• Proceedings of the Zoological Society Korea Conference
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• 2000.04a
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• pp.82.2-82
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• 2000

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.122-135
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• 2018

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of $CD8^+$ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.181-181
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• 1996

The (Na, K) ATPase is a membrane ion transporting ATPase composed of an ${\alpha}$ catalytic subunit and a ${\beta}$ glycoprotein subunit. The topology of the rat ${\alpha}$1 and ${\beta}$1 subunits has been studied by insertion of epitope(s) : at the NH2-terminus and COOH-terminus and between Glu117 and Glul18, Lys828 and Arg829, Gln900 and Trp901, and Va1939 and Phe940 of the ${\alpha}$ subunit; and at the NH2-terminus and COOH-terminus and between Glu228 and Tyr229 of the ${\beta}$ subunit. The epitope-tagged ${\alpha}$l, constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na, K)ATPase. All constructs, except for the one tagged between Gln900 and Trp901, resulted in ouabain-resistant colonies indicating that modified proteins retained functional integrity. The epitope-tagged ${\beta}$ constructs were transiently expressed in Cos-7 cells. The orientation of the epitopes with respect to the cell membrane was revealed by indirect immunofluorescence performed on permeabilized and non-permeabilized cells expressing the (Na, K)ATPase chains. The results indicate that the ${\alpha}$ subunit has 4 transmembrane segments in the COOH terminal membrane bound domain between residues 760 and 938, and that both the NH2-terminus and the COOH-terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH-terminus. The ${\beta}$ subunit has only one transmembrane spanning region with the NH2-terminus in the cytosol and the COOH-terminus on the extracytoplasmic surface of the plasma membrane.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.490-497
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• 2004

Although the E. coli heat-labile enterotoxin B subunit (LTB) is known to be a potent mucosal adjuvant towards co-administrated unrelated antigens and immunoregulator in T-helper 1-type-mediated autoimmune diseases, a more efficient and useful LTB is still required for prospective vaccine adjuvants. To determine whether a novel chimeric LTB subunit would produce an enhanced mucosal adjuvant activity and immune response, a number of LTB subunits were genetically fused with chimeric proteins using the epitope genes of the envelope glycoprotein E2 (gp51-54) from the classical swine fever virus (CSFV). It was found that the total serum immunoglobulin (Ig) levels of BALB/c mice orally immunized with chimeric proteins containing an N-terminal linked LTB subunit (LE1, LE2, and LE3) were higher than those of mice immunized with LTB, E2 epitope, and chimeric proteins that contained a C-terminal linked LTB subunit. In particular, immunization with LE1 markedly increased both the total serum Ig and fecal IgA level compared to immunization with LTB or the E2 epitope. Accordingly, the current results demonstrated that the LTB subunit in a chimeric protein exhibited a strong mucosal adjuvant effect as a carrier molecule, while the chimeric protein containing the LTB subunit stimulated the mucosal immune system by mediating the induction of antigen-specific serum Ig and mucosal IgA. Consequently, an LE1-mediated mucosal response may contribute to the development of effective antidiarrhea vaccine adjuvants.

• Biomedical Science Letters
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• v.13 no.4
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• pp.325-332
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• 2007

Cell-mediated immune response (CMI) is a major immune protective mechanism against tuberculosis (TB) infection. Among several components involved in CMI, recent studies suggest that CD8+ T cells are important in controlling TB infection. In our previous report, we defined four Mycobacterium tuberculosis (MTB) derived epiotpe peptides specific for HLA-A*0201-restricted CD8+ T cells. These four peptides are $PstAl_{75-83}$, $ThyA_{30-38}$, $RpoB_{127-135}$ and $85B_{15-23}$. In this study, these epitope peptides specific CD8+ T cell responses in tuberculous pleurisy were investigated using ex vivo $IFN-\gamma$ elispot assay and intracellular $IFN-\gamma$ staining method. As a result, we observed these epitope peptide specific CD8+ T cell responses are induced in all three patients with tuberculous pleurisy suggesting that CD8+ T cells are involved in protective immune mechanism against MTB infection in tuberculous pleurisy. However, the CMI to mitogens and MTB antigens from pleural fluids of patients with tuberculous pleurisy does not seem to correlate with that from peripheral blood, although the sample size is too small to make any conclusion. In sum, the MHC I restricted CD8+ T cell responses seem to be induced efficiently in the pleural fluids, at the site of TB infection, in which the CMI is actively induced. In addition, these experiments suggest that MHC I restricted CD8+ T cell mediated immune responses are also involved in protective mechanism against MTB infection in extra-pulmonary TB.

