Prediction and identification-based prediction of Chinese hepatitis C viral-specific cytotoxic T lymphocyte epitopes

Cytotoxic T lymphocytes (CTLs) play a critical role in the host immune response to infection by the Hepatitis C Virus (HCV). In the current study, a number of HCV CTL epitopes that represent the HLA polymorphisms found in the majority of Chinese people were predicted based on genomic and bioinformatic approaches. The predicted epitopes were evaluated for validity by examining the peptide-binding affinity for MHC class I molecules, the stability of peptide-MHC complexes, and frequencies of IFN γ-positive T cells. Among the predicted epitope peptides, HLA-A2 restricted epitopes [NS4B (1793-1801) SMMAFSAAL] and HLA-B7 restricted epitopes [P7 (774-782) AAWYIKGRL] were able to induce high frequencies of IFN γ-producing T cells, and the specific CTLs for other epitopes were not detected in peripheral blood lymphocytes from patients with HCV. Moreover, NS4B (1793-1801) exhibited high binding affinity for HLA-A2 molecules, and its stability of peptide-MHC class I complexes was sufficient, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. Primary analyses of the immunogenicity of predicted epitopes, such as in the current study, will contribute to the future design of an efficient vaccine that will be able to induce vigorous, sustainable, and broad HCV-specific CTL responses for the Chinese population.     

多CTL表位DNA疫苗诱导特异性CTL应答的研究

目的:构建多CTL表位DNA疫苗诱导特异性CTL应答。方法:将编码两个HCVCTL表位(H-2d)的基因序列插入真核表达载体pcDNA3.1中,构建多CTL表位DNA疫苗。用其免疫BALB/c小鼠后,以经不同CTL抗原肽冲击的P815细胞(H-2d)作为靶细胞,进行CTL杀伤试验。结果:多CTL表位DNA疫苗可诱导机体产生针对其编码的两种HCVCTL表位的特异性CTL免疫应答,且总的特异性CTL杀伤效应增强。结论:通过天然侧翼氨基酸相连的多个CTL表位为编码序列构建的多CTL表位DNA疫苗,不但能诱导机体产生针对各个CTL表位的特异性CTL应答,而且各特异性CTL应答的联合作用可显著增强总的CTL杀伤效应。     

丙型肝炎病毒CTL表位的HLA-A2限制性及其免疫学效应研究

目的 研究前期鉴定的5条丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位的HLA-A2限制性及其免疫学效应.方法 基于T2胞株,采用MHC-肽复合物稳定性试验研究前期鉴定的HCV特异性CTL表位与HLA-A2分子的亲和力;采用细胞内细胞因子染色(intracellular cy-tokine staining,ICS)和ELISPOT研究七述HLA-A2限制性CTL表位体外刺激HLA-A2刚性外周血单个核细胞(PBMC)产生肽特异性CTL的效应;采用CTL杀伤试验研究上述肽特异性CTL杀伤靶细胞(负载相同肽的T2细胞)的效应.结果 前期研究鉴定的5条CTL表位肽中,NS4b_78(SMMAF-SAAL)和NS5a_367(TVSSALAEL)与HLA-A2分子有高亲和力(FI1);ELISPOT结果显示NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)可诱导高水平的分泌IFN-γ的效应细胞[分别为(60±6)SFC/10~4PBMC vs(4±1)SFC/10~4PBMC,P<0.001;(10±3)SFC/10~4PBMC vs(2±1)SFC/10~4PBMC,P<0.001];ICS结果证实这两条肽刺激HLA-A2阳性PBMC后,在CD8~+T细胞中产生了高百分比的CD8~+IFN-γ~+T[分别为(2.33±0.22)%vs(0.05±0.01)%,P<0.001;(0.36±0.06)%vs(0.03±0.01)%,P<0.001];并且,肽特异性CTL可特异性杀伤负载相同肽的T2细胞.结论 NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)受HIA-A2限制,并具有较好的免疫原性.     

丙型肝炎病毒CTL表位的HLA-A2限制性及其免疫学效应研究

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/104 PBMC vs (4±1 ) SFC/104 PBMC, P < 0.01 ; (10 ± 3 ) SFC/104 PBMC vs (2±1 ) SFC/104 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8 + IFN-γ+ T cells in total CD8+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.     

