HBcrAg在慢性乙型肝炎中研究进展

2020-10-20 04:48:18 医学信息 2020年17期

党便利 孙永涛 康文臻

摘要:肝内共价闭合环状DNA(cccDNA)的存在导致乙型肝炎病毒(HBV)不能从感染的肝细胞中完全清除。乙型肝炎核心相关抗原(HBcrAg)作为非侵入性肝活检的替代物,与血清HBV DNA和肝内cccDNA相关,在慢性乙型肝炎(CHB)中具有重要作用。在血清HBV DNA或HBsAg不可检测临床病例中,仍可以检测到HBcrAg,且HBcrAg水平与CHB患者的预后显著相关。HBcrAg可预测自发或由治疗引起的乙肝炎e抗原(HBeAg)血清学转换、核苷类似物停药后的持续应答、潜在HBV再激活以及肝细胞癌进展或复发的风险。本文就HBcrAg的病毒学特点及其在CHB患者中的临床应用作一综述。

关键词:乙型肝炎核心相关抗原;共价闭合环状DNA;乙型肝炎病毒;慢性乙型肝炎

中图分类号:R512.62                                文献标识码:A                                DOI:10.3969/j.issn.1006-1959.2020.17.007

文章编号:1006-1959(2020)17-0023-04

Abstract:The presence of covalently closed circular DNA (cccDNA) in the liver prevents the hepatitis B virus (HBV) from being completely eliminated from infected liver cells. Hepatitis B core-associated antigen (HBcrAg), as a non-invasive liver biopsy alternative, is related to serum HBV DNA and intrahepatic cccDNA, and plays an important role in chronic hepatitis B (CHB). In clinical cases where serum HBV DNA or HBsAg is not detectable, HBcrAg can still be detected, and HBcrAg level is significantly related to the prognosis of CHB patients. HBcrAg can predict spontaneous or treatment-induced hepatitis B e antigen (HBeAg) seroconversion, sustained response after discontinuation of nucleoside analogs, potential HBV reactivation, and the risk of hepatocellular carcinoma progression or recurrence. This article reviews the virological characteristics of HBcrAg and its clinical application in CHB patients.

Key words:Hepatitis B core related antigen;Covalently closed circular DNA;Hepatitis B virus; Chronic hepatitis B

乙型肝炎病毒(HBV)感染可引起急、慢性肝病,也可能进展为肝硬化、肝细胞癌(hepatocellular carcinoma,HCC)等疾病[1,2]。由于感染的肝细胞核中存在共价闭合的环状DNA(covalently closed circular DNA,cccDNA),慢性乙型肝炎(chronic hepatitis B,CHB)无法完全根治[3]。cccDNA定量因侵入性、缺乏标准化检测方法等局限,而乙肝核心相关抗原(HBcrAg)作为CHB的疾病监测和预后的替代标志物,可反映肝内病毒复制活性[3]。本文综述了HBcrAg的结构、检测、与其他HBV生物标志物间的相关性、预测临床结果的能力以及其作为预测因子的作用,以期为CHB的临床实践应用提供理论参考。

1 HBcrAg结构及检测

HBcrAg由前核心/核心区编码的HBeAg、HBcAg及p22cr三种蛋白组成,具有相同的149个氨基酸序列[4]。HBcAg是HBV病毒衣壳的结构成分,而HBeAg和p22cr均是前核心蛋白的加工产物,同时,HBcAg和HBeAg又都是细胞毒性T细胞的靶点,因此,3种蛋白组成的HBcrAg可以诱导宿主细胞免疫应答[5,6]。2002年Kimura T等[7]首次发现HBcrAg与HBcAg和HBeAg特异性的敏感酶免疫分析有关,即使抗HBc或抗HBe阳性样本中仍可以检测HBcAg和HBeAg。HBcrAg浓度与HBV DNA浓度呈线性相关,用HBcrAg法测定HBV载量的准确性不受血清中HBeAg缺失或HBV基因组中前核心突变的影响[8],因此可作为监测CHB患者HBV DNA的补充指标。

