Tokushige K, Wakita T, Pachuk C, Moradpour D, Weiner D B, Zurawski V R, Wands J R
Molecular Hepatology Laboratory, Massachusetts General Hospital, Cancer Center, Charleston, MA 02129, USA.
Hepatology. 1996 Jul;24(1):14-20. doi: 10.1002/hep.510240104.
Hepatitis C virus (HCV) is a major worldwide cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The development of vaccines against HCV have been complicated by the high variability of the envelope region, and it is likely that the cellular immune responses to viral structural proteins may be important for eradicating persistent viral infection. Recently, it was reported that the injection into muscle cells of plasmids encoding viral genes resulted in the generation of strong cellular immune responses. We constructed vectors that express the highly conserved HCV core gene. In this regard, the pHCV 2-2 construct contained the entire HCV core region and pHCV 4-2 contained both the 5' noncoding region and the core gene. Cellular expression of HCV core protein was assessed following transfection into human and murine cell lines, and higher intracellular levels of the 21-kd core protein were observed with pHCV 2-2. These HCV core DNA constructs were used to immunize BALB/c mice and produced low-level anti-HCV core humoral immune responses. To assess cytotoxic T-lymphocyte (CTL) activity generated in vivo, a cloned syngeneic SP2/O myeloma cell line constitutively expressing HCV core protein was established and inoculated into BALB/c mice to produce growth of plasmacytomas. Strong CTL activity was generated because the tumor size and weight in pHCV 2-2-immunized mice were remarkably reduced compared with mice injected with mock DNA. Spontaneous CTL activity was also exhibited by splenocytes in an in vitro cytotoxicity assay. These investigations demonstrate that plasmid constructs expressing HCV core protein generate strong CTL activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents.
丙型肝炎病毒（HCV）是全球范围内急性和慢性肝炎、肝硬化及肝细胞癌的主要病因。针对HCV的疫苗研发因包膜区域的高度变异性而变得复杂，对病毒结构蛋白的细胞免疫反应可能对根除持续性病毒感染很重要。最近，有报道称将编码病毒基因的质粒注入肌肉细胞可产生强烈的细胞免疫反应。我们构建了表达高度保守的HCV核心基因的载体。在这方面，pHCV 2-2构建体包含整个HCV核心区域，而pHCV 4-2包含5'非编码区和核心基因。将其转染入人和鼠细胞系后评估HCV核心蛋白的细胞表达，发现pHCV 2-2可使细胞内21-kd核心蛋白水平更高。这些HCV核心DNA构建体用于免疫BALB/c小鼠，并产生了低水平的抗HCV核心体液免疫反应。为评估体内产生的细胞毒性T淋巴细胞（CTL）活性，建立了一个组成性表达HCV核心蛋白的克隆同基因SP2/O骨髓瘤细胞系，并将其接种到BALB/c小鼠体内以产生浆细胞瘤生长。由于与注射空载体DNA的小鼠相比，pHCV 2-2免疫的小鼠肿瘤大小和重量显著减小，因此产生了强烈的CTL活性。在体外细胞毒性试验中，脾细胞也表现出自发的CTL活性。这些研究表明，表达HCV核心蛋白的质粒构建体在体内和体外评估中均能产生强烈的CTL活性，有望成为抗病毒药物。
