Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.     

乙型肝炎病毒e抗原的生物学功能及血清学转换的免疫学基础

目前在临床乙型肝炎的治疗中,乙型肝炎病毒e抗原(HBeAg)消失及其抗体的出现已成为重要的疗效指标.本文回顾了HBeAg的发现及其生物学和医学意义,对HBeAg与乙型肝炎病毒核心抗原(HBcAg)的免疫原性进行了比较,阐述了HBeAg血清学转换的免疫学基础,并对出现HBeAg血清学转换的意义作了分析.     

含不同长度前C序列的乙型肝炎病毒核心抗原基因在重组痘苗病毒中的表达

核酸序列分析表明,乙型肝炎(乙肝)病毒核心抗原基因(C基因)编码区内存在两个ATG。目前认为第二个ATG为乙肝病毒C抗原的起始密码子,两个ATG之间的序列称为前C序列,共87bp。Uy、Rutter、Roossinck及景新等的研究表明,含前C序列时,C基因在大肠杆菌中的表达是形成具有HBeAg活性的P25~e膜蛋白,在哺乳动物细胞中的表达则是形成分泌性的HBeAg;不含前C序列时,在大肠杆菌和哺乳动物细胞中均形成P21~e     

免疫预防阻断乙型肝炎病毒母婴传播效果的观察

阻断乙型肝炎病毒(HBV)母婴传播是控制乙型肝炎的重大问题。为探讨免疫预防对阻断HBV母婴传播的效果及影响因素,对667例HBV表面抗原(HBsAg)阳性孕妇及其婴儿进行研究。这些孕妇按HBVe抗原(HBeAg)和HBVDNA检测结果,分为HBeAg阳性组及阴性组、DNA阳性组及阴性组;按是否于孕晚期注射乙型肝炎免疫球蛋白(HBIG),分为注射组及未注射组。婴儿于出生24h内均肌内注射HBIG100IU,并按0、1、6方案注射10μg重组酵母HBV疫苗;8～12月龄后随访婴儿,并进行HBV标志物(HBV-M)检测。667个婴儿中,20例感染HBV,免疫阻断失败率为3.0%。孕妇HBeAg阳性组免疫阻断失败率为8.7%,阴性组为0.2%,两组差异显著(P<0.001);两组婴儿对疫苗免疫应答率分别为83.0%和83.1%,无显著差异(P=0.988)。孕妇DNA阳性组免疫阻断失败率为8.1%,HBVDNA均≥6log10copies/ml。孕期注射与未注射HBIG组婴儿免疫阻断失败率分别为3.7%和2.7%,无显著差异(P=0.479);两组婴儿对疫苗免疫应答率分别为84.4%和82.4%,无显著差异(P=0.519)。孕妇HBeAg阳性注射HBIG组与未注射组的免疫阻断失败率分别为8.4%和8.9%,无显著差异(P=0.892)。孕妇HBeAg阴性注射与未注射HBIG组的免疫阻断失败率分别为0.0%和0.3%,也无显著差异(P=0.538)。11例免疫阻断失败的婴儿中,10例出生时血清HBsAg已为阳性;8～12个月后随访,HBsAg仍持续阳性,提示为宫内感染。本研究证实,孕期注射HBIG未能提高婴儿对HBV疫苗加HBIG的免疫阻断效果。宫内感染可能是疫苗加HBIG免疫阻断失败的主要原因。采用降低孕妇血清HBVDNA的措施,如对孕妇进行抗HBV治疗,也许能降低HBV宫内感染率。     

