Human cytotoxic T-cell responses against Epstein-Barr virus nuclear antigens demonstrated by using recombinant vaccinia viruses.

The potentially pathogenic effects of infection with Epstein-Barr virus (EBV), a B-lymphotropic agent with cell growth-transforming potential, are contained in healthy virus carriers by virus-specific cytotoxic T-lymphocyte (CTL) surveillance. The target antigens against which such CTL responses are directed are yet undefined, but the antigens probably derived from one or more of the EBV "latent" proteins constitutively expressed in virus-transformed B cells. We have analyzed target specificity of CTL responses from two EBV-immune donors that are preferentially reactive against autologous cells transformed with type A but not with type B virus isolates. Coding sequences for four EBV latent proteins with allelic polymorphism between A and B virus types--namely, the EBV nuclear antigens (EBNAs) EBNA 2, EBNA 3a, EBNA 3c, and EBNA leader protein--have been introduced into vaccinia virus vectors under control of vaccinia promoter P7.5 and used to express relevant EBNA proteins in appropriate target cells. Thus the CTL response from one donor has been mapped to type A EBNA 2 protein and from a second donor to type A EBNA 3a protein. Thereafter, a series of recombinant vaccinia viruses were constructed that carried specific internal deletions within the EBNA 2 type A coding sequence; by using these vectors, the above EBNA 2 type A-specific CTL response was shown to be directed against an epitope within a 100-amino acid fragment near the N terminus of the protein. This work clearly shows human CTL recognition of virus-coded nuclear antigens in the EBV system; moreover, it establishes an experimental approach that can be extended to all EBV latent proteins and to the more common CTL responses that cross-react against type A and type B virus isolates.     

Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens

Hepatitis C virus (HCV) chronically infects 2%‐3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV‐recombinant hepatoma cells formed large solid‐mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV‐specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti‐HCV‐specific vaccines based on two fundamentally different attenuated pathogen vaccine systems—attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV‐recombinant tumours when used in a therapeutic vaccination trial, replication‐competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non‐replicating nondisseminating adenovirus. Our results demonstrate a model with anti‐tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV‐associated cancers.     

Lymphocyte proliferative responses to recombinant hepatitis C virus antigens in patients with chronic hepatitis C

The purpose of the present study was to analyse lymphocyte proliferative responses to recombinant hepatitis C virus (HCV) antigens in chronic hepatitis C. Four recombinant peptides derived from the NS3, core, E1 and E2/NS1 regions of the HCV genome were used as antigens in lymphocyte proliferative responses. Forty-two patients, classified into various sub-groups, and 17 healthy control subjects were tested and the specific response was expressed as a stimulation index. Responses were analysed with alanine aminotransferase (ALT) level and histological diagnosis. NS3- and core-antigen specific responses in all patient groups were significantly higher than in the healthy control group. E1- and E2/NS1-antigen-specific responses in the patient group with ALT levels exceeding 100 IU/L were significantly higher than those in other patient groups. Histological diagnosis was not correlated to the intensity of the core- and NS3-specific responses. E1- and E2/NS1-antigens induced significantly elevated responses in patients with chronic active hepatitis and liver cirrhosis compared with results in the healthy control group and in patients with chronic persistent hepatitis. In conclusion, the significantly elevated responses to core- and NS3-antigens may be related to HCV infection and such responses to E1- and E2/NS1-antigens could be related to the severity and activity of the disease.     

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.     

A recombinant adenovirus encoding hepatitis C virus core and E1 proteins protects mice against cytokine-induced liver damage

Hepatitis C virus (HCV) infection has a strong tendency to evolve to chronicity despite up-regulation of proapoptotic cytokines in the inflamed liver. The mechanisms responsible for persistent viral replication in this inflammatory environment are obscure. It is conceivable that viral replication would be facilitated if the infected hepatocytes are rendered resistant to cytokine-induced cytotoxicity. In this study, we investigated if an adenovirus encoding HCV core and E1 (RAdCE1) could reduce liver cell injury in different in vivo models of cytokine-mediated hepatotoxicity in mice. We show that RAdCE1 markedly attenuates hepatocellular apoptosis and the increase in serum transaminase levels after concanavalin A (con A) challenge. This protective effect is accompanied by an inhibition of nuclear translocation of nuclear factor kappaB (NF-kappaB); reduced expression of inducible nitric oxide synthase (iNOS); decreased hepatic messenger RNA levels of chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10); and abrogation of liver leukocyte infiltration. RAdCE1 also causes a reduction in serum transaminase levels and inhibits hepatocellular apoptosis in mice given tumor necrosis factor (TNF)-alpha plus D-galactosamine. In conclusion, HCV structural antigens can protect liver cells against the proapoptotic effects of proinflammatory cytokines. The antiapoptotic status of infected liver cells may represent a mechanism favoring viral persistence. Our findings also suggest that, in chronic hepatitis C, the burden of hepatocellular damage mainly affects noninfected liver cells.     