丙型肝炎病毒非结构区5个细胞毒性T细胞表位的鉴定

目的 采用T细胞表位预测软件结合体外实验鉴定丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位.方法 采用T表位预测软件Rankpep预测HCV特异性CTL表位,选择候选CTL表位加以合成;用候选CTL表位肽分别刺激HCV感染者以及健康志愿者的外周血单个核细胞(PBMC),采用酶联免疫斑点试验(ELISPOT)检测PBMC中肽特异性分泌IFN-γ的斑点形成细胞(spots forming cells,SFC)的水平,采用细胞内细胞因子染色(intracellular cytokine staining,ICS)检测PBMC中肽特异性IFN-γ+CD8+T细胞的水平.结果 用5条候选CTL表位肽[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)和NS5a 367(TVSSALAEL)]分别刺激10个HCV感染者和2个健康者的PBMC后,健康者的PBMC不产生IFN-γ而7个HCV感染者的PBMC产生IFN-γ;HCV感染者的PBMC中肽特异性分泌IFN-γ的细胞的频率为(5-36)SFC/105 PBMC,肽特异性IFN-γ+CD8+T细胞占总CD8+T细胞的百分比为0.02%～0.25%.结论 ELISPOT结果和ICS结果证实5条肽NS3 450、NS3 594、NS4b 78、NSSa 416和NS5a 367为全新的HCV特异性CTL表位.     

Differential processing of HLA A2-restricted HIV type 1 cytotoxic T lymphocyte epitopes

Cytotoxic T lymphocytes (CTLs) play a key role in the control of persistent viral infections. Differences in the quality of this cellular immune response influence the long-term outcome of such infections, but the factors that determine which virus-derived peptide epitopes are targeted by CTLs remain poorly understood. Here, we examine the antigen-processing requirements of three human leukocyte antigen (HLA) A*0201-restricted HIV-1 CTL epitopes. Each of these three peptides appears to be generated by a distinct proteolytic pathway, despite presentation on the cell surface in association with the same HLA class I molecule. Presentation of the commonly immunodominant SLYNTVATL (HIV-1 p17 Gag; residues 77-85) epitope was unaffected by inhibition of the proteasome with lactacystin, but was dependent on the presence of the beta-subunit LMP7. These findings are consistent with emerging data on the complexity of peptide epitope generation, and suggest that differences in antigen processing might contribute to patterns of CTL recognition in vivo.     

HCV结构区DNA疫苗诱发小鼠特异性细胞免疫反应的研究

目的通过 HCV结构区 DNA疫苗直接免疫小鼠 ,诱发其体内特异性细胞介导的免疫反应的研究 ,为 HCVDNA疫苗的研制打下基础。方法用重组的 p BK- CMV质粒体外转染小鼠骨髓瘤细胞 SP2 / 0 ,建立了能体外表达 HCV结构区蛋白的 SP2 / 0 D细胞系 ,分别用 SP2 / 0和 SP2 / 0 D细胞攻击用空质粒或 p BK- CMV质粒免疫过的 Balb/ c小鼠 ,通过肿瘤形成、肿瘤重量、肿瘤组织淋巴细胞的浸润以及不同免疫组小鼠血清中 IL - 2和 IFN -γ含量来观察小鼠体内 T淋巴细胞的活性。结果用 SP2 / 0和 SP2 / 0 D攻击空质粒或 p BK- CMV质粒免疫过的小鼠 15 d后 ,SP2 / 0 D攻击的 p BK- CMV质粒免疫鼠肿瘤重量 ( 0 .9± 0 .12 ) g明显低于空白质粒免疫组和 SP2 / 0接种组 ( P<0 .0 0 1) ,且该免疫组小鼠血清中 IL- 2和 IFN- γ的浓度明显高于其它免疫组 ;此外 ,用 SP2 / 0 D攻击的 p BK- CMV免疫鼠 ,肿瘤病理切片中可见明显的 T淋巴细胞浸润。结论重组的 HCV结构区 DNA疫苗 ( p BK- CMV)能诱导小鼠体内特异性 T淋巴细胞反应 ,HCV DNA疫苗可能为临床疾病的治疗和预防提供一种新的途径。     

HCV结构区DNA疫苗诱发小鼠特异性细胞免疫反应的研究

目的 :通过HCV结构区DNA疫苗直接免疫小鼠 ,诱发其体内特异性细胞介导的免疫反应的研究 ,为HCVDNA疫苗的研制打下基础。方法 :用重组的pBK CMV质粒体外转染小鼠骨髓瘤细胞SP2 0 ,建立了能体外表达HCV结构区蛋白的SP2 0D细胞系 ,分别用SP2 0和SP2 0D细胞攻击用空质粒或pBK CMV质粒免疫过的Balb c小鼠 ,通过肿瘤形成、肿瘤重量、肿瘤组织淋巴细胞的浸润以及不同免疫组小鼠血清中IL 2和IFN γ含量来观察小鼠体内T淋巴细胞的活性。结果 :用SP2 0和SP2 0D攻击空质粒或pBK CMV质粒免疫过的小鼠 15d后 ,SP2 0D攻击的pBK CMV质粒免疫鼠肿瘤重量〔(0 9± 0 12 )g〕明显低于空白质粒免疫组和SP2 0接种组 (P <0 0 0 1) ,且该免疫组小鼠血清中IL 2和IFN γ的浓度明显高于其它免疫组 ;此外 ,用SP2 0D攻击的pBK CMV免疫鼠 ,肿瘤病理切片中可见明显的T淋巴细胞浸润。结论 :重组的HCV结构区DNA疫苗 (pBK CMV)能诱导小鼠体内特异性T淋巴细胞反应 ,HCVDNA疫苗可能为临床疾病的治疗和预防提供一种新的途径     

