Innate immune recognition of hepatitis B virus

Hepatitis B virus(HBV) is a hepatotropic DNA virus and its infection results in acute or chronic hepatitis. It is reported that the host innate immune system contributes to viral control and liver pathology, while whether and how HBV can trigger the components of innate immunity remains controversial. In recent years, the data accumulated from HBV-infected patients, cellular and animal models have challenged the concept of a stealth virus for HBV infection. This editorial focuses on the current findings about the innate immune recognition to HBV. Such evaluation could help us to understand HBV immunopathogenesis and develop novel immune therapeutic strategies to combat HBV infection.     

Innate and adaptive immune escape mechanisms of hepatitis B virus

Chronic hepatitis B virus (HBV) infection is an international health problem with extremely high mortality and morbidity rates. Although current clinical chronic hepatitis B (CHB) treatment strategies can partly inhibit and eliminate HBV, viral breakthrough may result due to non-adherence to treatment, the emergence of viral resistance, and a long treatment cycle. Persistent CHB infection arises as a consequence of complex interactions between the virus and the host innate and adaptive immune systems. Therefore, understanding the immune escape mechanisms involved in persistent HBV infection is important for designing novel CHB treatment strategies to clear HBV and achieve long-lasting immune control. This review details the immunological and biological characteristics and escape mechanisms of HBV and the novel immune-based therapies that are currently used for treating HBV.     

Innate immune targets of hepatitis B virus infection

Approximately 400 million people are chronically infected with hepatitis B virus(HBV) globally despitethe widespread immunization of HBV vaccine and the development of antiviral therapies. The immunopathogenesis of HBV infection is initiated and driven by complexed interactions between the host immune system and the virus. Host immune responses to viral particles and proteins are regarded as the main determinants of viral clearance or persistent infection and hepatocyte injury. Innate immune system is the first defending line of host preventing from virus invasion. It is acknowledged that HBV has developed active tactics to escape innate immune recognition or actively interfere with innate immune signaling pathways and induce immunosuppression, which favor their replication. HBV reduces the expression of pattern-recognition receptors in the innate immune cells in humans. Also, HBV may interrupt different parts of antiviral signaling pathways, leading to the reduced production of antiviral cytokines such as interferons that contribute to HBV immunopathogenesis. A full comprehension of the mechanisms as to how HBV inactivates various elements of the innate immune response to initiate and maintain a persistent infection can be helpful in designing new immunotherapeutic methods for preventing and eradicating the virus. In this review, we aimed to summarize different branches the innate immune targeted by HBV infection. The review paper provides evidence that multiple components of immune responses should be activated in combination with antiviral therapy to disrupt the tolerance to HBV for eliminating HBV infection.     

Innate immune recognition and modulation in hepatitis D virus infection

Hepatitis D virus(HDV) is a global health threat with more than 15 million humans affected. Current treatment options are largely unsatisfactory leaving chronically infected humans at high risk to develop liver cirrhosis and hepatocellular carcinoma. HDV is the only human satellite virus known. It encodes only two proteins, and requires Hepatitis B virus(HBV) envelope protein expression for productive virion release and spread of the infection. How HDV could evolve and why HBV was selected as a helper virus remains unknown. Since the discovery of Na+-taurocholate co-transporting polypeptide as the essential uptake receptor for HBV and HDV, we are beginning to understand the interactions of HDV and the immune system. While HBV is mostly regarded a stealth virus, that escapes innate immune recognition, HBV-HDV coinfection is characterized by a strong innate immune response. Cytoplasmic RNA sensor melanoma differentiation antigen 5 has been reported to recognize HDV RNA replication and activate innate immunity. Innate immunity, however, seems not to impair HDV replication while it inhibits HBV. In this review, we describe what is known up-to-date about the interplay between HBV as a helper and HDV's immune evasion strategy and identify where additional research is required.     

应用含CpG基序的寡核苷酸增强乙型肝炎病毒基因疫苗诱导的细胞免疫应答

目的 探讨人工合成的含ＣｐＧ基序的寡核苷酸（ＯＤＮ）作为佐剂对乙型肝炎病毒（ＨＢＶ）基因疫苗诱导小鼠产生细胞免疫应答的影响。方法 构建编码ＨＢＶ基因疫苗,人工合成含ＣｐＧ基序（ｍｏｔｉｆ）的硫代磷酸寡核苷酸作为佐剂,以Ｂａｌｂ／ｃ．１－Ｓ小鼠作为实验动物进行免疫接种;采用＾３Ｈ－ＴｄＲ法、＾５１Ｃｒ４ｈ释放法等分别检测免疫小鼠的淋巴细胞增殖和杀伤功能。结果 与空载体对照组相比较,ＨＢＶ基因疫苗诱发     

