Inulin Nanoparticles as Prebiotics.

J Microbiol Biotechnol 2018 Apr;28(4):510-519

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.

Synbiotics are a combination of probiotics and prebiotics, which lead to synergistic benefits in host welfare. Probiotics have been used as an alternative to antibiotics. Among the probiotics, (PA) has shown excellent antimicrobial activity against Gallinarum (SG) as a major poultry pathogen and has improved the production performances of animals. Inulin is widely used as a prebiotic for the improvement of animal health and growth. The main aim of this study is to investigate the effect of the antimicrobial activity of inulin nanoparticles (INs)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) in future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in phthalyl inulin were estimated by ¹H-NMR measurement as 25.1 mol.-%. The sizes of the PINs measured by DLS were approximately 203 nm. Internalization into PA was confirmed by confocal microscopy and flow cytometry. Antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by co-culture antimicrobial assays on SG. PIN-internalized probiotics had a higher antimicrobial ability than that of ACA MCs loaded with PA/inulin or PA. Interestingly, when PINs were treated with PA and encapsulated into ACA MCs, as a natural antimicrobial peptide, pediocin was produced much more in the culture medium compared with other groups inulin-loaded ACA MCs and PA-encapsulated into ACA MCs.
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http://dx.doi.org/10.4014/jmb.1712.12008DOI Listing
April 2018

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

PLoS One 2017 23;12(10):e0186671. Epub 2017 Oct 23.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186671PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653324PMC
November 2017

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

Tissue Eng Regen Med 2018 Feb 17;15(1):1-11. Epub 2017 Oct 17.

1Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanak-gu Seoul, 08826 Republic of Korea.

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.
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http://dx.doi.org/10.1007/s13770-017-0082-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171641PMC
February 2018

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

Appl Microbiol Biotechnol 2017 Jul 3;101(13):5353-5363. Epub 2017 May 3.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.
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http://dx.doi.org/10.1007/s00253-017-8293-6DOI Listing
July 2017

Pan-Genomic Approaches in Lactobacillus reuteri as a Porcine Probiotic: Investigation of Host Adaptation and Antipathogenic Activity.

Microb Ecol 2017 Oct 24;74(3):709-721. Epub 2017 Apr 24.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, reuterin-producing Lactobacillus reuteri is getting attention as an alternative to AGPs. In this study, we investigated genetic features of L. reuteri associated with host specificity and antipathogenic effect. We isolated 104 L. reuteri strains from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (group HS) and eight strains exhibiting the lower effect (group LS), were selected for genomic comparison. We generated draft genomes of the 16 isolates and investigated their pan-genome together with the 26 National Center for Biotechnology Information-registered genomes. L. reuteri genomes organized six clades with multi-locus sequence analysis, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein-coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host gastrointestinal epithelia and that these host-specific traits were acquired by horizontal gene transfer. We also identified differences between groups HS and LS in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. This study provides a deeper understanding of the relationship between probiotic properties and genomic features of L. reuteri.
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http://dx.doi.org/10.1007/s00248-017-0977-zDOI Listing
October 2017

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

Microb Cell Fact 2017 Feb 22;16(1):33. Epub 2017 Feb 22.

Department of Agricultural Biotechnology, Seoul National University, Seoul, 115-921, Republic of Korea.

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.
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http://dx.doi.org/10.1186/s12934-017-0648-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322615PMC
February 2017

Oral Delivery of Probiotics in Poultry Using pH-Sensitive Tablets.

J Microbiol Biotechnol 2017 Apr;27(4):739-746

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.

