表达HCV包膜蛋白的两种重组腺相关病毒疫苗的制备及免疫原性分析

Two HCV Vaccines Based on Adeno-associated Virus Vectors Elicited Durable and Effective Immune Responses in Mice

重组腺相关病毒载体(rAAV)可在动物体内高水平地持久表达外源基因,本研究采用两种rAAV载体(rAAV1与rAAV2)构建了表达丙型肝炎病毒中国分离株包膜糖蛋白(E1E2)的载体疫苗并以之免疫小鼠,分别采用免疫荧光证实其表达与总抗体,用HCV假病毒系统检测其中和抗体水平,用ELISpot分析其细胞免疫应答,结果表明:rAAV1-E1E2重组载体疫苗单针免疫激发的体液应答明显高于rAAV2-E1E2,rAAV1-E1E2单针注射后3个月可在肌肉组织中检出E2蛋白表达及特异性T细胞应答。上述结果提示HCV重组腺相关病毒载体疫苗单针免疫可引起明显持久的体液与细胞免疫应答。

Recombinant adeno-associated viruses （rAAV） vectors have been shown to mediate long-term transgene expression in mice and nonhuman primates. We have adapted viral vector system based on two rAAV vectors, namely rAAV1 and rAAV2. We have generated rAAV vectors expressing the envelope glycoprotein （El and E2） derived from Chinese HCV patient （genotype lb） and used these to immunize BALB/c mice. We detected the total antibody titer by IFA and neutralizing antibody（nAb） using in vitro HCV neutralizing assays based on HCV pseudotyped particles. Furthermore, IFN-7 ELISpot assay was used to assess the T cellular response against HCV at 12 weeks after rAAV1-EIE2 immunization. We also analyzed HCV envelope glycoprotein expression in muscle of rAAV1-EIE2 immunized mice. Our data showed： （i） rAAV1 directed long term expression of HCV genes in mice; （ii） immunized intramuscularly with a single dose of rAAV elicited durable and effective immune responses in mice; and（iii） Moreover, rAAV1 EIE2 induced higher total antibody and nAb titers than rAAV2-E1E2 did. These data suggest that rAAV1 vectors could stimulate robust, durable, and effective immune responses against HCV.