利用人摄金蛋白(METALLOTHIONEIN)启动子和牛乳突瘤病毒在哺乳动物细胞中表达乙型肝炎表面抗原

用人Metallothioenin-Ⅱ启动子、乙型肝炎表面抗原基因,SV40早期基因的编接位点和多聚A位点构成了表达组件,然后插入到经改造过的BpV-1质粒中。所得质粒pdMTsAg-5转染小鼠C127细胞得到转化克隆。Ausria Ⅱ检测证明13株中有12株能产生HBsAg。对其中一株进行HBsAg收率观察,隔天收获为292.6～525.8μg/升,每天收获为200.9～369.0μg/升,可连续收获60天以上。经重金属离子诱导后,收率增至583～854.4μg/升。培养液经超滤浓缩和两次密梯离心后,可集中为一个狭窄的峰,顶峰的Ausria Ⅱ cpm为1.05×10~7,密度为1.20g/ml。     

乙型肝炎病毒S-preS1-C融合基因在酵母中的表达及抗原性分析

以HBV全基因组质粒为模板,扩增出目的区段:S(1-222AA),preS1(10-50AA)和C(2-30AA),以酶切-PCR的方法进行连接,克隆入表达载体pPIC3.5k,电穿孔转化后利用G418加压筛选出多拷贝整合菌株,诱导表达并对表达产物进行检测。结果显示融合后的基因为ss1c,长度约为906bp。在酵母中成功地进行了表达,表达产物(SSIC)的大小约为31kDa,并形成直径约为22nm的病毒样颗粒。表达产物与HBs抗体、preS1抗体和HBc抗体均有特异反应。为进一步研究治疗性疫苗提供了重要的基础。     

乙型肝炎病毒表面抗原中蛋白基因在中国地鼠卵巢细胞中的高效表达及其产物活性的初步检测

构建了pSV2DHWS2S质粒,使dhfr扩增基因及乙型肝炎病毒的Pre-S_2+S基因分别在两个SV40早期启动子的调控之下。此质粒转化到CHO-dhfr~-细胞,经克隆、加氨甲喋呤(MTX)筛选、扩增,建立了3个高效分泌HBsAg中蛋白及主蛋白的克隆细胞系。检测了其中的M6细胞系生物学特性。结果表明,免疫电镜下可观察到22nm的颗粒;该细胞用转瓶连续培养60天,每2天收、换液1次,每升HBsAg平均产量为2.9mg。经初步纯化,在SDS-PAGE中显示23k、27k主蛋白带及33k、36k中蛋白带。主蛋白及中蛋白的反相血凝(RPHA)滴度分别为64和128;中蛋白的ELISA滴度为320。部分品系小鼠免疫后能产生滴度为8的抗Pre-S_2抗体。3只家免中仅有1只在免疫后第1、2周可测出Pre-S_2抗体,而3只兔的S抗体滴度都较高,持续时间也较长。     

乙型肝炎病毒表面抗原对细胞脂质合成作用的研究

乙型肝炎病毒(hepatitis B virus,HBV)合成的蛋白调节细胞脂质代谢的研究不断被报道,但乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)与脂质代谢的相互调控研究较少,且机制尚不明确。本研究通过对细胞转录组学的分析,揭示HBsAg对脂质代谢的调控机制。选用稳定表达HBsAg的细胞系HepG2-S-G2与其对照细胞系HepG2-neo-F4进行转录组学分析。利用定量聚合酶链反应(polymerase chain reaction,PCR)、蛋白质印迹法(Western blot,WB)分别检测重要差异基因OXCT1和CYP4F3在mRNA水平和蛋白水平的表达差异。为验证HBsAg促进脂质合成上调的表型,对两种细胞系进行油红O染色并检测细胞脂肪酸、总胆固醇水平。进一步对稳定转染HBV的细胞系HepG2.2.15进行降脂处理,以观察细胞上清液中HBsAg与脂质合成之间是否存在相互调控。结果显示,参与脂质代谢的差异基因发生显著变化,提示HBsAg引起了宿主细胞脂质合成途径的上调和消耗途径下调。定量PCR结果显示,相对于HepG2-neo-F4细胞,HepG2-S-G2细胞的3-酮酸辅酶A转移酶1(3-oxoacid CoA-transferase 1,OXCT1)mRNA水平升高约9倍,与转录组测序结果基本一致;CYP4F3基因在HepG2-S-G2细胞中转录相对下调。 WB结果显示,OXCT1和CYP4F3蛋白表达均出现相应的显著上调或下调,并且趋势与转录组分析一致。油红O染色以及细胞脂肪酸、总胆固醇水平检测结果证实HepG2-S-G2细胞中脂滴更明显,且游离脂肪酸和总胆固醇均显著升高。降脂处理结果显示细胞上清液中HBsAg显著降低。上述结果表明,HBsAg可上调脂质代谢、促进脂质合成,提示降脂可能成为抑制HBsAg的潜在有效途径。     