2 HBcrAg与其他HBV标记物间的相关性

2.1血清或肝内HBV DNA水平  HBcrAg濃度随着肝内HBV DNA水平变化而改变,且两者具有良好的相关性,也能直接反映血清HBV DNA浓度,与HBeAg状态无关[7,9]。

2.2肝内cccDNA水平  研究发现[10,11],血清HBcrAg水平与血清HBV DNA和肝内cccDNA水平均的相关性均良好。78%的接受抗病毒治疗的患者经常无法检测到血清HBV DNA,却可检测到血清HBcrAg,因此,血清HBcrAg可作为评估肝内cccDNA水平的病毒学标志物[9]。Hasegawa K等[12]建立了FBS-cres评分公式[3.1686-(0.0148×FBS)+(0.1982×HBcrAg)+(0.0008168×HBeAg)+(0.1761×log10 HBsAg)]预测CHB患者中cccDNA水平,该公式通过对未经核苷酸类似物(NA)治疗的患者的多元分析,FBS-cres得分显示HBcrAg与cccDNA水平之间存在相关性,证实了其预测的准确性。

2.3乙肝表面抗原  2014年的一项研究中发现HBsAg-HQ水平、HBV DNA、HBsAg和HBcrAg水平之间存在很强的相关性(P<0.001)。血清HBcrAg水平与血清HBV-DNA和HBsAg水平也显著相关(P<0.001)[13],这些结果表明HBcrAg与HBsAg-HQ的相关性比传统的HBsAg检测更为敏感。

2.4乙肝病毒RNA  前基因组RNA(pgRNA)作为HBV DNA聚合酶的模板,并介导病毒的持续复制。一些子代DNA被组装在内质网中形成完整的病毒并由肝细胞分泌[14]。Liu YY等[14]和Chen EQ等[15]相继证实,血清HBcrAg与肝内cccDNA的相关性均优于血清HBV RNA和HBsAg间相关性,且与HBeAg状况无关。

2.5乙肝病毒基因缺失  由于HBV基因组preS1/S2区域存在与CHB的进展相关的缺失[16],对preS定量分析可能会加深对CHB进展的了解[17]。Suzuki Y等[16]对90例未接受抗病毒治疗的CHB患者HBV的preS1/S2区域进行了深度测序,并剔除了受病毒标记影响的区域。对每位CHB患者的缺失区域频率分析表明,缺失区域最常见于preS2密码子132-141,其起始密码子突变与缺失显著相关于血清HBcrAg水平,提示preS密码子132-141的缺失对临床特征和病毒标志物有重要影响。

3 HBcrAg对CHB疾病进展的预测

3.1慢性感染不同阶段的HBcrAg  Misawa N等[18]发现,在HBeAg血清转化前,活性复制组的HBcrAg水平低于非活性复制组,而两组间HBV DNA水平相似;在HBeAg血清转化后,非活性复制组HBV DNA和HBcrAg均水平显著降低,而在活性复制组中两种指标均没有变化或略有下降,说明HBcrAg水平可能有助于HBeAg血清转化[18,19],提示血清HBcrAg也可以作为CHB的疾病监测和临床结果预测标记物。

3.2 HBeAg血清转化的预测  Testoni B等[20]发现,相对于未经抗病毒治疗的HBeAg阴性患者,HBeAg阳性患者的血清HBcrAg水平显著升高,且与血清HBV DNA、pgRNA和肝内HBV DNA、cccDNA水平以及转录活性相关。较高水平的HBcrAg与血清HBV DNA、肝内HBV DNA、pgRNA、cccDNA转录活性以及纤维化和坏死性炎症相关。对于HBeAg阴性患者,无活性携带者阶段的HBcrAg水平显着低于活性阶段,这与坏死性炎症和纤维化有关[13,21]。

