背景

2015年10月t-vec（talimogene laherparepvec），作为第二代i型溶瘤性单纯疱疹病毒，已被批准作为美国和欧洲的首个应用于临床的溶瘤病毒药物。单纯疱疹病毒作为溶瘤病毒应用于肿瘤治疗展现出其一定的优势和潜力。单纯疱疹病毒裂解感染的肿瘤细胞并释放相关抗原，活化抗原提呈细胞。更重要的是，溶瘤病毒治疗诱导活化肿瘤反应性免疫细胞，使逆转免疫抑制性的肿瘤微环境成为可能。而研究表明过继性抗肿瘤免疫疗法的效果可能取决于固有免疫反应的支持，那么联合过继性免疫治疗与溶瘤病毒治疗两种方法来治疗肿瘤可能会产生更有效的抗瘤反应。

方法

在表达卵清蛋白（ova）抗原的黑色素瘤细胞b16-ova基础上，转导单纯疱疹病毒受体（hvem），构建可被单纯疱疹病毒感染的新型细胞系b16-ova-hvem，体内外验证其目的基因的表达。验证细胞构建成功后，探究应用ⅱ型溶瘤单纯疱疹病毒（ohsv2）与过继性回输ot-1小鼠淋巴细胞联合治疗小鼠黑色素瘤的效果。b16-ova-hvem荷瘤c57bl/6小鼠，成瘤后分为四组，每组5只小鼠，分别给予ohsv2治疗、ot-1小鼠淋巴细胞回输治疗和联合治疗，荷瘤未治疗组为对照组。治疗后测量肿瘤大小，观察生存期；流式细胞术检测联合治疗组和对照组两组小鼠脾淋巴细胞中cd4+、cd8+t、nk、treg细胞和髓系来源的抑制性细胞（mdsc）比例变化。

结果

质粒dna测序和酶切鉴定结果均显示成功构建含hvem的质粒；荧光显微镜下可观察到筛选后细胞发绿色荧光；流式细胞术检测筛选后细胞gfp阳性率为98.3%；rt-pcr验证结果显示筛选后细胞含有目的基因hvem；ohsv2在感染复数（moi）为0.1、0.5和1的情况下均能明显感染筛选出的细胞；体内验证实验结果显示，ohsv2瘤内治疗后抑瘤效果显著（p<0.001）；流式检测结果显示ohsv2治疗后小鼠外周血中cd4+、cd8+t细胞比例明显高于对照组（p<0.01），而mdsc比例显著低于对照组（p<0.01）。动物实验结果显示，ohsv2与ot-1小鼠淋巴细胞联合治疗效果优于单独治疗，联合组小鼠脾淋巴细胞中cd4+、cd8+t细胞比例高于对照组，而mdsc、nk细胞和treg细胞比例低于对照组。

结论

成功构建b16-ova-hvem细胞系，其既可被ot-1小鼠t细胞特异性识别又可被单纯疱疹病毒感染。我们证明了联合过继性免疫细胞回输与溶瘤病毒治疗可以提高治疗效果，激发淋巴效应细胞增殖，为肿瘤特异性免疫治疗和溶瘤病毒联合治疗的研究奠定基础。

background

t-vec (talimogene laherparepvec), as the second generation of oncolytic herpes simplex virus (hsv-1), has been approved as the first application in the united states europe for clinical oncolytic virus drugs in october 2015. herpes simplex virus as a oncolytic virus used in cancer treatment has shown its certain advantages potential. herpes simplex virus infection can lead to the release of tumor-associated antigen (taa) from infected dead tumor cells activation of antigen-presenting cells. more importantly, the infection of oncolytic viruses changes the immune cell composition of the tumor, making it possible to overcome the immunosuppressive state in the tumor microenvironment. studies have shown that the effect of adoptive anti-tumor immunotherapy may depend on the support of the innate immune response, then the combination of adoptive immunotherapy oncolytic virus therapy to treat the tumor may produce more effective anti-tumor response.

methods

the aim of this study was transducting the herpes virus entry mediator (hvem) into mouse b16-ova cells, a melanoma cell line expressing ovalbumin on the cell membrane to construct a oncolytic herpes simplex virus permissive mouse melanoma cell line b16-ova-hvem.  then verify the expression of the target gene in vitro in vivo. to investigate the effect of type 2 oncolytic herpes simplex virus (ohsv2) we combined with adoptive ot-1 mouse lymphocytes therapies in the treatment of melanoma in mice. b16-ova-hvem tumor bearing c57bl/6 mice were divided into four groups, each group of 5 mice were given ohsv2 treatment, ot-1 mouse lymphocyte transfer therapy combination therapy, the control group was untreated. the percentage of cd4 +, cd8 + t, nk, treg cells myeloid-derived inhibitory cells (mdsc) in the spleen lymphocytes of the combined treatment group the control group were measured by flow cytometry variety.

results

we have successfully constructed the plasmid expressing hvem, which verified by plasmid dna sequencing enzyme digestion. green fluorescent can be viewed in the ed cells using fluorescence microscope. gfp-positive rate of the ed cells was 98.3% through flow cytometry. most of the ed cells were obviously infected by ohsv2 when moi were 0.1, 0.5 1.ohsv2 could significantly inhibit the growth of b16-ova-hvem in vivo (p<0.001). the percentage of cd4+ cd8+ t cells in the peripheral blood of the mice treated with ohsv2 were significantly higher than that of control group while the percentage of mdsc was lower (p<0.01). the results of animal experiments showed that the combination of ohsv2 ot-1 lymphocytes was better than that of the control group. the proportion of cd4 + cd8 + t cells in splenic lymphocytes of mice was higher than that of the control group, while the proportion of mdsc nk cells treg cells were lower than that of the control group.

conclusion

the new b16-ova-hvem cell line was successfully constructed, which can not only be specifically killed by the cd8+ t cell from ot -1 mice but also can be infected by herpes simplex virus. we demonstrated that combined adoptive immunotherapy oncolytic virus therapy can improve the therapeutic effect stimulate proliferation of lymphoid effector cells. this new cell line lays the foundation of the combination of tumor specific immune therapy oncolytic virus therapy.