HBsAg, HBcAg, and combined HBsAg/HBcAg-based therapeutic vaccines in treating chronic hepatitis B virus infection

BACKGROUND: As the host immunity is diminished in patients with chronic hepatitis B (CHB), different approaches have been used to up-regulate their immune responses to produce therapeutic effects. But, cytokines, growth factors and polyclonal immune modulators could not exhibit sufficient therapeutic effects in these patients. Immune therapy with HBV-related antigens (vaccine therapy) has been used in CHB patients. But there is a paucity of information about the design of HBV antigen-based immune therapy in these patients. DATA SOURCE: Preclinical and clinical studies on immune therapy with HBsAg-based vaccine, HBcAg and combination of HBsAg/HBcAg-based vaccines have been discussed. RESULTS: HBsAg-based prophylactic vaccine was used as an immune therapeutic agent in CHB patients; however, monotherapy with HBsAg-based immune therapy could not lead to sustained control of HBV replication and/or liver damages. HBsAg-based vaccine was used as a combination therapy with cytokines, growth factors, and antiviral drugs. HBsAg-based vaccine was also used for cell-based therapy. However, satisfactory therapeutic effects of HBsAg-based vaccine could not be documented in CHB patients. In the mean time, evidences have supported that HBcAg-specific immunity is endowed with antiviral and liver protecting capacities in CHB patients. Recent data concentrate on the clinical use of combined HBsAg- and HBcAg-based vaccines in CHB patients.CONCLUSION: Antigen-based immune therapy with HBV- related antigens may be an alternative method for the treatment of CHB patients but proper designs of antigens, types of adjuvants, dose of vaccinations, and routes of administration need further analyses for the development of an effective regimen of immune therapy against HBV.

New therapeutic vaccination strategies for the treatment of chronic hepatitis B

Chronic hepatitis B virus(CHB) is currently treated with either interferon-based or nucleot(s)idebased antiviral therapies.However,treatment with pegylated interferon alpha results in a durable antiviral response in only about 30%patients and is associated with side effects.Most patients receiving nucleot(s)ide analogue treatment do not establish long-term,durable control of Infection and have rebounding viremia after cessation of therapy.Thus,novel therapy strategies are necessary to achieve the induction of potent and durable antiviral immune responses of the patients which can maintain long-term control of viral replication.Therapeutic vaccination of HBV carriers is a promising strategy for the control of hepatitis B.Here the authors review new therapeutic vaccination strategies to treat chronic hepatitis B which may be introduced for patient treatment in the future.

Toward a new era of hepatitis B virus therapeutics:The pursuit of a functional cure

Hepatitis B virus(HBV)infection,although preventable by vaccination,remains a global health problem and a major cause of chronic liver disease.Although current treatment strategies suppress viral replication very efficiently,the optimal endpoint of hepatitis B surface antigen(HBsAg)clearance is rarely achieved.Moreover,the thorny problems of persistent chromatin-like covalently closed circular DNA and the presence of integrated HBV DNA in the host genome are ignored.Therefore,the scientific community has focused on developing innovative therapeutic approaches to achieve a functional cure of HBV,defined as undetectable HBV DNA and HBsAg loss over a limited treatment period.A deeper understanding of the HBV life cycle has led to the introduction of novel direct-acting antivirals that exert their function through multiple mechanisms,including inhibition of viral entry,transcriptional silencing,epigenetic manipulation,interference with capsid assembly,and disruption of HBsAg release.In parallel,another category of new drugs aims to restore dysregulated immune function in chronic hepatitis B accompanied by lethargic cellular and humoral responses.Stimulation of innate immunity by pattern-recognition receptor agonists leads to upregulation of antiviral cytokine expression and appears to contribute to HBV containment.Immune checkpoint inhibitors and adoptive transfer of genetically engineered T cells are breakthrough technologies currently being explored that may elicit potent HBV-specific T-cell responses.In addition,several clinical trials are attempting to clarify the role of therapeutic vaccination in this setting.Ultimately,it is increasingly recognized that elimination of HBV requires a treatment regimen based on a combination of multiple drugs.This review describes the rationale for progressive therapeutic interventions and discusses the latest findings in the field of HBV therapeutics.

