Identification of hepatitis C virus 1b-derived peptides recognized by both cellular and humoral immune systems in HCV1b(+) HLA-A24(+) patients.

Despite scientific advances, the therapeutic options for hepatitis C virus (HCV) are limited by poor response rates. HCV1b is particularly resistant to standard interferon therapy. The inhibition of the progression of chronic hepatitis and liver cirrhosis and the prevention of the occurrence and recurrence of hepatocellular carcinoma (HCC) are important and thus, there is a need for new therapeutic modalities for HCV1b infection. We, therefore, investigated highly immunogenic peptides and report in this study three novel candidate peptides (at positions 711-720 in envelope 2 protein, 885-893 in non-structural protein 2 and 1716-1724 in non-structural protein 4B) among 35 peptides of conserved regions of HCV1b proteins containing HLA-A24 binding motifs tested. Namely, HCV(711-720), HCV(885-893) and HCV(1716-1724) induced HLA-A24-restricted and peptide-specific cytotoxic T lymphocytes (CTLs) activity in peripheral blood mononuclear cells (PBMCs) from 7, 6 and 5 of 12 patients and also were recognized by plasma of 8, 5 and 7 of 12 HCV1b(+) patients, respectively. These results may provide new insight into the development of a peptide-based specific immunotherapy for HCV1b(+) HLA-A24(+) patients.     

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 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.     

Characterization of an immunologically conserved epitope from hepatitis C virus E2 glycoprotein recognized by HLA-A2 restricted cytotoxic T lymphocytes

BACKGROUND/AIMS: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins. METHODS: Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays. RESULTS: Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL. CONCLUSIONS: These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.     

HIV/HCV混合感染者与HCV感染者感染HCV基因型分析*

目的 使用二代测序技术检测广州地区人类免疫缺陷病毒（HIV）/丙型肝炎病毒（HCV）混合感染者和HCV感染者HCV基因型,了解本地区HCV基因型的流行特点。方法 在233例HCV感染者中,包括95例HIV/HCV混合感染者和138例HCV感染者,使用Illumina Miseq平台PE300模式对HCV Core和NS5B片段进行测序,采用生物信息学分析确定HCV基因型,对测序结果提示混合基因型感染的样本,使用NS5A基因型特异引物进行验证。结果 HIV/HCV混合感染者静脉吸毒感染构成比显著高于HCV感染者（77.7%对19.6% P<0.001）,HCV感染者输血感染的构成比显著高于HIV/HCV混合感染者（48.6%对3.2% P<0.001）;HIV/HCV混合感染者HCV Core基因片段平均数据量为（15456±6689） reads,HCV感染者为（14323±5321） reads,两组无显著差异（P>0.05）;HIV/HCV混合感染者HCV NS5B基因片段平均数据量为（16432±3467） reads,HCV感染者为（17611±5632） reads,也无显著差异（P>0.05）;本组228例（97.9%）HCV感染者为单一HCV基因型感染,HIV/HCV混合感染者HCV 6a型和3b型感染率显著高于HCV感染者（分别为47.4%对26.8%,P=0.001和13.7%对3.6%,P=0.005）,HCV 1b型感染率显著低于HCV感染者（20.0%对59.4%,P<0.001）;在233例入组的HCV感染者中,发现5例（2.1%）为混合基因型感染,其中4例为HIV/HCV混合感染者,1例为HCV感染者。结论 广州地区HIV/HCV混合感染者与HCV感染者感染HCV基因型构成比存在差异,其临床意义还需要探讨。     

The viral clearance in interferon-treated chronic hepatitis C is associated with increased cytotoxic T cell frequencies.

