【作者】 刘妍；

【导师】 徐东平；

【作者基本信息】 中国人民解放军军事医学科学院 ， 免疫学， 2012， 博士

【摘要】 研究背景：乙型肝炎病毒（hepatitis B virus, HBV）感染可引起多种临床表现，包括无症状HBV携带状态、急性乙型肝炎、轻中度或重度慢性乙型肝炎，并可进一步发展为肝硬化、肝细胞癌和慢加急性肝衰竭。我国有9300万慢性HBV感染者，其中慢性乙型肝炎患者约2000万人，乙肝肝硬化患者约400万人，每年约28万人死于肝细胞癌。而慢加急性肝衰竭患者每年可造成2万多人死亡，其发生与机体对感染病毒产生过强的免疫应答相关，而病毒基因变异可能是引起机体免疫应答异常的一个重要因素。HBV具有高变异特性，虽是DNA病毒，其复制却与逆转录病毒类似存在一个RNA中间体的逆转录过程，而参与此过程的HBV DNA多聚酶/逆转录酶（reverse transcriptase,RT）缺乏严格的校对功能，使病毒复制过程中的碱基错配率明显高于其他DNA病毒。HBV也是高复制率病毒，每天病毒基因组中每个碱基都有发生所有碱基替换的可能。此外，HBV的复制模板共价闭合环状DNA（covalently closed circular DNA, cccDNA）半衰期长，稳定存在于感染的肝细胞核中，是HBV持续感染的根本原因。在自然感染状态下HBV是以野生株为优势株的准种群形式存在，根据HBV基因组序列差异分为8种不同的基因型（≥8%）和若干基因亚型（≥4%）。各种基因型HBV的病毒学特点不同，也可影响肝病的临床转归。HBV基因组最常见的热点变异是基本核心启动子（basal core promoter, BCP）区和前核心（precore, PC）区变异。HBV PC区编码的e抗原（hepatitis B e antigen, HBeAg）是一种免疫耐受抗原，而BCP区转录调节HBeAg表达，发生在这两个区的变异可终止或减低HBeAg表达，增强病毒复制力或逃避宿主免疫应答。另一方面，由于HBeAg与HBV核心抗原（HBcAg）共有相同的免疫表位，该区变异也增加了免疫系统对HBcAg的攻击，可能与乙肝进展及重症化相关。研究表明HBV BCP/PC区变异可能与急性重型肝炎的发生相关，但也有不同的研究结果。基因型又可以影响BCP/PC区变异的发生率，增加结果分析的复杂性。关于HBV BCP/PC变异和基因型与慢加急性肝衰竭发生的相关性分析少见报道。本研究的第一部分系统分析了我国临床大样本急性乙肝、轻中度慢乙肝、重度慢乙肝和慢加急性肝衰竭患者的BCP/PC区变异和基因型特点以及临床意义，结果有助于理解HBV病毒学特点在乙型肝炎慢性化、重症化机制中的作用。当前，核苷（酸）类似物是临床最常用的抗HBV感染治疗药物，包括三种核苷类，即拉米夫定（lamivudine, LAM）、恩替卡韦（entecavir, ETV）和替比夫定（tebivudine, LdT），两种核苷酸类，即阿德福韦酯（adefovir dipivoxil, ADV）和富马酸替诺福韦酯（tenofovirdisoproxil fumarate, TDF），前四种已在我国临床批准使用。这些药物通过直接靶向HBV RT区而发挥抗病毒作用，但均不能从根本上清除感染肝细胞核中持续存在的HBV cccDNA，一旦停药反跳率高，因此需要长期抗病毒治疗。然而长期抗病毒治疗会使病毒在药物压力下获得适应性变异或使预存于准种群中的极少量变异株获得选择性扩增，产生耐药病毒导致治疗失败，成为临床面临的棘手问题。发生在HBV逆转录酶区的耐药变异分为直接降低药物敏感性的原发耐药变异和增强病毒复制力的补偿变异，还有一些与ADV耐药相关的有争议的变异。随着临床上可选择的药物增多，耐药及交叉耐药的风险增加，变异形式也更加多样化。此外，不适当的核苷（酸）类药物序贯使用促进了多重耐药病毒株的产生。已有的关于耐药变异病毒的研究大多来源于一些临床试验研究，用药方式相对固定，研究的样本量有限，但实际上临床用药方式复杂，引起的HBV耐药变异谱有其特有规律，以往尚缺少大样本分析数据。本研究的第二部分系统分析了我国大样本经核苷（酸）类药物治疗的慢性HBV感染者HBV基因耐药谱，并对1例我们新鉴定的针对LAM、ADV和ETV的新型多重耐药HBV变异株的临床演变特点和感染的临床治疗进行动态分析。结果为全面了解我国临床实际治疗中的HBV耐药谱提供新的信息，对于帮助我国HBV耐药管理的规范化具有重要意义。目的：（1）分析临床较大样本急性乙肝、轻中度慢乙肝、重度慢乙肝和慢加急性肝衰竭患者HBV基因型和BCP/PC区变异特点，揭示HBV基因型和BCP/PC区变异在不同病程HBV感染患者的表现特点和与疾病进展的相关性。（2）分析临床较大样本慢性HBV感染者RT区多位点核苷（酸）类似物耐药相关变异及其临床意义，揭示我国临床HBV耐药发生频率、四种核苷（酸）类似物基因型耐药变异特点及多重耐药HBV变异株的演变特点和感染的临床治疗策略。方法：（1）收集182例急性乙肝、325例轻中度慢乙肝、170例重度慢乙肝和298例慢加急性肝衰竭患者血清。提取HBV DNA，应用单管巢式聚合酶链反应（polymerase chain reaction,PCR），扩增HBV RT（含S）基因和BCP/PC区基因，对PCR产物进行双向测序。根据RT/S区序列用MEGA4软件进行分子进化树分析确定HBV基因型，用Vector NTI软件比对分析文献报道的具有（潜在）临床意义的10个位点的核苷酸变异，包括BCP区1753、1754、1758、1762、1764、1766和1768位点变异和PC区的1862、1896和1899位点变异。（2）收集1803例经核苷（酸）类似物长期治疗的慢性HBV感染患者血清，扩增HBVRT区全长基因，对PCR产物进行双向测序，分析rt80、rt84、rt173、rt180、rt181、rt184、rt194、rt202、rt204、rt214、rt215、rt217、rt233、rt236和rt250共15个位点的耐药相关变异特点。并进一步对1例新型多重耐药HBV感染患者系列血清样本的耐药变异及HBVDNA载量、转氨酶（alanine aminotransferase, ALT）等指标进行检测，结合克隆测序（>20个克隆/样本/时间点）结果，分析多重耐药HBV株的动态演变规律、表型耐药特点和感染的临床治疗策略。结果：（1）急性乙肝患者HBV B基因型与C基因型比例和BCP/PC区野生株比例显著高于慢性乙肝患者。急性乙肝患者HBV BCP区A1762T、G1764A和前C区G1896A、G1899A的检出率显著低于慢性乙肝患者，但BCP区T1758C的检出率却显著高于慢性乙肝患者，且T1758C变异与A1762T/G1764A变异有相互排斥倾向。与C基因型HBV相比，B基因型HBV BCP区变异频率更低，PC区变异频率相似。在含四种基本BCP/PC变异模式的急性乙肝患者间ALT水平相似，而含PC区变异的慢性乙肝患者ALT水平更高；含有BCP/PC区双变异的急性乙肝患者HBV DNA载量更高，而相应的慢性乙肝患者HBV DNA载量更低；急性乙肝患者的HBeAg阴性率高于慢性乙肝患者，且BCP/PC区双变异的急性乙肝患者HBeAg阴性率更高，而慢性乙肝患者随着BCP/PC区变异的累积HBeAg阴性率呈逐级增高趋势。