3.3 HBsAg血清转化的预测  Loggi E等[22]研究发现,相对于75例临床上不活跃的HBV携带者,85例CHB患者中HBcrAg水平明显更高。同时,鉴定临床上不活跃的携带者HBcrAg最佳临界值为2.5 log U/ml,其预测准确性可与血清HBsAg水平相媲美。对于血清HBsAg自发性清除的患者,79%患者的HBcrAg未检出,而剩余21%的患者仍可检测到血清HBcrAg(中位数为2.7 log U/ml)[19,23],提示HBcrAg可能进一步用于预测CHB的疾病阶段,尽管其最佳临界值仍有待确定。

4抗病毒治疗

NA可有效降低CHB患者肝炎活性并抑制其血清HBV DNA,却存在耐药或停药复发的风险[24]。如果CHB患者满足《JSH HBV感染管理指南》中评估NA停药时机的3个实验室标准:①NA治疗2年以上;②使用实时PCR无法检测到血清HBV DNA;③停药时血清HBeAg呈阴性,则可根据HBsAg和HBcrAg水平进一步预测停药复发风险[25]。

4.1 NA治疗过程中HBcrAg和其他指标的变化  研究发现[25],在接受NA治疗(中位时间为126个月)43名患者中,98%患者血清HBV DNA呈陰性,其中51%患者仍可检出肝内cccDNA。在接受聚乙二醇干扰素(PEG-IFN)和阿德福韦(ADV)联合治疗后再进行ADV单药治疗的患者中也有类似的结果[27]。因此,抗HBV治疗后血清中HBV DNA的减少与肝内cccDNA的减少并不相关。值得注意的是,HBcrAg减少与肝内cccDNA水平变化具有良好的相关性[9,28]。即使在血清HBsAg未检出的患者中,21%仍可检测到血清HBcrAg,而仅有2.1%的患者可检测到的HBV DNA[23]。对于HBcrAg基线水平高于8 log U/ml的PEG-IFN治疗的患者,可在治疗12周时实现HBeAg血清转化和HBV DNA抑制, 预测值达94.4%[29]。在另一项研究中,50例接受PEG-IFN联合NA治疗4周,再接受PEG-IFN连续治疗20周,治疗开始时HBcrAg水平高于 4.5 log U/ml,预测治疗结束24个月后HBcrAg无应答,且HBeAg无血清转化[30]。因此,基线及抗HBV治疗时血清HBcrAg的水平也可能预测CHB患者疾病进展。Tanaka E等[31]比较了HBcrAg和血清HBV DNA在预测81名CHB患者发生LAM耐药性风险的临床价值,其中31%患者在随访期间(中位数为 19.3个月)出现了LAM耐药性。虽然开始服用LAM后HBcrAg和HBV DNA均会降低,但HBcrAg比HBV DNA的下降幅度更大;治疗6个月后HBcrAg水平小于4.6 log U/ml的19例患者未发生LAM耐药,但在两年内仍有50%的患者发生耐药。由于HBeAg血清转化与未转化的患者之间的HBsAg和HBcrAg有所不同,因此,可以结合分析HBsAg和HBcrAg来判断患者HBeAg是否发生血清转化[32]。         4.2 NA停用时机  研究发现[33,34],当停药时血清HBcrAg水平大于 3.7 log U/ml,预测停药1年内出现病毒学复发。另一项研究也有相似发现[35],CHB患者NA停用时HBcrAg呈高水平(中位数为4.9 log U/ml),预测停药6个月后出现病毒学复发。所有停用LAM时HBcrAg 小于3.0 log U/ml的患者均无ALT复升,而停药时较高水平的HBcrAg,复发者与非复发者分别为(4.5±1.0)log U/ml、(3.4±0.9)log U/ml,P=0.0145,是重要的复发预测指标;另外其他NA[如恩替卡韦(ETV)或TDF]停用时的HBcrAg水平也可作为预测复发独立的指标[36]。Hsu YC等[37]建立TDF停药复发率评分公式(SCALE-B):35×HBsAg(logIU/mL)+20×HBcrAg(logU/ml)+2×年龄(年)+ALT(U/L)+40,得出停药1~5年的临床复发率基本一致,分别为0.87、0.88、0.87、0.85和0.90。因此,血清HBcrAg可能作为NA停用时机和重新激活风险标志物。