BACKGROUND/AIMS: Cytotoxic T lymphocytes have been demonstrated in peripheral blood and liver tissue of patients with chronic hepatitis C virus infection, but their significance for viral clearance is unknown. Therefore, we analyzed hepatitis C virus-specific cytotoxic T lymphocyte precursor frequencies in chronic hepatitis C virus carriers during interferon-alpha treatment. METHODS: Blood mononuclear cells or CD8+ T cells from HLA-A2 positive and negative patients and controls were analyzed in chromium-release assays using a panel of 18 synthetic peptides from the HCV core, E1 and NS4 antigens bearing HLA-A2 binding motifs. Specific cytotoxic T lymphocyte precursor frequencies were studied within CD8+ T cells derived from interferon-alpha-treated patients using a TNF-alpha-based ELISPOT assay and compared to viremia levels. RESULTS: T cells from 16 of 24 HLA-A2+ but none of the six HLA-A2- patients with chronic hepatitis C and six HLA-A2+ healthy controls lysed targets pulsed with peptide cocktails. Fine specificity revealed four very immunogenic epitopes in the core (C36-44, C132-140) and the envelope regions (E332-340, E363-372). Cytotoxic T lymphocyte precursor frequencies were prospectively analyzed in 11 interferon-alpha-treated HLA-A2+ hepatitis C virus patients. Four sustained and two transient therapy responders showed lower pretreatment viremia levels and significantly higher specific cytotoxic T lymphocyte precursor frequencies during viral clearance compared to five therapy non-responders and untreated controls. CONCLUSIONS: The quantitative induction of HLA-class I restricted responses by interferon-alpha could contribute to a beneficial outcome of hepatitis C virus infections. Furthermore, it appears that the balance between viral load and specific cellular immune responses is critical for successful viral clearance.     

Magnitude of CD8+ T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8⁺ T-cell responses and the clinical course of acute HCV infection.
Methods:  Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8⁺ T-cell responses was performed using an interferon-γ-based enzyme-linked immunospot assay using peripheral CD8⁺ T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b.
Results:  Five patients presented detectable HCV-specific CD8⁺ T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8⁺ T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8⁺ T-cells correlated with maximum serum alanine aminotransferase level during the course ( r  = 0.841, P  = 0.036).
Conclusions:  HCV-specific CD8⁺ T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8⁺ T-cell responses, but without development of antibody against HCV.     

Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection

Spontaneous resolution of hepatitis C virus (HCV) infection is a relatively infrequent event, and these individuals provide a unique opportunity to characterize correlates of protective immunity as an important first step in the development of vaccine candidates. The aim of this study was to directly and comprehensively enumerate HCV-nonstructural protein 3 (NS3) specific CD4(+) and CD8(+) T cells ex vivo from HLA diverse individuals who had been successful in spontaneously resolving HCV infection. We measured interferon gamma (IFN-gamma) production with an ELISPOT assay using magnetic bead-separated CD4(+) or CD8(+) T cells in response to autologous DCs that had been pulsed with 15mer per peptides overlapping by 11 amino acids and spanning all of the NS3 protein (150 total peptides). All subjects with spontaneously recovered HCV infection demonstrated vigorous and multispecific CD4(+) T-cell responses to NS3 peptides, and 6 of 10 subjects demonstrated CD8(+) T-cell responses. More importantly, we identified novel, previously unpredicted antigenic regions, which in most cases elicited high frequencies within a given individual. In conclusion, subjects who have spontaneously eradicated HCV infection up to 35 years earlier demonstrate persistent CD4(+) and CD8(+) T-cell responses specific to NS3. By providing a comprehensive screening of all potential T-cell epitopes contained in the NS3 region, our strategy defines the breadth of the T-cell response and identifies novel, unpredicted specificities.     

CD8+ cell responses to hepatitis C virus (HCV) in the liver of persons with HCV-HIV coinfection versus HCV monoinfection

OBJECTIVE: Cellular immune responses are difficult to detect in the peripheral blood of persons with chronic hepatitis C virus (HCV) infection. We sought to determine whether T cell responses were present in the liver of patients with human immunodeficiency virus (HIV) and HCV coinfection. METHODS: T cells were expanded from liver-biopsy samples from 10 patients coinfected with HIV and HCV (median CD4(+) cell count, 456 cells/mm(3)) and 8 patients infected with HCV alone. CD8(+) cell responses were detected by use of a modified enzyme-linked immunospot (ELISpot) assay with recombinant vaccinia virus, and CD4(+) cell responses were detected by use of ELISpot with recombinant HCV proteins core, nonstructural (NS) 3, and NS5. RESULTS: Intrahepatic CD8(+) cell responses to HCV were detected in 7 of 10 patients coinfected with HCV and HIV (median frequency, 638 spot-forming cells [sfc]/1 x 10(6) cells) and were similar to those observed in patients singly infected with HCV (7/8; median, 647 sfc/1 x 10(6) cells). Intrahepatic HCV-specific CD4(+) cell responses were also comparable in both groups and correlated with the intrahepatic CD8(+) cell responses (r=0.59; P=.03). CONCLUSION: HCV-specific CD8(+) cell responses are present in the liver of persons with chronic HCV infection even when they are coinfected with HIV; these correlate with intrahepatic HCV-specific CD4(+) cell responses.     