（2）与慢性乙肝患者相比，慢加急性肝衰竭患者HBV BCP区T1753C/A/G、A1762T、G1764A、C1766T变异频率和前C区G1862T、G1896A、G1899A变异频率显著增高。而且，A1762T/G1764A双联变异和G1896A热点变异频率以及10个分析位点的平均碱基替代数目随着疾病进展（轻中度慢乙肝＜重度慢乙肝＜慢加急性肝衰竭）逐级增高。与C基因型HBV相比，B基因型HBV的BCP区变异频率明显降低，而PC区变异频率明显增高。与HBV BCP区变异不同的是，在上述三种疾病类型中HBV PC区变异均显著影响HBeAg向抗HBe的血清学转换。此外，感染了PC变异株的慢加急性肝衰竭患者比感染了PC野生株的患者病死率明显增高。（3）1803例患者中有560例检出耐药相关变异，其中214例来自490例接受LAM单一治疗的患者，35例来自428例接受ADV单一治疗的患者，5例来自18例接受LdT单一治疗的患者以及306例来自794例接受序贯或联合抗病毒治疗的患者，而在73例接受ETV单一治疗的患者中未检出耐药变异。381例接受LAM耐药后换成ADV继续治疗患者中有36例（9.4%）检出ADV耐药变异，而LAM耐药后加用ADV继续治疗的82例患者中仅1例（1.2%）检出ADV耐药变异。ETV耐药变异不仅在LAM和ETV经治的患者中检出，而且在LAM单一治疗的患者中也检出。tL180M+rtM204I双变异株检出频率在C基因型HBV较B基因型HBV更常见，并且感染了此种双变异株的患者ALT水平明显高于感染了rtM204I单一变异HBV株的患者。值得注意的是，在8例患者中检出多重耐药HBV变异株。（4）我们对1例新型多重耐药HBV变异株感染的慢乙肝患者进行随访，该患者自2002年6月起先后序贯接受了长达116个月的抗病毒治疗，即10个月的LAM与干扰素-α2b联合治疗、23个月的LAM单一治疗、13个月的ADV单一治疗、12个月的ETV单一治疗、12个月的ADV与干扰素-α2b联合治疗、6个月的ADV单一治疗、40个月的ADV加用LAM联合治疗。目前HBV DNA维持检测不到水平，ALT水平正常。克隆测序显示病毒准种池中多重耐药HBV株的动态演变过程，即由最初的野生株，LAM耐药株rtM204I，ADV耐药株rtA181V和rtA181T， LAM耐药株rtL180M+rtM204V， ETV耐药株rtL180M+rtS202G+rtM204V， ADV耐药株rtN236T±rtA181T，多重耐药株rtL180M+rtA181V+rtS202G+rtM204V+rtN236T和rtL180M+rtS202G+rtM204V+rtN236T，到ETV耐药株rtL180M+rtS202G+rtM204V。结论:(1)感染了B基因型HBV、BCP/PC区野生型HBV、BCP区T1758C变异型HBV更易发展为急性乙肝，而慢性乙肝的发生与BCP/PC区变异的累积密切相关。(2) HBV BCP/PC区变异与慢性乙肝的疾病进展呈正相关，BCP/PC区变异HBV感染的慢乙肝患者更易发生重症肝炎以及慢加急性肝衰竭，具有PC变异株的慢加急性肝衰竭患者具有更高死亡风险。(3)虽然仅四种核苷（酸）类药物在我国临床使用，临床上实际抗病毒治疗组合方案多达40余种，HBV耐药变异形式也复杂多样，长期不规范用药促进了耐药及多重耐药HBV变异株的产生，为治疗带来更大的挑战。HBV基因型耐药谱的数据分析对于我国乙肝耐药管理具有重要临床意义。(4)多重耐药病毒是在病毒准种池中复杂的单药和双药耐药病毒基础上产生的。该病例中鉴定的三种新型多重耐药HBV株为国际上首次报道，并且在我国目前TDF未上市的情况下应用LAM与ADV联合治疗可抑制多重耐药HBV株的复制。结果为我国治疗多重耐药HBV感染提供了借鉴。更多还原

【Abstract】 Background:Hepatitis B virus (HBV) infection leads to a wide spectrum of clinical manifestations,ranging from asymptomatic carrier state, acute hepatitis B (AHB), mild or severe chronichepatitis B (CHB-M, CHB-S), liver cirrhosis (LC), hepatocellular carcinoma (HCC), toacute-on-chronic liver failure (ACLF). In China, HBV infected chronically approximate93million people, of whom hepatitis B-related LC patients account for4million, and about280thousand patients died of HCC each year. Hepatitis B-related ACLF is reported to result in morethan20thousand deaths annually. The development of hepatitis B-related ACLF may beassociated with stronger immune response. HBV gene mutation may be one of the key factorscontributing to abnormal immune response.HBV is a highly variable DNA virus. Like retrovirus, HBV replicates its genome via anRNA intermediate using reverse transcription. However, due to the lack of the rigorousproofreading function of reverse transcriptase, errors in HBV DNA replication occur at a muchhigher rate than other DNA viruses. HBV is also a highly replicable virus and all possiblenucleotide (nt) mutations might emerge per site per day in HBV genome. In addition, the maintemplate for viral transcription, HBV covalently closed circular DNA (cccDNA), has a longhalf-life and stably exists in the infected