T lymphocyte responses against hepatitis B virus-related hepatocellular carcinoma induced by adenovirus vaccine encoding HBx

HBx is an oncogenic tumor-associated antigen and is dominantly expressed in hepatitis and hepatoma tissues, the induction of active cellular responses against HBx should be a promising approach for the treatment of hepatitis B virus-related hepatocellular carcinoma. The present study was designed to test whether a replication-defective adenovirus vaccine expressing HBx antigen could be effectively used in the immunotherapy of hepatocellular carcinoma. To validate the possibility, we developed a novel HBx-positive hepatocellular carcinoma in mice by inoculated the pcDNA-HBx transfected Hepa1-6 cells subcutaneously into the right flank of mice. We found that immunotherapy with Ad-HBx was effective at both protective and therapeutic antitumor immunity in the hepatoma models in immune-competent mice. Histological examination revealed that Ad-HBx treatment led to significantly increased induction of apoptosis, tumor necrosis, and elevated CD8+ lymphocyte infiltration. In addition, the induction efficacy of the CTL response is dramatically enhanced by immunotherapy. Cytokine analysis confirmed that the antitumor efficacy of Ad-HBx may mostly result from cellular immunity. Our findings may prove useful in development of adenovirus vaccine based on HBx antigen to the treatment of HBV-associated hepatocellular carcinoma.     

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute-phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I-restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8(+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi-species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute-phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.     

Definition of two new epitopes on human immunodeficiency virus type 1 gag protein recognized by human CD8+ cytotoxic T lymphocyte clones.

We have established 3 CD8+ cytotoxic T lymphocyte (CTL) clones from the peripheral blood mononuclear cells of two HIV-1-seropositive asymptomatic donors. The epitopes recognized by these CTL clones were defined using synthetic peptides. The epitopes were located on HIV-1gag protein between amino acid (a.a.) 145 and 155 (QAISPRTLNAW), a.a.193 and 201 (GHQAAMQML), and a.a.260 and 267 (EIYKRWII), and were presented by HLA-A25, HLA-B38 and HLA-B8, respectively. The former 2 epitopes have not been previously defined.The HLA-A25-restricted epitope overlapped with HLA-B57-restricted and HLA-Cw3-restricted epitopes previously reported. In addition, this epitope overlapped with an HLA-DQ-restricted epitope recognized by CD4+ CTL. The HLA-B38-restricted epitope overlapped with HLA-A2-restricted and HLA-Bw52-restricted epitopes that were previously reported. The HLA-B38-restricted epitope between a.a.193 and 201 was highly conserved among HIV-1 strains. The results demonstrate that two new epitopes were defined in a region of gag protein that includes multiple epitopes presented by multiple HLA.     

Mechanisms of induction of primary virus-specific cytotoxic T lymphocyte responses.

We have investigated the ability of various antigen-presenting cell (APC) types to induce primary anti-viral cytotoxic T lymphocyte (CTL) responses by single in vitro stimulation. Of these APC types, only dendritic cells (DC) and RMA-S lymphoma cells could induce primary CTL responses, but by divergent mechanisms. DC were capable of generating primary virus-specific CTL, either by presenting viral peptide or processed infectious virus. In contrast, RMA-S cells could not present endogenous antigen, e.g. after virus infection, but this cell line very efficiently presented exogenous viral peptides to induce primary virus-specific CTL in vitro. Spleen cells, lipopolysaccharide-induced B cell blasts or the non-mutated RMA cells did not have the ability to trigger unprimed T cells by single in vitro stimulation. We have investigated several characteristics important for primary CTL response induction by DC and RMA-S cells (summarized in Fig. 6). Primary CTL response induction by DC or RMA-S cells was blocked by anti-LFA-1 or anti-CD8 monoclonal antibodies (mAb). DC rapidly aggregated with unprimed T cells, which was independent of LFA-1 and CD8 molecules. RMA-S cells did not form conjugates with unprimed T cells. Despite their abundant major histocompatibility complex (MHC) class I cell-surface expression, DC did not bind much exogenously added viral peptide. In contrast, the MHC class I molecules on RMA-S cells bound a large quantity of exogenously administered peptide. Powerful adhesion by DC and high expression of relevant MHC/peptide complexes on RMA-S cells are important features in the initial contact with unprimed T lymphocytes. In a later stage of contact, both DC and RMA-S cells activate LFA-1 (and CD8) molecules at the T cell surface to strengthen and maintain the contact between T cell and APC.     