Clinical significance of in vitro replication-enhancing mutations of the hepatitis C virus (HCV) replicon in patients with chronic HCV infection

BACKGROUND: Mutations in nonstructural (NS) hepatitis C virus (HCV) proteins enhance replication in HCV-1a/b replicons. The prevalence of such mutations and their clinical significance in vivo are unknown. METHODS: Parts of HCV NS3 and NS4B-NS5B genes that included 31 in vitro replication-enhancing sites were sequenced for 26 patients with chronic HCV genotype 1 infection. RESULTS: Five patients showed specific mutations within NS3 at sites enhancing replication in the replicon. Those mutations were associated with a slower decrease in HCV RNA concentration during interferon (IFN)- alpha -based therapy (P = .007). Neither specific nor other mutations within NS3 and NS4B-NS5B were associated with baseline HCV RNA concentrations. Within NS5A, fewer mutations in the major HCV strain (P = .001) and increased quasi-species complexity (P = .02) and diversity (P = .02) correlated with increasing baseline HCV RNA concentrations. In silico analyses of NS3 protein structures suggested that the majority of observed mutations did not lead to major conformational changes. CONCLUSIONS: Specific mutations leading to enhanced replication in the replicon system were detected in 5 of 26 patients in vivo and were not associated with baseline HCV RNA concentrations but were associated with a slower decrease in HCV RNA concentration during IFN- alpha -based therapy. Quasi-species heterogeneity of NS5A correlated with baseline HCV RNA concentrations.     

Clinical responders to antiviral therapy of chronic HCV infection show elevated antiviral CD4+ and CD8+ T-cell responses

Summary. Chronic hepatitis C virus (HCV) infection is characterized by attenuated antiviral T‐cell responses, making their detection and characterization a technological challenge. The role and the dynamics of antiviral T‐cell responses during antiviral therapy are incompletely understood. To assess HCV‐specific T‐cell responses during antiviral therapy of genotype‐1‐infected patients, we adopted a flow cytometric approach to comprehensively evaluate virus‐specific CD4+ and CD8+ T‐cell proliferative responses against pools of genotype‐ and subtype‐specific serial, overlapping peptides spanning the entire virus. Studies in cross‐sectional cohorts of treatment‐naïve (TN) patients , early and sustained clinical virological responders (EVRs and SVRs) or clinical nonresponders (NRs) showed that this proliferative assay had significantly greater sensitivity in detecting HCV‐specific responses, compared with ex vivo cytokine flow cytometry. At the same time, it could be used to detect and quantify both CD4+ and CD8+ responses simultaneously. EVRs and SVRs showed significantly more HCV‐specific CD4+ and CD8+ responses, compared with either TN patients or NRs. This corresponded to a higher magnitude of responses as well as a greater breadth of reactivity with higher responses against the core/E1, NS3, NS4 and NS5b regions of the virus. Interestingly, both clinical responders and NRs showed higher cytomegalovirus‐specific CD4+ responses, compared with TN patients. These results demonstrate an association between clinically successful antiviral therapy and enhanced magnitude and breadth of antiviral responses. Moreover, the study demonstrates the clinical relevance of this flow cytometric proliferation assay system, in combination with an unbiased library of viral peptides, in evaluating the biology of antiviral T‐cell responses during infection and therapy.     

Identification of two new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from colorectal carcinoma-associated antigen PLAC1/CP1

Background

To explore the potential application of placenta-specific PLAC1/Cancer Placenta (CP) 1 antigen for immunotherapy in CRC patients, further identification of the cytotoxic T lymphocyte epitopes from this antigen is necessary.

