• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2095-2105
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• 2018

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.500-506
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• 2006

An experiment was conducted with twenty crossbred male calves (7-15 days old) divided into 4 different experimental groups on the basis of body weights to study the effect of vitamin E supplementation on the serum ${\alpha}$-tocopherol concentration and immune response of the calves. All the calves were fed on milk and calf starter up to 13 weeks and afterwards, they were fed on concentrate mixture and oat hay up to 32 weeks of age. In addition, the calves in groups I, II, III and IV were supplemented with 0, 125, 250 and 500 IU feed grade DL-${\alpha}$-tocopheryl acetate, respectively. Blood samples were collected at 0 day and subsequently at 1, 2, 3, 4, 6 and 8 months of age to monitor the serum ${\alpha}$-tocopherol concentration in crossbred calves. After 24 weeks of experimental feeding, 4 animals from each group were intramuscularly inoculated with single dose (3 ml) of Haemorrhagic septiceaemia (Pasteurella multocida P52 strain) oil adjuvant vaccine. The cumulative group mean serum ${\alpha}$-tocopherol concentration (${\mu}g/100ml$) was 88.12, 210.11, 235.21 and 294.02 in-groups I, II, III and IV, respectively and differed significantly (p<0.001) among the four groups. Lymphocyte stimulation indices (LSI) did not differ among the groups significantly. The pooled mean ELISA antibody titer against Pasteurella multocida (P52 strain) was 788.02, 926.85, 1,214.00 and 1,109.51 for group I, II, III and IV, respectively, which indicated higher antibody titer in groups supplemented with vitamin E as compared to the control group. It may be concluded that vitamin E supplementation increased the ${\alpha}$-tocopherol concentration in serum and dietary supplementation of vitamin E at higher level has a humoral immune enhancing effect against killed bacterial antigen.

• Biomolecules & Therapeutics
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• v.13 no.2
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• pp.101-106
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• 2005

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

• Biomolecules & Therapeutics
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• v.11 no.3
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• pp.157-162
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• 2003

The generation of secretory IgA antibodies(Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHIctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was analyzed. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/CTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of CTXB. This study also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin/CTXA2B chimeric protein might be a potential antigen for oral immunization against UPEC.

• Korean Journal of Veterinary Research
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• v.35 no.1
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• pp.87-94
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• 1995

Western immunoblot analysis of antigen of T sergenti merozoite revealed that the immunodominant proteins of this organism were characterized as the 18KD, 29KD, 34KD, 45KD and 105KD in Korea. The 34KD and 45KD among those immunodominant proteins of the parasite were isolated and their amino acid sequences from the $NH_2$-terminus were determined and synthesized. They respective polypeptides were cationized to enhance their antigenicity, fortified with Freund's adjuvant and tested for immunogenicity in rabbits and cattle. The results obtained were as follows; 1. Theileria sergenti merozoite antigen was shown in 120KD, 100KD, 66KD, 45KD, 34KD and 30KD in western immunoblot using serum of rabbits immunized with 34KD synthetic polypeptide and 70KD, 58KD, 55KD and 45KD using bovine serum. In western immunoblot, 45KD, 34KD and 30KD were recognized by immunized rabbits, and 50KD and 45KD by cattle sera immunized with 45KD synthetic polypeptide, respectively. 2. The ELISA utilizing the synthetic polypeptides demonstrated significant antibody response to the respective peptides. After the 2nd booster injection, an OD of 0.760(preimmunization 0.132) in rabbits and an OD of 0.645(preimmunization 0.488) to 34KD synthetic polypeptide in cattle were observed. In animals immunized with 45KD synthetic polypeptide, after the 2nd booster injection, an OD of 0.640(preimmunization 0.144) in rabbit, and an OD of 0.776 (preimmunization 0.477) in cattle were measured. 3. After the 2nd booster the reciprocal IFA titer was 1:64 in rabbits and 1:512 in cattle immunized with the 34KD synthetic polypeptide. The IFA titre was observed as 1:512 in rabbit and 1:1,024 in cattle in immunized with the 45KD synthetic polypeptide.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1444-1452
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• 2019

The conventional prophylactic vaccines for human papillomavirus (HPV) efficiently prevent infection with high-risk HPV types, but they do not promote therapeutic effects against cervical cancer. Previously, we developed HPV16 E7-expressing Lactobacillus casei (L. casei-E7) as a therapeutic vaccine candidate for cervical cancer, which induces antitumor therapeutic effects in a TC-1 murine cancer model. To improve the therapeutic effect of L. casei-E7, we performed co-treatment with poly-gamma-glutamic acid (${\gamma}-PGA$), a safe and edible biomaterial naturally secreted by Bacillus subtilis. We investigated their synergistic effect to improve antitumor efficacy in a murine cancer model. The treatment with ${\gamma}-PGA$ did not show in vitro cytotoxicity against TC-1 tumor cells; however, an enhanced innate immune response including activation of dendritic cells was observed. Mice co-administered with ${\gamma}-PGA$ and L. casei-E7 showed significantly suppressed growth of TC-1 tumor cells and an increased survival rate in TC-1 mouse models compared to those of mice vaccinated with L. casei-E7 alone. The administration of ${\gamma}-PGA$ markedly enhanced the activation of natural killer (NK) cells but did not increase the E7-specific cytolytic activity of $CD8^+$ T lymphocytes in mice vaccinated with L. casei-E7. Overall, our results suggest that oral administration of ${\gamma}-PGA$ induces a synergistic antitumor effect in combination with L. casei-E7.

