Gallo Pasquale, Dharmapuri Sridhar, Nuzzo Maurizio, Maldini Daniele, Cipriani Barbara, Forni Guido, Monaci Paolo
Molecular and Cell Biology Department, I.R.B.M.P. Angeletti, Pomezia, Roma, Italy.
Int J Cancer. 2007 Feb 1;120(3):574-84. doi: 10.1002/ijc.22274.
The transforming rat HER2/neu oncogene (neu), when embedded in the genome of transgenic BALB/c (neuT) mice, provokes the development of an invasive carcinoma in each of their 10 mammary glands. We used the neuT mice model system to evaluate the immunization efficiency and the protective effect of intramuscular injection of adenovirus (Ad) and/or of DNA with electrostimulation (DNA+ES), both expressing the rat p185(neu) protein. A neu cDNA sequence, which exclusively contains codons preferred by highly expressed mammalian genes, was used in this study. This "optimized" cDNA displayed higher expression in cultured cells and greater cell-mediated response than the original gene when injected as DNA+ES. Ad expressing the optimized sequence (Ad5-neu.opt) induced a higher immune response, as measured by the frequency of IFN-gamma-secreting spleen cells and antibody titers. Different Ad/DNA combinations and immunization schedules confirmed the superiority of Ad5-neu.opt in inducing a strong Th1-skewed humoral and CD8(+) cell-mediated response. Two Ad5-neu.opt injections of 10(9) viral particles at week 10 and 12 were sufficient to induce the highest response, which persisted at detectable levels up to 33 weeks of age. Anti-Ad5 antibodies elicited by previous injections neutralized the effect of an additional Ad5-neu.opt immunization at week 19. A group, which received 3 injections of DNA+ES at week 23, 27 and 31, in addition to the 3 Ad injections at week 10, 12 and 19 showed an increased frequency of IFN-gamma(+), CD8(+) PBMC at week 25, which persisted at detectable levels till week 38. Ad5-neu.opt administration at 10 and 12 weeks of age had a significant impact on tumor progression. At 44 weeks, 40% of the mice were completely protected from tumors with a mean tumor of 3.8. In contrast, control mice developed 10 tumors and died by week 27. Vaccination blocked the tumor development at the atypical hyperplasia stage present at the time of treatment. Tumors developing at later times express reduced levels of rat p185(neu) protein.
将转化型大鼠HER2/neu癌基因（neu）嵌入转基因BALB/c（neuT）小鼠基因组后，会在其10个乳腺中的每个乳腺引发浸润性癌的发展。我们使用neuT小鼠模型系统来评估肌肉注射腺病毒（Ad）和/或经电刺激的DNA（DNA+ES）（二者均表达大鼠p185(neu)蛋白）的免疫效率和保护效果。本研究使用了一个neu cDNA序列，该序列专门包含高表达哺乳动物基因偏爱的密码子。当作为DNA+ES注射时，这个“优化的”cDNA在培养细胞中表现出更高的表达水平以及更强的细胞介导反应。通过分泌IFN-γ的脾细胞频率和抗体滴度测量发现，表达优化序列的腺病毒（Ad5-neu.opt）诱导产生了更高的免疫反应。不同的Ad/DNA组合和免疫方案证实了Ad5-neu.opt在诱导强烈的Th1偏向性体液和CD8(+)细胞介导反应方面的优越性。在第10周和第12周注射两次10⁹病毒颗粒的Ad5-neu.opt足以诱导出最高反应，该反应在33周龄时一直保持在可检测水平。先前注射引发产生的抗Ad5抗体中和了第19周额外一次Ad5-neu.opt免疫的效果。除了在第10周、第12周和第19周进行3次腺病毒注射外，在第23周、第27周和第31周接受3次DNA+ES注射的一组小鼠，在第25周时IFN-γ(+)、CD8(+)外周血单个核细胞的频率增加，该频率在第38周时一直保持在可检测水平。在10周龄和12周龄时给予Ad5-neu.opt对肿瘤进展有显著影响。在44周时，40%的小鼠完全免受肿瘤侵害，平均肿瘤大小为3.8。相比之下，对照小鼠长出了10个肿瘤，并在第27周死亡。疫苗接种在治疗时存在的非典型增生阶段阻断了肿瘤发展。在后期出现的肿瘤中，大鼠p185(neu)蛋白的表达水平降低。
