一个新的HBcAg之H-2K～d限制性表位的鉴定及其功能特性研究
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摘要
CD8~+ T淋巴细胞是机体免疫系统的主要组成部分,在针对病毒、细胞内细菌和真菌等病原性微生物以及恶性肿瘤等的免疫监视、免疫防御以及免疫治疗等过程中发挥关键性的作用。在实验室研究及临床实践过程中通常设计疫苗或者其它的相关制剂,诱导或增强机体的CD8~+ T淋巴细胞应答,以便预防或者治疗感染性疾病或肿瘤。设计疫苗诱导细胞免疫应答进行感染性疾病或肿瘤的免疫治疗有多种思路,基于表位的疫苗设计(EBVD)是其中的重要方面。基于表位的疫苗设计的前提和基础是鉴定大量的MHC-I类分子限制性的CTL表位,并且对各表位特异性CTL的效应特征有明确的了解。
慢性HBV感染是一全球性的重大健康问题。HBV感染的慢性化主要是因为机体对HBV的免疫应答,特别是细胞免疫应答,不能清除病原,导致感染的持续存在。因为在急性自限性HBV感染时,机体存在大量的针对HBV多个抗原表位的多特异性、多克隆的CTL应答,而在慢性感染时这些CTL应答非常微弱甚至检测不到。因此采取恰当的免疫方式,打破机体对HBV的免疫耐受,重建活跃的免疫应答,有望清除病毒,终止慢性HBV感染。目前已经鉴定出了大量的来自HBV的CTL表位,包括人HLA-I类分子限制性的和小鼠H-2-I类分子限制性的表位。但是这些表位都是首先通过细胞毒性实验鉴定出来的,即这些表位特异性的CTL能够杀伤表达相应抗原的靶细胞,这也是目前通用的CTL表位鉴定标准。但是遇到抗原性刺激时分泌IFNν和TNFα等细胞因子也是CTL的重要效应机制之一,而且这些细胞因子往往是CTL发挥作用的关键性介质。已经有文献表明CTL的细胞毒性和产生细胞因子是两个独立调节的过程,并非总是伴随发生。因此在进行CTL表位鉴定和评价时很有必要分析肽诱导的效应细胞分泌细胞因子的能力。HBV及其复制产物本身是非细胞病理性的,HBV感染后的肝细胞损伤是机体免疫系统对HBV相关抗原发生应答,特异性的CTL识别
CD8~+ T lymphocytes are one of the most important components of the immune system and usually play critical roles on the immune surveillance, immune defence and immune therapy for many kinds of disease, including cancer and infectious diseases caused by virus, intracellular bacteria or fungi. Vaccines or other reagents, which work to induce or enhance antigen-specific CD8~+T cell response, are hopeful to cure these diseases. There are many ways through which we can design vaccine for the immune therapy of infectious diseases and cancer. Epitope-based vaccine design (EBVD) is one of the most important and rational choices. The groundwork of EBVD is identifying lots of MHC-I-restricted CTL epitope and understanding the detailed knowledge about the functional effects of epitope-specific CTLs.Chronic hepatitis B virus (HBV) infection is one of the most important healthy problems worldwide. The key reason of persistently infection of HBV is the host's immune response, especially the cellular response, is too weak or too limited to control or to eliminate virus. Multispecific antiviral CD8~+ T cell responses with a type I profile of cytokine production are detected in subjects with self-limited acute hepatitis B, but patients with chronic HBV infections are usually characterized by weak and even undetectable virus-specific CD8~+ T cell response. Therefore breaking the immune tolerance and resuming the active immune response are hopeful to clear the virus and terminate the infection. At present, a lot of HBV-derived CTL epitopes, including human HLA-I-restricted and mouse H-2-I-restricted CTL epitopes, have been identified. All of these epitope were identified through cytotoxicity assay first, which is to say, these epitop-specific CTLs could kill the corresponding target cells, which express the correct
epitope peptide bound with proper MHC-I molecules. This property is the commonly used standard for CTL epitope. It is well known that the production of cytokines such as IFNγ and TNFα is also the important functional mechanism through which CTLs can exert its effects. What is more, the cytotoxicity and cytokines production are separately regulated during this process instead of being always associated with each other. It is necessary to analysis the ability of epitope-specific CTLs to produce cytokines when identifying and evaluating CTL epitopes. HBV itself and its replicating products are not cytopathic and the injury of hepatocytes after HBV infection is mainly due to that the host's HBV specific CTLs recognize and kill hepatocytes which express HBV antigens and clear the virus inside the hepatocytes, of course, cause hepatitis simultaneously. The cytokines produced by HBV-specific CTLs can inhibit HBV replication without hepatocytes killing. Obviously, the cure of HBV infection depends more on cytokines than on cytotoxicity, that is to say, the cytokines secreted by CTLs are more important than cytotoxicity of CTLs in elimination of HBV infection. So we have to understand the precise functional mechanisms (secreting cytokines and/or cytotoxicity) and the functional capacities of epitope-specific CTLs for the selection of proper epitope and epitope combination to design vaccine, to induce protective immune response without the injury of host.HBcAg is a structural protein and has strong immunogenicity. We want to know if there are any novel mouse H-2 K~d-restricted CTL epitopes within this antigen. Both