hepatocyte nuclear, resulting in persistent infection. Innatural infection, wild-type HBV strains predominantly exist in the viral quasispecies pool.HBV has been classified into at least eight genotypes (≥8%) and multiple subgenotypes (≥4%)according to the differences in HBV entire genomic sequences or small surface gene sequences.Virologic features vary in different genotype HBV and may associate with the clinical outcomesof liver diseases.The most common hotspot mutations in HBV genome are located in the basal corepromoter (BCP) and precore (PC) regions. Hepatitis B e antigen (HBeAg), encoded by precoremRNA, is considered to be an immune tolerogen and its expression is transcriptionally regulatedby BCP. Therefore mutations in the two regions may abrogate or decrease the translation ofHBeAg, leading to the increase of the viral replication capacity in vitro and evasion of the hostimmune response. On the other hand, as HBeAg and hepatitis B core antigen (HBcAg) sharingthe same immune epitopes, mutations in PC region may enhance the immune attack on HBcAgof the infected hepatocytes when lacking of HBeAg. This may be associated with the diseaseprogression and the severity of hepatitis B.Several studies have reported that BCP/PC mutations may be associated with acute fulminant hepatic failure, while some other studies have debated on this. HBV genotypes mayinfluence the incidence of BCP/PC mutations and complicate the result analysis. Additionally,there is still a paucity of data on the association of HBV BCP/PC mutations and genotypefeatures with ACLF occurrence. The first part of this study systemically investigated the clinicalimplications and virologic features of HBV BCP/PC mutations and genotypes in a large numberof AHB, CHB-M, CHB-S and ACLF patients, which will help understand the virologicmechanisms of chronicity and severity of hepatitis B.Currently, nucleos(t)ide analogs (NAs) are the commonly used anti-HBV therapy drugs inclinic, including three nucleoside analogs, i.e. lamivudine (LAM), entecavir (ETV), andtelbivudine (LdT); and two nucleotide analogs, i.e. adefovir dipivoxil (ADV) and tenofovirdisoproxil fumarate (TDF), and the former four ones have been approved for clinical use inChina. These medications act primarily through blocking reverse transcription of pregenomicRNA to HBV DNA. However, they are unable to eradicate HBV infection because of thepersistence of HBV cccDNA in the infected hepatocytes. High virus rebound and/or hepaticflare will occur once the therapy is discontinued. Herein, a long-term antiviral therapy is neededfor sustained suppression of HBV DNA replication.However, drug pressure may select fitness mutants with a survival advantage compared towild-type virus or make minute quantity of preexisting mutants in viral quasispecieses poolacquire selectively amplified during long-term antiviral treatment of NAs. Drug-resistant HBVmutants may lead to treatment failure and progression of liver diseases, which has been anintractable issue in clinical practice. The