慢性HCV是一种隐性疾病,目前影响世界上大约3%的人口,在初次感染几十年后可能导致肝功能衰竭和癌症。然而,目前尚无可用于预防慢性HCV的疫苗.从急性缓解HCV感染的患者中,显然,强且广泛的细胞毒性T淋巴细胞(CTL)应答在HCV清除中是重要的。DNA疫苗是编码病原体抗原以诱导病原体特异性免疫应答的裸质粒DNA分子。它们的生产成本低廉,并且在动物和人类中具有出色的安全性。此外,DNA疫苗能够诱导强烈的CTL反应,使它们非常适合HCV疫苗。我们的目标是最大限度地提高疫苗接受者的机会,以一种新的抗原序列诱导广泛的T细胞应答,多抗原疫苗策略。我们已经产生了编码HCV基因型1a和1b非结构蛋白NS3/4a的共有序列的DNA质粒,NS4b,NS5a,和NS5b。恒河猴用于研究这些构建体的免疫原性。4只动物免疫3次,相隔6周,在每个抗原构建体1.0mg的剂量下,作为肌内注射,然后进行体内电穿孔,这大大增加了局部细胞对DNA的摄取。在免疫恒河猴免疫方案(PIR)后2周测量免疫应答,并显示对多种HCV非结构抗原的广泛应答,通过干扰素-γELISpot测量,每百万外周血单核细胞(PBMC)具有高达4680个斑点形成单位。此外,多参数流式细胞术通过细胞内细胞因子染色检测HCV特异性CD4+和CD8+T细胞反应,并检测HCV特异性CD107a+/GrzB+CD8+T细胞,表明与基线测量相比,2周PIR有抗原特异性细胞溶解反应。在最后的研究时间点,6周PIR,HCV特异性CD45RA-记忆样T细胞在外周血中保持可检测。本手稿中提供的数据支持以下观点:使用猕猴模型进行疫苗免疫原性研究可用于描述关键的抗HCV非结构性抗原细胞免疫反应,并支持基于DNA的预防性HCV疫苗的开发。
Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world\'s population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients\' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding
consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.