Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Arboviruses and Viral Hemorrhagic Fevers (National Reference Laboratory), Pasteur Institute of Iran, Tehran, Iran.
IUBMB Life. 2019 Oct;71(10):1645-1652. doi: 10.1002/iub.2109. Epub 2019 Jul 12.
Hepatitis C virus (HCV) is a major health problem all over the world. Among HCV proteins, nonstructural protein 3 (NS3) is one of the most promising target for anti-HCV therapy and a candidate for vaccine design. DNA vaccine is an efficient approach to stimulate antigen-specific immunity but the main problem with that is less immunogenic efficiency in comparison with traditional vaccines. Several approaches have been applied to enhance the immunogenicity of DNA. Recently, bacteria-derived substances are considered as one of the most attractive adjuvants for vaccines, which among them, Listeriolysin O (LLO) of Listeria monocytogenes is a toxin with an extremely immunogenic feature. We investigated detoxified form of LLO gene as genetic adjuvant to modulate NS3 DNA vaccine potency. Immunogenic truncated NS3 gene sequence of HCV (1095-1380aa) and detoxified LLO gene region (5-441aa) were amplified by PCR and cloned into the pcDNA3.1 plasmid separately. The expression of recombinant proteins (pc-NS3, pLLO) was confirmed in HEK293T cell line by western blotting. BALB/c mice models received three doses of different formula of plasmids in two-week intervals and two weeks after the final immunization, the immune responses were evaluated by specific total antibody level, lymphocyte proliferation, cytotoxicity, and cytokine levels assays. To evaluate in vivo cytotoxic activity, tumor challenge was performed. The recombinant plasmids were successfully expressed in mammalian cell line, and coadministration of pc-NS3 with pLLO induced the highest titer of total IgG against NS3 antigen compared with other controls. Determination of IgG subclasses confirmed the efficient increase in mixed responses with Th1 dominancy. Furthermore, significant levels of cytokines (p < .05) and lymphocyte proliferation responses (p < .05) indicated the superiority of this regimen. The findings may have important implication for LLO gene application as genetic adjuvant in immune response against HCV.
丙型肝炎病毒（HCV）是全世界的一个主要健康问题。在 HCV 蛋白中，非结构蛋白 3（NS3）是抗 HCV 治疗的最有希望的靶标之一，也是疫苗设计的候选者。DNA 疫苗是刺激抗原特异性免疫的有效方法，但与传统疫苗相比，其主要问题是免疫原性效率较低。已经应用了几种方法来增强 DNA 的免疫原性。最近，细菌衍生的物质被认为是疫苗最有吸引力的佐剂之一，其中李斯特菌溶素 O（LLO）是一种具有极强免疫原性特征的毒素。我们研究了李斯特菌 LLO 基因的解毒形式作为调节 NS3 DNA 疫苗效力的遗传佐剂。通过 PCR 扩增 HCV 的免疫原性截断 NS3 基因序列（1095-1380aa）和解毒 LLO 基因区域（5-441aa），并分别将其克隆到 pcDNA3.1 质粒中。Western blot 证实了重组蛋白（pc-NS3、pLLO）在 HEK293T 细胞系中的表达。BALB/c 小鼠模型在两周的间隔内接受三种不同配方的质粒，在最后一次免疫接种后两周，通过特异性总抗体水平、淋巴细胞增殖、细胞毒性和细胞因子水平测定来评估免疫反应。为了评估体内细胞毒性活性，进行了肿瘤挑战。重组质粒在哺乳动物细胞系中成功表达，与 pc-NS3 共给药诱导针对 NS3 抗原的总 IgG 滴度最高，与其他对照组相比。IgG 亚类的测定证实了 Th1 优势的混合反应的有效增加。此外，细胞因子（p < .05）和淋巴细胞增殖反应（p < .05）的显著水平表明该方案具有优势。这些发现对于 LLO 基因作为 HCV 免疫反应的遗传佐剂的应用可能具有重要意义。
