Inoculation of Plasmids Encoding Japanese Encephalitis Virus PrM-E Proteins with Colloidal Gold Elicits a Protective Immune Response in BALB/c Mice

We established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti-JEV antibodies and a protective response against JEV challenge in BALB/c mice. When we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit stronger and more sustained neutralizing immune responses than intramuscular or intraperitoneal injection. After being inoculated twice, mice were found to resist challenge with 100,000 times the 50% lethal dose (LD(50)) of JEV (Beijing-1 strain) even when immunized with a relatively small dose of 0.5 μg of plasmid DNA. Protective passive immunity was also observed in SCID mice following transfer of splenocytes or serum from plasmid DNA- and colloidal gold-immunized BALB/c mice. The SCID mice resisted challenge with 100 times the LD(50) of JEV. Analysis of histological sections detected expression of proteins encoded by plasmid DNA in the tissues of intravenously, intradermally, and intramuscularly inoculated mice 3 days after inoculation. DNA immunization with colloidal gold elicited encoded protein expression in splenocytes and might enhance immune responses in intravenously inoculated mice. This approach could be exploited to develop a novel DNA vaccine

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles

Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine

Effects of rVV-HCV treatment on the CN2-29^(+/−)/MxCre^(+/−) mice.

<p>(<b>A)</b> HCV gene structure in the CN2-29^(+/−)/MxCre^(+/−) mice and recombinant vaccinia viruses (rVV-HCV). MxCre/CN2-29 cDNA contains the core, E1, E2, and NS2 regions. The rVV-CN2 cDNA contains the core, E1, E2, and NS2 regions. The rVV-N25 cDNA contains the NS2, NS3, NS4A, NS4B, NS5A, and NS5B regions. The rVV-CN5 cDNA contains the entire HCV region. (<b>B</b>) Four groups of CN2-29^(+/−)/MxCre^(+/−) mice were inoculated intradermally with rVV-CN2, rVV-N25, rVV-CN5, or LC16m8 90 days after the poly(I:C) injection. Blood, liver, and spleen tissue samples were collected 7 and 28 days after the inoculation. (<b>C</b>) Liver sections from the four groups of CN2-29^(+/−)/MxCre^(+/−) mice 7 days after the inoculation. The sections were stained with H&E, silver, oil-red-O, or PAS. The scale bars indicate 50 µm. (<b>D</b>) Histological evaluation of piecemeal necrosis in the four groups of CN2-29^(+/−)/MxCre^(+/−) mice 7 days after inoculation. (<b>E</b>) Histological evaluation of steatosis in the four groups of CN2-29^(+/−)/MxCre^(+/−) mice 7 days after inoculation. Significant relationships are indicated by a P-value.</p

Immunization with rVV-N25 induced CD8 T-cell degranulation, a marker for cytotoxicity, and IFN-γ production.

<p>(<b>A</b>) The numbers represent the percentage of CD107a positive cells and negative cells (left two columns) and IFN-γ-positive cells and negative cells (right two columns). (<b>B, C</b>) The ratio of CD8⁺IFN-γ⁺ cells to all CD8 T cells for rVV-N25-treated mice was significantly higher than that for mice treated with any other rVV. Splenocytes (4 × 10⁶ per well) were cultured with EL-4CN2 or EL-4NS2 cell lines in RPMI 1640 complete medium including 3% T-STIM™ with ConA for 2 weeks. Harvested cells were incubated for 4 h with EL-4, EL-4CN2, or EL-4NS2 in combination with PE-labeled anti-CD107a mAb and monensin in RPMI 1640 complete medium with 50 IU/mL IL-2, according to the manufacturer's instruction. After incubation, cell suspensions were washed with PBS, and the cells were further stained with APC-labeled anti-IFN-γ mAb and Pacific blue-labeled anti-CD8 mAb. Harvested cells were stained with anti-CD107a-PE, anti-IFN-γ-APC, or anti-CD8-Pacific blue. Results that are representative of three independent experiments are shown. Significant relationships are indicated by P-value.</p

Pathogenesis in immunocompetent mice with persistent HCV expression.