As alternatives to antibiotics in livestocks, probiotics have been used, although most of them in the form of liquid or semisolid formulations, which show low cell viability after oral administration. Therefore, suitable dry dosage forms should be developed for livestocks to protect probiotics against the low pH in the stomach such that the products have higher probiotics survivability. Here, in order to develop a dry dosage forms of probiotics for poultry, we used hydroxypropyl methylcellulose phthalate 55 (HPMCP 55) as a tablet-forming matrix to develop probiotics in a tablet form for poultry. Here, we made three different kinds of probiotics-loaded tablet under different compression forces and investigated their characteristics based on their survivability, morphology, disintegration time, and kinetics in simulated gastrointestinal fluid. The results indicated that the probiotics formulated in the tablets displayed higher survival rates in acidic gastric conditions than probiotics in solution. Rapid release of the probiotics from the tablets occurred in simulated intestinal fluid because of fast swelling of the tablets in neutral pH. As a matrix of tablet, HPMCP 55 provided good viability of probiotics after 6 months under refrigeration. Moreover, after oral administration of probiotics-loaded tablets to chicken, more viable probiotics were observed, than with solution type, through several digestive areas of chicken by the tablets.
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http://dx.doi.org/10.4014/jmb.1606.06071DOI Listing
April 2017

Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding.

Protein Expr Purif 2016 10 31;126:77-83. Epub 2016 May 31.

Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea; Department of Animal Science, Tianjin Agricultural University, Tianjin, 300-384, People's Republic of China. Electronic address:

The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 μg of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains.
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http://dx.doi.org/10.1016/j.pep.2016.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173294PMC
October 2016

Nasal immunization with mannan-decorated mucoadhesive HPMCP microspheres containing ApxIIA toxin induces protective immunity against challenge infection with Actinobacillus pleuropneumoiae in mice.

J Control Release 2016 07 14;233:114-25. Epub 2016 May 14.

Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; Department of Animal Science, Tianjin Agricultural University, Tianjin 300-384, China. Electronic address:

The development of subunit mucosal vaccines requires an appropriate delivery system or an immune modulator such as an adjuvant to improve antigen immunogenicity. The nasal route for vaccine delivery by microparticles has attracted considerable interest, although challenges such as the rapid mucociliary clearance in the respiratory mucosa and the low immunogenicity of subunit vaccine still remain. Here, we aimed to develop mannan-decorated mucoadhesive thiolated hydroxypropylmethyl cellulose phthalate (HPMCP) microspheres (Man-THM) that contain ApxIIA subunit vaccine - an exotoxin fragment as a candidate for a subunit nasal vaccine against Actinobacillus pleuropneumoniae. For adjuvant activity, mucoadhesive thiolated HPMCP microspheres decorated with mannan could be targeted to the PRRs (pathogen recognition receptors) and mannose receptors (MR) of antigen presenting cells (APCs) in the respiratory immune system. The potential adjuvant ability of Man-THM for intranasal immunization was confirmed by in vitro and in vivo experiments. In a mechanistic study using APCs in vitro, it was found that Man-THM enhanced receptor-mediated endocytosis by stimulating the MR of APCs. In vivo, the nasal vaccination of ApxIIA-loaded Man-THM in mice resulted in higher levels of mucosal sIgA and serum IgG than mice in the ApxIIA and ApxIIA-loaded THM groups due to the specific recognition of the mannan in the Man-THM by the MRs of the APCs. Moreover, ApxIIA-containing Man-THM protected immunized mice when challenged with strains of A. pleuropneumoniae serotype 5. These results suggest that mucoadhesive Man-THM may be a promising candidate for a nasal vaccine delivery system to elicit systemic and mucosal immunity that can protect from pathogenic bacteria infection.
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http://dx.doi.org/10.1016/j.jconrel.2016.05.032DOI Listing
July 2016

Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli.

BMC Biotechnol 2016 05 4;16(1):39. Epub 2016 May 4.

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.

Background: Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.

Results: We firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.

Conclusions: In this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.
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http://dx.doi.org/10.1186/s12896-016-0268-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855837PMC
May 2016

Systemic administration of RANKL overcomes the bottleneck of oral vaccine delivery through microfold cells in ileum.

Biomaterials 2016 Apr 23;84:286-300. Epub 2016 Jan 23.

