Yazdanian Maryam, Memarnejadian Arash, Mahdavi Mehdi, Sadat Seyed Mehdi, Motevali Fatemeh, Vahabpour Rouhollah, Khanahmad Hossein, Siadat Seyed Davar, Aghasadeghi Mohammad Reza, Roohvand Farzin
Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, IR Iran.
Virology Department, Pasteur Institute of Iran, Tehran, IR Iran.
Hepat Mon. 2013 Oct 23;13(10):e14178. doi: 10.5812/hepatmon.14178. eCollection 2013.
A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses.
To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses.
Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals.
Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. "Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses".
Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines.
用于丙型肝炎病毒（HCV）感染的理想疫苗应引发强烈的以Th1为主导的细胞免疫反应。在缺乏Th1特异性佐剂的情况下，通过蛋白质抗原进行免疫通常会诱导Th2型和较弱的细胞免疫反应。
比较卡介苗（BCG）与作为经典佐剂的非离子共聚物普朗尼克F127（F127）在将HCV核心蛋白（HCVcp）配制成诱导Th1免疫反应的候选疫苗中的佐剂效果。
在阿拉伯糖诱导型（araBAD）启动子控制下，携带相应基因的pIVEX2.4a-core载体在大肠杆菌BL21-AI菌株中表达N端带有His标签的HCVcp（1-122），并通过应用次氮基三乙酸（Ni-NTA）层析进行纯化。在第0、3、6周，用100μl免疫原（F127 + HCVcp或BCG + HCVcp；5μg HCVcp/小鼠/剂量）或对照制剂（PBS、BCG、F127）对小鼠尾根部进行皮下免疫。比较每组免疫动物中IgG的总量和亚型，以及细胞免疫反应（增殖、体内细胞毒性T淋巴细胞[CTL]和针对一种强优势的H2-d限制性、CD8 +表位肽、HCVcp的核心39-48；RRGPRLGVRA的干扰素-γ/白细胞介素-4酶联免疫斑点试验）。
通过蛋白质印迹法确认了预期大小（21 kDa）左右的核心蛋白的表达和纯化。与接种HCVcp + F127的动物相比，接种HCVcp + BCG的小鼠表现出显著更高的淋巴细胞增殖和干扰素-γ产生，但细胞裂解水平较低（在CTL试验中分别为45%对62%）。此外，与接种HCVcp + BCG的动物相比，接种HCVcp + F127的小鼠中总的抗核心IgG和IgG1水平显著更高，表明F127在刺激体液免疫和以Th2为主导的免疫反应方面具有相对更高的效力。
结果表明，HCVcp + BCG诱导了适度的CTL以及混合的Th1/Th2免疫反应，具有更高水平的细胞增殖和干扰素-γ分泌，表明在基于HCVcp的亚单位疫苗中配制时，卡介苗可能具有更好的效果。
