Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer（EC）. The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses （Ad-GP, Ad-Apoptin, Ad-EGFP） in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro. In 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assays, the growth of EC-109 cells was slightly inhibited by Ad-GP, Ad-Apoptin and Ad-EGFE However, Ad-VP induced a significant cytotoxic effect. Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro, detected by 4＇,6-diamidino-2-phenylindole（DAPI） or acridine orange and ethidium bromide staining. The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential（Aψm）, the release of eytochrome c and the activation of caspase-3, 6 and 7 in Ad-VP infected EC-109 cells. In contrast, all these assays show almost no effects of the recombinant adenoviruses on L02 cells. These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells. Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

Activity of T Cells Stimulated by Hemagglutinin-neuraminidase of Newcastle Disease Virus in vivo

To investigate the stimulated activity of T cells and the anti-tumor properties of hemagglutinin-neuraminidase（HN） of Newcastle disease virus（NDV） strain Changchun（NDVcc）, the expression of HN gene in hepatoma cells（human HepG-2 and mouse H22 cells） infected with the recombinant adenovirus（Ad-HN） was identified by Western blot analysis and flow cytometry. Sialidase activity of NDVcc HN expressed by Ad-HN was assayed by the periodate-resorcinol method. The in vivo anti-tumor effects of NDVcc HN were evaluated in the H22 solid tumor model. Regional lymph nodes of the mouse model treated with Ad-HN were removed to harvest T lymphocytes and evaluating the specific cytotoxicity of cytotoxic T lymphocyte（CTL） and natural killer（NK） cells by an L-lactate dehydrogenase（LDH） assay, in the mean time, the secretion of cytokines was analyzed by enzyme linked immunosorbent assays（ELISA）. The results show that NDVcc HN was effectively expressed by Ad-HN in HepG-2 and H22 cells. The sialidase activity assay showed that Ad-HN significantly reduced sialic acid level of the hepatoma cells compared with the cells infected the empty adenovirus vector（Ad-mock）. When treated with Ad-HN, the growth of subcutaneous H22 primary tumors in C57BL/6 mice was suppressed, and the mean mice survival increased. In addition, the treatment of Ad-HN elicited strong NK and CTL responses, and high levels of Th1 cytokines, such as IL-2 and IFN-γ. In conclusion, NDVcc HN effectively elicits T cell-mediate anti-tumor cytotoxicity via sialidase activity and may be a novel strategy for cancer immunotherapy.

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichia pastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.

Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

Hemagglutinin-neuramidinase（HN） is one of the most important surface structure proteins of the Newcastle disease virus（NDV）. HN not only mediates receptor recognition but also possesses neuraminidase（NA） activity, which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma（LLC） solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene（Ad-HN）, and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Th1/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4＋ and CD8＋ T cell populations and cytotoxic T lymphocyte（CTL） and killer cell response, but also skews the immune response toward Th1. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.

Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

Hemagglutinin-neuramidinase (HN ) ，纽卡斯尔疾病导出病毒的蛋白质，不是仅仅调停受体识别而且拥有 neuraminidase (NA ) 活动，劈开那些受体的一个部件的能力， N-acetylneuraminic 酸(NAcneu， sialic ) 。包括人，在哺乳动物的种类的这蛋白质有有趣的反肿瘤以及有免疫力的刺激性质，这被知道。在癌症基因治疗探索 HN 基因的使用，我们构造了表示 HN 蛋白质(vFV-HN ) 的一个 recombinant 家禽痘病毒并且在 vivo 并且在 vitro 把 recombinant 病毒的 theanti 肿瘤活动与野类型的家禽痘病毒(FPV ) 的作比较。这里，我们发现尽管 B16 房间对野类型的家禽痘病毒的基础细胞毒素的效果有点抵抗，有 vFV-HN 的感染引起了显著细胞毒素的效果并且，与 vFV-HN 使免疫的忍受肿瘤的老鼠的幸存显著地与独自与 FPV 使免疫的老鼠的幸存相比被增加。而且，有 vFV-HNelicited 的老鼠的免疫 B16 肿瘤特定的细胞毒素的 T 淋巴细胞(CTL ) 回答和在 vivo 的 bothCD4+ 和 CD8+ T 房间人口的同种细胞的扩大。另外，从老鼠的淋巴节点的 T 房间种牛痘， Th1 cytokine IL-2 和 IFN-v 高级的 vFV-HN 藏匿了，显示肿瘤房间的回归与 Th1 类型主导的有免疫力的回答有关。这些结果证明有 vFV-HN 的种痘可以是为癌症基因治疗的潜在的策略。

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines.In this study,we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen(HA) from the strains A/New Caledonia/20/99(H1N1)(pV1A5) and A/California/04/2009(H1N1)(pVEH1) ,respectively.After verifying antigen expression,the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model.Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies.HA-specific T-cells were also measured in IFN-γELISpot assays.The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD50 of mouse-adapted A/New Caledonia/20/99(H1N1) .Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses(P<0.05) than the pVAX1 control group.Additionally,the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100%compared with a 40%survival rate in the pVEH1 vaccinated group(P<0.05) .Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus(H1N1) ,while the pandemic influenza DNA vaccine only partially protects against this virus.