HBV抗原基因修饰树突状细胞疫苗体外抗HBV的作用

目的：探讨腺病毒载体介导HBV抗原基因修饰的树突状细胞(DCs)诱导抗HBV特异性CTL反应方法：制备携带HBsAg、HBeAg和HBcAg基因的3种重组腺病毒Ad-HBs,Ad-HBe,Ad- HBc,分别转染自脐带血体外诱导培养的DCs,观察腺病毒转染DCs效率和DCs中HBV抗原的表达;混合淋巴细胞反应(MLR)测定HBV抗原基因修饰DCs刺激同种异体T淋巴细胞增殖能力;乳酸脱氢酶释放法检测特异性CTL细胞对HepG_222．1．5靶细胞的杀伤能力．结果：腺病毒载体能够高效介导HBV三个抗原基因在DCs中表达,90%以上DCs表达示踪基因EGFP,且DCs细胞形态完整：感染后72 h HBsAg和HBeAg含量分别为0．919和0．328(吸光度A值)．MLR实验显示,HBV抗原基因修饰DCs仍然具有刺激同种异体T细胞的增殖能力,Ad-HBs转染DCs组、Ad-HBe转染DCs组、Ad-HBc转染DCs组和未转染DCs组之间刺激T细胞的增殖水平无明显差异(F=1．194,P=0．389);在E：T比例为2：1,10：1和25：1时,Ad-HBs转染DC组、Ad-HBe转染DCs组和Ad-HBc转染DCs组对HepG_222．1．5细胞的杀伤率均明显高于未转染DCs组(P＜0．001);以Ad- HBc转染DC组对HepG_222．1．5细胞杀伤率最高．结论：HBV抗原基因修饰DCs疫苗具有刺激同种异体T细胞增殖能力,同时能增强抗HBV特异性CTL反应的能力,可能发展为一种新型抗病毒疫苗．     

丙型肝炎病毒核心蛋白致癌作用的研究

研究表明丙型肝炎病毒(HCV)核心(core)蛋白可能具有潜在的直接致癌作用。应用腺相关病毒载体介导的病毒重组技术,成功观察到HCV核心蛋白诱发的肝癌形成。 1.材料和方法:(1)HCV core区基因克隆及测序:按文献[1]方法进行,各种工具酶等购自美国Promega公司。②含HCV core区重组腺相关病毒的制备:将腺相关病毒载体质粒PSSHG(西安交通大学医学院解剖教研室构建惠赠)和HCV     

表达丙型肝炎病毒非结构蛋白3的重组腺病毒免疫小鼠的研究

目的构建表达HCV非结构蛋白3（NS3）的重组腺病毒RAd／NS3，探讨其免疫小鼠后诱导机体产生特异性免疫应答的能力。方法PCR扩增编码NS3蛋白（329～935位氨基酸）的基因片段，定向克隆至重组腺病毒AdEasy-1系统的穿梭质粒pAdTrack-CMV上，与腺病毒基因组质粒pAdEasy-1共转化BJ5183菌，利用细菌内同源重组获得重组腺病毒质粒，转染293细胞，以包装、扩增重组腺病毒。将重组腺病毒以1×10^8 pfu剂量经皮下注射免疫BALB／c小鼠，并以100μg重组质粒pRC／NS3经肌内注射途径免疫小鼠为对照。ELISA法检测免疫小鼠血清抗体水平，^51 Cr释放法检测免疫小鼠细胞毒性T淋巴细胞体外杀伤功能。结果重组腺病毒经皮下免疫小鼠后可刺激机体产生较强的特异性体液免疫应答和细胞免疫应答，且免疫效果强于重组质粒pRC／NS3组，初次免疫后2周，前者20只小鼠抗-HCV全部阳转，而后者10只小鼠仅3只阳转，未观察到明显的不良反应。结论表达HCV NS3抗原的重组腺病毒载体疫苗能够诱导机体产生有效的体液和细胞免疫应答。     