SYFPEITHI and BIMAS programs were used to predict putative H-2 K~d -restricted T cell epitopes from within the amino acid sequences of HBcAg. The four highest scored 9-amino acids or 10-amino acids candidate epitope peptides in both algorithms were selected as candidates for further identification. The binding affinity of these peptides with H-2 K~d was measured through stably transfected cell line RMA-S/K~d and FACS after the peptides synthesis, purifying and identification. The HBcAg-expression plasmid pcHBc3.1+ were constructed and identified through restricted enzyme digestion and DNA sequencing. The plasmid was transferred into cell line P815 and the transfected cell line P815C that stably express HBcAg was established and identified. Balb/C mice were immunized i.m. with plasmid pcHBc3.1+ and the splenocytes of these immunized mice were separated and induced with each candidate peptide and mIL2. After 5 days of incubation, the cultural supernatant and splenocytes were collected and the IFNγ production
and cytotoxicity were analyzed through ELISPOT, ELISA and 51~Cr releasing assay, respectively. The results showed that peptide HBcAg (131-139) could bind H-2 K~d efficiently and this peptide-induced mouse splenocytes could produce IFNy but have no cytotoxicity, no matter the transfected cell line P815C or the peptide-pulsed P815 cell were used as target cells. Furthermore, we proved that this peptide-induced splenocytes were almost CD8~+ and the IFNy secretion by these cells was H-2 K~d-restricted and CD8 dependent because that the anti-K~d and anti-CD8 monoclone antibody can block this cytokine secretion significantly. The peptide-stimulated and expanded mice splenocytes were administered into HBV transgenic mice and the serum ALT activity, liver histological changes, HBcAg expressed in liver and HBV DNA load were analyzed. HBcAg (131-139)- induced splenocytes could reduce the HBV DNA load and the expression of HBcAg in liver significantly but did not cause hepatocytes necrosis or increase the sALT activity obviously of HBV transgenic mice. On the contrary, peptide HBcAg ((87-95))-specific CTLs, which were used as positive control in this experiment, could produce IFNy and kill target cells in vitro, could reduce the serum HBV DNA load and the HBcAg expression, but at the same time, killed hepatocytes, increased serum ALT level as well. These results showed that peptide HBcAg (131-139) was an unusual HBV-derived mice H-2 K~d-restricted CD8~+ T cell epitope whose specific CD8~+ T cells could produce IFNy without cytotoxicity encountering antigenic stimulation. This result is coincident with the viewpoint that the cytotoxicity and cytokines-production of CTL are separately regulated events. Furthermore, this result suggests preliminarily that this separately regulation between cytotoxicity and cytokine-production of CTLs is determined by, at least to some degree, the CTLs' epitope specificity.We selected 5 HBV-derived mice H-2~d-restriced CTL epitopes, including the newly discovered epitope HBcAg(131-139), to analyze the functional characteristics of their specific CTLs, including the IFNy production and cytotoxicity. The HBsAg-expression plasmid pcHBs3.1+ was constructed and identified. Balb/C mice were immunized i.m. with plasmid pcHBc3.1+ or pcHBs3.1+ and the splenocytes were separated and incubated with each peptide and mIL2 to induce epitope-specific CTLs. The IFNy production and cytotoxicity were measured as described above. After stimulation and expansion in vitro the epitope specific CTLs were transferred into HBV transgenic mice to evaluate the effect in
vivo. The results showed that the two sides of the functional mechanism of CTLs were not always parallel to each other, some of them produced more cytokines and some of them had more cytotoxicity. When one certain function was considered, there were significant differences among these epitope-specific CTLs. The sequence of the ability to produce IFNy among these epitopes-specific CTLs was HBcAg ((87-95)) > HBsAg (28-39) > HBcAg (131-139) > HBsAg (199-208) (?) HBsAg (201-209) and the sequence of cytotoxocity was HBcAg (87-95)> HBsAg (199-208)(?) HBsAg (201-209) > HBsAg (28-39) > HBcAg (131-139). The effects in vivo was similar with the effects in vitro: the more IFNy production, the more reduction of HBV DNA load; the more