Nucleic acid vaccines: A taboo broken and prospect for a hepatitis B virus cure

Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.

Effect of Qinling granule in treatment of 102 patients with chronic hepatitis B

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Phage display creates innovative applications to combat hepatitis B virus

Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.

Induced immunity against hepatitis B virus

Prevention of hepatitis B virus(HBV) infection with its consequent development of HBV chronic liver disease and hepatocellular carcinoma is a global mandatory goal. Fortunately, safe and effective HBV vaccines are currently available. Universal hepatitis B surface antigen HBV vaccination coverage is almost done. Growing knowledge based upon monitoring and surveillance ofHBV vaccination programs has accumulated and the policy of booster vaccination has been evaluated. This review article provides an overview of the natural history of HBV infection, immune responses and the future of HBV infection. It also summarizes the updated sources, types and uses of HBV vaccines, whether in the preclinical phase or in the post-field vaccination.

Overcoming HBV immune tolerance to eliminate HBsAg-positive hepatocytes via pre-administration of GM-CSF as a novel adjuvant for a hepatitis B vaccine in HBV transgenic mice

Granulocyte-macrophage colony-stimulating factor （GM-CSF） is known to be a potential vaccine adjuvant despite contradictory results from animal and human studies. The discrepancies may be due to the different doses and regimens of GM-CSF that were used, given that either mature or immature dendritic cells （DCs） could be induced under different conditions. To test the hypothesis that GM-CSF can be used as a novel adjuvant for a hepatitis B virus （HBV） therapeutic vaccine, we administered GM-CSF once per day for three days prior to vaccination with recombinant HBV vaccine （rHBVvac） in mice. We observed greater DC maturation in these pre-treated animals at day 3 as compared to day 1 or day 2 of daily GM-CSF administration. This strategy was further investigated for its ability to break the immune tolerance established in hepatitis B surface antigen-transgenic （HBsAg-Tg） animals. We found that the levels of induced anti-HBsAg antibodies were significantly higher in animals following three days of GM-CSF pre-treatment before rHBV vaccination after the third immunization. In addition to the increase in anti-HBsAg antibody levels, cell-mediated anti-HBsAg responses, including delayed-type hypersensitivity, T-cell proliferation, interferon-y production, and cytotoxic T lymphocytes, were dramatically enhanced in the three-day GM-CSF pre-treated group. After adoptive transfers of CD8＋ T cells from immunized animals, antigen-specific CD8＋ T cells were observed in the livers of recipient HBsAg-Tg animals. Moreover, the three-day pre-treatments with GM-CSF prior to rHBVvac vaccination could significantly eliminate HBsAg-positive hepatocytes, suggesting beneficial therapeutic effects. Therefore, this protocol utilizing GM-CSF as an adjuvant in combination with the rHBVvac vaccine has the potential to become a novel immunotherapy for chronic hepatitis B patients.

Vaccine therapies for chronic hepatitis B： can we go further？

Chronic hepatitis B is a major health burden worldwide. In addition to the recent progress in antiviral treatment, therapeutic vaccination is a promising new strategy for the control of chronic hepatitis B. On the basis of the major specific and non-specific immune dysregulations and defects in chronic hepatitis B patients, this paper presents the peptide and protein-based, DNA-based, cell-based, and antigen-antibody-based therapeutic vaccines, which have undergone clinical trials. The advantages, disadvantages, and future perspectives for these therapeutic vaccines are discussed.