通过乙型肝炎病毒核心基因5''''端的修饰使核心抗原在大肠杆菌中高效表达

为了使乙型肝炎病毒核心抗原(HBcAg)在大肠杆菌中获得高效表达,本文首次采用一种新方法对核心基因前区进行改造与修饰,即用限制性内切酶Taq I从核心基因内部5′端切开,去除核心基因起始信号ATG及ATG 5′端上游的全部前核心区(Precore),再化学合成一段既包含核心基因起始信号又具有多种功能的DNA片段。将两者拼接重组到表达载体pUC9上,转化受体菌,成功地获得高效表达HBcAg的菌株。用ELISA法检测,表达滴度为1:80000。表达产量占菌体总蛋白的16％。其菌体裂解液经免疫电镜观察,可见到成堆聚集的典型HBcAg颗粒。抗原单体分子量约为22000道尔顿,双体为44000。与目前国内外所普遍采用的方法比较,本文的方法有许多明显的优点,可用于其它基因改造。     

乙型肝炎病毒S-preS1-C融合基因在酵母中的表达及抗原性分析

以HBV全基因组质粒为模板,扩增出目的区段S(1-222AA),preS1(10-50AA)和C(2-30AA),以酶切-PCR的方法进行连接,克隆入表达载体pPIC3.5k,电穿孔转化后利用G418加压筛选出多拷贝整合菌株,诱导表达并对表达产物进行检测.结果显示融合后的基因为ss1c,长度约为906bp.在酵母中成功地进行了表达,表达产物(SSIC)的大小约为31kDa,并形成直径约为22nm的病毒样颗粒.表达产物与HBs抗体、preS1抗体和HBc抗体均有特异反应.为进一步研究治疗性疫苗提供了重要的基础.     

抗原抗体复合物型治疗性疫苗(乙克)临床研究中患者血清乙型肝炎病毒表面抗原下降的分析与启示

近年来全球慢性乙型肝炎(chronic hepatitis B,CHB)防治指南提出了“功能性治愈”(functional cure)的概念,即患者经过治疗达到血清乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)消失,但现有抗病毒治疗很难实现这一目标。本研究对既往临床试验中经抗原抗体复合物型治疗性疫苗(乙克)治疗后的CHB患者HBsAg下降情况进行了归纳分析,结果显示,经乙克治疗随访后达到乙型肝炎e抗原(hepatitis B e antigen,HBeAg)血清学转换者的HBsAg下降高达0.95log₁₀IU/mL,显著高于未达到HBeAg血清学转换者的0.32log₁₀IU/mL(P<0.01),而经氢氧化铝佐剂治疗随访后发生HBeAg血清学转换(0.49log₁₀IU/mL)者与未发生HBeAg血清学转换者(0.36log₁₀IU/mL)之间HBsAg下降无统计学差异。乙克组治疗过程中,丙氨酸氨基转移酶(alanine aminotransferase,ALT)骤升(ALT flare)在HBsAg下降>1.0log₁₀IU/mL者中较多见,氢氧化铝组未观察到此现象。回归分析显示,乙克治疗后HBsAg下降的影响因素有患者出现HBeAg血清学转换、感染的HBV为B基因型、治疗过程中ALT出现10倍增高,以及基线血清HBsAg为高水平。结果提示,乙克诱导的特异性免疫对降低CHB患者血清HBsAg水平有一定效果,采用“抗病毒药物治疗＋针对HBsAg的中和性抗体被动免疫＋乙克主动免疫”的“三明治”治疗策略可能会提高“功能性治愈”率。     