5 HBcrAg對HCC发展的预测

研究证实[38],HBcrAg对HCC复发有预测价值。在1031例未经NA治疗的CHB患者随访期间(中位:10.7年),有78位患者(7.6%)发展为HCC。使用Cox比例风险模型对HBV基因型状态、HBV DNA水平、HBcrAg水平、HBeAg状态和基础核心启动子(BCP)状态进行多因素分析,结果显示HBcrAg大于2.9 log U/ml和基础核心启动子(BCP)突变与HCC发生相关,且对未经治疗的CHB患者的HCC进展预测能力优于HBV DNA[38,39]。另有发现[40],血清HBcrAg可监测肝癌中的cccDNA,可能是肝癌患者预后的良好指标。89例HCC患者肝脏标本的HBcrAg和HBV DNA均与患者的cccDNA相关,HBcrAg水平为4.0 log U/ml时,可识别出中等病毒载量的HCC高危患者[40]。NA治疗可降低CHB患者HCC发生风险,但不能完全消除[41]。接受NA治疗两年以上的109例CHB患者的HBcrAg可作为肝癌发展的独立危险因素(HR,3.53)[42]。接受NA治疗的76例CHB患者血清HBV-DNA虽已检测不到,但肝癌患者HBcrAg基线水平(治疗前)明显高于对照组。另外,治疗前HBcrAg大于 4.67 log U/ml可独立预测HCC发生率[43]。治疗后,HBcrAg大于3.89 log U/ml时,预测肝癌的优势比为3.27,而非肝硬化患者HBcrAg大于3.90 log U/ml时,预测肝癌优势比为5.95[43]。因此,血清HBcrAg水平可能是CHB患者的肝癌发生风险及术后复发的有效预测指标。

6总结

本文总结了HBcrAg的特征及临床应用,说明HBcrAg可作为肝内cccDNA良好的替代生物标记。所有CHB疾病状态下血清HBcrAg水平均与HBV DNA含量有关。提示HBcrAg有助于区分HBeAg阴性CHB和非活动携带者状态,这两种疾病状态具有不同的临床意义和预后。无法检测到血清HBV DNA和HBsAg(即达到“功能性治愈”)的患者仍会出现HBV重新激活和HCC等严重并发症,同时,一定比例的“功能治愈”患者可检测到HBcrAg,因此,有必要对HBcrAg阳性或阴性患者的长期预后进行前瞻性研究。血清HBcrAg可预测HBeAg和HBsAg的血清转化,并对CHB的疾病阶段预测的准确性可与HBsAg相当,因此,其可作为CHB的疾病监测和临床结果预测标志物。由于HBcrAg对隐匿性的HBV重新激活具有良好的预测价值,因此,对中、低危人群应做更多研究并进行停药风险分析。此外,HBcrAg可预测HCC的发生,且与抗病毒治疗状态无关。总之,HBcrAg能反映肝内cccDNA水平和复制活性,对CHB的疾病进展监测、预测停药时机和病毒重新激活风险均有重要作用。为了将HBcrAg用于CHB临床实践的众多方面,在临床实践中常规使用之前仍需更广泛的临床验证。

参考文献:

[1]Kao JH.Hepatitis B vaccination and prevention of hepatocellular carcinoma[J].Best Pract Res Clin Gastroenterol,2015,29(6):907-917.

[2]Lai CL,Yuen MF.The natural history and treatment of chronic hepatitis B:A critical evaluation of standard treatment criteria and end points[J].Ann Intern Med,2007,147(1):58-61.

[3]Raimondo G,Allain JP,Brunetto M,et al.Statements from the Taormina expert meeting on occult hepatitis B virus infection[J].J Hepatol,2008,49(4):652-657.

[4]Hadziyannis E,Laras A.Viral Biomarkers in Chronic HBeAg Negative HBV Infection[J].Genes,2018,9(10):469.

[5]Maasoumy B,Wiegand SB,Jaroszewicz J,et al.Hepatitis B core-related antigen(HBcrAg)levels in the natural history of hepatitis B virus infection in a large European cohort predominantly infected with genotypes A and D[J].Clin Microbiol Infect,2015,21(6):606,e1-e10.