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. Electronic address:

A successful delivery of antigen through oral route requires to overcome several barriers, such as enzymatic barrier of gastrointestinal tract and epithelial barrier that constitutes of microfold cells (M cells) for antigen uptake. Although each barrier represents a critical step in determining the final efficiency of antigen delivery, the transcytosis of antigen by M cells in the follicle-associated epithelium (FAE) to Peyer's patches appears to be a major bottleneck. Considering the systemic administration of receptor activator of nuclear factor (NF)-ĸB ligand (RANKL) induces differentiation of receptor activator of nuclear factor (NF)-ĸB (RANK)-expressing enterocytes into M cells, here, we illustrated a promising approach of antigen delivery using full length transmembrane RANKL (mRANKL). The results showed that the intraperitoneal injection of mRANKL increased the population of dendritic cells and macrophages in mesenteric lymph nodes and spleen. Subsequently, systemic administration of mRANKL resulted in significantly higher number of functional GP2(+) M cells leading higher transcytosis of fluorescent beads through them. To corroborate the effect of mRANKL in antigen delivery through M cells, we orally delivered microparticulate antigen to mice treated with mRANKL. Oral immunization induced strong protective IgA and systemic IgG antibody responses against orally delivered antigen in mRANKL-treated mice. The higher antibody responses are attributed to the higher transcytosis of antigens through M cells. Ultimately, the higher memory B cells and effector memory CD4 T cells after oral immunization in RANKL-treated mice confirmed potency of RANKL-mediated antigen delivery. To the best of our knowledge, this is the first study to demonstrate significant induction of mucosal and humoral immune responses to M cell targeted oral vaccines after the systemic administration of RANKL.
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http://dx.doi.org/10.1016/j.biomaterials.2016.01.043DOI Listing
April 2016

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

Curr Microbiol 2016 Mar 28;72(3):259-66. Epub 2015 Nov 28.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

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.
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http://dx.doi.org/10.1007/s00284-015-0946-zDOI Listing
March 2016

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

BMC Immunol 2015 Nov 25;16:71. Epub 2015 Nov 25.

Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South Korea.

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.
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http://dx.doi.org/10.1186/s12865-015-0132-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659156PMC
November 2015

Mannan-decorated thiolated Eudragit microspheres for targeting antigen presenting cells via nasal vaccination.

Eur J Pharm Sci 2015 Dec 28;80:16-25. Epub 2015 Sep 28.

Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; Department of Animal Science, Tianjin Agricultural University, Tianjin 300-384, China. Electronic address:

Mucosal vaccination of protein as an antigen requires appropriate delivery or adjuvant systems to deliver antigen to mucosal immune cells efficiently and generate valid immune responses. For successful nasal immunization, the obstacles imposed by the normal process of mucociliary clearance which limits residence time of applied antigens and low antigen delivery to antigen presenting cells (APCs) in nasal associated lymphoid tissue (NALT) need to be overcome for the efficient vaccination. Here, we prepared mucoadhesive and mannan-decorated thiolated Eudragit microspheres (Man-TEM) as a nasal vaccine carrier to overcome the limitations. Mucoadhesive thiolated Eudragit (TE) were decorated with mannan for targeting mannose receptors (MR) in antigen presenting cells (APCs) to obtain efficient immune responses. The potential adjuvant ability of Man-TEM for intranasal immunization was confirmed by in vitro and in vivo experiments. In mechanistic study using APCs in vitro, we obtained that Man-TEM enhanced the receptor-mediated endocytosis by stimulating the MR receptors of APCs. The nasal vaccination of OVA-loaded Man-TEM in mice showed higher levels of serum IgG and mucosal sIgA than the soluble OVA group due to the specific recognition of MR of APCs by the mannan in the Man-TEM. These results suggest that mucoadhesive and Man-TEM may be a promising candidate for nasal vaccine delivery system to elicit systemic and mucosal immunity.
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http://dx.doi.org/10.1016/j.ejps.2015.09.014DOI Listing
December 2015

Combinatorial Approach of Antigen Delivery Using M Cell-Homing Peptide and Mucoadhesive Vehicle to Enhance the Efficacy of Oral Vaccine.