治疗型双质粒HBV DNA疫苗的构建及其鉴定

为构建以人白细胞介素 2 /γ干扰素(hIL 2 /hIFN γ)融合基因为佐剂的治疗型HBVDNA疫苗 ,采用DNA重组技术分别构建含有HBV包膜中蛋白 (preS2 ·S)抗原和hIL 2 /hIFN γ融合蛋白的真核表达质粒即pcDNAS2 ·S和pcDNAIIF ,酶谱和测序分析表明克隆的preS2 ·S和hIL 2 /hIFN γ融合蛋白基因片段的方向、序列与预期相符。用脂质体转染试剂转染COS 7细胞 ,并用ELISA检测转染细胞培养上清中目的基因表达水平。结果显示 ,质粒转染后 4 8h达峰值 ,分别为HBsAg(P/N) =7.6 3、IL 2 =1 0 .35ng/ml、IFN γ =7.90ng/ml。以CTLL 2依赖细胞株/MTT比色法和微量细胞病变抑制法 (WISH VSV)检测pcDNAIIF转染后 4 8h培养上清中IL 2及IFN γ活性 ,结果分别为 998U/ml和 2 4 9U/ml。证明构建的重组质粒pcDNAS2 ·S和pcDNAIIF结构正确 。

HBV DNA疫苗诱导健康及HBV转基因小鼠细胞免疫应答

采用HBVCTL表位多肽体外刺激或冲击免疫效 (E)、靶(T)细胞 ,以观察DNA疫苗诱导健康及HBV转基因 (Tg)小鼠细胞免疫效果。结果发现 ,DNA疫苗能有效诱导健康BALB/c小鼠CTL活性 ,活性强弱与E/T比值及其上清液中IFN γ分泌水平有一定关系。HBsAg表位多肽(pp2 0 )体外刺激DNA疫苗免疫组效应细胞 ,培养上清IL 1 2释放水平 (2 1 1 3± 39 8pg/ml)明显较对照组 (86 7±2 7 1 pg/ml)高(P <0 0 5 ,t=4 4 82 )。DNA疫苗免疫HBVTg小鼠诱导CTL活性 (1 2 7%± 6 7% )较蛋白疫苗免疫对照组(1 7%±3 2 % )高(P <0 0 1 ,t=3 6 2 9) ;其效应细胞体外受 pp2 0刺激后分泌IL 1 2水平 (4 0 0± 30 1pg/ml)明显高于同期空载体免疫对照组 (3 8±3 0 pg/ml,P <0 0 5 ,t=2 376 )。采用在体电脉冲法DNA疫苗接种的 5只HBVTg小鼠中 ,于 4周时有 2只血清HBsAg阴转 ,并于 8周时出现抗 HBs阳性 ,而对照组中无一例血清HBsAg发生变化。表明HBVDNA疫苗能有效诱导健康及HBVTg小鼠细胞免疫应答 。