Hepatitis C virus-specific cytotoxic T cell response restoration after treatment-induced hepatitis C virus control

Hepatitis C virus(HCV)-specific cytotoxic T cell(CTL) response plays a major role in viral control during spontaneous infection resolution. These cells develop an exhausted and pro-apoptotic status during chronic onset, being unable to get rid of HCV. The role of this response in contributing to sustained viral response(SVR) after anti-HCV is controversial. Recent studies show that after successful interferon-based anti-HCV treatment, HCV traces are still detectable and this correlates with a peak of HCV-specific CTL response activation, probably responsible for maintaining SVR by subsequent complete HCV clearing. Moreover, SVR patients’ serum is still able to induce HCV infection in na?ve chimpanzees, suggesting that the infection could be under the control of the immune system after a successful treatment, being transmissible in absence of this adaptive response. At least theoretically, treatmentinduced viral load decrease could allow an effective HCV-specific CTL response reestablishment. This effect has been recently described with anti-HCV interferonfree regimes, based on direct-acting antivirals. Nevertheless, this is to some extent controversial with interferon-based therapies, due to the detrimental immunoregulatory α-interferon effect on T cells. Moreover, HCV-specific CTL response features during anti-HCV treatment could be a predictive factor of SVR that could have clinical implications in patient management. In this review, the recent knowledge about the role of HCV-specific CTL response in the development of SVR after anti-HCV treatment is discussed.     

Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response

Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. CONCLUSION: Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis.     

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection

BACKGROUND/AIMS: It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS: Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS: An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS: Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.     

Induction of murine neonatal tolerance against Graves' disease using recombinant adenovirus expressing the TSH receptor A-subunit

Graves' disease is a common organ-specific autoimmune disease. The identity of its autoantigen, the TSH receptor (TSHR), was established and used to induce a typical animal model. A-subunit, the shed portion of TSHR, either initiates or amplifies the autoimmune response of the thyroid gland, thereby causing Graves' disease in humans. In the present study, we investigate the effect of the TSHR A-subunit on the induction of murine neonatal tolerance for the development of Graves' disease. Female BALB/c mice were pretreated with different doses of adenovirus expressing the A-subunit of TSHR (Ad-TSHR289) by either ip or im injection within the first 24 h after their birth. Graves' disease was induced after the animals reached adulthood. Nearly all mice pretreated with the high dose of Ad-TSHR289 failed to develop TSHR antibodies, detected by the TSH-binding inhibition assay, hyperthyroidism, and thyroid follicular hyperplasia. The mice preimmunized im with the lower doses of Ad-TSHR289 developed a relatively low level of TSH-binding inhibition and the low incidence of hyperthyroidism. Accordingly, the percentages of splenic CD4+CD25+/CD4+ and CD25+Foxp3+/CD4+ Treg cells were increased in mice pretreated with the high dose of Ad-TSHR289. Taken together, our data strongly indicate that the immunotolerance against Graves' disease could be induced in neonatal mice using a specific TSHR antigen in a high dose either by ip or im injection, preventing the development of Graves' disease.     

Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.

BACKGROUND & AIMS: The prospective comparison of patients with acute hepatitis C virus (HCV) who spontaneously clear the virus with those who cannot achieve viral elimination and progress to chronic hepatitis offers the unique opportunity to analyze natural mechanisms of viral elimination. METHODS: We studied the HCV-specific CD4(+) T-cell response in 38 patients with acute HCV and correlated the clinical course with the antiviral immune response. The individual HCV-specific T-cell response was assessed in a proliferation assay ((3)H-thymidine uptake) and an enzyme-linked immunospot assay. RESULTS: Patients were classified according to their clinical course and pattern of CD4(+) T-cell responses in 3 categories: first, patients mounting a strong and sustained antiviral CD4(+)/Th1(+) T-cell response who cleared the virus (HCV RNA-negative; n = 20); second, patients who were unable to mount an HCV-specific CD4(+) T-cell response and developed chronic disease (n = 12); and third, patients who initially displayed a strong CD4(+) T-cell response and eliminated the virus (HCV PCR-negative) but subsequently lost this specific T-cell response (n = 6). The loss of the HCV-specific CD4(+) T-cell response was promptly followed by HCV recurrence. CONCLUSIONS: The results indicate that a virus-specific CD4(+)/Th1(+) T-cell response that eliminates the virus during the acute phase of disease has to be maintained permanently to achieve long-term control of the virus. The induction and/or maintenance of virus-specific CD4(+) T cells could represent a promising therapeutic approach in HCV infection.     