乙型肝炎病毒表面抗原aa126 Ile→Ser变异株HBsAg的暂时表达和抗原性鉴定

曾在一个儿童患者体内,发现一个新的乙型肝炎病毒（ＨＢＶ）变异株,其ＨＢｓＡｇ主蛋白ａａ１２６发生Ｉｌｅ（ＡＴＴ）到Ｓｅｒ（ＡＧＴ）的取代。已知ａｄｒ／ａｙｒ亚型ＨＢｓＡｇ１２６位为Ｉｌｅ,而ａｄｗ／ａｙｗ亚型ＨＢｓＡｇ１２６位为Ｔｈｒ,表明ＨＢｓＡｇ１２６Ｓｅｒ是一个新的变异株。用计算机做结构分析的结果表明,突变体ＨＢｓＡｇ１２６Ｓｅｒ主蛋白ａａ１２０－ａａ１３０区段的二级结构与野生型ａｄｒＨＢＶＨＢｓＡｇ１２６Ｉｌｅ相比发生明显的改变。这种构象的变化可能会影响ａ抗原决定簇（ａ１２４－ａａ１４７）的抗原性。为了证实这一点,构建了突变Ｓ基因表达质粒,在ＳＶ４０早期启动子的控制下进行抗原表达。利用ＨＢｓＡｇ９肽（Ｔｈｒ－Ｉｌｅ１２６－Ｐｒｏ－Ａｌａ－Ｇｌｎ－Ｇｌｙ－Ｔｈｒ－Ｓｅｒ－Ｍｅｔ）抗ｉ单克隆抗体和９肽（Ｔｈｒ－Ｔｈｒ１２６－Ｐｒｏ－Ａｌａ－Ｇｌｎ－Ｇｌｙ－Ｔｈｒ－Ｓｅｒ－Ｍｅｔ）抗ｔ单克隆抗体进行放射免疫测定,结果表明,这种Ｓｅｒ１２６突变蛋白对抗ｉ单克隆抗体的反应性比野生蛋白减弱,对抗ｔ单克隆抗体的反应性则与ＨＢｓＡｇ１２６Ｔｈｒ接近。但用三种抗－ａ单克隆抗体的检测结果揭示,突变蛋白ＨＢｓＡｇ１２６     

Characterization of T cell clones specific to a determinant of hepatitis B virus core and e antigens in chronic type B hepatitis: Implication for a T cell mechanism of HBV immunopathogenesis

T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130–140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) chain variable (V) gene was found: V12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR chain joining region (J) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V gene usage and the expression of conserved residues in junctional-region sequences of TCR chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis.     

4例乙肝表面抗原阴性乙肝病毒DNA阳性标本结果分析

目的 探讨4例乙肝病毒HBV-DNA阳性标本乙肝表面抗原(HbsAg)阴性的原因.方法 4例HbsAg阴性HBV-DNA阳性标本和1例HbsAg阳性HBV-DNA阳性标本,碱裂解法提取乙肝病毒基因组,采用巢式PCR扩增s基因全长片段,T-A克隆后测序,根据s基因序列推导氨基酸序列,分析乙肝表面抗原α决定簇区域的氨基酸变异情况.结果 与参考株比较,5例血清中HBV-DNAs基因均出现不同位点碱基突变,导致乙肝表面抗原α决定簇区域的氨基酸突变主要为:1号标本R122K、I126A、S143T;2号标本R122K、I126N、Q129N;3号标本R122K、I126S、T131N、M133T;4号标本R122K、I126S、T131N、M133T及5号标本的R122K.结论 乙肝表面抗原α决定簇区域126位氨基酸突变可能会影响HbsAg的检测结果.     