Clinical immune characterization of hepatitis B virus infection and implications for immune intervention: Progress and challenges

The host immune response plays an important role in mediating hepatitis B virus (HBV) control and induction of liver damage, which determines the outcome of infection. However, interactions between HBV, the immune system, and the liver microenvironment, remain poorly understood. This review briefly outlines what we know about innate and adaptive immune responses to HBV, as well as the liver immunology in infected patients. It addresses how our knowledge of the anti-HBV immune response might aid the development of adoptive immune therapeutic strategies against HBV. This review also highlights the challenges we are facing in understanding the cellular and molecular mechanisms bywhich the innate, adaptive and liver immune responses exert a synergistic antiviral function and influence disease progression. It concludes by addressing future directions and unanswered questions regarding the use of clinical immunotherapy. We hope this review will help hepatologists and gastroenterologists to understand the anti-HBV immune response, as well as current challenges and potential immunotherapeutic strategies against this disease.     

HBV感染免疫耐受期患者应积极抗病毒治疗

随着乙型肝炎研究的不断深入,越来越多的研究和数据发现免疫耐受期患者有较高的比例存在活动性炎症或纤维化。近年来国内外指南关于乙型肝炎抗病毒治疗的适应证范围也逐步放宽,这也是基础研究、临床科研以及社会经济和医保等多方面因素共同的结果。研究发现HBV DNA持续高水平复制是肝癌发生的独立危险因素,对于口服抗病毒治疗的人群低病毒血症的存在同样会增加肝硬化/肝癌的发生。     

Innate and adaptive immune responses to herpes simplex virus

Immune responses against HSV-1 and HSV-2 are complex and involve a delicate interplay between innate signaling pathways and adaptive immune responses. The innate response to HSV involves the induction of type I IFN, whose role in protection against disease is well characterized in vitro and in vivo. Cell types such as NK cells and pDCs contribute to innate anti-HSV responses in vivo. Finally, the adaptive response includes both humoral and cellular components that play important roles in antiviral control and latency. This review summarizes the innate and adaptive effectors that contribute to susceptibility, immune control and pathogenesis of HSV, and highlights the delicate interplay between these two important arms of immunity.     

HBV/HCV相关性肝细胞癌抗病毒治疗专家建议

乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染在肝细胞癌(HCC)的发生发展中起重要作用.我国近年发布的《慢性乙型肝炎防治指南(2010版)》和《原发性肝癌诊疗规范(2011版)》都强调了肝癌患者抗病毒治疗的重要性,但未作深入具体阐述.《丙型肝炎防治指南(2004版)》也注意到抗病毒治疗延缓HCC的发生.有鉴于此,中华医学会肝病学分会肝癌学组召开了三次专题讨论会,系统收集分析了现有HCC综合治疗中抗病毒治疗的临床研究文献,回顾了HCC治疗中抗病毒药物临床应用进展,依据现有病毒相关性HCC抗病毒治疗的循证医学临床资料,综合部分专家的意见,按照循证医学证据分级的GRADE系统(表1)进行细化和补充,针对这些患者抗病毒治疗的应用达成共识,提出如下具体建议,供国内同道参考,以期在临床实践过程中依据新的临床医学证据进行修改和更新,进一步完善《原发性肝癌诊疗规范》、《慢性乙型肝炎防治指南》和《丙型肝炎防治指南》的实施.     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.     

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM： To establish a cell model harboring replicative clinical hepatitis B virus （HBV） isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B （CHB） patients.
METHODS： The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction （PCR）. All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel.
RESULTS： A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent.
CONCLUSION： The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.     

拉米夫定治疗其他方法抗病毒治疗失败的慢性乙型肝炎患者的疗效

目的观察拉米夫定对复发性慢性乙型肝炎患者的疗效和安全性。方法选择27例其他方法抗病毒治疗失败的慢性乙型肝炎患者,给予拉米夫定100mg口服,每日一次,连续服用12个月。结果治疗后12个月时ALT和血清总胆红素平均值(分别为52.7±26.5U/L和19.7±21.1μmol/L),与治疗前平均值(211.3±182.4U/L和54.6±28.8μmol/L)相比显著下降(P<0.01);其ALT复常率为88.9%(24/27),HBV DNA阴转率77.8%(21/27),HBeAg阴转率29.6%(8/27),HBeAg/抗-HBe血清转换率18.5%(5/27)。停药后6个月内有7例复发。治疗全程未见严重不良反应。结论拉米夫定治疗其他方法抗病毒治疗失败的慢性乙型肝炎患者也能够迅速抑制病毒的复制,使肝功能复常,而且安全、方便。     