• Korean Journal of Veterinary Research
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• v.25 no.1
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• pp.7-17
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• 1985

Many investigators have been pursuing various attempts so far to produce hepatitis B surface antigen(HBsAg) vaccines using the techniques such as isolation from plasma of chronic HBsAg carrier, recombinant DNA technique or preparation of synthetic peptides specific for immunogenic determinants. Hepatitis B virus can not grow on any cell lines by the tissue culture technique at the present time. The plasma of chronic HBsAg carrier is expensive and its source is limited. The HBsAg from the recombinant DNA technique gave still very low yield. Another approach, therefore, has been initiated to develop a synthetic hepatitis B virus vaccine. The possible use of several distinct synthetic vaccines in prophylaxis can be facilitated by availability of full synthetic immunogens. Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins, although the free synthetic peptide can be immunogenic. To understand basic knowledges on the antigenicity and immunogenicity of a synthetic peptide specific for major immunogenic determinant of HBsAg, a nonapeptide, $H_2N^{139}Cys-Thr-Lys-Pro-Thr-Asp-Gly-^{146}Asn-Aba$ COOH, which corresponds to HBsAg amino acid residues 139 to 147, was synthesized by the Merrifield's solid-phase method with a slight modification. The antigenicity and immunogenicity of this specific synthetic peptide were examined comparing with purified plasma-derived natural HBsAg. The results obtained are as follows; 1. The peptide synthesized showed the identical amino acid composition to the theoretical value. The degree of purification and molecular weight were acertained by methods of high performance liquid chromatography and mass spectrometry. 2. Using m-maleimidobenzoyl-N-hydroxysuccinimide ester as a conjugating agent, the synthetic peptide was conjugated to rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin. Their conjugation yields were 8.3, 9.5, 15.8, 13.5, and 11.2%, respectively. 3. The natural HBsAg was purified from plasma of chronic HBsAg carrier. By the electron microscopic observation of the purified natural HBsAg preparation, no Dane particles were observed and the preparation showed negative DNA polymerase activity. 4. Antigenicity of the synthetic peptide and the plasma-derived natural HBsAg was determined by competition radioimmunoassay using $^{125}I$-natural HBsAg. Their 50% inhibitions appeared as $90{\mu}g/ml$ and $0.12{\mu}g/ml$ for the synthetic peptide and the natural HBsAg, respectively. This indicates that the former was about 750-fold less antigenic than the latter. 5. Immunogenicity of the synthetic peptide was determined by administering the peptide-carrier conjugates into rabbits with and without Freund's complete adjuvant. Regardless the carrier proteins and adjuvant, positive immune responses to the synthetic peptide were observed. The higher antibody titers, however, were shown in the groups administered with Freund's complete adjuvant. 6. Immunizing dose 50% in mice of the various peptide-carrier conjugates was 5.47, 6.00, 65.16, 31.25 and $13.03{\mu}g/dose$ for rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin, respectively, while the natural HBsAg showed $0.65{\mu}g/dose$. 7. It was postulated that homologous proteins prefer to heterologous ones as the carriers.

• IMMUNE NETWORK
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• v.11 no.1
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• pp.79-94
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• 2011

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-${\kappa}B$. Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. Results: DPT promoted the activation of $CD8^+$ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-${\gamma}$ production and induction of cytotoxic T lymphocyte activity. Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

• Korean Journal of Poultry Science
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• v.8 no.2
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• pp.69-75
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• 1981

The experiment was carried out to observe whether the route of administration of allantoic aminiotic fluid obtained from the chicken embryo infected with $B_1$ virus would affect the protectivity of brids against the challenge exposure of a virulent strain of ND virus. Four groups of birds of 30 days of age were immunized intranassally (0.1 $m\ell$), intramuscularly (1.0 $m\ell$), by spray administration (0.00015 $m\ell$/1㎤) or via drinking water(10.0 $m\ell$), with 1 in 100 dilution of th. fluid containing $B_1$ virus titre of 10$\^$8.5/ELD$\_$50/ per $m\ell$ and all the immunized birds, after 15 days of vaccination, were challenged intramuscularly with 1.0$m\ell$ of 10,000 MLD per $m\ell$ of a virulent ND virus. The results obtained are summerized as follows: 1. Good immunity was induced when 1 in 100 dilution of allantoaminiotic fluid with $B_1$ virus titre of 10$\^$8.5/ELD$\_$50/$m\ell$was applied to 30 day old chicks intramuscularly, intranasally and by spray application, but it was not the case when the allantomiotic fluid was diluted to 1 in 1,000. The ID$\_$50/ of birds immunized with 1 in 100 dilution of allantoaminiotic fluid by various routes of administration such as intramuscular Injection, spray application and intranasal instillation were 10$\^$2.8/＞10$\^$4.l and/＞10$\^$4.2/ 2. The high protectivity against the challenge exposure with a virulent Newcastle disease virus with 10,000 MLD/$m\ell$ were observed when the birds were immunized with a live vaccine of 10$\^$8.5/ ELD$\_$50/$m\ell$ by intramuscular injection, intranasal instillation or spray application, and the rates by different routes of application were 92.62%, 95.33% and 93.75%, respectively. On the contrary, no good immunity was induced in the groups of birds immunized via drinking water with the live vaccine, the rate of protection against the challenge exposure being 47.18%.