Construction Of Non - Replicative Recombinant Vaccinia Virus Expressing Mouse GM - CSF And Preliminary Evaluation Of Its Adjuvant Effect

Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) is a cytokine secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts. As a a white blood cell growth factor, GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes, playing a great role in innate immunity. Since1990s, in various studies of tumor therapy, GM-CSF, as an adjuvant, expressed by viral vectors alone or in combination with some tumor targeting genes, suicide genes or immunomodulatory molecules coexpressed by viral vectors were widely used. It is thought to stimulate the recruit-ment, proliferation and maturation of DCs, enhancing antigen presentation to MHC class-I-and-â…¡-restricted T cells,mediating tumor destruction, and thereby enhancing immuno-therapeutic effects.Viral vectors can enhance the specific immune effects by expressing tumor associated antigens (TAAs). There are3main kinds of TAAs-tumor associated viral antigens, oncofetal antigens and mutated proto-oncogenes/tumor suppressors. But as tumor are derived from normal tissues, most TAAs cannont be regarded as "exogenous" antigens and recognized by the immune system. Howerer, tumor associated viral antigen is one of the most promising TAAs. Because they only exist n tumor tissues, with no expression in normal tissues. In this study we used Human Papilloma Virus (HPV)16E6E7protein, which is a specific tumor antigen described above. We had previously investigated the immune effects of a non-replicating vaccinia virus expressing HPV16E6E7in mice, and found a limited level of cellular mmune response. To further enhance its immune effects, viral vectors expressing;ome cytokines, which function as adjuvants, could be used.Therefore, we chose non-replicating vaccinia virus Tiantan Strain as a vector, to onstruct a recombinant non-replicating vaccinia virus expressing mouse GM-CSF lone. Then we made a primary evaluation of its influence on the specific cellular mmune reponse against HPV16E7, by covaccinating recombinant vaccinia virus xpressing HPV16E6E7, laying the foundation for studies using GM-CSF as an ajuvant for viral vector vaccines.We mainly got the following results:I. We successfully constructed4recombinant vaccinia Tiantan plasmids:â‘ pJSBH6LacZ expressing LacZ gene under the control of H6promoter. The other3plasminds were construced using this vector;â‘¡pJSB75GMCSFH6LacZ expressing mouse GM-CSF under the control of7.5promoter;â‘¢pJSB11E67H6LacZ expressing HPV16E6E7fusion gene under the control of11promoter;â‘£pJSB11E6775GMCSFH6LacZ expressing mouse GM-CSF under the control of7.5promoter and HPV16E6E7fusion gene under the control of11promoter;II. We successfully contructed rNTVJGMCSFLacZ, a recombinant non-replicating vaccinia virus Tiantan Strain expressing mouse GM-CSF. Identified by PCR and Western Blot, mGM-CSF gene was correctly inserted into rNTVJGMCSFLacZ, and the virus can express and secret mGM-CSF protein of bioactivity in vitro. Furthermore, the result of genetic stability experiments showed that mouse GM-CSF gene were stable in rNTVJGMCSFLacZ;â…¢. In vaccinated mice, we observed a trend that rNTVJGMCSFLacZ covaccinated with RVVJE67could promote the latter one to induce specific cellular response, while had no obvious enhancement of the humoral immune response.In conclusion, we used non-replicating vaccinia virus Tiantan Strain, of which we own the independent intellectual property rights, as a vector, to express GM-CSF with bioactivity. And then we preliminarily showed an enhancement in specific cellular immune reponse in rNTVJGMCSFLacZ vaccinated mice, which lays the foundation for future studies using GM-CSF as an ajuvant for viral vector vaccines.