乙肝病毒感染者淋巴细胞亚群失衡与细胞免疫功能紊乱的临床研究

目的 研究不同临床类型乙型肝炎病毒（HBV）感染淋巴细胞亚群变化情况,探讨HBV感染与细胞免疫紊乱关系.方法 以364例HBV感染患者作为研究对象,41例健康体检人员作为对照,比较急性肝炎组（78例）、慢性肝炎组（244例）、肝硬化组（42例）淋巴细胞亚群变化及不同HBV DNA载量HBV感染患者淋巴细胞亚群变化.结果 慢性肝炎组、肝硬化组CD3＋、CD4＋、CD8＋均明显降低,其中肝硬化组下降较为显著,组间差异有统计学意义（P＜0.05）,慢性肝炎组CD4 ＋/CD8＋明显降低与对照组和急性肝炎组差异均有统计学意义（P＜0.05）,急性肝炎组CD8＋明显高于对照组,差异有统计学意义（P＜0.05）,慢性肝炎组、肝硬化组CD3、CD4＋、CD8＋与急性肝炎组、对照组比较差异有统计学意义（P＜0.05）;HBV-DNA高载量组与低载量组淋巴细胞亚群比较,CD8＋较高,CD4＋/CD8＋较低,差异有统计学意义（P＜0.05）.结论 慢性肝炎及肝硬化均存在一定程度的细胞免疫功能低下,对患者淋巴细胞亚群的监测及患者免疫功能评估有重要的临床应用价值.     

Engineering immune therapy against hepatitis B virus

Approximately 350–400 million people worldwide are chronically infected with the hepatitis B virus (HBV). These individuals harbor the virus for their whole life and they transmit the virus to uninfected individuals. In addition, considerable numbers of chronic HBV carriers develop progressive liver diseases like chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma. At present, antiviral agents like type-1 interferons, lamivudine, adefovir and entacavir are used to treat a selected population of chronic HBV carriers. These antiviral treatments are not satisfactory in that they are unable to eradicate HBV, only partially efficient in less than 30% subjects, expensive, can have debilitating side-effects and require constant monitoring. In addition, once treatment is stopped, the virus and clinical conditions return in many patients. Recent advancements in cellular and molecular biology indicate that the host's immune responses to HBV play cardinal roles during acquisition, pathogenesis, progression, and complications of chronic HBV infection. Immune responses are also important in the context of antiviraltherapy and clinical recovery. This explains why the efficacy of antiviral drugs is limited even in some selected patients with chronic HBV infection. Various published work now state that HBV-specific immunity may be beneficial for patients with chronic HBV infection and non-HBV-specific immunity may be related to flare up of liver diseases. Accordingly, a new few field of immunological research and clinical application of prophylactic vaccines (vaccine therapy) has been started in chronic HBV carriers. Vaccine therapy has inspired optimism as a new therapeutic approach, but it is unlikely that the present regimen of vaccine therapy will stand the test of time. Based on present understandings about vaccine/host interactions, we provide herein an outline for engineering more potent regimen of HBV-specific immune therapy against HBV.     

乙型病毒性肝炎的免疫动力学

乙型肝炎的发病机制主要是机体清除乙型肝炎病毒(hepatitis B virus,HBV)而引发的细胞免疫病理改变,机体对病毒的免疫应答有赖于一系列免疫活性细胞的相互作用.目前国内外主要就HBV感染控制者和感染持续者间的免疫差异原因进行研究.本文就乙型肝炎病毒感染状态下机体控制HBV感染下各相关免疫细胞间的作用机制以及...     