Methods

We assessed the protein expression of PLAC1/CP1 using a tissue chip and immunochemistry staining in CRC samples. Simultaneously, we predicted four PLAC1/CP1-derived HLA-A*0201-restricted peptides by using reverse immunology methods. Peptide-specific CD8⁺ T cell responses were assessed by an IFN-γ release ELISPOT assay. Effector CD8⁺ T cells lyse HLA-A*0201 CRC cell line SW620 was detected in a granzyme-B release ELISPOT cytotoxicity assay.

Results

Our results indicated that PLAC1/CP1 was highly expressed in 56.7 % (55/97) of adenocarcinomas. PLAC1/CP1 protein expression was associated with CRC tumor differentiation, the tumor/node/metastasis stage, and lymph node metastasis. Two of four peptides showed high affinities in an HLA-A2 binding assay. In 66.7 % (6/9) of peripheral blood mononuclear cells of CRC samples with PLAC1/CP1 protein-positive expression, these two peptides, PLAC1/CP1 p41-50 (FMLNNDVCV) and PLAC1/CP1 p69-77 (HAYQFTYRV), were immunogenic in the induction of peptide-specific CD8⁺ T cell responses as assessed by an IFN-γ release ELISPOT assay. Furthermore, the generated effector CD8⁺ T cells could specifically lyse the PLAC1/CP1 HLA-A*0201 CRC cell line SW620 in a granzyme-B release ELISPOT cytotoxicity assay.

Conclusions

These results show that the PLAC1/CP1 antigen is a possible prognostic marker of CRC and that PLAC1/CP1 p41-50 and PLAC1/CP1 p69-77 are novel HLA-A*0201-restricted CD8⁺ T cell epitopes and potential targets for peptide-based immunotherapy in CRC patients.     

CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity

The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.     

Quantitative analysis of hepatitis C virus-specific CD8+ T cells in peripheral blood and liver using peptide-MHC tetramers

It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.     

Association between HCV amino acid substitutions and outcome of peginterferon and ribavirin combination therapy in HCV genotype 1b and high viral load

Background and Aim: We prospectively compared the sensitivity to interferon (IFN) and the efficacy of antiviral combination therapy with peginterferon (PEG‐IFN) and ribavirin for chronic hepatitis C virus (HCV) genotype 1b infection according to the amino acid sequences of the HCV core, E1, and NS5A regions reported to be associated with the outcome of antiviral therapy. Methods: A total of 107 patients with HCV genotype 1b were investigated. All patients received combination therapy with PEG‐IFN alpha‐2b and ribavirin. Amino acids 70 and 91 (core), 139 (E1), and 2209–2248 (NS5A) of HCV were analyzed by direct nucleotide sequencing. Results: The reduction in HCV RNA concentration at 24 h after a single administration of conventional IFN‐alpha and after the start of combination therapy was significantly less marked, and rates of complete early virologic response, end‐of‐treatment response, and sustained virologic response (SVR) were significantly lower (all P < 0.0001) in patients with glutamine at amino acid 70 (n = 29) than in those with arginine at that position (n = 70). We found no differences associated with the other amino acid positions. Amino acid 70 was an independent factor for the responses to the therapy in multivariate analysis. Conclusion: The identity of amino acid 70 of the HCV core region affected the sensitivity to IFN; patients with glutamine at amino acid 70 of HCV showed resistance to IFN. Consequently, it strongly affected the outcome of combination therapy with PEG‐IFN and ribavirin in Japanese patients with HCV genotype 1b.     