resistance mutations are located in the HBV RT domain.The primary resistance mutations can directly decrease the viral susceptibility to NAs and thesecondary or compensatory mutations can restore or enhance the replication capacity of resistantmutants. Some proposed mutations may be associated with ADV resistance, although it is stillcontroversial. As more antiviral strategies are available for anti-HBV therapy, the risks andpatterns of drug-resistance and cross-resistance are higher. In addition, nonoptimal sequentialuse of NAs may promote the development of multidrug resistant HBV.To date, data on HBV resistance mutations are largely derived from some clinical trials andcohorts with relatively fixed NAs schedules, limited samples and drug resistance profiling.However, the NAs schedules are more complex in real clinical practice resulting in specificregularity in HBV drug resistance profile and there is a paucity of data on resistance profile inChinese patients. Therefore, the second part of this study investigated the HBV genotypicresistance profiles in a large number of NAs-experienced Chinese patients with chronic HBVinfection based on our previously-established HBV resistance database. The analysis of clinicaland experimental data from a rare and novel multidrug resistance (LAM, ADV and ETV) patientreveals the dynamic evolution and suppression of the multidrug resistant HBV strains underlong-term antiviral treatment. The results will provide new information on HBV genotypicresistance profiles in real clinical practices and may have important clinical implications for HBV drug resistance management in China.Objective:(1) To investigate the features of HBV BCP/PC mutations and genotypes in a large numberof AHB, CHB-M, CHB-S and ACLF patients, and uncover the clinical implications of thevirologic features and help understand the virologic mechanisms of chronicity and severity ofhepatitis B.(2) To investigate the HBV genotypic resistance profiles in a large number ofNAs-experienced Chinese patients with chronic HBV infection and to further analyze a dynamicevolution and suppression of HBV strains with rare and novel multidrug resistance to LAM,ADV and ETV in a patient and provide new information on HBV genotypic resistance profilesin real clinical practices and help standardise HBV drug resistance management in China.Methods:(1) Serum samples from182patients with AHB,325patients with CHB-M,170patientswith CHB-S, and298patients with ACLF were collected and HBV DNA was extracted. TheHBV sequences of695base pair (bp)-long BCP/PC gene and1225bp-long RT/S gene weredetermined by direct sequencing method after nested polymerase chain reaction (PCR)amplification which has been improved by our group. HBV genotype assignment was based onphylogenetic analysis of the RT/S sequence using MEGA4software. Ten sites of interest wereanalyzed based on their clinical or potential clinical significance suggested in previouspublications, namely, nt1753,1754,1758,1762,1764,1766,1768in BCP region and nt1862,1896,1899in PC region using Vector NTI Suite software.