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.

Regulation of noncoding region for expression of Sendai virus hemagglutinin-neuraminidase (HN) gene

Hemagglutinin-neuraminidase (HN) protein was expressed in COS-7 cells, indicating that the expression of HN protein driven by SRα promoter is higher than that driven by chicken β-actin promoter. Moreover, with 5’ noncoding region (NCR) of HN gene, the expression was enhanced. Northern blotting demonstrated that this phenomenon was caused by the difference of HN mRNA transcription. To know the regulatory function of 5’ NCR, HN gene 5’ NCR was replaced by 5’ NCR of keratin gene or cytochrome P-450 gene and the 3’ NCR was deleted by site-directed mutagenesis. By using CAT gene as a reporter, S1 nuclease assay was done to quantitate the HN mRNA transcript in the COS-7 cells co-transfected with the reporter and mutated plasmids, indicating that 5’ NCR is non-specific to the enhancement of HN protein expression, and the 3’ NCR also has a special regulatory function.

Antitumor Effects of Newcastle Disease Virus Hemagglutinin-neuraminidase Used as a Molecular Adjuvant

Gene therapy is a potentially powerful tool used in cancer therapy. The strength of immune responses in- duced by some strategies is usually low, therefore, the development of agents capable of enhancing these responses is highlighted. The authors investigated the potential of an approach based on the hemagglutinin-neuraminidase（HN） of Newcastle disease virus（NDV） as a potential immune adjuvant. It was found that recombinant adenovirus（Ad） in- fected SGC7901 cells expressing HN exhibited both hemagglutinin（HA） and neuraminidase（NA） activities. It was demonstrated that administration of HN induced higher levels of the effector cytokines TNF-a, IFN-a and IFN-y and increased natural killer（NK） cell activity. Based on the therapeutic tumor model, the results show that the administra- tion of HN with Apoptin led to improved survival and tumor suppression. In conclusion, this study indicates that HN stimulates innate immune responses to make the activity of NK cells increased, which highlights the potential adju- vant activity of HN in cancer gene therapy.

Induction of Effective Antitumor Immune Response by Combined Administration of hIL-18 and NDV HN

To analyze the antitumor potential and mechanism of action of simultaneous Newcastle disease virus （NDV） hemagglutinin-neuraminidase（HN） and human interleukin 18（hIL-18） gene transfer in C57BL/6 mice with H22 hepatoma,the mouse model with H22 hepatoma was established in C57BL/6 mice, and the antitumor effects of the combined application of NDV HN and hIL-18 were evaluated in vivo. The results show that the growth of established tumors in mice immunized with adenovirus（Ad）-HN in conjunction with Ad-hIL-18 was significantly inhibited compared with that in mice immunized with Ad-HN, Ad-hIL-18 alone, or the empty vector（Ad-mock）. Furthermore, the immunization of mice with Ad-HN in conjunction with Ad-hIL-18 elicited strong natural killer activity and H22 tumor-specific cytotoxic T lymphocyte（CTL） responses in vivo. In addition, T cells from the lymph nodes of mice immunized with Ad-hIL-18 or Ad-HN＋Ad-hIL-18 secreted high levels of the Th1 cytokine IL-2 and interferon-γ （IFN-γ）, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with NDV HN together with hIL-18 may be a novel and powerful strategy for cancer immunotherapy.