Hepatitis C virus‐specific CD8+ T cell frequencies are associated with the responses of pegylated interferon‐α and ribavirin combination therapy in patients with chronic hepatitis C virus infection

Aim: Hepatitis C virus (HCV)‐specific cytotoxic T lymphocytes (CTLs) play critical roles in elimination of the HCV‐infected hepatocytes. However, the mechanism of HCV elimination by pegylated interferon‐α (peg‐IFNα) plus ribavirin is not fully understood. We examined HCV‐specific CTL responses during this combination therapy. Methods: CD8+ T cells were isolated from 16 HCV infected patients treated by this combination therapy and were subjected to IFN‐γ enzyme‐linked immunospot (ELISPOT) assay. Results: The numbers of IFN‐γ spots against HCV Core or NS3 protein‐derived peptides in HCV patients before treatment were similar to those in healthy donors, and those in HCV patients significantly increased 4 weeks after the initiation of combination therapy. All HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses in pre‐treated patients were not associated with ALT levels and HCV viral loads of HCV patients before treatment. And those in pre‐treated patients were similar between sustained virologic responder (SVR) patients and non‐SVR patients. Significant increase of HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses between before and 4 weeks after this combination therapy were observed in SVR patients, but not in non‐SVR patients. Conclusions: These results demonstrated that significant increase of HCV‐specific CD8+ T cells at 4 weeks after the initiation of IFN treatment might be associated with the elimination of HCV. Our findings suggest that the reactivity against HCV Core and NS3 proteins‐derived peptides might be useful in predicting the clinical outcome of the combination therapy of peg‐IFNα and ribavirin.     

树突状细胞的非特异性免疫功能降低对HBV、HCV清除及细胞毒性T淋巴细胞应答无影响

目的 观察HBV、HCV感染者树突状细胞(DC)的非病毒特异性免疫功能状态与细胞毒性T淋巴细胞(CTL)免疫应答以及病毒清除的关系。方法 对25例成人慢性HBV和HCV合并感染者进行了间隔8年的两次调查、依据临床转归分为HBV和HCV均清除组(A组)14例、单独HCV清除者(B组)6例，单独HBV消除者(C组)3例，HBV和HCV均未清除者(D组)2例，对照组(N组)为同一地区健康献血员11例。体外分离培养DC，检测其表型及抗原摄取功能、刺激异体淋巴细咆增殖能力和4组感染者的CTL免疫应答情况。结果 B、C、D组与A组、N组比较，DC的非病毒特异性免疫功能降低，表现为CD86表达的降低、刺激异体淋巴细胞增殖的能力下降以及抗原摄取能力降低。A组对HBV和HCV的4条抗原表位多肽均有较高的CTL应签率(11／12)；B组对HCV的两条抗原表位多肽均有应答(5／5)，但无对HBV两条表位多肽均应签者、仅有1例对P2有反应；C组对HBV的抗原表位多肽均有应答，但无对两条HCV表位多肽均应答者；D组及N组对HBV或HCV所有实验多肽均无应答。结论 HBV和HCV的清除与病毒特异性的CTL应答相关。HBV和(或)HCV持续存在可能是导致DC功能异常的原因。     

HIV/HCV合并感染者和HCV单纯感染者NS3/4A蛋白酶、 NS5A抑制剂的天然耐药变异分析

目的 通过对抗病毒治疗前HIV/HCV合并感染者和HCV单纯感染者HCV NS3/4A蛋白酶、NS5A抑制剂相关耐药位点进行检测,探讨上述患者天然耐药变异的差异。方法 收集2016年1月—2020年1月在广州医科大学附属市八医院住院或门诊就诊的246例HIV/HCV合并感染者和HCV单纯感染者的血清标本,使用Illumina二代测序平台进行测序,比较已在中国获批准的NS3/4A蛋白酶、NS5A抑制剂相关耐药变异在两组患者的差异,纳入分析的药物包括阿舒瑞韦/达拉他韦（ASV/DCV,基因1b型）,艾尔巴韦/格拉瑞韦（EBR/GZR,基因1b型）和格卡瑞韦/哌仑他韦（GLE/PIB,泛基因型）。符合正态分布的计量资料两组间比较采用t检验,不符合正态分布的计量资料两组间比较采用Mann-Whitney U检验。计数资料两组间比较采用χ²检验或Fisher精确检验。结果 本研究纳入的246例患者的血清样本中,239例（97.2%）成功进行PCR扩增并测序,包括102例HIV/HCV合并感染者和137例HCV单纯感染者。对ASV/DCV和EBR/GZR相关耐药变异分析结果提...