• Parasites, Hosts and Diseases
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• v.51 no.3
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• pp.327-334
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• 2013

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an ${\alpha}$-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

• Parasites, Hosts and Diseases
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• v.46 no.4
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• pp.243-246
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• 2008

Bla g 2 is a cockroach allergen of great importance, This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique, Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2), Only 5,3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.587-594
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• 2000

Monoclonal antibodies against glutamate dehydrogenase (GDH) from Sulfolobus solfataricus were produced and characterized using epitope mapping and biosensor technology, Five monoclonal antibodies raised against S. solfataricus GDH were each identified as a single protein band that comigrated with purified S. solfataricus GDH on the SDS-polyacrylamide gel electrophoresis and immunoblot. Epitope mapping analysis showed that only one subgroup among the antibodies tested recognized the same peptide fragments of GDH. Using the anti-S. solfataricus GDH antibodies as probes, the cross-reactivities of GDHs from various sources were investigated and it was found that the mammalian GDH is not immunologically related to S. solfataricus GDH. The structural differences between the microbial and mammalian GDHs were further investigated using biosensor technology (Pharmacia BIAcore) and monoclonal antibodies against S. solfataricus and bovine brain. The binding affinity of S. solfataricus glutamate dehydrogenase anti-S. solfataricus for GDH ($K_D$=11 nM) was much tighter than that of anti-bovine for GDH ($K_D$=450 nM). These results, together with the epitope mapping analysis, suggest that there may be structural differences between the two GDH species, in addition to their different biochemical properties.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.211-217
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• 2020

Molecular mimicry is the most common mechanism that breaches self-tolerance. We previously identified autoantibodies to aquaporin-5 (AQP5) in the sera of patients with Sjögren's syndrome and found that the aquaporin of Prevotella melaninogenica (PmAqp), an oral commensal, is highly homologous to human AQP5. This study aimed to test whether PmAqp can induce anti-AQP5 autoantibodies via molecular mimicry. From the amino acid sequence of PmAqp, an immunizing peptide; i.e., PmE-L, was designed, which contained both the B cell epitope "E" and T cell epitope. C57BL/6 and BALB/c mice were subcutaneously immunized with linear or cyclic forms of PmE-L emulsified in incomplete Freund's adjuvant. The concentrations of the antibodies in sera were measured using enzyme-linked immunosorbent assays. Both linear and cyclic PmE-L induced high levels of antibodies against not only the immunized peptides but also autoantibodies against AQP5E and antibodies against PmE, a Pm homolog of AQP5E. In C57BL/6 mice; however, the cyclic form of PmE-L was more efficient than the linear form in inducing autoantibodies against AQP5E that contained a cyclic epitope. The levels of anti-PmE antibodies and anti-AQP5E autoantibodies showed a strong positive correlation (r = 0.95, p < 0.0005), suggesting molecular mimicry. Collectively, the mice produced anti-AQP5E autoantibodies in response to a PmAqp-derived peptide. This model proved to be useful for studying the mechanisms of autoantibody production by molecular mimicry.

• Journal of Periodontal and Implant Science
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• v.47 no.5
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• pp.292-311
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• 2017

Purpose: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance. Methods: Human polyclonal $CD4^+CD25^+CD127^{lo-}$ Tregs (127-Tregs) and $na\ddot{i}ve$ $CD4^+CD25^+CD45RA^+$ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an $NOD/scid/IL-2R{\gamma}^{-/-}$ mouse model of collagen-induced arthritis. Results: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of $CD4^+CD25^+$ Tregs at the articular joints in a mechanistic and orchestrated way. Conclusions: We propose human $na\ddot{i}ve$ $CD4^+CD25^+CD45RA^+$ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.