[6]Lin CL,Kao JH.New perspectives of biomarkers for the management of chronic hepatitis B[J].Clin Mol Hepatol,2016,22(4):423-431.

[7]Kimura T,Rokuhara A,Sakamoto Y,et al.Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load[J].J Clin Microbiol,2002,40(20):439-445.

[8]Locarnini S,Zoulim F.Molecular genetics of HBV infection[J].Antivir Ther,2010,15(Suppl 3):3-14.

[9]Wong DK,Seto WK,Cheung KS,et al.Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA[J].Liver Int,2017,37(7):995-1001.

[10]Wong DK,Yuen MF,Yuan H,et al.Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients[J].Hepatology,2004,40(3):727-737.

[11]Wang L,Cao X,Wang Z,et al.Correlation of HBcrAg with Intrahepatic Hepatitis B Virus Total DNA and Covalently Closed Circular DNA in HBeAg-Positive Chronic Hepatitis B Patients[J].J Clin Microbiol,2019,57(1):e01303-e01318.

[12]Hasegawa K,Nishikawa H,Enomoto H,et al.Proposed model for the prediction of intrahepatic covalently closed circular DNA level in patients with chronic hepatitis B[J].Hepatol Res,2018,49(3):271-283.

[13]Seto WK,Wong DK,Fung J,et al.Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B[J].Clin Microbiol Infect,2014,20(11):1173-1180.

[14]Liu YY,Liang XS.Progression and status of antiviral monitoring in patients with chronic hepatitis B:From HBsAg to HBV RNA[J].World J Hepatol,2018,10(9):603-611.

[15]Chen EQ,Wang ML,Tao YC,et al.Serum HBcrAg is better than HBV RNA and HBsAg in reflflecting intrahepatic covalently closed circular DNA[J].J Viral Hepat,2019,26(5):586-595.

[16]Suzuki Y,Maekawa S,Komatsu N,et al.HBV preS deletion mapping using deep sequencing demonstrates a unique association with viral markers[J].PLoS One,2019,14(2):e0212559.

[17]Suzuki F,Miyakoshi H,Kobayashi M,et al.Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients[J].J Med Virol,2009,81(1):27-33.

[18]Misawa N,Matsumoto A,Tanaka E,et al.Patients with and without loss of hepatitis B virus DNA after hepatitis B e antigen seroconversion have different virological characteristics[J].J Med Virol,2006,78(1):68-73.

[19]Seto WK,Wong DK,Fung J,et al.Linearized hepatitis B surface  antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B[J].Clin Microbiol Infect,2014,20(11):1173-1180.

[20]Testoni B,Lebosse F,Scholtes C,et al.Serum hepatitis B core-related antigen(HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients[J].J Hepatol,2019,70(4):615-625.

[21]Zhang ZQ,Lu W,Wang YB,et al.Measurement of the hepatitis B core-related antigen is valuable for predicting the pathological status of liver tissues in chronic hepatitis B patients[J].J Virol Methods,2016,235:92-98.

[22]Loggi E,Vukotic R,Conti F,et al.Serum hepatitis B core-related antigen is an effffective tool to categorize patients with HBeAg-negative chronic hepatitis[J].B J Viral Hepat,2019,26(5):568-575.

[23]Seto WK,Tanaka Y,Wong DK,et al.Evidence of serologic activity in chronic hepatitis B after surface antigen(HBsAg)seroclearance documented by conventional HBsAg assay[J].Hepatol Int,2012,7(1):98-105.

[24]Jeng WJ,Chen YC,Sheen IS,et al.Clinical Relapse After Cessation of Tenofovir Therapy in Hepatitis B e Antigen-Negative Patients[J].Clin Gastroenterol Hepatol,2016,14(12):1813-1820.

[25]JSH Guidelines for the Management of Hepatitis B Virus Infection[J].Hepatol Res,2014,44 (Suppl.S1):1-58.

[26]Lai CL,Wong D,Ip P,et al.Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B[J].J Hepatol,2017,66(2):275-281.

