Tsoneva Desislava, Stritzker Jochen, Bedenk Kristina, Zhang Qian, Frentzen Alexa, Cappello Joseph, Fischer Utz, Szalay Aladar A
Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.
Genelux Corporation, San Diego Science Center, San Diego, CA, United States of America; Biocenter, University of Würzburg, Würzburg, Germany.
PLoS One. 2015 Sep 8;10(9):e0137573. doi: 10.1371/journal.pone.0137573. eCollection 2015.
Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.
血液检测是转化研究中监测溶瘤病毒疗法和其他生物疗法的必要、易于实施且低成本的替代方法。在此，我们评估了三种有潜力用作生物流体生物标志物的候选蛋白：来自大肠杆菌的两种葡萄糖醛酸酶（GusA）和葡萄球菌属RLH1的（GusPlus），以及来自王子高斯水母的荧光素酶（GLuc）。编码这些蛋白的三个基因分别在相同启动子的控制下插入痘苗病毒GLV-1h68基因组。三种产生的重组病毒用于感染培养中的肿瘤细胞和裸鼠体内的人肿瘤异种移植物。与主动分泌的GLuc不同，细胞质葡萄糖醛酸酶GusA和GusPlus仅因病毒介导的溶瘤作用而释放到上清液中。在受控的检测条件下，GusPlus在含有低至1 pg/ml GusPlus的样品中对酶活性的检测最为灵敏，其次是GusA（25 pg/ml）和GLuc（≥375 pg/ml）。出乎意料的是，尽管与GusPlus相比GusA具有较低的比活性，但注射了编码GusA病毒株的荷瘤小鼠血清中的底物转化率却明显高于GusPlus。这归因于GusA在血流中的半衰期分别比GusPlus和GLuc长3.2倍和16.2倍，因此是比其他两种酶更灵敏的病毒复制监测指标。由于表达的标记基因的酶活性与病毒滴度之间具有良好的相关性，我们得出结论，体液中生物标志物蛋白的量半定量地代表了感染肿瘤中的病毒量，这通过微光成像得到了证实。我们发现GusA是三种测试标志物中监测溶瘤病毒疗法最可靠的生物标志物。
