DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins

DNA vaccination was employed to study immune responses to hepatitis C virus (HCV) proteins. As an immunizing strategy, we studied immune responses of BALB/c (H-2d) and C57BL/6 mice (H-2b) to HCV genes delivered intramuscularly as a polycistronic construct capsid/E1/E2/NS2/NS3 (pRC/C-NS3) encoding 5 structural and nonstructural proteins. We also evaluated canarypox virus containing the same HCV genes as a means for potentiating immune responses to naked DNA. Our results indicate that mice that received a polycistronic pRC/C-NS3 with canarypox booster had enhanced antibody and cellular responses to HCV proteins. Immunodominant CD8(+) T cell responses to several HCV structural and nonstructural proteins, characterized by cytotoxicity and interferon (IFN)-gamma production or IFN-gamma production without significant cytotoxicity, were observed in both strains of mice. The combination of naked DNA with a nonreplicating canarypox booster encoding HCV polycistronic pRC/C-NS3 genes appears to diversify and enhance T cell responses to HCV proteins.     

丙型肝炎病毒核心基因免疫诱生细胞免疫应答研究

目的 研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫在诱生特异性细胞免疫应答中的作用。方法 将包含ＨＣＶ Ｃ基因片段的重组真核表达质粒ｐｃＣＮＡ ＨＣＶ Ｃ,在主宰其可以在小鼠骨髓瘤ＳＰ２／０（Ｈ－２^d）中表达之后,注射ＢＡＬＢ／ｃ小鼠股四头肌。ＥＬＩＳＡ法检测血清中抗体水平;^3Ｈ－ＴｄＲ掺入法测定免疫小鼠脾细胞特异性增殖能力;^51Ｃｒ释放法检测免疫小鼠细胞毒性Ｔ淋巴细胞（ＣＴＬｓ）体外杀伤功能。结     

Immune and non-immune responses to hepatitis C virus infection

The host innate and adaptive immune systems are involved in nearly every step of hepatitis C virus(HCV) infection. In patients,the outcome is determined by a series of complex host-virus interactions,whether it is a natural infection or results from clinical intervention. Strong and persistent CD8+ and CD4+ T-cell responses are critical in HCV clearance,as well as cytokineinduced factors that can directly inhibit virus replication. Newly available direct-acting antivirals(DAAs) are very effective in viral clearance in patients. DAA treatment may further result in the down-regulation of programmed death-1,leading to rapid restoration of HCV-specific CD8+ T cell functions. In this review,we focus on recent studies that address the host responses critical for viral clearance and disease resolution. Additional discussion is devoted to the prophylactic vaccine development as well as to current efforts aimed at understanding the host innate responses against HCV infection. Current theories on how the ubiquitin system and interferon-stimulated genes may affect HCV replication are also discussed.     

Immune responses in hepatitis C virus infection and mechanisms of hepatitis C virus persistence

Immune responses against hepatitis C virus (HCV) play a crucial role in the pathogenesis of chronic hepatitis C. HCV infection often persists and leads to chronic hepatitis and eventually cirrhosis. Accumulated data suggest that HCV proteins suppress host immune responses through the suppression of functions of immune cells, such as cytotoxic T lymphocytes, natural killer cells, and dendritic cells. They also suppress the type 1 interferon signaling system. The resulting insufficient immune responses against HCV lead to the sustained infection. The appropriate control of immune responses would contribute to the eradication of HCV and the improvement of hepatitis, but there are still many issues to be clarified. This review describes the scientific evidence to support these emerging concepts, and will touch on the implications for improving antiviral therapy.     

Host antibody response to viral structural and nonstructural proteins after hepatitis A virus infection.

Subgenomic hepatitis A virus (HAV) RNA sequences were translated in vitro to produce proteins representing the structural (P1) and nonstructural (P2 and P3) domains of the viral polyprotein. These proteins were used as antigens to detect the presence of antibodies in sera from acute and convalescent humans and an experimentally infected chimpanzee. All infected individuals tested had antibodies that recognized uncleaved P1 proteins as well as nonstructural proteins. Antibodies in sera from infected individuals recognized conformation-dependent epitopes that were sensitive to SDS and heat treatment. Time-course studies of the experimentally infected chimpanzee showed that antibodies to the HAV proteins were detectable between 24 and 31 days after infection and persisted for greater than 6 months. Human sera remained positive for antibodies to both structural and nonstructural antigens for at least 2 1/2 years. The data suggest that HAV nonstructural proteins could be used as serologic markers for HAV diagnosis and for evaluating field trials of inactivated vaccines.     

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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丙型肝炎病毒非结构蛋白与宿主细胞蛋白相互作用的研究进展

丙型肝炎病毒(hepatitis C virus,HCV)是继乙型肝炎病毒(hepatitis B virus,HBV)之后另一个导致慢性肝炎、肝硬化和肝癌的常见原因.目前HCV致病及致癌机制仍不清楚,也缺乏针对HCV有效的治疗方法及疫苗预防.肝炎病毒蛋白与受染细胞蛋白之间相互作用是肝炎发病机制研究的热点内容.近年研究...     