DHBV感染鸭特异免疫应答比较

目的 研究嗜肝病毒感染病毒清除向慢性化转变的免疫机理。方法 通过接种不同龄鸭、不同感染方案建立DHBV急性、慢性感染组及免疫组模型,并检测各组病毒复制及特异体液和细胞介导免疫反应。结果 成年鸭接种DHBV导致一过性病毒血症的形成,感染后抗－DHBs和抗-DHBc的水平比慢性感染组高（P＜0．05）,而免疫组抗－DHBs、抗－DHBc的水平又比急感染组高（P＜0．01）。特异细胞介导免疫反应（CMI）的分析提示急性感染组感染后10d外周血单个核细胞（PBMC）对特异抗原的体外增殖反应强于慢性感染组,但在感染5周内快速下降。而免疫组CMI与急性感染组比差异无显著意义。结论 感染宿主免疫应答特别是特异免疫反应是决定感染转归的重要因素。     

Modification of the immune response against hepatitis B virus by the human immunodeficiency virus

Summary Hepatitis B virus and the human immunodeficiency virus are similarly transmitted. Individuals with preexisting HIV infection have a higher chance to become HBsAg carriers than do anti-HIV negative persons. Cytotoxic T cells with specificity for HBcAg, that are under the control of HBcAg-specific helper T cells, are responsible for liver injury. There is good evidence that HIV infection lowers inflammatory activity, is associated with milder liver histology, high levels of viral replication and low seroconversion rates. In addition interferon alpha therapy is less effective in anti-HIV positive subjects. The immune response against HBsAg is helper T-cell dependent and vaccination against hepatitis B is of low effectiveness. In addition, vaccination against hepatitis B may activate the HIV disease and is, therefore, presently not to be recommended.     

HBV/HCV重叠感染的抗病毒治疗

从全球范围看,乙型肝炎病毒(hepatitis B virus,HBV)和丙型肝炎病毒(hepatitis C virus,HCV)重叠感染估计约有700-2000万人口感染.重叠感染和单一HBV或HCV感染比较,更易发展为肝硬化、肝细胞癌甚至肝衰竭的比例也高,HBV和HCV重叠感染可有四种不同的临床模式,即HCV活动...     

不同剂量乙肝免疫球蛋白联合乙肝疫苗阻断乙肝病毒母婴传播的疗效比较

目的比较不同剂量乙肝免疫球蛋白(HBIG)联合乙肝疫苗阻断乙肝病毒母婴传播的疗效。方法将HBs Ag阳性孕妇分娩的新生儿124例分为双阳组60例和单阳组64例。双阳组按家长的意愿分为双阳大剂量组30例和双阳小剂量组30例,单阳组亦分为单阳大剂量组和单阳小剂量组各32例。新生儿出生后6 h内,大剂量组予肌注HBIG 200 U,小剂量组予肌注HBIG 100 U;同时联合应用乙肝疫苗。一周岁时检测HBV血清学标志物(乙肝五项)。结果在双阳组中大剂量和小剂量HBIG的乙肝母婴阻断率分别为93.3%和83.3%;在单阳组中大剂量和小剂量HBIG的乙肝母婴阻断率分别为100.0%和96.9%,差异无统计学意义(P0.05)。结论孕妇HBs Ag阳性时,新生儿出生后要及时注射HBIG,用量100 U或200 U均可。     

慢性乙型肝炎患者抗病毒治疗中的病毒准种演变

目的 探讨拉米夫定耐药后换用恩替卡韦补救治疗的慢性乙型肝炎患者的病毒准种演变.方法 提取1例慢性乙型肝炎患者治疗中的7个不同时间点(0、24、48、60、72、96、152周)血清中的HBV DNA,巢式聚合酶链反应法扩增HBV DNA聚合酶基因逆转录酶区,克隆测序法对逆转录酶区氨基酸替换形式及准种分布进行分析,并采用扩增耐药突变系统聚合酶链反应法对患者病毒种群中野毒株与病毒总量进行定量检测.结果 患者在治疗过程中主要存在rtM204V、rtL180M+rtM204V和rtM204I 3种拉米夫定耐药相关的病毒株变异形式,各病毒株所占比例不断发生变化,基线时HBV野毒株为优势病毒株,在病毒学突破时,种群中全部为耐药突变株;换用恩替卡韦治疗后,随着病毒载量的下降,拉米夫定耐药突变株被抑制,野毒株在种群中比例逐渐上升,并成为优势病毒株(79.3%).扩增耐药突变系统聚合酶链反应检测结果显示,在基线和发生病毒学突破时,野毒株在种群中的比例分别为68.55%和0.21%,换药治疗后24周,野毒株比例开始上升,此后野毒株占种群的比例波动于16.01%～26.93%.结论 慢性乙型肝炎患者在核苷(酸)类药物序贯治疗过程中,HBV种群的准种分布一直处于动态变化中.不同的HBV准种演变模式可能在恩替卡韦补救治疗中对恩替卡韦耐药发生的作用也不相同.

Abstract：

Objective To investigate the evolution of hepatitis B virus (HB V) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment. Methods Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96,152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR). Results Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%. Conclusions The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.