Effects of interaction between genetic variants in human leukocyte antigen DQ and granulysin genes in Chinese Han subjects infected with hepatitis B virus

Single nucleotide polymorphisms (SNPs) of HLA‐DQ and granulysin (GNLY) are reportedly associated with HBV infection. The aim of this study was to investigate the effects of interactions between SNPs in HLA‐DQ and GNLY on the outcome of hepatitis B virus (HBV) infection in Chinese Han subjects. HLA‐DQ (rs9275572) and GNLY (rs1866139 and rs11127) were genotyped in 310 subjects with HBV‐related chronic liver disease, 295 in whom spontaneous clearance of HBV had occurred and 316 who had not been exposed to HBV. HLA‐DQ rs9275572 was significantly correlated with HBV clearance (dominant genetic model: OR, 1.84; 95% CI, 1.30–2.61; adjusted P = 0.001). There was no statistical association of GNLY rs1866139 and rs11127with HBV infection outcomes. However, significant sex‐specific associations with HBV susceptibility were observed in men who carried rs1866139 CG or rs11127 TC and in women who carried rs1866139 GG or rs11127 CC. The findings were the same in the validation cohort, which was composed of 829 subjects. Based on a multifactor dimensionality reduction test with permutation correction, a three‐way interaction between SNPs in HLA‐DQ and GNLY was identified in terms of HBV clearance. In conclusion, additional evidence for an association of HLA‐DQ and GNLY SNPs with HBV infection outcomes has been identified and a SNP‐SNP interaction between HLA‐DQ and GNLY on HBV clearance observed.     

大肠杆菌表达的乙型肝炎病毒核心抗原的某些理化特性

1979年Pasek等用大肠杆菌表达乙型肝炎病毒核心抗原(HBcAg)成功以来,国内外也有类似的报道,但对其理化特性的报道不多。为了更有效地保存和应用此抗原,本文研究了从表达HBcAg的大肠杆菌菌液中纯化抗原的方法,以及抗原的某些特性。 HBcAg是由pHBV-1的C基因与pTL载体重组后在大肠杆菌BMH71-18内表达的,     

Divergent Primary Immune Responses Induced by Human Immunodeficiency Virus-1 gp120 and Hepatitis B Surface Antigen Determine Antibody Recall Responses

The development of a vaccine based on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) that elicits potent protective antibodies against infection has been challenging. Recently, we compared the antibody production patterns of HIV-1 Env gp120 and hepatitis B virus surface antigen (HBsAg) to provide insights into how we may improve the protective efficacy of Env-based immunogens. Our previous study showed that HIV Env and HBsAg display different mechanisms of antibody elicitation and that T cells facilitate the responses to repeated immunizations. Here, to elucidate the detailed roles of primary immunization in immune memory response formation and antibody production, we immunized C57BL/6 mice with each antigen and evaluated the development of T follicular helper (Tfh) cells, germinal centers, and the memory responses involved in prime and boost immunizations. We found that after prime immunization, compared with HBsAg, gp120 induced higher frequencies of Tfh cells and programmed death (PD)-1⁺ T cells, greater major histocompatibility complex II expression on B cells, comparable activated B cells, but weaker germinal center (GC) reactions and memory B cell responses in the draining lymph nodes, accompanied by slower antibody recall responses and poor immune memory responses. The above results suggested that more PD-1⁺ T cells arising in primary immunization may serve as major contributors to the slow antibody recall response elicited by HIV-1 Env.     

Sodium dodecylsulfate polyacrylamide gel electrophoresis of recombinant hepatitis B surface antigen particles

To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles. On the other hand, the results of alkylation experiments indicated the presence of free sulfhydryl group(s) in a little portion of freshly-purified HBsAg which was retarded on the size-exclusion chromatographic column and had significant antigenicity. This fraction of HBsAg was shown to be oligomeric and capable of spontaneous assembly into higher-order structures during aging.     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.