治疗型HBV DNA疫苗的研究与应用

综述治疗型乙型肝炎病毒 (HBV)疫苗研究概况 ,重点阐述治疗型HBVDNA疫苗研究进展、治疗乙型肝炎的依据、作用机制和与传统疫苗的比较等。

HBV^+自噬小体疫苗治疗HBV急性感染的实验研究

目的:探讨转染HBV基因组的Hep G2.2.15细胞自噬小体(HBV+DRibbles)诱导HBV特异性免疫应答及其对HBV急性感染的治疗作用。方法:ELISA法检测HBV+DRibbles体外再刺激HBs Ag特异性效应细胞产生IFN-γ的水平;对HBV急性感染模型小鼠实施HBV+DRibbles、HBV+DRibbles联合DC免疫,ELISA法检测HBV抗原及抗原肽刺激免疫小鼠淋巴细胞产生IFN-γ的含量;ELISA法、荧光定量PCR法和酶法分别检测小鼠血清HBe Ag、HBV DNA、ALT和AST的水平;免疫组织化学法检测小鼠肝组织HBc Ag表达及免疫病理损伤。结果:与培养液对照组和HBs Ag蛋白刺激组相比,HBV+DRibbles刺激HBs Ag特异性效应细胞能够产生更高水平的IFN-γ(P=0.004);与未负载抗原的DC细胞对照组和负载HBs Ag的DC刺激组相比,DC负载HBV+DRibbles再刺激HBs Ag特异性CD4+、CD8+T细胞均能产生更高水平的IFN-γ(P<0.001)。与PBS对照组相比,HBV+DRibbles疫苗组和HBV+DRibbles联合DC免疫组均能诱导HBV急性感染模型小鼠产生HBV特异性免疫应答,明显降低血清HBe Ag、HBV DNA水平,减低HBc Ag+肝细胞比例,而ALT及AST水平未见明显差异,肝组织结构基本正常;但HBV+DRibbles疫苗组与HBV+DRibbles联合DC免疫组之间差异无统计学意义。结论:HBV+DRibbles作为HBV抗原载体,能够有效诱导DC对HBV抗原的交叉递呈;HBV+DRibbles诱导的HBV特异性细胞免疫应答对HBV急性感染具有一定的治疗作用。

慢性HBV感染的免疫治疗

目前现有的抗病毒药物对慢性HBV感染的疗效不甚满意.HBV感染的控制和清除有赖于机体的免疫系统,其感染慢性化与机体特异性免疫,尤其是细胞免疫功能低下密切相关.旨在通过增强或恢复机体抗HBV免疫功能以控制和清除HBV感染的免疫治疗是被人们寄予厚望的一种治疗策略,备受关注.本文将从树突状细胞、调节性T细胞、CD8^+T细胞、治疗性疫苗等几个方面对近几年慢性HBV感染的免疫学和免疫治疗研究进展进行综述.

胸腺五肽联合乙肝疫苗对HBsAg低滴度慢性无症状携带者疗效分析

目的观察胸腺五肽联合乙肝疫苗对HBsAg低滴度慢性无症状携带者的疗效。方法将低HBsAg滴度(<20ng/ml)的慢性乙肝病毒感染者治疗组分为A组HBsAg滴度<500ng/ml(n=29)、A1组500ng/ml<HBsAg滴度<20ng/ml(n=14);将对照组分为相应的B组(n=23)和B1组(n=20)。治疗组采用胸腺五肽注射液1mg肌肉注射,初始20日为1次/d,继后为隔日1次,疗程140天,并予乙肝疫苗40μg,上臂三角肌皮下注射,每月1次,疗程4个月。对照组口服维生素等药物护肝治疗。应用时间分辨免疫荧光分析法(TRF)定量检测治疗前后患者乙肝病毒标志物的滴度变化。结果用TRF法检测,A组HBsAg有14例(483%)<05ng/ml,HBsAb有17例(586%)>10mIU/ml;对照B组只有2例(2/23,87%)感染者同时出现HBsAg<05ng/ml和HBsAb>10mIU/ml。治疗后A组与B组HBsAg和HBsAb滴度相比较,P值均<005,差异有显著性意义。治疗后A1和B1组HBsAg和HBsAb滴度相比较,P值均>005,差异无显著性意义。结论胸腺五肽联合乙肝疫苗对HBsAg滴度<500ng/ml的慢性无症状携带者,有明显降低HBsAg和升高HBsAb滴度的作用,有部分感染者可清除乙肝病毒。

重组乙肝疫苗联合干扰素对慢性乙型肝炎患者抗HBV及改善肝功能的临床研究

目的通过重组乙肝疫苗联合干扰素抗HBV研究,探讨其治疗乙型肝炎的可行性及作用机制。方法两组慢性乙型肝炎患者均常规给予保肝降酶药物,联合组每周3次肌注IFNα2b3MU,另加重组乙肝疫苗20μg每月1次三角肌处肌注,疗程为6个月。对照组只单独肌注IFNα2b,分别观察其抗HBV及改善肝功能情况。结果联合组抗HBV及改善肝功能效果与对照组比较有显著差异。结论重组乙肝疫苗联合干扰素抗HBV显示出良好的治疗前景。