<p>(<b>A</b>) Structure of CN2-29^(+/−)/MxCre^(+/−) and the Cre-mediated activation of the transgene unit. R6CN2 HCV cDNA was cloned downstream of the CAG promoter, neomycin-resistant gene (<i>neo</i>), and poly A (pA) signal flanked by two <i>loxP</i> sequences. This cDNA contains the core, E1, E2, and NS2 regions. (<b>B</b>) Cre-mediated genomic DNA recombination. After poly(I:C) injection, genomic DNA was extracted from liver tissues and analyzed by quantitative RTD-PCR for Cre-mediated transgenic recombination. The transgene was almost fully recombined in transgenic mouse livers 7 days after the injection. In all cases, n = 3 mice per group. (<b>C</b>) HCV core protein expression was sustained for at least 600 days after poly(I:C) injection. (<b>D</b>) Immunohistochemical analysis revealed that most hepatocytes expressed the HCV core protein within 6 days after injection. (<b>E</b>) Liver sections from CN2-29^(+/−)/MxCre^(+/−) mice after the poly(I:C) injection. Infiltrating lymphocytes (arrows) were observed on days 6 and 180; Hepatocellular carcinoma (HCC) was observed on day 360. In contrast, these pathological changes were not observed in CN2-29^(+/−)/MxCre^(−/−) mice after the injection. The inset image shows abnormal mitosis in a tumor cell. (<b>F</b>) Hepatocyte swelling and abnormal architecture of liver-cell cords (silver staining), as well as abnormal glycogen accumulation (PAS staining) were observed on day 90 in CN2-29^(+/−)/MxCre^(+/−) mice. We observed steatosis (oil-red-O staining) on day 180 and, subsequently, fibrosis (Azan staining) on day 480. The scale bars indicate 50 µm.</p

Role of CD4 and CD8 T cells in rVV-N25-treated mice.

<p>(<b>A</b>) Schematic diagram depicts depletion of CD4 and CD8 T cells via treatment with monoclonal antibodies. (<b>B</b>) Comparison of HCV core protein expression in control, CD4-depleted, and CD8-depleted mice 28 days after immunization with LC16m8 or rVV-N25. (<b>C, D</b>) Histological analysis of liver samples from CD4-depleted or CD8-depleted CN2-29^(+/−)/MxCre^(+/−) mice 28 days after immunization with LC16m8 or rVV-N25. The scale bars indicate 100 µm (<b>C</b>) and 50 µm (<b>D</b>). (<b>E</b>) Histological evaluation of steatosis in liver samples from CD4-depleted or CD8-depleted CN2-29^(+/−)/MxCre^(+/−) mice 28 days after immunization with LC16m8 or rVV-N25. Significant relationships are indicated by a P-value.</p

Immunization with rVV-N25 suppresses serum inflammatory cytokine levels.

<p>(<b>A</b>) Daily cytokine levels in the serum of CN2-29^(+/−)/MxCre^(+/−) mice during the week following immunization with LC16m8, rVV-CN2, rVV-N25, or rVV-CN5. Values represent means ± SD (n  = 3) and reflect the concentrations relative to those measured on day 0. The broken lines indicate the baseline data from wild-type mice. In all cases, n  = 6 mice per group. (<b>B</b>) Liver sections from CN2-29^(+/−)/MxCre^(+/−) and CN2-29^(+/−)/MxCre^(−/−) mice. (<b>C</b>) Histology activity index (HAI) scores of liver samples taken from CN2-29^(+/−)/MxCre^(+/−), or CN2-29^(+/−)/MxCre^(−/−) mice. (<b>D</b>) Liver sections from CN2-29^(+/−)/MxCre^(+/−) mice in which TNF-α was neutralized and the IL-6 receptor was blocked. The scale bars indicate 50 µm. (<b>E</b>) HAI scores of liver samples taken from CN2-29^(+/−)/MxCre^(+/−) in which TNF-α was neutralized and the IL-6 receptor was blocked. Tg and non-Tg indicate CN2-29^(+/−)/MxCre^(+/−) and CN2-29^(+/−)/MxCre^(−/−), respectively. (F) Macrophages were the main producers of TNF-α and IL-6 in CN2-29^(+/−)/MxCre^(+/−) mice following poly(I:C) injection. (G) Immunization with rVV-N25 reduced the number of macrophages in liver samples from CN2-29^(+/−)/MxCre^(+/−) mice and suppressed TNF-α and IL-6 production from macrophages (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051656#pone-0051656-g006" target="_blank">Figure 6G</a>). Significant relationships are indicated by a P-value.</p