• BMB Reports
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• v.28 no.3
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• pp.243-248
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• 1995

Cholesteryl ester transfer protein (CETP), a hydrophobic glycoprotein promoting transfer of cholesteryl esters (CE) from high-density lipoproteins (HDL) to lower-density lipoproteins in the plasma, has been recognized a potent atherogenic factor during the development of coronary artery diseases. This study demonstrated a possible utilization of antisense RNA to inhibit expression of the CETP gene using vaccinia virus as an expression system. The CETP cDNA was inserted into a transfer vector (pSC11) in sense and antisense orientations and used to generate recombinant viruses. Recombinants containing sense or antisense orientations of the CETP cDNA were isolated by $TK^-$ selection and X-gal test. The inserted CETP cDNAs in the recombinants were identified by Southern blot analysis and allowed to transcribe in host cells (CV-1). Expressions of the exogenous CETP mRNA, extracted from the CV-1 cells coinfected with viruses containing sense and antisense DNAs, were monitored by Northern blot analysis using the CETP cDNA probe, by Western blot analysis using monoclonal antibody against the C-terminal active region of the CETP and by the CETP assay. Decreased expressions of the exogenous CETP cDNA were clearly evident in the Northern and Western blot analyses as the dose of antisense expression increased. In the CETP assay, the CETP activities decreased compared to the activity obtained from the cell extracts infected with sense construct only.

• IMMUNE NETWORK
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• v.12 no.3
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• pp.118-125
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• 2012

CD40-CD40L-mediated help from CD4 T cells is essential to induce primary CD8 T cell responses specific to the non-inflammatory cell-based antigen H60. In this study, using H60 as a model antigen, we generated recombinant vaccinia viruses (rVVs) expressing the H60 CD8 epitope and investigated whether CD4 help was required to activate the CD8 T cell response specific to the virally expressed H60. The immune response after infection with rVVs expressing H60 was similar to that after immunization with H60 congenic splenocytes, with a peak frequency of H60-specific CD8 T cells detected in the blood on day 10 post-infection. A CD8 T cell response specific for virally derived H60 was not induced in CD4-depleted mice, but was in CD40-deficient mice. These results provide insights into the characterization of the CD8 T cell response specifically for antigens originating from cellular sources compared to viral sources.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.1022-1029
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• 2018

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, $IFN-{\gamma}$-producing splenocytes, $CD69^+CD8^+$ T cells, and $IFN-{\gamma}$ secretion when compared with bacillus Calmette-$Gu{\acute{e}}rin$ (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more $IFN-{\gamma}$-producing splenocytes and $CD69^+CD8^+$ T cells, and higher levels of $IFN-{\gamma}$ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of $IFN-{\gamma}$-producing splenocytes sensitive to $CD8^+$ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines ($IFN-{\gamma}$ and $TNF-{\alpha}$) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

• IMMUNE NETWORK
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• v.16 no.4
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• pp.233-241
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• 2016

DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that the VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), enhance antiviral CTL responses by increasing MHC class I-restricted viral antigen presentation in dendritic cells (DCs). Bone marrow-derived DCs (BM-DCs) were infected with a recombinant vaccinia virus (VV) expressing OVA (VV-OVA), and then treated with CP or RTX. Both CP and RTX increased MHC class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Oral administration of CP or RTX significantly increased MHC class I-restricted OVA presentation by splenic and lymph node DCs in VV-OVA-infected mice, as assessed by directly measuring OVA peptide SIINFEKL-K^b complexes on the cell surface and by performing functional assays using OVA-specific CD8 T cells. Accordingly, oral administration of CP or RTX elicited potent OVA-specific CTL activity in VV-OVA-infected mice. The results from this study demonstrate that VR1 agonists enhance anti-viral CTL responses, as well as a neuro-immune connection in anti-viral immune responses.

• Journal of Microbiology
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• v.43 no.6
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• pp.537-545
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• 2005

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used $H-2K^b$ restricted T-cell epitopes of NP. The NP-specific $CD8^+$ T cell response was analyzed using a $^{51}Cr-release$ assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific $CD8^+$ T cell response at eight days after infection. We also found that several different methods to check the NP-specific $CD8^+$ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited $2\~4$ weeks after immunization and maximized at $6\~8$ weeks. NP-specific $CD8^+$ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

• IMMUNE NETWORK
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• v.14 no.4
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• pp.219-225
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• 2014

We examined the immunogenicity of H-2 class I-restricted and HLA-A2-restricted epitopes through peptide immunization of HLA-A2-transgenic mice that also express mouse H-2 class I molecules. All four of the tested epitopes restricted by H-2 class I robustly elicited T-cell responses, but four of seven epitopes restricted by HLA-A2 did not induce T-cell responses, showing that HLA-A2-restricted peptide epitopes tend to be poorly immunogenic in HLA-A2-transgenic mice. This finding was confirmed in HLA-A2-transgenic mice infected with a recombinant vaccinia virus expressing hepatitis C virus proteins. We examined the precursor frequency of epitope-specific naïve $CD8^+$ T cells in HLA-A2-transgenic and conventional C57BL/6 mice and found that the poor immunogenicity of HLA-A2-restricted peptide epitopes is related to the paucity of naïve $CD8^+$ T-cell precursors in HLA-A2-transgenic mice. These results provide direction for the improvement of mouse models to study epitope repertoires and the immunodominance of human T-cell responses.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.41-48
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• 2002

Background: Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8+ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/$K^b$ transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge. Methods: HBx-peptides encapsulated by pH-sensitive liposomes were prepared. $A2K^b$ transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with $1{\times}10^7pfu$/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK-143B cells. IFN-${\gamma}$ secretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A ($2{\mu}g/ml$), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with $20{\mu}g/ml$ of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard $^{51}Cr$-release assay and intracellular IFN-${\gamma}$ assay. Results: Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8+ T cells. Mice immunized with $50{\mu}g/head$ were much better protected against viral challenge compared to those immunized with $5{\mu}g$/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with $5{\mu}g/head$ than $50{\mu}g/head$ group. Increase of the number of cells that intracellular IFN-${\gamma}$ secreting cell among CD8+ T cells showed similar result. Conclusion: Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by $^{51}Cr$-release assay and the percentage of accumulated intracellular IFN-${\gamma}$ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of $^{51}Cr$-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of $50{\mu}g/head$, while in the in vitro restimulation, it showed more spectific response in $5{\mu}g$/head group.