Mol Pharm 2015 Nov 2;12(11):3816-28. Epub 2015 Oct 2.

Department of Agricultural Biotechnology, Seoul National University , Seoul 151-921, Korea.

Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer's patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4(+) T cell immune responses but also generated strong CD8(+) T cell responses with enhanced production of IFN-γ in spleen. Given that M cells are considered a promising target for oral vaccination, this study could provide a new combinatorial method for the development of M-cell-targeted mucosal vaccines.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00265DOI Listing
November 2015

Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

J Nanosci Nanotechnol 2015 Jan;15(1):606-10

Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 μ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.
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http://dx.doi.org/10.1166/jnn.2015.8781DOI Listing
January 2015

Mucoadhesive Chitosan Derivatives as Novel Drug Carriers.

Curr Pharm Des 2015 ;21(29):4285-309

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.

Chitosan on its own is a well-established natural polymer and is widely regarded as a biodegradable, biocompatible and nontoxic material for drug delivery applications. Although unmodified chitosan has some mucoadhesive properties on its own, its bioavailability is limited due to its short retention time in the body. Moreover, the high solubility of chitosan at acidic pH levels limits its use for mucosal drug delivery (especially through the oral route). Chemically-modified mucoadhesive chitosan, especially thiolated chitosan, has arisen as an alternative to create novel mucosal drug delivery systems. The mucoadhesive properties that are conferred to the thiolated chitosan certainly set this novel class of second or third-generation thiomers apart. To understand the significance of mucoadhesive chitosan, we first present the mechanism of mucoadhesion and provide comprehensive coverage of description of a variety of chemical modifications to prepare mucoadhesive thiolated chitosan derivatives. We then present the plethora of applications of these modified chitosan variants in a wide range of drug delivery fields, including the delivery of antigens, proteins and genes through a variety of routes, including oral, nasal, pulmonary, vaginal and others. By presenting the range of applications for mucoadhesive chitosan drug carriers we herein demonstrate that chemically-modified thiolated chitosan is a versatile and effective material for a new class of drug delivery vehicles.
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http://dx.doi.org/10.2174/1381612821666150901103819DOI Listing
August 2016

Attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum.

Biomaterials 2015 Aug 15;59:144-59. Epub 2015 May 15.

Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea; Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. Electronic address:

Orally delivered proteins or antigens are taken up by epithelial microfold cells (M cells) in Peyer's patches, especially abundant in the ileum of small intestine. However, several barriers including gastric pH, enzymatic degradation, rapid transit and lack of specificity of proteins towards M cells, has made the goal of oral delivery of proteins very challenging. To overcome the problems, we developed an ileum targeted protein delivery system using hydroxypropyl methylcellulose phthalate (HPMCP). Initially, we attuned pH-sensitive property of HPMCP for controlled dissolution at ileum pH (≥7.4) by thiolation. Thiolation also improved mucoadhesive property of HPMCP to prolong the particles transit time through the gastrointestinal tract. Typically, thiolated HPMCP (T-HPMCP) prevented protein release in acidic pH in stomach and duodenum but released the proteins at ileal pH in a controlled manner. To evaluate the effectiveness of an oral delivery vehicle, T-HPMCP was used to deliver an M cell targeting protein antigen to mice through oral route. The antigens were mostly delivered and located in Peyer's patches in the ileum demonstrating the higher uptake of antigens through M-cells. Importantly, oral delivery of the antigen with T-HPMCP not only induced strong antibody mediated immune responses but also generated memory T cells in the spleen as adaptive immunity indicating a direct evidence of an effective delivery system. Thus, this study represents the first demonstration of HPMCP for ileum-specific delivery of protein vaccine through oral route.
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http://dx.doi.org/10.1016/j.biomaterials.2015.04.017DOI Listing
August 2015
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