基于病毒载体的乙型肝炎治疗性疫苗研究进展

慢性乙型肝炎是危害全球的重大公共卫生问题。临床上目前使用药物可暂时控制疾病进程,使小部分患者达到“功能性治愈”。慢性乙型肝炎久治不愈的原因是机体针对乙型肝炎病毒免疫耐受的结果,破坏免疫耐受环境、恢复特异性免疫反应有助于免疫系统清除乙型肝炎病毒,实现“功能性治愈”。以病毒为载体的乙型肝炎治疗性疫苗在诱导人体产生乙型肝炎病毒特异性T细胞方面有极大效果。本文主要综述近几年国内外基于病毒载体的乙型肝炎治疗性疫苗的研究进展。

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

钙网蛋白融合HBsAg基因重组腺病毒新型载体疫苗的构建与鉴定

目的:构建表达钙网蛋白(calreticulin,CRT)与乙型肝炎病毒表面抗原(hepatitis B surface antigen,HBsAg)融合基因重组腺病毒载体(Ad-CRT/HBsAg),为研发新型乙型肝炎病毒(hepatitis B virus,HBV)治疗性疫苗奠定基础。方法:采用腺病毒表达系统(ViraPowerTM Adenoviral Expression System)构建重组腺病毒表达载体。首先利用RT-PCR的方法扩增CRT基因,并进一步构建CRT与HBsAg基因融合重组的pJW4303表达载体,在构建过程中给融合基因加上特定的CACC接头,再克隆入载体pEN-TR/D-TOPO以获得入门克隆,经PCR及测序鉴定正确后,用重组酶(LR ClonaseTMⅡEnzyme Mix)进行入门克隆与表达载体(pAd-CMV/V5-DEST)间的重组反应,以获得表达克隆Ad-CRT/HBsAg。表达克隆鉴定后,用限制性内切酶PacⅠ线性化后转染HEK293A包装细胞得到重组腺病毒。经过扩增后,用极限稀释法检测病毒滴度,用Western blot法检测Ad-CRT/HBsAg载体是否能正确表达目的蛋白。结果:构建的含有CRT/HBsAg融合基因的腺病毒表达克隆,经PCR和测序鉴定构建正确。重组表达克隆转染HEK293A细胞并扩增,获得的病毒滴度为2.68×1011 pfu/ml,且这个重组病毒载体能正确表达CRT/HBsAg融合蛋白。结论:成功构建了CRT/HBsAg融合基因重组腺病毒载体(rAd-CRT/HBsAg),为此重组载体用于治疗HBV慢性感染以及HBsAg阳性肝癌奠定基础。

HBsAg蛋白疫苗对HBV感染免疫耐受患者的治疗作用

目的:探索乙肝治疗性疫苗(即HBsAg蛋白疫苗,由乙肝疫苗60μg、卡介苗素1mg、花生油水乳化剂0·1ml组成,混合使用),对慢性乙型肝炎免疫耐受患者的治疗效果。方法:选择120例HBsAg+、HBeAg+、抗-HBc+、HBVDNA+,ALT、AST正常者,垂直传播、水平传播各半,分3组:治疗1组(垂直传播)40例,治疗2组(水平传播)40例,对照组(水平垂直各半)40例。给药方法:HBsAg蛋白疫苗每3周双侧三角肌注射1次共12个月,HBIG200IU每3周1针肌注,共12个月。结果:治疗1组:HBeAg转阴率为50%,HBVDNA阴转率为57·5%;治疗2组:HBeAg阴转率50%,HBVDNA阴转率57·5%;对照组HBeAg转阴率为5%,HBVDNA阴转率为20%。结论:HBsAg蛋白疫苗对慢性乙型肝炎免疫耐受有较好的治疗作用,可进一步研究试用。