• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.718-724
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• 2017

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and half-life. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

• IMMUNE NETWORK
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• v.14 no.4
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• pp.207-218
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• 2014

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, ${\alpha}$-galactosylceramide (${\alpha}GC$) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific $CD8^+$ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by ${\alpha}GC$ can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after ${\alpha}GC$-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 $CD8^+$ T cells in vivo, thereby allowing the proliferation of functional $CD8^+$ T cells. Importantly, when ${\alpha}GC$ and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific $CD8^+$ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between ${\alpha}GC$-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• IMMUNE NETWORK
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• v.21 no.6
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• pp.44.1-44.15
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• 2021

Tumor peptides associated with MHC class I molecules or their synthetic variants have attracted great attention for their potential use as vaccines to induce tumor-specific CTLs. However, the outcome of clinical trials of peptide-based tumor vaccines has been disappointing. There are various reasons for this lack of success, such as difficulties in delivering the peptides specifically to professional Ag-presenting cells, short peptide half-life in vivo, and limited peptide immunogenicity. We report here a novel peptide vaccination strategy that efficiently induces peptide-specific CTLs. Nanoparticles (NPs) were fabricated from a biodegradable polymer, poly(D,L-lactic-co-glycolic acid), attached to H-2K^b molecules, and then the natural peptide epitopes associated with the H-2K^b molecules were exchanged with a model tumor peptide, SIINFEKL (OVA_257-268). These NPs were efficiently phagocytosed by immature dendritic cells (DCs), inducing DC maturation and activation. In addition, the DCs that phagocytosed SIINFEKL-pulsed NPs potently activated SIINFEKL-H2K^b complex-specific CD8⁺ T cells via cross-presentation of SIINFEKL. In vivo studies showed that intravenous administration of SIINFEKL-pulsed NPs effectively generated SIINFEKL-specific CD8⁺ T cells in both normal and tumor-bearing mice. Furthermore, intravenous administration of SIINFEKL-pulsed NPs into EG7.OVA tumor-bearing mice almost completely inhibited the tumor growth. These results demonstrate that vaccination with polymeric NPs coated with tumor peptide-MHC-I complexes is a novel strategy for efficient induction of tumor-specific CTLs.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.9039-9043
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• 2014

There is a continuing need for innovative alternative therapies for liver cancer. DNA vaccines for hormone/growth factor immune deprivation represent a feasible and attractive approach for cancer treatment. We reported a preventive effect of a DNA vaccine based on six copies of the B cell epitope GRP18-27 with optimized adjuvants against H22 hepatocarcinoma. Vaccination with pCR3.1-VS-HSP65-TP-GRP6-M2 (vaccine) elicited much higher level of anti-GRP antibodies and proved efficacious in preventing growth of transplanted hepatocarcinoma cells. The tumor size and weight were significantly lower (p<0.05) in the vaccine subgroup than in the control pCR3.1-VS-TP-HSP65-TP-GRP6, pCR3.1-VS-TP-HSP65-TP-M2 or saline subgroups. In addition, significant reduction of tumor-induced angiogenesis associated with intradermal tumors of H22 cells was observed. These potent effects may open ways towards the development of new immunotherapeutic approaches in the treatment of liver cancer.

• Genomics & Informatics
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• v.20 no.1
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• pp.11.1-11.20
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• 2022

Vibrio harveyi belongs to the Vibrio genus that causes vibriosis in marine and aquatic fish species through double-stranded DNA virus replication. In humans, around 12 Vibrio species can cause gastroenteritis (gastrointestinal illness). A large amount of virus particles can be found in the cytoplasm of infected cells, which may cause death. Despite these devastating complications, there is still no cure or vaccine for the virus. As a result, we used an immunoinformatics approach to develop a multi-epitope vaccine against most pathogenic hemolysin gene of V. harveyi. The immunodominant T- and B-cell epitopes were identified using the hemolysin protein. We developed a vaccine employing three possible epitopes: cytotoxic T-lymphocytes, helper T-lymphocytes, and linear B-lymphocyte epitopes, after thorough testing. The vaccine was developed to be antigenic, immunogenic, and non-allergenic, as well as having a better solubility. Molecular dynamics simulation revealed significant structural stiffness and binding stability. In addition, the immunological simulation generated by computer revealed that the vaccination might elicit immune reactions in the actual life after injection. Finally, using Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher codon adaptation index value, which was then included in the cloning vector pET2+ (a). Altogether, our experiment implies that the proposed peptide vaccine might be a good option for vibriosis prophylaxis.

• BMB Reports
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• v.33 no.1
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• pp.92-95
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• 2000

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALB/c mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1p24(202-221) sequence, and antibody-secreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: ${\kappa}$) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.121-124
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• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• Korean Journal of Veterinary Research
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• v.46 no.2
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• pp.127-133
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• 2006

Pasteurella multocida is a terrible veterinary pathogen that causes widespread infections in husbandry. To induce homologous and/or heterologous immunity against the infections, outer membrane protein Hs (OmpH) in the envelope of different strains of P. multocida are thought to be attractive vaccine candidates. Previously we cloned and characterized a gene for OmpH from pathogenic P. multocida (A : 3) (In Press, Korean J. Microbiol. Biotechnol. 2005, 33, December). The gene is composed of 1,047 nucleotides (nt) coding 348 amino acids (aa) with signal peptide of 20 aa. The truncated ompH, a gene without nt coding for the signal peptide, was generated using pRSET A to name "pRSET A/OmpH-F2". This truncated ompH was well expressed in Escherichia coli BL21 (DE3). Truncated OmpH was purified for induction of immunity against live pathogen of fowl cholera (P. multocida A : 3) in mice. Some $50{\mu}g$ of the purified polypeptide was intraperitoneally injected into mice two times with 10 day interval. Lethal dose ($25{\mu}l$) of live P. multocida A : 3 was determined by directly injecting the pathogen into wild mice (n = 25). To demonstrate the vaccine candidate of the truncated OmpH, the live pathogen ($25{\mu}l$) was challenged with the OmpH-immunized mouse group as well as positive & negative controls (n = 80). The results show that the truncated OmpH can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (A : 3).

• IMMUNE NETWORK
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• v.7 no.4
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• pp.186-196
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• 2007

Background: Although IL-12 has been widely accepted to playa central role in the control of pathogen infection, the use of recombinant IL-12 (rIL-12) as a vaccine adjuvant has been known to be ineffective because of its rapid clearance in the body. Methods: To investigate the effect of sustained release of IL-12 in vivo in the peptide and protein vaccination models, rIL-12 was encapsulated into poly ($A_{DL}$-lactic-co-glycolic acid) (PLGA). Results: We found that codelivery of IL-12-encapsulated microspheres (IL-12EM) could dramatically increase not only antibody responses, but also antigen-specific $CD4^+\;and\;CD8^+$ T cell responses. Enhanced immune responses were shown to be correlated with protective immunity against influenza and respiratory syncytial virus (RSV) virus challenge. Interestingly, the enhancement of $CD8^+$ T cell response was not detectable when $CD4^+$ T cell knockout mice were subjected to vaccination, indicating that the enhancement of the $CD8^+$ T cell response by IL-12EM is dependent on $CD4^+$ T cell "help". Conclusion: Thus, IL-12EM could be applied as an adjuvant of protein and peptide vaccines to enhance protective immunity against virus infection.