cytotoxicity, the more increasement if sALT activity. Once more, these results prove that the viewpoint HBV DNA load of HBV transgenic mice is mainly decreased by the cytokines such as IFNy and the sALT activity is mainly elevated by cytotoxicity of CTLs. These results also suggest preliminarily the viewpoint "epitope determines effect".In conclusion, we found and identified an unusual HBV-derived mice H-2 K~d-restricted CD8~+ T cell epitope whose specific CD8~+ T cells produced IFNy but almost had no cytotoxicity encountering antigenic stimulation. We compared the production of IFNy and cytotoxicity in vitro, the killing of hepatocytes and inhibition of HBV replication in HBV transgenic mice among 5 epitope-specific CTLs. We found that CTLs with different epitope specificity had different functional mechanisms and different functional ability. These results indicate preliminarily that the functional characteristics of CTLs can be determined by their epitope specificity. After this, we can select proper epitope and epitope combination to design vaccine, to induce proper immune response, which can cure disease without injury of hosts.
慢性HBV感染是一全球性的重大健康问题。HBV感染的慢性化主要是因为机体对HBV的免疫应答,特别是细胞免疫应答,不能清除病原,导致感染的持续存在。因为在急性自限性HBV感染时,机体存在大量的针对HBV多个抗原表位的多特异性、多克隆的CTL应答,而在慢性感染时这些CTL应答非常微弱甚至检测不到。因此采取恰当的免疫方式,打破机体对HBV的免疫耐受,重建活跃的免疫应答,有望清除病毒,终止慢性HBV感染。目前已经鉴定出了大量的来自HBV的CTL表位,包括人HLA-I类分子限制性的和小鼠H-2-I类分子限制性的表位。但是这些表位都是首先通过细胞毒性实验鉴定出来的,即这些表位特异性的CTL能够杀伤表达相应抗原的靶细胞,这也是目前通用的CTL表位鉴定标准。但是遇到抗原性刺激时分泌IFNν和TNFα等细胞因子也是CTL的重要效应机制之一,而且这些细胞因子往往是CTL发挥作用的关键性介质。已经有文献表明CTL的细胞毒性和产生细胞因子是两个独立调节的过程,并非总是伴随发生。因此在进行CTL表位鉴定和评价时很有必要分析肽诱导的效应细胞分泌细胞因子的能力。HBV及其复制产物本身是非细胞病理性的,HBV感染后的肝细胞损伤是机体免疫系统对HBV相关抗原发生应答,特异性的CTL识别
CD8~+ T lymphocytes are one of the most important components of the immune system and usually play critical roles on the immune surveillance, immune defence and immune therapy for many kinds of disease, including cancer and infectious diseases caused by virus, intracellular bacteria or fungi. Vaccines or other reagents, which work to induce or enhance antigen-specific CD8~+T cell response, are hopeful to cure these diseases. There are many ways through which we can design vaccine for the immune therapy of infectious diseases and cancer. Epitope-based vaccine design (EBVD) is one of the most important and rational choices. The groundwork of EBVD is identifying lots of MHC-I-restricted CTL epitope and understanding the detailed knowledge about the functional effects of epitope-specific CTLs.Chronic hepatitis B virus (HBV) infection is one of the most important healthy problems worldwide. The key reason of persistently infection of HBV is the host's immune response, especially the cellular response, is too weak or too limited to control or to eliminate virus. Multispecific antiviral CD8~+ T cell responses with a type I profile of cytokine production are detected in subjects with self-limited acute hepatitis B, but patients with chronic HBV infections are usually characterized by weak and even undetectable virus-specific CD8~+ T cell response. Therefore breaking the immune tolerance and resuming the active immune response are hopeful to clear the virus and terminate the infection. At present, a lot of HBV-derived CTL epitopes, including human HLA-I-restricted and mouse H-2-I-restricted CTL epitopes, have been identified. All of these epitope were identified through cytotoxicity assay first, which is to say, these epitop-specific CTLs could kill the corresponding target cells, which express the correct
epitope peptide bound with proper MHC-I molecules. This property is the commonly used standard for CTL epitope. It is well known that the production of cytokines such as IFNγ and TNFα is also the important functional mechanism through which CTLs can exert its effects. What is more, the cytotoxicity and cytokines production are separately regulated during this process instead of being always associated with each other. It is necessary to analysis the ability of epitope-specific CTLs to produce cytokines when identifying and evaluating CTL epitopes. HBV itself and its replicating products are not cytopathic and the injury of hepatocytes after HBV infection is mainly due to that the host's HBV specific CTLs recognize and kill hepatocytes which express HBV antigens and clear the virus inside the hepatocytes, of course, cause hepatitis simultaneously. The cytokines produced by HBV-specific CTLs can inhibit HBV replication without hepatocytes killing. Obviously, the cure of HBV infection depends more on cytokines than on cytotoxicity, that is to say, the cytokines secreted by CTLs are more important than cytotoxicity of CTLs in elimination of HBV infection. So we have to understand the precise functional mechanisms (secreting cytokines and/or cytotoxicity) and the functional capacities of epitope-specific CTLs for the selection of proper epitope and epitope combination to design vaccine, to induce protective immune response without the injury of host.HBcAg is a structural protein and has strong immunogenicity. We want to know if there are any novel mouse H-2 K~d-restricted CTL epitopes within this antigen. Both SYFPEITHI and BIMAS programs were used to predict putative H-2 K~d -restricted T cell epitopes from within the amino acid sequences of HBcAg. The four highest scored 9-amino acids or 10-amino acids candidate epitope peptides in both algorithms were selected as candidates for further identification. The binding affinity of these peptides with H-2 K~d was measured through stably transfected cell line RMA-S/K~d and FACS after the peptides synthesis, purifying and identification. The HBcAg-expression plasmid pcHBc3.1+ were constructed and identified through restricted enzyme digestion and DNA sequencing. The plasmid was transferred into cell line P815 and the transfected cell line P815C that stably express HBcAg was established and identified. Balb/C mice were immunized i.m. with plasmid pcHBc3.1+ and the splenocytes of these immunized mice were separated and induced with each candidate peptide and mIL2. After 5 days of incubation, the cultural supernatant and splenocytes were collected and the IFNγ production
and cytotoxicity were analyzed through ELISPOT, ELISA and 51~Cr releasing assay, respectively. The results showed that peptide HBcAg (131-139) could bind H-2 K~d efficiently and this peptide-induced mouse splenocytes could produce IFNy but have no cytotoxicity, no matter the transfected cell line P815C or the peptide-pulsed P815 cell were used as target cells. Furthermore, we proved that this peptide-induced splenocytes were almost CD8~+ and the IFNy secretion by these cells was H-2 K~d-restricted and CD8 dependent because that the anti-K~d and anti-CD8 monoclone antibody can block this cytokine secretion significantly. The peptide-stimulated and expanded mice splenocytes were administered into HBV transgenic mice and the serum ALT activity, liver histological changes, HBcAg expressed in liver and HBV DNA load were analyzed. HBcAg (131-139)- induced splenocytes could reduce the HBV DNA load and the expression of HBcAg in liver significantly but did not cause hepatocytes necrosis or increase the sALT activity obviously of HBV transgenic mice. On the contrary, peptide HBcAg ((87-95))-specific CTLs, which were used as positive control in this experiment, could produce IFNy and kill target cells in vitro, could reduce the serum HBV DNA load and the HBcAg expression, but at the same time, killed hepatocytes, increased serum ALT level as well. These results showed that peptide HBcAg (131-139) was an unusual HBV-derived mice H-2 K~d-restricted CD8~+ T cell epitope whose specific CD8~+ T cells could produce IFNy without cytotoxicity encountering antigenic stimulation. This result is coincident with the viewpoint that the cytotoxicity and cytokines-production of CTL are separately regulated events. Furthermore, this result suggests preliminarily that this separately regulation between cytotoxicity and cytokine-production of CTLs is determined by, at least to some degree, the CTLs' epitope specificity.We selected 5 HBV-derived mice H-2~d-restriced CTL epitopes, including the newly discovered epitope HBcAg(131-139), to analyze the functional characteristics of their specific CTLs, including the IFNy production and cytotoxicity. The HBsAg-expression plasmid pcHBs3.1+ was constructed and identified. Balb/C mice were immunized i.m. with plasmid pcHBc3.1+ or pcHBs3.1+ and the splenocytes were separated and incubated with each peptide and mIL2 to induce epitope-specific CTLs. The IFNy production and cytotoxicity were measured as described above. After stimulation and expansion in vitro the epitope specific CTLs were transferred into HBV transgenic mice to evaluate the effect in
vivo. The results showed that the two sides of the functional mechanism of CTLs were not always parallel to each other, some of them produced more cytokines and some of them had more cytotoxicity. When one certain function was considered, there were significant differences among these epitope-specific CTLs. The sequence of the ability to produce IFNy among these epitopes-specific CTLs was HBcAg ((87-95)) > HBsAg (28-39) > HBcAg (131-139) > HBsAg (199-208) (?) HBsAg (201-209) and the sequence of cytotoxocity was HBcAg (87-95)> HBsAg (199-208)(?) HBsAg (201-209) > HBsAg (28-39) > HBcAg (131-139). The effects in vivo was similar with the effects in vitro: the more IFNy production, the more reduction of HBV DNA load; the more cytotoxicity, the more increasement if sALT activity. Once more, these results prove that the viewpoint HBV DNA load of HBV transgenic mice is mainly decreased by the cytokines such as IFNy and the sALT activity is mainly elevated by cytotoxicity of CTLs. These results also suggest preliminarily the viewpoint "epitope determines effect".In conclusion, we found and identified an unusual HBV-derived mice H-2 K~d-restricted CD8~+ T cell epitope whose specific CD8~+ T cells produced IFNy but almost had no cytotoxicity encountering antigenic stimulation. We compared the production of IFNy and cytotoxicity in vitro, the killing of hepatocytes and inhibition of HBV replication in HBV transgenic mice among 5 epitope-specific CTLs. We found that CTLs with different epitope specificity had different functional mechanisms and different functional ability. These results indicate preliminarily that the functional characteristics of CTLs can be determined by their epitope specificity. After this, we can select proper epitope and epitope combination to design vaccine, to induce proper immune response, which can cure disease without injury of hosts.
引文
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50. Goulder PJR, Altfeld MA, Rosenberg ES et al. Substantial Differences in Specificity of
HIV-specific Cytotoxic T Cells in Acute and Chronic HIV Infection. J.Exp.Med. 2001;193:181-94.
51. Gallimore A, Dumrese T, Hengartner H, Zinkernagel RM, Rammensee HG. Protective Immunity Does Not Correlate with the Hierarchy of 燰 irus-specific Cytotoxic T Cell Responses to Naturally Processed Peptides. J.Exp.Med. 1998; 187:1647-57.
52. Larkin J, Clayton M, Sun B et al. Hepatitis B virus transgenic mouse model of chronic liver disease. Nat.Med. 1999;5:907-12.
53. Ando K, Moriyama T, Guidotti LG et al. Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J.Exp.Med. 1993;178:1541-54.
54. Mancini M, Hadchouel M, Tiollais P, Michel ML. Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-gamma-secreting T cells after DNA-based immunization. J.Immunol. 1998;161:5564-70.
55. Chisari FV. Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis. Curr.Top.Microbiol.Immunol. 1996;206:149-73.
56. Singh M, Kumar V. Transgenic mouse models of hepatitis B virus-associated hepatocellular carcinoma. Rev.Med.Virol. 2003;13:243-53.
57. Wieland SF, Vega RG, Muller R et al. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J.Virol. 2003;77:1227-36.
58. Sette AD, Oseroff C, Sidney J et al. Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice. J Immunol 2001;166:1389-97.
59. Davis HL, Brazolot Millan CL, Mancini M et al. DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice. Vaccine 1997;15:849-52.
60. Hilleman MR. Critical overview and outlook: pathogenesis, prevention, and treatment of hepatitis and hepatocarcinoma caused by hepatitis B virus. Vaccine 2003;21:4626-49.
61. Guidotti LG, Morris A, Mendez H et al. Interferon-regulated pathways that control
hepatitis B virus replication in transgenic mice. J Virol. 2002;76:2617-21.
62. Guidotti LG, Chisari FV. Cytokine-mediated control of viral infections. Virology 2000;273:221-7.
63. Betts MR, Price DA, Brenchley JM et al. The Functional Profile of Primary Human Antiviral CD8+ T Cell Effector Activity Is Dictated by Cognate Peptide Concentration. J Immunol 2004;172:6407-17.
64. Webster GJM, Reignat S, Brown D et al. Longitudinal Analysis of CD8+ T Cells Specific for Structural and Nonstructural Hepatitis B Virus Proteins in Patients with Chronic Hepatitis B: Implications for Immunotherapy. J.Virol. 2004;78:5707-19.