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.     

Variable intensity of purifying selection on cytotoxic T-lymphocyte epitopes in hepatitis C virus

In an analysis of the patterns of nucleotide diversity in 26 datasets providing population-level data on different genomic regions of different hepatitis C virus (HCV) subtypes, known cytotoxic T-lymphocyte (CTL) epitope regions in most cases showed evidence of the occurrence of purifying selection. Two main factors were found to be associated with the strength of purifying selection: (1) purifying selection was stronger in CTL epitopes in non-envelope proteins than in envelope proteins and (2) purifying selection was stronger when the epitope was "matched", i.e., when the described or "canonical" epitope sequence was present unaltered in at least one sequence in the dataset. Of all polymorphic sites, non-synonymous sites in matched CTL epitopes in non-envelope proteins had the lowest gene diversities, implying that these variants are subject to ongoing purifying selection. This in turn suggests that the population frequency of such variants may of be the result of a balance between opposing forces: on the one hand, positive selection favoring escape mutants in hosts that express the presenting MHC molecule and, on the other hand, purifying selection acting, in the absence of the presenting MHC molecule, to reduce the frequency of slightly deleterious variants.     

Protective cytotoxic T lymphocyte responses induced by DNA immunization against immunodominant and subdominant epitopes of Listeria monocytogenes are noncompetitive

Taking advantage of the fact that plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope can induce CTLs, we examined the influence of T-cell responses to dominant epitopes on those to a subdominant epitope derived from Listeria monocytogenes. Our data suggest that interaction between T cells against dominant and subdominant epitopes does not operate in the generation of the hierarchy. Furthermore, we found that a single dominant epitope is sufficient for the induction of protective immunity.     

Natural variation of equine infectious anemia virus Gag protein cytotoxic T lymphocyte epitopes.

Two defined cytotoxic T lymphocyte (CTL) epitopes from equine infectious anemia virus (EIAV)-infected horses, equine leukocyte alloantigen (ELA)-A5.1-restricted epitope 18a, and ELA-A9-restricted epitope 28b-1 were evaluated for conservation among three wild-type EIAV strains. Epitope 18a variation occurred in all three wild-type EIAV strains, while epitope 28b-1 varied in one strain. Further, 12% amino acid changes occurred in the Gag proteins of a recently isolated wild-type strain, documenting a much greater Gag protein variation than previously reported. Evaluation of epitope 18a among two virus isolates from sequential disease episodes in a single horse, H513 (ELA-A5.1/A8), demonstrated that no variation that affected CTL recognition occurred. H513 PBMC had CTLm to epitope 18a before the occurrence of disease episodes caused by viruses expressing epitope 18a; however, the frequencies were low (5-15/10(6) PBMC). Later in infection there was an absence of disease episodes associated with an increase in CTLm frequency to EIAV(WSU5)-infected targets, but not epitope 18a-pulsed targets. Therefore, if CTLm to EIAV epitopes were involved in maintaining the carrier state in H513, they recognized epitopes other than 18a.     

Effective induction of type 1 helper IgG2a and cytotoxic T-cell responses in mice following immunization with human papillomavirus type 16 E2 in MF59

Human papillomavirus (HPV) 16 E2-specific cell-mediated immunity to the early viral antigen E2 is associated with regression of natural infection in patients with cervical dysplasia. Vaccination strategies that activate this type of immune response may have application in the immunotherapeutic treatment of pre-existing HPV infections. The objective of this study was to test if cell-mediated immunity to E2 could be activated when delivered with the already licensed adjuvant MF 59.We found that immunization of mice with E2 in MF 59 stimulated T-cell responses when administered either intraperitoneally (IP) or subcutaneously (SC), and that the response was polarized to a Th-1 type IgG2a response in the IP immunized mice. The magnitude of the lymphoproliferative response was augmented by reducing the time interval between the primary and secondary immunizations from 12 to 4 wk. Stronger responses to the C-terminal third of E2 were detected, suggesting that one or more immunodominant epitopes were localized to this region. Significantly, immunization with E2 in MF 59 IP was sufficient to stimulate an E2-specific cytotoxic T-cell response.This immunization regimen activates the components of a cell-mediated immunity that are predicted to be efficacious in clearance of pre-existing infection, and supports its testing in a papillomavirus challenge model, as the next step in the progression toward its development as an immunotherapeutic vaccine for use in humans.