[27]Lutgehetmann M,Volzt T,Quaas A,et al.Sequential combination therapy leads to biochemical and histological improvement despite low ongoing intrahepatic hepatitis B virus replication[J].Antivir Ther,2008,13(1):57-66.

[28]Wong DK,Tanaka Y,Lai CL,et al.Hepatitis B virus core-related antigens as markers for monitoring chronic hepatitis B infection[J].J Clin Microbiol,2007,45(12):3942-3947.

[29]Chuaypen N,Posuwan N,Payungporn S,et al.Serum hepatitis B core-related antigen as a treatment predictor of pegylated interferon in patients with HBeAg-positive chronic hepatitis B[J].Liver Int,2016,36(6):827-836.

[30]Matsumoto A,Yatsuhashi H,Nagaoka S,et al.Factors associated with the effffect of interferon-alpha sequential therapy in order to discontinue nucleoside/nucleotide analog treatment in patients with chronic hepatitis B[J].Hepatol Res,2015,45(12):1195-1202.

[31]Tanaka E,Matsumoto A,Suzuki F,et al.Measurement of hepatitis B virus core-related antigen is valuable for identifying patients who are at low risk of lamivudine resistance[J].Liver Int,2006,26(1):90-96.

[32]Wang B,Carey I,Bruce M,et al.HBsAg and HBcrAg as predictors of HBeAg seroconversion in HBeAg-positive patients treated with nucleos(t)ide analogues[J].J Viral Hepat,2018,25(8):886-893.

[33]Caviglia GP,Abate ML,Noviello D,et al.Hepatitis B core-related antigen kinetics in chronic hepatitis B virus genotype D-infected patients treated with nucleos(t)ide analogues or pegylated-interferon-alpha[J].Hepatol Res,2017,47(8):747-754.

[34]Jung KS,Park JY,Chon YE,et al.Clinical outcomes and predictors for relapse after cessation of oral antiviral treatment in chronic hepatitis B patients[J].J Gastroenterol,2016,51(8):830-839.

[35]Matsumoto A,Tanaka E,Minami M,et al.Low serum level of hepatitis B core-related antigen indicates unlikely reactivation of hepatitis after cessation of lamivudine therapy[J].HepatolRes,2007,37(8):661-666.

[36]Shinkai N,Tanaka Y,Orito E,et al.Measurement of hepatitis B virus core-related antigen as predicting factor for relapse after cessation of lamivudine therapy for chronic hepatitis B virus infection[J].Hepatol Res,2006,36(4):272-276.

[37]Hsu YC,Nguyen MH,Mo LR,et al.Combining hepatitis B core-related and surface antigens at end of nucleos(t)ide analogue treatment to predict off-therapy relapse risk[J].Aliment Pharmacol Ther,2019,49(1):107-115.

[38]Ganem D,Prince AM.Hepatitis B virus infection–natural history and clinical consequences[J].N Engl J Med,2004,350(11):1118-1129.

[39]Liu S,Zhou B,Valdes JD,et al.Serum HBV RNA:A New Potential Biomarker for Chronic Hepatitis B Virus Infection[J].Hepatology,2019,69(4):1816-1827.

[40]Schadler S,Hildt E.HBV life cycle:Entry and morphogenesis[J].Viruses,2009,1(2):185-209.

[41]Misawa N,Matsumoto A,Tanaka E,et al.Patients with and without loss of hepatitis B virus DNA after hepatitis B e antigen seroconversion have different virological characteristics[J].Journal of Medical Virology,2010,78(1):68-73.

[42]Honer Zu Siederdissen C,Maasoumy B,Cornberg M.What is new on HBsAg and other diagnostic markers in HBV infection Best Pract[J].Res Clin Gastroenterol,2017,31(3):281-289.

[43]Liu YY,Liang XS.Progression and status of antiviral monitoring in patients with chronic hepatitis B:From HBsAg to HBV RNA[J].World J Hepatol,2018,10(9):603-611.

收稿日期:2020-06-21;修回日期:2020-07-03

編辑/肖婷婷