(2) Serum samples were collected from1803NAs-experienced Chinese patients withchronic HBV infection and HBV DNA was extracted.1225bp-long HBV RT full gene wasamplified by a high-sensitive direct PCR sequencing. Mutations at15locations (including rt80,rt84, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt214, rt215, rt217, rt233, rt236, rt250) in theRT domain were analyzed. Furthermore, serially dynamic serum samples were collected fromone CHB patient who successively experienced116-month NAs antiviral treatment. The PCRproducts of HBV RT gene were directly sequenced and also cloned into the pGEM-Teasy vectorfor clonal sequencing (more than20clones per sample per time-point). The dynamicallyevolutionary regulation, phenotypic resistance characteristics and efficient treatment of thenovel multidrug-resistant HBV strains was analyzed.Results:(1) AHB patients had a significantly higher ratio of genotype B to C and a higherprevalence of BCP/PC wild-type virus than CHB patients. Significantly lower prevalence ofA1762T, G1764A, G1896A, and G1899A but higher prevalence of T1758C was found in AHBpatients. Interestingly, T1758C and A1762T/G1764A appeared mutual restraint. Compared to genotype C virus, genotype B virus had a lower BCP mutation frequency and a similar PCmutation frequency. AHB patients harboring four basic BCP/PC mutations had similar alanineaminotransferase (ALT) levels, while CHB patients harboring PC mutations had higher ALTlevels. AHB patients harboring basic BCP+/PC+mutations had higher viral loads, while lowerviral loads in CHB patients with the same mutations. AHB patients had a higher percentage ofHBeAg negativity than CHB patients and the percentage was even higher in those harboringbasic BCP+/PC+mutations. In CHB patients, accumulation of BCP/PC mutations wasaccompanied with escalation of HBeAg negativity.(2) Compared to CHB patients, ACLF patients had a significantly higher mutationfrequency of T1753V (C/A/G), A1762T, G1764A, and C1766T in the BCP region and G1862T,G1896A, and G1899A in the PC region. Moreover, the frequencies of A1762T, G1764A andG1896A hotspot mutations and the average substitution number at the10sites of interest of theviral sequences increased in a stepwise manner in the order of CHB-M＜CHB-S＜ACLFpatients. Genotype B virus had significantly lower BCP mutation frequencies and higher PCmutation frequencies than genotype C virus. Different from the BCP mutants, the PC mutantsshowed a strong positive influence on HBeAg seroconversion for all three groups of patients.Notably, ACLF patients infected with the PC mutants had a significantly higher mortality thanthose infected with the wild-type viruses.