表达乙型肝炎核心抗原、干扰素γ重组质粒免疫小鼠诱导的免疫应答

本研究通过HBV核心基因DNA重组质粒联合干扰素(IFN)γ基因表达质粒同时免疫动物,了解经联合细胞因子DNA免疫后,免疫小鼠T细胞增殖、自然杀伤(NK)细胞杀伤活性及细胞因子释放等免疫效应.探讨联合IFN γ基因表达质粒用于DNA免疫的应用价值.     

Immune responses to hepatitis C and non-hepatitis C antigens in hepatitis C virus infected and HIV-1 coinfected patients

OBJECTIVE: To characterize immune phenotype and function in hepatitis C virus (HCV) infection in the presence and absence of HIV-1 infection. DESIGN: Cross-sectional comparison among controls (group A), patients with HCV infection (group B), HCV-HIV-1 coinfected patients (group C), coinfected patients receiving treatment for HIV-1 (group D), and untreated HIV-1 infected patients (group E). METHODS: Flow cytometric analysis for lymphocyte phenotypes, lymphocyte proliferation and cytokine production by ELISPOT. Results: HCV infected patients tended to have an increased percentage of activated (CD38, HLA-DR) CD8 cells (group A, 2+/-1.4%; group B, 6+/-3.9%; P=0.08). Proliferative responses to non-HCV antigens were comparable in group A and group B subjects. A greater proportion of group B patients had stimulation indices (SI) > 3 to the HCV protein NS3 compared to group C and D patients (67%, 0%, and 11% respectively; P < 0.003), but only two patients in group B had SI > or = 5. The SI to NS3 was significantly higher in group B patients [median, 4; interquartile range (IQR), 3-9) than in group C (median, 2; IQR, 1-3; P < 0.04) or group D (median, 1; IQR, 1-4; P < 0.009) patients. Plasma HCV RNA levels correlated directly with alanine aminotransferase levels (p, 0.52; P < 0.05) and inversely with the number of CD4 lymphocytes (rho, -0.55; P < 0.009) and proliferation to NS3 (p, -0.55; P < 0.009). CONCLUSIONS: Lymphocytes of HCV infected patients show weak proliferative responses to HCV antigens while responses to other antigens are preserved. Infection with HIV-1 potentiates this deficiency. Poor CD4 T cell responses to HCV are associated with and may determine the failure to control HCV propagation.     

Immune responses in hepatitis B virus infection

Hepatitis B virus infection is one of the most frequent causes of chronic liver disease worldwide. Even though a preventive vaccine is available, the search for a cure for chronically infected patients remains a high priority to reduce the morbidity and mortality from liver cirrhosis and hepatocellular carcinoma. This review summarizes the immune response in acute, self-limited and chronic hepatitis B; its differential effects on viral replication and liver injury; and prospects for immunotherapy.     

Sensitivity of hepatitis C virus to cyclosporine A depends on nonstructural proteins NS5A and NS5B

HCV re-occurs after liver transplantation and increases mortality. Cyclosporine, but not tacrolimus, has potent antiviral effects against HCV replication in cell culture. To determine the conditions, if any, under which HCV is susceptible to cyclosporine in vivo, we selected for cyclosporine-resistant mutant HCV in vitro. The resulting mutations were mapped to x-ray crystallographic structures and sequence databases. Mutations selected by cyclosporine were clustered in the nonstructural (NS) proteins NS5A and NS5B. Different sets of mutations in NS5A, paired with the same 2 NS5B mutations, conferred different levels of cyclosporine resistance when engineered back into the HCV replicon. Mutations in NS5B are structurally consistent with a proposed model of regulation of RNA binding by cyclophilin B (CyPB). These mutations also highlight a natural polymorphism between different HCV genotypes that correlates with the variation in response to cyclosporine A (CsA) noted in some clinical trials. Replicons engineered to have mutations in only NS5A (P < or = 0.0001) or only NS5B (P = 0.002) suggest that while both NS5A or NS5B variants alter cyclosporine susceptibility, NS5A has the largest effect. CONCLUSION: Preexisting sequence variation could alter the effect of cyclosporine on HCV in vivo.     

Immune modulation by the hepatitis C virus core protein

Hepatitis C virus (HCV) infection is currently the most important cause of chronic viral hepatitis in the world and one of the most frequent indications for liver transplantation. HCV uses different strategies to evade the innate and adaptive immune response, and this evasion plays a key role in determining viral persistence. Several HCV viral proteins have been described as immune modulators. In this review, we will focus on the effect of HCV nucleocapsid core protein in the function of immune cells and its correlation with the findings observed in HCV chronically infected patients. Effects on immune cell function related to both extracellular and intracellular HCV core localization will be considered. This review provides an updated perspective on the mechanisms involved in HCV evasion related to one single HCV protein, which could become a key tool in the development of new antiviral strategies able to control and/or eradicate HCV infection.