Perspectives for a vaccine against hepatitis C virus

There is no vaccine for HCV and the only available treatment, IFNalpha alone or in combination with ribavirin, has proven efficacious in less than 50% of patients. Given that approximately 200 million chronic HCV infections have been estimated worldwide, there is a pressing need to develop vaccination strategies aimed at preventing and possibly eradicating HCV infection. However, several major practical and scientific problems arise in designing an HCV vaccine. First, HCV is only readily detected as RNA by PCR. Second, the only species that can be infected by HCV are humans and chimpanzees. Third, the virus does not replicate efficiently in vitro. Fourth, some viral proteins have very high mutability. Last, there is little information on correlates of immunity. Although an ideal vaccine should protect from infection, in that it should elicit sterilizing immunity, this is quite an ambitious goal in the PCR era. In the case of HCV, where acute HCV infection is a very limited health problem and infection can only be assessed by PCR, a more realistic goal might be to look for vaccines capable of protecting from chronic infection. We have preliminary evidence in chimpanzees that an HCV vaccine consisting of recombinant envelope proteins can elicit antibodies and inflammatory CD4+ T cell responses which can prevent chronic infection in the majority of vaccinees. Although the scientific and clinical challenges that need to be addressed are still substantial, advances in recombinant protein technology, novel adjuvants, and DNA vaccines, will be key in developing strategies to generate protective immunity against chronic HCV infection.     

Medicinal plants against hepatitis C virus

Hepatitis C virus(HCV)is a global health concern which is responsible for most of the liver diseases.Currently,there is no vaccine available for prevention of HCV infection due to the high degree of strain variation.The current standard of care is a combination of pegylated interferonαwith ribavirin and boceprevir/telaprevir.This treatment was partially effective and had significant side effects.Hence,there is a need to develop new antiviral agents that interfere with different stages of the HCV life cycle.Recent advances in the understanding of both the cellular and molecular mechanisms of HCV replication have provided the basis for novel therapeutic strategies.Several hundred plant species and their phyto-constituents have been isolated for screening against HCV,and some have been shown to have great medicinal value in preventing and/or ameliorating viral diseases in pre-clinical and clinical trials.This review summarizes medicinal plants and their phytochemicals which inhibit different stages of HCV life cycle and discuss their potential use in HCV therapy.     

丙型肝炎病毒重组蛋白的免疫保护性

目的 研究两种HCV重组蛋白联合免疫小鼠所诱导的免疫应答及免疫保护作用.方法 用两种重组蛋白HCV-T和HCV-第一高变区多片段重组融合蛋白(F4HVR1)分别与联合免疫BALB/c小鼠,共免疫3次,用ELISA方法测定血清特异性抗体;末次免疫后14 d处死5只小鼠,分离小鼠脾细胞.体外检测IFN-γ、IL-4和行CTL杀伤实验;剩余的小鼠背部皮下注射1.0×106个SP2/O-NS3细胞,观察其保护作用.组间均数差异采用LSD-t检验.结果 与PBS组相比,用HCV-T和HCV-F4HVR1联合免疫诱导了针对HCV-F4HVR1的特异性的IgG(t=3.815,3.762,P＜0.05)、高水平的HCV-NS3特异性的CTL效应(t=3.971,P＜0.05)和高水平的IL-4(t=3.813,3.426,3.671,P＜0.05)和IFN-γ(t=3.512,3.417,P＜0.05)的分泌.结论 用HCV-T和HCV-F4HVR1联合免疫小鼠可诱导出高水平的特异性体液免疫和细胞免疫,能有效地预防SP2/O-NS3细胞的攻击.