(3) Drug-resistant mutations were detected in560of the1803patients, including214of490patients who received LAM,35of428patients who received ADV,5of18patients whoreceived LdT monotherapy and306of794patients who received various sequential/combinedNA therapies. No drug-resistant mutations were detected in the73patients who received ETVmonotherapy. ADV resistant mutations were detected in36of381patients who received LAMand then switched-to ADV in contrast to one of82patients who received ADV add-on LAM.ETV-resistant mutations were detected not only in LAM-and ETV-treated patients but also inLAM-treated ETV-naive patients. Double mutations rtM204I and rtL180M were detected morefrequently in genotype C than in genotype B virus, and patients infected with this mutant hadhigher alanine aminotransferase levels than those infected with mutant containing the rtM204Isubstitution alone. Notably, multidrug-resistant HBV strains were identified in eight patients.(4) A representative CHB patient who received116months of successively antiviraltherapy from June,2002was followed up. He experienced10months of LAM plus interferon(IFN)-α2b combination therapy,23months of LAM monotherapy,13months of ADVmonotherapy,12months of ETV monotherapy,12months of ADV plus interferon (IFN)-α2bcombination therapy,6months of ADV monotherapy, and40months of ADV plus LAMcombination therapy. HBV DNA maintained undetectable level and normalized ALT level wasachieved after serial fluctuation.Clonal sequencing showed that the multidrug-resistant HBV strains in viral quasispeciesdynamically evolved from initial wild-type strain, to LAM-resistant rtM204I strains, ADV-resistant rtA181V and rtA181T strains, LAM-resistant rtL180M+rtM204V strains,ETV-resistant rtL180M+rtS202G+rtM204V strains, ADV-resistant rtN236T±rtA181T strains,multidrug-resistant rtL180M+rtA181V+rtS202G+rtM204V+rtN236T and rtL180M+rtS202G+rtM204V+rtN236T strains, and finally to ETV-resistant rtL180M+rtS202G+rtM204V strains.Conclusions:(1) Patients infected genotype B virus, BCP/PC wild-type virus or BCP T1758C mutantHBV are more likely to develop AHB. In contrast, the development of CHB is closelyassociated with the accumulation of BCP/PC mutations.(2) HBV BCP/PC mutations are positively associated with the progression of CHB. CHBpatients with BCP/PC mutants are prone to develop CHB-S or ACLF. Notably, ACLF patientswith PC mutants have a higher risk of mortality.(3) Although only four NAs are approved for clinical use in China, there are more than40antiviral schedules in real clinical practice, adding the complexity and diversity of drug-resistantmutational patterns in China. The long-term use of NAs with nonoptimal responses promotesarising of the drug-resistant and multidrug-resistant HBV, which brings larger challenges toclinical treatment. The data on HBV genotypic resistance profiles in this study may havesignificant implications for HBV drug resistance management in China.(4) The emergence of multidrug-resistant HBV is based on complex single-anddouble-drug-resistant viruses in viral quasispecies. This is the first time that multidrug-resistantHBV strains were identified in a clinical case and were suppressible by LAM plus ADV whenTDF was not available in China, which provide reference for management of multidrug-resistantHBV in China.更多还原