Earnest-Silveira L, Chua B, Chin R, Christiansen D, Johnson D, Herrmann S, Ralph S A, Vercauteren K, Mesalam A, Meuleman P, Das S, Boo I, Drummer H, Bock C-T, Gowans E J, Jackson D C, Torresi Joseph
Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia.
Department of Surgery, Austin Hospital, University of Melbourne, Australia.
J Gen Virol. 2016 Aug;97(8):1865-1876. doi: 10.1099/jgv.0.000493. Epub 2016 May 4.
An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.
针对丙型肝炎病毒（HCV）的有效免疫反应需要多特异性1类CD8 +和II类CD4 + T细胞的早期发育以及广泛的中和抗体反应。我们已经生产出了包含HCV 1a基因型的核心、E1和E2的哺乳动物细胞衍生的HCV病毒样颗粒（VLP），以产生这种免疫反应。在此，我们描述了HCV VLP的生化和形态学特征，并研究了HCV核心特异性T细胞对这些颗粒的反应。HCV VLP中的E1和E2糖蛋白形成非共价异二聚体，并与核心蛋白一起组装成浮力密度为1.22至1.28 g·cm-3的VLP。HCV VLP可用抗ApoE和抗ApoC进行免疫沉淀。在电子显微镜下，VLP具有异质性形态，大小范围为40至80 nm。HCV VLP表现出与鼠源树突状细胞的剂量依赖性结合，并且抗CD81抗体可阻断HCV VLP进入Huh7细胞。用从碘克沙醇梯度中纯化的HCV VLP对BALB / c小鼠进行疫苗接种可产生中和抗体反应，而对人源化MHC I类转基因小鼠进行疫苗接种则可产生HCV核心特异性CD8 + T细胞反应。此外，从慢性感染HCV 1a和3a基因型的患者血清中纯化的IgG可阻断HCV VLP与Huh7细胞的结合和进入。这些结果表明，我们的哺乳动物细胞衍生的HCV VLP可诱导体液免疫和HCV特异性CD8 + T细胞反应，这将对HCV预防性疫苗的开发具有重要意义。
