Molecular Approaches To Vaccine Development For Hepatitis C

本实验室建立了一种在昆虫细胞中将丙型肝炎病毒结构蛋白高效组装成丙型肝炎病毒样颗粒的系统。这些非传染性的丙型肝炎病毒样颗粒与从丙型肝炎病毒感染者中分离的假定的病毒粒子具有相似的形态、血清学和生物物理特性。与重组亚单位疫苗相比，类丙型肝炎病毒颗粒的病毒蛋白可能以天然的类病毒粒子构象存在，因此在诱导保护性体液和细胞免疫反应方面可能更具优势。丙型肝炎病毒低密度脂蛋白的体液和细胞免疫原性已在小鼠和狒狒模型中得到证实。此外，我们还研究了丙型肝炎病毒蛋白在黑猩猩体内的免疫原性和保护性免疫诱导作用。用丙型肝炎病毒Lp或丙型肝炎病毒Lp佐剂ASO1B免疫黑猩猩，每组两只。在8个月的4次免疫后，所有动物都产生了强烈的丙型肝炎病毒特异性细胞免疫反应，包括针对CORE、E1和E2的干扰素-伽马和IL-2、CD4和CD8 T细胞以及增殖淋巴细胞反应。两组黑猩猩都接种了100CID50的丙型肝炎病毒CG1B疫苗。用丙型肝炎病毒攻击后，一只黑猩猩在攻击后第三周和第四周出现一过性病毒血症，病毒滴度较低(10E3-4拷贝/毫升)。另外三只黑猩猩感染了更高水平的病毒血症(10E4-5拷贝/毫升)，但它们的病毒水平在10周后变得无法量化(&lt；1000拷贝/毫升)。在丙型肝炎病毒攻击后，所有四只黑猩猩的外周干扰素-γT细胞和增殖反应均显著增加，肝内T细胞对丙型肝炎病毒结构蛋白的反应也显著增加。这些T细胞反应与丙型肝炎病毒RNA水平的下降相一致。与之相比，4只幼年黑猩猩感染了相同的丙型肝炎病毒，其中3只出现了持续感染，病毒血症在10E5-6拷贝/毫升范围内。我们的研究表明，在黑猩猩模型中，丙型肝炎病毒-LP免疫诱导了强烈的丙型肝炎病毒特异性细胞免疫反应，并对丙型肝炎病毒攻击具有部分保护作用。 经过我们目前的纯化程序，丙型肝炎病毒-LP的纯度仅为10%左右，如果要将该方法开发为临床候选疫苗，还需要显著提高纯度。基于我们对病毒颗粒中丙型肝炎病毒包膜蛋白结构特征的了解，我们在E2蛋白的N端设计了一个组氨酸X6标签，使该标签能够在表面暴露。组氨酸序列不应干扰丙型肝炎病毒的结构组装，并有助于亲和纯化。该标记的E2在丙型肝炎病毒脂蛋白背景下的表达证明了该方法的可行性，并正在进行进一步的工作以改进丙型肝炎病毒脂蛋白的纯化。 为了提高和扩大丙型肝炎病毒LP的免疫原性，我们报道了一种新型嵌合丙型肝炎病毒样颗粒的产生，该颗粒携带具有重要保护性免疫诱导作用的T细胞表位的丙型肝炎病毒非结构蛋白序列。合成了6个与人类病毒清除相关的高度保守的丙型肝炎病毒CD8+T细胞非结构表位，并将其与丙型肝炎病毒脂蛋白E2蛋白的C末端融合。E_2 C-端信号肽序列的疏水性降低，证实为多表位序列。在昆虫细胞中扩增、纯化、生化、免疫学和电子显微镜鉴定了携带非结构多表位的嵌合型丙型肝炎病毒蛋白。在表达人HLA-A2.1分子的AAD转基因小鼠中检测了嵌合的丙型肝炎病毒LP的免疫原性。与丙型肝炎病毒蛋白类似，嵌合型丙型肝炎病毒蛋白诱导针对核心和包膜的丙型肝炎病毒特异性体液和细胞免疫反应。此外，嵌合的丙型肝炎病毒-LP可激发针对非结构表位的强大T细胞反应。嵌合的丙型肝炎病毒-LP多表位策略显著改善和扩大了丙型肝炎病毒的免疫原性，有望成为人类抗丙型肝炎病毒感染的候选疫苗。此外，我们正在将丙型肝炎病毒-脂蛋白的方法与其他免疫形式，如质粒DNA，在主增强方案中相结合。

A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Chimpanzees, two in each group, were immunized with HCV-LP or HCV-LP adjuvant ASO1B. After four immunizations over an eight-month period, all animals developed strong HCV-specific cellular immune response including IFN-gamma and IL-2, CD4 and CD8 T-cell and proliferative lymphocyte responses against core, E1 and E2. The chimpanzees in both groups were challenged with a 100 CID50 of HCV CG1B inoculum. Upon challenge with HCV, one chimpanzee developed transient viremia with low HCV RNA titers (10E3-4 copies/ml) in the third and fourth weeks post-challenge. The three other chimpanzees became infected with higher levels of viremia (10E4-5 copies/ml) but their viral levels became unquantifiable (< 1000 copies/ml) 10 weeks post-challenge. After HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral IFN-gamma T-cell and proliferative responses as well as the presence of intrahepatic T-cell response against the HCV structural proteins. These T-cell responses coincided with the fall in HCV RNA level. In comparison, four nave chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with viremia in the range of 10E5-6 copies/ml. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. The purity of HCV-LP after our current purification procedure is only about 10%, which needs to be significantly improved if this approach is going to be developed clinically as a vaccine candidate. Based on what we know about the structural features of HCV envelope proteins in the viral particles, we engineered a histidineX6 tag at the N-terminus of E2 protein, which allows surface exposure of this tag. The histidine sequences should not interfere with the structural assembly of HCV and would facilitate affinity purification. Expression of this tagged E2 in the context of HCV-LP demonstrated feasibility of this approach and further work is being performed to improve HCV-LP purification. In an effort to improve and broaden the immunogenicity of HCV-LP, we report the generation of novel chimeric hepatitis C virus-like particles carrying HCV nonstructural protein sequences with T-cell epitopes important for induction of protective immunity. Six highly-conserved HCV CD8+ T-cell nonstructural epitopes associated with viral clearance in humans were synthesized as a polytope construct and fused to the C-terminus of the E2 protein of HCV-LP. Hydrophobicity of the signal peptide sequence in the C-terminus of E2 was reduced and the polytope sequence was confirmed. Chimeric HCV-LP carrying the HCV nonstructural polytope were amplified in insect cells, purified, and characterized biochemically, immunologically and by electron microscopy. The immunogenicity of the chimeric HCV-LP was tested in AAD transgenic mice expressing the human HLA-A2.1 molecule. Similar to HCV-LP, chimeric HCV-LP induced HCV-specific humoral and cellular immune responses against the core and envelope. In addition, chimeric HCV-LP elicited robust T-cell responses against the nonstructural epitopes. The chimeric HCV-LP polytope strategy substantially improved and broadened the immunogenicity of HCV-LP and holds promise as a vaccine candidate against HCV infection in humans.In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen.