乙型肝炎病毒变异与慢加急性肝衰竭发病关系的研究
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摘要
背景：
乙型肝炎病毒(hepatitis B virus, HBV)感染是世界范围内导致病毒相关性慢性肝脏疾病最常见的原因之一。据估计全世界约有3亿慢性HBV携带者,我国的HBV感染者约为1.2亿。慢性HBV感染可引起包括无症状携带、慢性乙型肝炎(chronic hepatitis B, CHB)、肝硬化和肝癌,或进展为肝衰竭(liver failure, LF)的一系列肝脏疾病谱。慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)是在慢性乙肝病毒感染基础上,由于病毒复制、感染、内毒素血症及劳累饮酒等诱因,引起的肝脏急性炎症加重,肝脏发生大块或亚大块坏死,导致病情急剧加重,引起肝衰竭。中国80%的ACLF患者与HBV感染相关。ACLF的死亡率很高,在未进行肝移植的患者中高达(60-80)%。
在CHB基础上进展为肝衰竭的机制复杂,HBV病毒和机体免疫的因素都起着重要的作用。既往文献报道HBV病毒变异,特别是前C区终止密码子(G1896A,PC)和基本核心启动子(basie core promoter, A1762T/G1764A, BCP)的变异率在急性肝衰竭的患者中明显升高；PC和BCP变异下调了作为免疫耐受原的HBeAg,血清中HBeAg下降或缺如,导致HBcAg表达增加,使Th1辅助细胞发动细胞毒性T细胞(cytoxic T lymphocyte, CTL)攻击感染的肝细胞,从而导致肝细胞坏死。
HBcAg作为CTL靶抗原,包含有多个能被CTL识别的表位(epitope)。表位是特异性细胞免疫应答得以激发的主要原动力,处于免疫识别和免疫激活的核心位置。核苷酸的转换、插入和缺失可导致氨基酸的有义突变,当这种突变影响到病毒的特异性T淋巴细胞和B淋巴细胞表位的序列和序列构象时,进一步会影响与HLA分子的结合力或与B、T淋巴细胞受体的结合力,从而影响宿主针对病毒的免疫应答能力。在一些肿瘤和其他病毒细胞免疫应答方面,研究已证实基因变异会导致T细胞表位漂移(epitope drift),从而改变了表位的免疫原性,进而影响疾病的发展与预后。
目的：
以中国广东地区ACLF患者为主要对象,从HBV变异影响基因转录调控和影响宿主免疫应答两个方面,探讨病毒变异与ACLF患者发病的关系。
方法：
39例ACLF患者和38例严重肝炎活动患者(severe hepatitis B, SHB)作为主要研究对象。其中ACLF诊断标准：在慢性肝病的基础上急性起病,15天至26周出现以下临床表现者：①极度乏力,有明显的消化道症状；②黄疸迅速加深,血清总胆红素(TB)≥正常值上限10倍,或每日上升≥17.1μmol／1；③凝血酶原时间明显延长,凝血酶原活动度(PTA)≤40%。SHB定义参照文献的报道：丙氨酸转氨酶(ALT)≥600U／l、TB≥51.3μmol／l和PTA≤50%。作为对照组的44例CHB患者定义为：HBV表面抗原(HBsAg)阳性半年以上,出现的轻度或中度肝炎活动,即ALT波动于(80-400)U／l和TB≤17.1μmol／l。以上患者临床和实验室检查均无明显肝硬化征象。ACLF和SHB的患者平均随访10.6月(最长17个月,最短3个月)。采集患者的临床资料和血清标本。抽提DNA,通过聚合酶链反应(polymerase chain reaction, PCR)扩增和测序HBV前C／C区,通过克隆测序的方法对HBV混合株感染加以验证。通过生物预测网站SYFPEITHI和BIMAS分析线性Th细胞表位和CTL表位发生变异后与HLA分子的结合能力,用HBcAg X线衍射3D结构分析B细胞表位变异后构型的改变。用五聚体染色(pentamer staining)技术探讨表位免疫原性的改变。
结果：
1)临床资料的比较：ACLF组与SHB组比较TB的水平明显升高,而ALB及PTA均降低；差异均有统计学意义；反映肾功能的指标血肌酐(Cr)(99.4±94.7vs.75.0±14.1；P<0.001)和判断是否合并感染的指标白细胞总数(WBC)[8.0±3.2(×109／l)vs.6.2±2.1(×109／l)；P=0.005]在ACLF组也升高。两组之间HBV基因型分布和BCP、PC变异发生率均无明显差异。ACLF中死亡组与存活组相比：PTA明显降低,WBC明显升高,差异均有统计学意义；Cr也较存活组高,但无显著性差异(P=0.072)。
2)HBV基因型：ACLF和SHB两组均以基因B型和C型为主,有1例患者为基因D型；5例患者由于HBV DNA定量低,PCR扩增失败未鉴定基因型,未纳入统计。其中基因B型比率分别高达(26／35,72.2%)和(24／36,68.6%),均高于CHB组(22／44,50%)。SHB组与CHB比较有统计学差异(P=0.044)；ACLF组也较CHB组升高,但无统计学意义(P=0.069)。
3)C基因调控区变异
(1) BCP(A1762T/G1764A)的变异率在ACLF中明显高于CHB组(56.7% vs.32.4%,P=0.046),PC(G1896A)变异率也显著高于CHB组(50% vs.15.9%,P=0.003)。用“-”表示未变异,用“+”代表变异来进行简化；ACLF组HBV发生BCP-/PC+和BCP+/PC+的比例明显高于CHB组,而BCP-／PC-的比例明显低于CHB组。
(2)在BCP+/PC-、BCP-/PC-、BCP-/PC+和BCP+／PC+模式中HBeAg的阴性率逐步升高,分别为在40%、53%、76.5%和83.3%；而且BCP-／PC+和BCP+/PC+模式下调HBeAg的作用比其他两种模式明显增强；在单变异模式中PC变异下调HBeAg的作用较BCP变异明显(P=0.001)。
(3)nt1846 A-T变异的发生率在ACLF组明显高于SHB组[80.6%(25／31)vs.16.7%(5／30),P<0.001]及CHB组[80.6%(25／31)vs.11.1%(4／36),P<0.001]。而该变异位点与本研究中患者的基因型分布无相关性。
(4) BCP-/PC-模式中HBeAg阴性率在四种模式中并非最低；进一步分析在这种模式中ACLF的患者均为HBeAg阴性。而这部分ACLF的患者HBV DNA定量与该模式中SHB的患者无差异。
4) C基因编码区变异
(1)C基因编码区氨基酸发生变异的频率在ACLF组明显高于CHB组。在ACLF中氨基酸变异率超过10%,且与CHB组比较有统计学差异的共有7个位点。这些高频率的变异位点绝大部分位于表位区域：aa5、aa35和aa60分别位于的Thl-20、Th35-45和Th49-69辅助细胞表位区,aa27位于CTL18-27细胞毒性T细胞表位区,aa83和aa135分别位于B76-87和B130-135表位区。进一步统计以上表位区变异的频率显示：ACLF在各个表位区变异频率均高于CHB组。
(2)aa5和aa60变异存在明显的相关性,Pearson相关系数为0.632(P<0.001)。并且这两点在随访中表现为协同共变异：即aa5位氨基酸发生P-T的变异,同时观察到aa60发生L-V的变异；或aa5 T-P协同aa60 V-L。HBcAg晶体3D结构显示,aa5和aa60在空间构型上位置相邻,分享同一个四螺旋束(four-helix bundle)。网站预测aa5和aa60所在的肽段作为潜在的CTL表位与HLA分子的结合力,显示aa5发生P-T的变异后引起所在的肽段与HLA-A*0201分子的结合分数升高了3分(13 vs.16)；而aa60 L-V的变异后所在肽段与HLA-A*0201分子的结合分数降低。
(3)对于高变异位点所位于的已知线性Th细胞表位和CTL表位通过生物学网站预测其免疫原性的改变；对于空间构象型B细胞表位用HBcAg X衍射3D结构分析其构型的改变。通过HBcAg晶体结构分析构象性B细胞表位显示,高变异位点所在的B细胞表位均位于HBcAg结构中向外侧突出的位置；而在B细胞表位HBcAg130-135中,aa135处于最向外突出的点。CTL表位HBcAg18-27发生27位I-V的变异后,SYFPEITHI评分显示与HLA-A*0201分子的结合分数升高了2分(24 vs.22)；BIMAS评分显示HBcAg18-27(V27)与HLA-A*0201结合的分数为2309.961,而I27为346.494。
5)表位HBcAg18-27
(1)HLA-A*0201限制性特异性CTL表位HBcAg18-27,在中国I27的背景下,ACLF组V27变异率高。在ACLF组V27的发生率明显高于CHB组,差异有统计学意义[10／39(25.6%)vs.3／44(6.8%)；P=0.019]；同样,SHB组V27的比率也高于CHB组[8／38(21.1%)vs.3／44(6.8%)；P=0.059]。
(2)为了探讨表位HBcAg18-27的动态变化,随访了SHB和ACLF组中10例发病时V27单一病毒株感染或V27和I27混合株感染的患者,及19例发病时I27单一病毒株感染的患者。在4例HLA-A2阳性的患者中无论发病时是V27单一病毒株感染或是V27和I27混合株感染,随访后均变异成I27单一病毒株感染；而在5例HLA-A2阴性患者中,随访后包含V27的病毒仍能检测出。进一步的克隆测序,验证了V27向I27转换的变异模式。而18例发病时感染I27单一病毒株的患者随访后仍然为单一I27病毒株感染。
(3)用五聚体技术,分析HBcAg18-27表位变异后免疫原性的差异。分离HLA-A2阳性ACLF和SHB患者的PBMC,用HBcAg18-27两种形式(I27和V27)的特异性五聚体直接检测分析特异性CD8细胞的频数；直接检测的结果显示特异性CD8细胞的频数较低,两组之间无统计学差异。对于直接检测后仍有余留PBMC的患者,将其PBMC增殖培养13天后再进行五聚体染色：在案例报道中显示HBcAg18-27(V27)特异性CD8细胞的频数高于HBcAg18-27(127)特异性CD8细胞的频数。
(4)在ACLF和SHB的患者中,aa27V患者HBV DNA定量低于aa27I的患者,尽管差异无统计学意义(P=0.093)。而ACLF组HBV DNA定量明显低于CHB组[(5.75±1.73 vs.9.16±1.72)log10 copies/ml；P<0.001],SHB组也低于CHB组[(5.55±1.61 vs.9.16±1.72)1og10copies/ml；P<0.001].
结论：
1)与从SHB进展为ACLF相关的因素包括：TB、Cr和WBC的升高,ALB和PTA的降低。在ACLF中,PTA下降及WBC升高与不良结局相关。
2)基因B型在ACLF和SHB两组比率均高于CHB组,且SHB组与CHB组比较有统计学意义。这表明在中国南方地区基因B型与严重的肝脏炎症活动发病相关。
3) BCP(A1762T/G1764A)和PC(G1896A)变异率在ACLF组明显高于CHB组。ACLF组HBV发生BCP-／PC+和BCP+／PC+的比例明显高于CHB组,而BCP-／PC-比例明显低于CHB组。这个结果证实了A1762T／G1764A和G1896A高变异与ACLF发病的相关性。
4)在BCP+/PC-、BCP-/PC-、BCP-/PC+和BCP+/PC+模式中HBeAg的阴性率逐步升高；而且BCP-／PC+和BCP+／PC+模式下调HBeAg的作用与其他两种模式相比明显增强；在单变异模式中PC变异下调HBeAg作用强于BCP变异；且在ACLF组BCP+／PC+和PC单变异的比例明显高于CHB组。这表明PC+和BCP+／PC+模式导致的HBeAg与ACLF发病相关。
5)C基因编码区发生变异的频率在ACLF组明显高于CHB组。ACLF中氨基酸变异率明显升高的位点绝大部分位于表位区域。统计各个表位区变异的频率,在ACLF组均高于CHB组。这说明表位变异可能在ACLF发病中起着重要的作用。
6)aa5和aa60变异存在明显的协同关系。两位点尽管在线性位置相隔远,但在HBcAg空间构型上位置相邻,分享HBcAg四聚体中同一个螺旋束。通过预测分析肽段发生aa5 P-T的变异后与HLA-A*0201结合力增强；提示aa5变异后可能提高表位的免疫原性、诱导更强的免疫反应；而aa5 P-T的变异在ACLF中明显升高(52.63%)；表明aa5T与ACLF的发病相关。aa60由于与aa5空间位置相邻,可能在极性、PH值、亲水性等多个因素的影响下,出现协同变异。
7)HLA-A2限制性CTL表位HBcAg18-27(V27)在ACLF组的比率明显升高,且随访后ACLF患者中,HLA-A2阳性的患者均变异I27；且HBcAg18-27(V27)特异性CD8细胞的频数高于HBcAg18-27(127)特异性CD8细胞的频数。这表明HBcAg18-27(27V)在HLA-A2阳性患者中引起较强的免疫应答,在清除了aa27V病毒的同时,导致了肝脏炎症的发生,与ACLF的发病相关。
Hepatitis B virus (HBV) is responsible for chronic infection. There are approximately 300 million chronic HBV carriers worldwide and about 120 million HBV carriers in China. HBV infection can lead to a series of spectrums of liver diseases including asymptomatic carrier, chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) or acute-on-chronic liver failure (ACLF). According to the Asian Pacific Association for the Study of the Liver including acute hepatic insult, ACLF was characterized by jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. Similarly, in China, the diagnosis of ACLF was based on recent development of jaundice [total bilirubin (TB)≥171.0μmol/l] or rapidly rising levels of TB (≥17.1μmol/L/day) and plasma prothrombin activity (PTA)≤40%. These were accompanied by the development of complications such as hepatic encephalopathy (≥grade 2), abrupt and obvious increase in ascites or spontaneous bacterial peritonitis, or hepatorenal syndrome. In China, HBV-related ACLF cases account for more than 80% of ACLF patients. ACLF has been as a result of the high incidence of chronic HBV infection and was reported to have a high mortality rate (60-80%) in the absence of liver transplantation.
The pathogenesis of ACLF remains unclear. Both viral and immune factors may play a role. HBV mutations in the basal core promoter (BCP) and precore (PC) have attracted special attention. Previous studies have clarified that a higher prevalence of the BCP double mutations and PC mutation were in patients with acute liver failure. Because the BCP mutations may enhance HBV replication in vitro and the PC mutation abrogates translation of HBeAg, which is considered a tolerogen buffering any immune attack on the infected hepatocytes.
HBV DNA contains four open reading frames. One of them is the C gene, which encodes a core peptide (HBcAg). HBcAg has been postulated to be an immunological target of cytotoxic T lymphocytes (CTL). Studies on endogenously processed viral peptides demonstrated that a peptide could be recognized by CTL and is the principal motive power which can activate specific cell immune response. When the mutate site just located in the epitope sequence, it will be possible to change the immunological characteristics and then have an impact on the host responsibility to HBV. Investigation of changes in such regions may help to predict the occurrence of ACLF.
Purpose
In this study, the nucleotide sequences of the precore and core region of HBV were analyzed to investigate the relationship of HBV mutations with ACLF. Moreover, we looked into the association between ACLF and the epitopes in HBcAg, who have prototype and variant.
Methods
Thirty-nine patients with ACLF and 38 with severe hepatitis B (SHB) were involved in our study. SHB was defined as ALT≥600 IU/1 associated with TB≥3.0 mg/dl and PTA≤50%. Forty four chronic hepatitis B (CHB) patients with ALT level within the range of 80-400 IU/1 and TB≤1.0 mg/dl were enrolled as control. The patients with ACLF and SHB were followed up for average 10.6 months (ranged for 3-17m).Sequences of PreC/C region, HBV genotype was determined. The line epitopes, including Th epitope and CTL epitope, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The 3D data of B cell epitopes were presented from the X-ray structure of the HBcAg by Chimera software. The pentamer staining was performed to analyze epitope-specific CTL.
Results
1) There were significant differences in TB, serum albumin (ALB) and PTA between patients with ACLF and SHB. Creatinine (CR) and white blood cell (WBC) were significantly high in ACLF compared with SHB. Of the 39 patients with ACLF,18 had a fatal outcome and 21 survived more than 3 months after onset of liver failure. Lower PTA and higher WBC were detected as risk factors, while a high CR was only probable as a risk factor for mortality.
2) The distribution of genotype B was statistically higher in SHB Patients than that in CHB patients (72.2% vs.50%, P=0.044). And genotype B was found more often in ACLF compared with in CHB, although there was no statistically significant difference (P=0.069).
3) The prevalence of BCP (A1762T/G1764A) mutations were found significantly more often in ACLF than in CHB (56.7% vs.32.4%, P=0.046). The rate of PC (G1896A) mutation was higher in ACLF compared with CHB (50% vs.15.9%, P=0.003). Moreover, compared with the distribution of HBV BCP/PC mutation patterns in investigated groups, the patterns of BCP-/PC+and BCP+/PC+ occurred more frequently in ACLF patients than that in CHB patients.
4) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-(40%), BCP-/PC-(53.3%), BCP-/PC+(76.5%) and BCP+/PC+ (83.3%) viruses. And the rate of HBeAg negativity was not lowest in patients with BCP-/PC-virus. Furthermore, the rate of HBeAg negativity was compared among CHB, SHB and ACLF. The rate of HBeAg negativity was significantly high in ACLF patients..
5) The prevalence of T1846 was found significantly high in ACLF than in SHB [80.6%(25/31) vs.16.7%(5/30), P<0.001] or than in CHB [80.6%(25/31) vs.11.1% (4/36),P<0.001].
6) In ACLF patients, the sites with amino acid variability of more than 10% in PreC/C protein included aa5, aa60, aa97, aa83, aa27, aa98 and aa35. And there were statistically significant difference in above amino acids variability compared with ACLF and CHB. Th epitopes and CTL epitopes, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The affinity of CTL18-27 to HLA-A*0201 was enhanced when I varied into V at the site of aa27. From the 3D structure of HBc tetramers, the observed major B epitopes (HBcAg76-87 and HBcAg130-135) appeared predominantly on the outer surface of the HBc protein. The one of most variable positions aa135 within the B epitope HBcAg130-135 was the most externally exposed.
7) Amino acid mutation in aa5 always accompanied with aa60; and the index of Pearson correlation was 0.632 (P<0.001). aa5 P-T accompanied with aa60 L-V and aa5 T-P with aa60 V-L in followed-up patients. Shown in the 3D structure of HBc tetramers, these two sites closely contacted within the four-helix bundle. Moreover, the affinity of epitope with variant in aa5 to HLA-A*0201 was enhanced, while the affinity of epitope with mutant in aa60 was decreased by epitope prediction WEB.
8) The prevalence of V27 was found significantly more often in ACLF than in CHB [10/39 (25.6%) vs.3/44 (6.8%); P=0.019], although there was no statistically significant difference between SHB and CHB [8/38 (21.1%) vs.3/44 (6.8%); P=0.059].
9) Ten patients primarily infected with pure V27 or the mixture of 127 and V27 and 19 primarily infected with pure 127 were followed up for at least 3 months in SHB and ACLF groups. In two cases (2/29), the sequence of the HBcAg18-27 encoding region could not be amplified. The remaining 27 patients were including 9 with pure V27 or the mixture of 127 and V27 and 19 with pure 127. These 9 patients with pure V27 or the mixture of 127 and V27 were classified into two groups according to HLA-A2 type. Among 4 HLA-A2 positive cases, V27 was not detected and it turned into 127. On contrary, V27 could still be detected among 5 HLA-A2 negative cases. Amino acid at position 27 was unchanged in 18 cases originally infected with 127, in whom the longest period of follow-up was 17 months. We used clones sequence to verify the virus change.
10) The low frequency of epitope-specific CD8+T cells by pentamer staining ex vivo was unavailable in our study, thus the residue PBMCs were stimulated with I27/V27 peptide mixture, and then the pentamer staining was performed. The results of the pentamer staining showed V27 specific CD8 T cells were detected at high level, while the 127 specific CD8 T cells were found to be much less common.
11) Mean HBV DNA level was significantly lower in ACLF than in CHB [(5.75±1.73 vs.9.16±1.72) log10 copies/ml; P<0.001], and a significant difference was also found between SHB and CHB [(5.55±1.61 vs.9.16±1.72) log10 copies/ml; P< 0.001]. HBV DNA level was lower in patients with V27 than in ones with 127 in ACLF and SHB, although there was no statistically significant difference [(5.14±1.31 vs.5.80±1.74) log10 copies/ml; P=0.093].
Conclusions
1) In this study, we found the prevalence of genotype B was 72% and 74% in ACLF and SHB, respectively, which was much higher in CHB. And BCP and PC mutations were more often in ACLF than in CHB. These suggested HBV genotype B and BCP/PC mutations were significantly associated with ACLF.
2) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-, BCP-/PC-, BCP-/PC+ and BCP+/PC+ viruses. The loss of HBeAg caused by BCP+/PC+ and BCP-/PC+ was related with the pathogenesy of ACLF.
3) Amino acid mutation in aa5 accompanied with aa60. These two sites closely contacted within the four-helix bundle in the 3D structure of HBc tetramers. The affinity of epitope with variant(T) in aa5 to HLA-A*0201 was enhanced by epitope prediction WEB. The above data suggested aa5T was associated with ACLF.
4) In HBcAg, the epitope drift or up regulation of immunodominance at multiply sites play a role of the development of ACLF.
5) HBcAg18-27 epitope with V27 may be associated with the development of ACLF and CTL response produced by the epitope involves the process of controlling the virus replication.
乙型肝炎病毒(hepatitis B virus, HBV)感染是世界范围内导致病毒相关性慢性肝脏疾病最常见的原因之一。据估计全世界约有3亿慢性HBV携带者,我国的HBV感染者约为1.2亿。慢性HBV感染可引起包括无症状携带、慢性乙型肝炎(chronic hepatitis B, CHB)、肝硬化和肝癌,或进展为肝衰竭(liver failure, LF)的一系列肝脏疾病谱。慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)是在慢性乙肝病毒感染基础上,由于病毒复制、感染、内毒素血症及劳累饮酒等诱因,引起的肝脏急性炎症加重,肝脏发生大块或亚大块坏死,导致病情急剧加重,引起肝衰竭。中国80%的ACLF患者与HBV感染相关。ACLF的死亡率很高,在未进行肝移植的患者中高达(60-80)%。
在CHB基础上进展为肝衰竭的机制复杂,HBV病毒和机体免疫的因素都起着重要的作用。既往文献报道HBV病毒变异,特别是前C区终止密码子(G1896A,PC)和基本核心启动子(basie core promoter, A1762T/G1764A, BCP)的变异率在急性肝衰竭的患者中明显升高；PC和BCP变异下调了作为免疫耐受原的HBeAg,血清中HBeAg下降或缺如,导致HBcAg表达增加,使Th1辅助细胞发动细胞毒性T细胞(cytoxic T lymphocyte, CTL)攻击感染的肝细胞,从而导致肝细胞坏死。
HBcAg作为CTL靶抗原,包含有多个能被CTL识别的表位(epitope)。表位是特异性细胞免疫应答得以激发的主要原动力,处于免疫识别和免疫激活的核心位置。核苷酸的转换、插入和缺失可导致氨基酸的有义突变,当这种突变影响到病毒的特异性T淋巴细胞和B淋巴细胞表位的序列和序列构象时,进一步会影响与HLA分子的结合力或与B、T淋巴细胞受体的结合力,从而影响宿主针对病毒的免疫应答能力。在一些肿瘤和其他病毒细胞免疫应答方面,研究已证实基因变异会导致T细胞表位漂移(epitope drift),从而改变了表位的免疫原性,进而影响疾病的发展与预后。
目的：
以中国广东地区ACLF患者为主要对象,从HBV变异影响基因转录调控和影响宿主免疫应答两个方面,探讨病毒变异与ACLF患者发病的关系。
方法：
39例ACLF患者和38例严重肝炎活动患者(severe hepatitis B, SHB)作为主要研究对象。其中ACLF诊断标准：在慢性肝病的基础上急性起病,15天至26周出现以下临床表现者：①极度乏力,有明显的消化道症状；②黄疸迅速加深,血清总胆红素(TB)≥正常值上限10倍,或每日上升≥17.1μmol／1；③凝血酶原时间明显延长,凝血酶原活动度(PTA)≤40%。SHB定义参照文献的报道：丙氨酸转氨酶(ALT)≥600U／l、TB≥51.3μmol／l和PTA≤50%。作为对照组的44例CHB患者定义为：HBV表面抗原(HBsAg)阳性半年以上,出现的轻度或中度肝炎活动,即ALT波动于(80-400)U／l和TB≤17.1μmol／l。以上患者临床和实验室检查均无明显肝硬化征象。ACLF和SHB的患者平均随访10.6月(最长17个月,最短3个月)。采集患者的临床资料和血清标本。抽提DNA,通过聚合酶链反应(polymerase chain reaction, PCR)扩增和测序HBV前C／C区,通过克隆测序的方法对HBV混合株感染加以验证。通过生物预测网站SYFPEITHI和BIMAS分析线性Th细胞表位和CTL表位发生变异后与HLA分子的结合能力,用HBcAg X线衍射3D结构分析B细胞表位变异后构型的改变。用五聚体染色(pentamer staining)技术探讨表位免疫原性的改变。
结果：
1)临床资料的比较：ACLF组与SHB组比较TB的水平明显升高,而ALB及PTA均降低；差异均有统计学意义；反映肾功能的指标血肌酐(Cr)(99.4±94.7vs.75.0±14.1；P<0.001)和判断是否合并感染的指标白细胞总数(WBC)[8.0±3.2(×109／l)vs.6.2±2.1(×109／l)；P=0.005]在ACLF组也升高。两组之间HBV基因型分布和BCP、PC变异发生率均无明显差异。ACLF中死亡组与存活组相比：PTA明显降低,WBC明显升高,差异均有统计学意义；Cr也较存活组高,但无显著性差异(P=0.072)。
2)HBV基因型：ACLF和SHB两组均以基因B型和C型为主,有1例患者为基因D型；5例患者由于HBV DNA定量低,PCR扩增失败未鉴定基因型,未纳入统计。其中基因B型比率分别高达(26／35,72.2%)和(24／36,68.6%),均高于CHB组(22／44,50%)。SHB组与CHB比较有统计学差异(P=0.044)；ACLF组也较CHB组升高,但无统计学意义(P=0.069)。
3)C基因调控区变异
(1) BCP(A1762T/G1764A)的变异率在ACLF中明显高于CHB组(56.7% vs.32.4%,P=0.046),PC(G1896A)变异率也显著高于CHB组(50% vs.15.9%,P=0.003)。用“-”表示未变异,用“+”代表变异来进行简化；ACLF组HBV发生BCP-/PC+和BCP+/PC+的比例明显高于CHB组,而BCP-／PC-的比例明显低于CHB组。
(2)在BCP+/PC-、BCP-/PC-、BCP-/PC+和BCP+／PC+模式中HBeAg的阴性率逐步升高,分别为在40%、53%、76.5%和83.3%；而且BCP-／PC+和BCP+/PC+模式下调HBeAg的作用比其他两种模式明显增强；在单变异模式中PC变异下调HBeAg的作用较BCP变异明显(P=0.001)。
(3)nt1846 A-T变异的发生率在ACLF组明显高于SHB组[80.6%(25／31)vs.16.7%(5／30),P<0.001]及CHB组[80.6%(25／31)vs.11.1%(4／36),P<0.001]。而该变异位点与本研究中患者的基因型分布无相关性。
(4) BCP-/PC-模式中HBeAg阴性率在四种模式中并非最低；进一步分析在这种模式中ACLF的患者均为HBeAg阴性。而这部分ACLF的患者HBV DNA定量与该模式中SHB的患者无差异。
4) C基因编码区变异
(1)C基因编码区氨基酸发生变异的频率在ACLF组明显高于CHB组。在ACLF中氨基酸变异率超过10%,且与CHB组比较有统计学差异的共有7个位点。这些高频率的变异位点绝大部分位于表位区域：aa5、aa35和aa60分别位于的Thl-20、Th35-45和Th49-69辅助细胞表位区,aa27位于CTL18-27细胞毒性T细胞表位区,aa83和aa135分别位于B76-87和B130-135表位区。进一步统计以上表位区变异的频率显示：ACLF在各个表位区变异频率均高于CHB组。
(2)aa5和aa60变异存在明显的相关性,Pearson相关系数为0.632(P<0.001)。并且这两点在随访中表现为协同共变异：即aa5位氨基酸发生P-T的变异,同时观察到aa60发生L-V的变异；或aa5 T-P协同aa60 V-L。HBcAg晶体3D结构显示,aa5和aa60在空间构型上位置相邻,分享同一个四螺旋束(four-helix bundle)。网站预测aa5和aa60所在的肽段作为潜在的CTL表位与HLA分子的结合力,显示aa5发生P-T的变异后引起所在的肽段与HLA-A*0201分子的结合分数升高了3分(13 vs.16)；而aa60 L-V的变异后所在肽段与HLA-A*0201分子的结合分数降低。
(3)对于高变异位点所位于的已知线性Th细胞表位和CTL表位通过生物学网站预测其免疫原性的改变；对于空间构象型B细胞表位用HBcAg X衍射3D结构分析其构型的改变。通过HBcAg晶体结构分析构象性B细胞表位显示,高变异位点所在的B细胞表位均位于HBcAg结构中向外侧突出的位置；而在B细胞表位HBcAg130-135中,aa135处于最向外突出的点。CTL表位HBcAg18-27发生27位I-V的变异后,SYFPEITHI评分显示与HLA-A*0201分子的结合分数升高了2分(24 vs.22)；BIMAS评分显示HBcAg18-27(V27)与HLA-A*0201结合的分数为2309.961,而I27为346.494。
5)表位HBcAg18-27
(1)HLA-A*0201限制性特异性CTL表位HBcAg18-27,在中国I27的背景下,ACLF组V27变异率高。在ACLF组V27的发生率明显高于CHB组,差异有统计学意义[10／39(25.6%)vs.3／44(6.8%)；P=0.019]；同样,SHB组V27的比率也高于CHB组[8／38(21.1%)vs.3／44(6.8%)；P=0.059]。
(2)为了探讨表位HBcAg18-27的动态变化,随访了SHB和ACLF组中10例发病时V27单一病毒株感染或V27和I27混合株感染的患者,及19例发病时I27单一病毒株感染的患者。在4例HLA-A2阳性的患者中无论发病时是V27单一病毒株感染或是V27和I27混合株感染,随访后均变异成I27单一病毒株感染；而在5例HLA-A2阴性患者中,随访后包含V27的病毒仍能检测出。进一步的克隆测序,验证了V27向I27转换的变异模式。而18例发病时感染I27单一病毒株的患者随访后仍然为单一I27病毒株感染。
(3)用五聚体技术,分析HBcAg18-27表位变异后免疫原性的差异。分离HLA-A2阳性ACLF和SHB患者的PBMC,用HBcAg18-27两种形式(I27和V27)的特异性五聚体直接检测分析特异性CD8细胞的频数；直接检测的结果显示特异性CD8细胞的频数较低,两组之间无统计学差异。对于直接检测后仍有余留PBMC的患者,将其PBMC增殖培养13天后再进行五聚体染色：在案例报道中显示HBcAg18-27(V27)特异性CD8细胞的频数高于HBcAg18-27(127)特异性CD8细胞的频数。
(4)在ACLF和SHB的患者中,aa27V患者HBV DNA定量低于aa27I的患者,尽管差异无统计学意义(P=0.093)。而ACLF组HBV DNA定量明显低于CHB组[(5.75±1.73 vs.9.16±1.72)log10 copies/ml；P<0.001],SHB组也低于CHB组[(5.55±1.61 vs.9.16±1.72)1og10copies/ml；P<0.001].
结论：
1)与从SHB进展为ACLF相关的因素包括：TB、Cr和WBC的升高,ALB和PTA的降低。在ACLF中,PTA下降及WBC升高与不良结局相关。
2)基因B型在ACLF和SHB两组比率均高于CHB组,且SHB组与CHB组比较有统计学意义。这表明在中国南方地区基因B型与严重的肝脏炎症活动发病相关。
3) BCP(A1762T/G1764A)和PC(G1896A)变异率在ACLF组明显高于CHB组。ACLF组HBV发生BCP-／PC+和BCP+／PC+的比例明显高于CHB组,而BCP-／PC-比例明显低于CHB组。这个结果证实了A1762T／G1764A和G1896A高变异与ACLF发病的相关性。
4)在BCP+/PC-、BCP-/PC-、BCP-/PC+和BCP+/PC+模式中HBeAg的阴性率逐步升高；而且BCP-／PC+和BCP+／PC+模式下调HBeAg的作用与其他两种模式相比明显增强；在单变异模式中PC变异下调HBeAg作用强于BCP变异；且在ACLF组BCP+／PC+和PC单变异的比例明显高于CHB组。这表明PC+和BCP+／PC+模式导致的HBeAg与ACLF发病相关。
5)C基因编码区发生变异的频率在ACLF组明显高于CHB组。ACLF中氨基酸变异率明显升高的位点绝大部分位于表位区域。统计各个表位区变异的频率,在ACLF组均高于CHB组。这说明表位变异可能在ACLF发病中起着重要的作用。
6)aa5和aa60变异存在明显的协同关系。两位点尽管在线性位置相隔远,但在HBcAg空间构型上位置相邻,分享HBcAg四聚体中同一个螺旋束。通过预测分析肽段发生aa5 P-T的变异后与HLA-A*0201结合力增强；提示aa5变异后可能提高表位的免疫原性、诱导更强的免疫反应；而aa5 P-T的变异在ACLF中明显升高(52.63%)；表明aa5T与ACLF的发病相关。aa60由于与aa5空间位置相邻,可能在极性、PH值、亲水性等多个因素的影响下,出现协同变异。
7)HLA-A2限制性CTL表位HBcAg18-27(V27)在ACLF组的比率明显升高,且随访后ACLF患者中,HLA-A2阳性的患者均变异I27；且HBcAg18-27(V27)特异性CD8细胞的频数高于HBcAg18-27(127)特异性CD8细胞的频数。这表明HBcAg18-27(27V)在HLA-A2阳性患者中引起较强的免疫应答,在清除了aa27V病毒的同时,导致了肝脏炎症的发生,与ACLF的发病相关。
Hepatitis B virus (HBV) is responsible for chronic infection. There are approximately 300 million chronic HBV carriers worldwide and about 120 million HBV carriers in China. HBV infection can lead to a series of spectrums of liver diseases including asymptomatic carrier, chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) or acute-on-chronic liver failure (ACLF). According to the Asian Pacific Association for the Study of the Liver including acute hepatic insult, ACLF was characterized by jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. Similarly, in China, the diagnosis of ACLF was based on recent development of jaundice [total bilirubin (TB)≥171.0μmol/l] or rapidly rising levels of TB (≥17.1μmol/L/day) and plasma prothrombin activity (PTA)≤40%. These were accompanied by the development of complications such as hepatic encephalopathy (≥grade 2), abrupt and obvious increase in ascites or spontaneous bacterial peritonitis, or hepatorenal syndrome. In China, HBV-related ACLF cases account for more than 80% of ACLF patients. ACLF has been as a result of the high incidence of chronic HBV infection and was reported to have a high mortality rate (60-80%) in the absence of liver transplantation.
The pathogenesis of ACLF remains unclear. Both viral and immune factors may play a role. HBV mutations in the basal core promoter (BCP) and precore (PC) have attracted special attention. Previous studies have clarified that a higher prevalence of the BCP double mutations and PC mutation were in patients with acute liver failure. Because the BCP mutations may enhance HBV replication in vitro and the PC mutation abrogates translation of HBeAg, which is considered a tolerogen buffering any immune attack on the infected hepatocytes.
HBV DNA contains four open reading frames. One of them is the C gene, which encodes a core peptide (HBcAg). HBcAg has been postulated to be an immunological target of cytotoxic T lymphocytes (CTL). Studies on endogenously processed viral peptides demonstrated that a peptide could be recognized by CTL and is the principal motive power which can activate specific cell immune response. When the mutate site just located in the epitope sequence, it will be possible to change the immunological characteristics and then have an impact on the host responsibility to HBV. Investigation of changes in such regions may help to predict the occurrence of ACLF.
Purpose
In this study, the nucleotide sequences of the precore and core region of HBV were analyzed to investigate the relationship of HBV mutations with ACLF. Moreover, we looked into the association between ACLF and the epitopes in HBcAg, who have prototype and variant.
Methods
Thirty-nine patients with ACLF and 38 with severe hepatitis B (SHB) were involved in our study. SHB was defined as ALT≥600 IU/1 associated with TB≥3.0 mg/dl and PTA≤50%. Forty four chronic hepatitis B (CHB) patients with ALT level within the range of 80-400 IU/1 and TB≤1.0 mg/dl were enrolled as control. The patients with ACLF and SHB were followed up for average 10.6 months (ranged for 3-17m).Sequences of PreC/C region, HBV genotype was determined. The line epitopes, including Th epitope and CTL epitope, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The 3D data of B cell epitopes were presented from the X-ray structure of the HBcAg by Chimera software. The pentamer staining was performed to analyze epitope-specific CTL.
Results
1) There were significant differences in TB, serum albumin (ALB) and PTA between patients with ACLF and SHB. Creatinine (CR) and white blood cell (WBC) were significantly high in ACLF compared with SHB. Of the 39 patients with ACLF,18 had a fatal outcome and 21 survived more than 3 months after onset of liver failure. Lower PTA and higher WBC were detected as risk factors, while a high CR was only probable as a risk factor for mortality.
2) The distribution of genotype B was statistically higher in SHB Patients than that in CHB patients (72.2% vs.50%, P=0.044). And genotype B was found more often in ACLF compared with in CHB, although there was no statistically significant difference (P=0.069).
3) The prevalence of BCP (A1762T/G1764A) mutations were found significantly more often in ACLF than in CHB (56.7% vs.32.4%, P=0.046). The rate of PC (G1896A) mutation was higher in ACLF compared with CHB (50% vs.15.9%, P=0.003). Moreover, compared with the distribution of HBV BCP/PC mutation patterns in investigated groups, the patterns of BCP-/PC+and BCP+/PC+ occurred more frequently in ACLF patients than that in CHB patients.
4) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-(40%), BCP-/PC-(53.3%), BCP-/PC+(76.5%) and BCP+/PC+ (83.3%) viruses. And the rate of HBeAg negativity was not lowest in patients with BCP-/PC-virus. Furthermore, the rate of HBeAg negativity was compared among CHB, SHB and ACLF. The rate of HBeAg negativity was significantly high in ACLF patients..
5) The prevalence of T1846 was found significantly high in ACLF than in SHB [80.6%(25/31) vs.16.7%(5/30), P<0.001] or than in CHB [80.6%(25/31) vs.11.1% (4/36),P<0.001].
6) In ACLF patients, the sites with amino acid variability of more than 10% in PreC/C protein included aa5, aa60, aa97, aa83, aa27, aa98 and aa35. And there were statistically significant difference in above amino acids variability compared with ACLF and CHB. Th epitopes and CTL epitopes, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The affinity of CTL18-27 to HLA-A*0201 was enhanced when I varied into V at the site of aa27. From the 3D structure of HBc tetramers, the observed major B epitopes (HBcAg76-87 and HBcAg130-135) appeared predominantly on the outer surface of the HBc protein. The one of most variable positions aa135 within the B epitope HBcAg130-135 was the most externally exposed.
7) Amino acid mutation in aa5 always accompanied with aa60; and the index of Pearson correlation was 0.632 (P<0.001). aa5 P-T accompanied with aa60 L-V and aa5 T-P with aa60 V-L in followed-up patients. Shown in the 3D structure of HBc tetramers, these two sites closely contacted within the four-helix bundle. Moreover, the affinity of epitope with variant in aa5 to HLA-A*0201 was enhanced, while the affinity of epitope with mutant in aa60 was decreased by epitope prediction WEB.
8) The prevalence of V27 was found significantly more often in ACLF than in CHB [10/39 (25.6%) vs.3/44 (6.8%); P=0.019], although there was no statistically significant difference between SHB and CHB [8/38 (21.1%) vs.3/44 (6.8%); P=0.059].
9) Ten patients primarily infected with pure V27 or the mixture of 127 and V27 and 19 primarily infected with pure 127 were followed up for at least 3 months in SHB and ACLF groups. In two cases (2/29), the sequence of the HBcAg18-27 encoding region could not be amplified. The remaining 27 patients were including 9 with pure V27 or the mixture of 127 and V27 and 19 with pure 127. These 9 patients with pure V27 or the mixture of 127 and V27 were classified into two groups according to HLA-A2 type. Among 4 HLA-A2 positive cases, V27 was not detected and it turned into 127. On contrary, V27 could still be detected among 5 HLA-A2 negative cases. Amino acid at position 27 was unchanged in 18 cases originally infected with 127, in whom the longest period of follow-up was 17 months. We used clones sequence to verify the virus change.
10) The low frequency of epitope-specific CD8+T cells by pentamer staining ex vivo was unavailable in our study, thus the residue PBMCs were stimulated with I27/V27 peptide mixture, and then the pentamer staining was performed. The results of the pentamer staining showed V27 specific CD8 T cells were detected at high level, while the 127 specific CD8 T cells were found to be much less common.
11) Mean HBV DNA level was significantly lower in ACLF than in CHB [(5.75±1.73 vs.9.16±1.72) log10 copies/ml; P<0.001], and a significant difference was also found between SHB and CHB [(5.55±1.61 vs.9.16±1.72) log10 copies/ml; P< 0.001]. HBV DNA level was lower in patients with V27 than in ones with 127 in ACLF and SHB, although there was no statistically significant difference [(5.14±1.31 vs.5.80±1.74) log10 copies/ml; P=0.093].
Conclusions
1) In this study, we found the prevalence of genotype B was 72% and 74% in ACLF and SHB, respectively, which was much higher in CHB. And BCP and PC mutations were more often in ACLF than in CHB. These suggested HBV genotype B and BCP/PC mutations were significantly associated with ACLF.
2) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-, BCP-/PC-, BCP-/PC+ and BCP+/PC+ viruses. The loss of HBeAg caused by BCP+/PC+ and BCP-/PC+ was related with the pathogenesy of ACLF.
3) Amino acid mutation in aa5 accompanied with aa60. These two sites closely contacted within the four-helix bundle in the 3D structure of HBc tetramers. The affinity of epitope with variant(T) in aa5 to HLA-A*0201 was enhanced by epitope prediction WEB. The above data suggested aa5T was associated with ACLF.
4) In HBcAg, the epitope drift or up regulation of immunodominance at multiply sites play a role of the development of ACLF.
5) HBcAg18-27 epitope with V27 may be associated with the development of ACLF and CTL response produced by the epitope involves the process of controlling the virus replication.
引文
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[19]Stuyver L, De Gendt S, Van Geyt C, et al. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness [J]. J Gen Virol,2000,81(Pt 1): 67-74.
[20]Arauz-Ruiz P, Norder H, Robertson BH, et al. Genotype H:a new Amerindian genotype of hepatitis B virus revealed in Central America[J]. J Gen Virol,2002, 83(Pt 8):2059-2073.
[21]Bowyer SM, Sim JG. Relationships within and between genotypes of hepatitis B virus at points across the genome:footprints of recombination in certain isolates[J]. J Gen Virol,2000,81(Pt 2):379-392.
[22]Huy TT, Ushijima H, Quang VX, et al. Genotype C of hepatitis B virus can be classified into at least two subgroups[J]. J Gen Virol,2004,85(Pt 2):283-292.
[23]Norder H, Courouce AM, Coursaget P, et al. Genetic diversity of hepatitis B virus strains derived worldwide:genotypes, subgenotypes, and HBsAg subtypes[J]. Intervirology,2004,47(6):289-309.
[24]Yuen MF, Tanaka Y, Mizokami M, et al. Role of hepatitis B virus genotypes Ba and C, core promoter and precore mutations on hepatocellular carcinoma:a case control study[J]. Carcinogenesis,2004,25(9):1593-1598.
[25]Hasegawa K, Huang JK, Wands JR, et al. Association of hepatitis B viral precore mutations with fulminant hepatitis B in Japan[J]. Virology,1991, 185(1):460-463.
[26]Kosaka Y, Takase K, Kojima M, et al. Fulminant hepatitis B:induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen[J]. Gastroenterology,1991,100(4):1087-1094.
[27]Laskus T, Rakela J, Nowicki MJ, et al. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B[J]. Gastroenterology, 1995,109(5):1618-1623.
[28]Liang TJ, Hasegawa K, Rimon N, et al. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis[J]. N Engl J Med,1991,324(24): 1705-1709.
[29]Omata M, Ehata T, Yokosuka O, et al. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis [J]. N Engl J Med,1991,324(24):1699-1704.
[30]Sato S, Suzuki K, Akahane Y, et al. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis [J]. Ann Intern Med,1995, 122(4):241-248.
[31]Hsu HY, Chang MH, Lee CY, et al. Precore mutant of hepatitis B virus in childhood fulminant hepatitis B:an infrequent association[J]. J Infect Dis,1995, 171(4):776-781.
[32]Laskus T, Persing DH, Nowicki MJ, et al. Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States [J]. Gastroenterology,1993,105(4):1173-1178.
[33]Teo EK, Ostapowicz G, Hussain M, et al. Hepatitis B infection in patients with acute liver failure in the United States[J]. Hepatology,2001,33(4):972-976.
[34]Ren X, Xu Z, Liu Y, et al. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis[J]. J Viral Hepat.
[35]Imamura T, Yokosuka O, Kurihara T, et al. Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan[J]. Gut,2003,52(11): 1630-1637.
[36]Hasegawa K, Huang J, Rogers SA, et al. Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis[J]. J Virol,1994, 68(3):1651-1659.
[37]Baumert TF, Yang C, Schurmann P, et al. Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes[J]. Hepatology,2005,41(2):247-256.
[38]Hou J, Lin Y, Waters J, et al. Detection and significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis[J]. J Gen Virol, 2002,83(Pt 9):2291-2298.
[39]Pult I, Chouard T, Wieland S, et al. A hepatitis B virus mutant with a new hepatocyte nuclear factor 1 binding site emerging in transplant-transmitted fulminant hepatitis B[J]. Hepatology,1997,25(6):1507-1515.
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[41]Pollicino T, Zanetti AR, Cacciola I, et al. Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis[J]. Hepatology,1997,26(2): 495-499.
[42]Sterneck M, Kalinina T, Gunther S, et al. Functional analysis of HBV genomes from patients with fulminant hepatitis[J]. Hepatology,1998,28(5):1390-1397.
[43]Kalinina T, Riu A, Fischer L, et al. A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis[J]. Hepatology,2001,34(2):385-394.
[44]Wai CT, Fontana RJ, Polson J, et al. Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States[J]. J Viral Hepat,2005,12(2):192-198.
[45]Ozasa A, Tanaka Y, Orito E, et al. Influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis B virus infection[J]. Hepatology,2006,44(2):326-334.
[46]Joh R, Hasegawa K, Ogawa M, et al. Genotypic analysis of hepatitis B virus from patients with fulminant hepatitis:comparison with acute self-limited hepatitis[J]. Hepatol Res,2003,26(2):119-124.
[47]Sainokami S, Abe K, Sato A, et al. Initial load of hepatitis B virus (HBV), its changing profile, and precore/core promoter mutations correlate with the severity and outcome of acute HBV infection[J]. J Gastroenterol,2007,42(3): 241-249.
[48]Ganem D, Prince AM. Hepatitis B virus infection--natural history and clinical consequences[J]. N Engl J Med,2004,350(11):1118-1129.
[49]Bertoletti A, Chisari FV, Penna A, et al. Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein[J]. J Virol,1993,67(4):2376-2380.
[50]Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection[J]. J Virol,2007, 81(8):4215-4225.
[51]Webster GJ, 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]. J Virol,2004, 78(11):5707-5719.
[52]Sendi H, Mehrab-Mohseni M, Shahraz S, et al. CTL escape mutations of core protein are more frequent in strains of HBeAg negative patients with low levels of HBV DNA[J]. J Clin Virol,2009,46(3):259-264.
[53]Bertoletti A, Costanzo A, Chisari FV, et al. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope[J]. J Exp Med,1994, 180(3):933-943.
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