G145R突变后HBsAg"a"决定簇合成肽的免疫学特性分析

目的研究G145R突变后乙肝表面抗原(HBsAg)"a"决定簇合成肽的免疫学特性改变情况. 方法首先合成2条短肽P1-wt和P2-145R,分别代表野毒株和G145R突变后HBsAg"a"决定簇合成肽.然后用β-巯基乙醇(2-ME)变性试验以及用乙肝疫苗标准品制备的小鼠多抗血清研究2条合成肽的空间构象以及抗原性异同.最后用等量合成肽免疫小鼠,用酶免疫法、竞争抑制试验和Western blot试验等研究G145R突变后"a"决定簇合成肽的免疫原性改变.结果合成肽P1-wt和P2-145R用2-ME温和变性后,PAGE结果表现为相对分子质量(Mr)为4×103左右的单一条带,而变性前2条合成肽均表现为Mr是从4×103到30×103的弥漫条带,主带位置在5×103和10×103,分别相当于二聚体和四聚体位置;用HBsAg的多克隆抗体(anti-HBs)检测合成肽的抗原性,结果在固定抗原量的前提下,在抗体1∶32 000稀释时仍可检测到P1-wt的阳性结果,而当同一抗体稀释到1∶8000时,P2-145R检测结果即为阴性.合成肽P2-145R免疫小鼠产生的抗体与P1-wt合成肽的反应滴度比与P2-145R合成肽本身的反应低4～8倍. 结论针对HBsAg"a"决定簇合成肽可自发形成一定的空间构象,G145R突变株的HBsAg"a"决定簇的抗原性和免疫原性与野毒株相比发生了明显改变,为正确评价G145R变异株的流行危害以及现行疫苗的保护效果提供了实验依据.     

乙型肝炎病毒表面抗原基因点突变对HBsAg抗原性的影响

目的 研究乙型肝炎病毒表面抗原(HBsAg)常见基因点突变对HBsAg抗原性的影响,了解我国目前常用的HBsAg检测试剂对HBV S基因突变株的检测灵敏性,减少漏检,有效控制乙肝病毒(HBV)感染的传播.方法 构建HBsAg重组野毒株和重组变异株表达质粒,分别将重组野毒株HBsAg表达质粒pSS1adw2及pSS1adr和重组变异株HBsAg表达质粒pSS1adw2-145Arg、pSS1adr-126 Ser和pSS1adr-126 Asn转染COS-7细胞,进行瞬时转染.采用市售HBsAg ELISA检测试剂盒对细胞上清进行抗原性检测.结果 野毒株HBsAg和两种126位变异株HBsAg具有较好的抗原性;145位点突变后、导致HBsAg的抗原性下降.结论 推测是由于145位点变异影响了"a"抗原决定簇的空间结构,从而降低了其与抗-HBs的结合能力.     

G145R变异乙型肝炎表面抗原诱导的抗体性质研究

乙型肝炎表面抗原（HBsAg）变异可导致抗原性改变。HBsAga决定簇最常见的变异位点是G145R。G145R变异可能改变原来的抗原表位，并形成新的抗原表位。本研究探讨G145RHBsAg能否刺激机体产生抗体及免疫应答的情况。     

乙型肝炎病毒S基因129Leu和145Arg变异株的抗原表达与DNA免疫研究

目的 研究我国发现的乙肝病毒S基因突变株(129Leu和145Arg)表达HBsAg及DNA免疫的特性. 方法 用自乙肝疫苗免疫无保护婴儿血清中克隆的2株变异S基因(其中"a"决定簇突变,分别为nt540A→T,aa129Gln→Leu和nt587G→A,aa145Gly→Arg)片段,置换已知核苷酸序列并可表达及复制的HBV1.2个拷贝全基因野毒株重组质粒p3.8Ⅱ的S基因.将重组质粒转染HepG2细胞,用Abbott试剂和24种单抗检测转染细胞培养上清中HBsAg的结合力.用重组质粒DNA肌肉免疫小鼠. 结果 129Leu变异株转染细胞上清对HBsAg酶联免疫反应检测的吸光度(A)值高于p3.8Ⅱ野毒株,而145Arg变异株的A值低于p3.8Ⅱ;用24种单抗测定的总趋势为129Leu表达的HBsAg 的S/C值高于p3.8Ⅱ,而145Arg的结果则低于p3.8Ⅱ.三者表达的HBeAg水平相似.用DNA免疫经129Leu诱生的抗-HBs水平低于野毒株p3.8Ⅱ,但略高于145Arg. 结论 129Leu变异株表达的HBsAg与HBs多抗及单抗的结合力较野毒株明显增高,但129Leu 变异株DNA免疫诱生的抗-HBs水平却低于野毒株p3.8Ⅱ.免疫原性低下可能有利于129Leu致持续性感染.     

常见乙型肝炎病毒表面抗原突变体的抗原性分析

目的：研究乙型肝炎病毒（HBV）表面抗原突变株和野毒株对表面抗体结合能力的差异,探讨免疫漏检基因变异株的机制及对策。方法：应用基因重组技术构建HBsAg常见基因突变株和野毒株表达质粒,分别在COS－7细胞中瞬时表达,定量后用常规HBsAg免疫诊断试剂盒检测,观察重组抗原与不同抗－HBs的结合能力。结果：T126 S变异与单克隆抗体的结合力增高,G145R,K141 E和2株三位点同时变异株则明显下降,对M133T变异与单克隆抗体的结合力影响不大。变异株与多克隆抗体的结合力也有变化,但仍能检测到大部分变异株抗原。结论:“a”抗原决定簇氨基酸的置换影响了HBsAg与抗－HBs的结合能力,并且氨基酸的置换位置和特性对抗原性的影响各有不同。因此,建议应用HBsAg多克隆抗体或开发研制“免疫逃逸突变株”的特异性抗体,以完善HBsAg免疫诊断试剂的抗体组成,减少免疫诊断对常见基因变异株的漏检。     

HBsAg、HBsAb共存乙型肝炎患者S基因"a"决定簇变异检测分析

目的:研究HBsAg、HBsAb共存慢性乙型肝炎患者HBV S基因“a”决定簇变异情况及对HBsAg抗原性的影响。方法:对7例HBsAg、HBsAb同时阳性慢性乙型肝炎患者的8份血清,采用竞争PCR微流芯片法定量检测HBV DNA;用套式PCR法扩增HBV S基因,对PCR产物进行直接测序,比较S基因的核苷酸和推导出的氨基酸序列的差异,采用ELISA/MEIA法检测HBVM;以15例HBsAg、HBeAg/HBeAb、HBcAb阳性乙肝疫苗免疫失败儿童、9例终末期乙肝患者肝移植术后接受HBIG、拉米夫定治疗患者作为对照。结果:7例患者HBsAb含量均低于80mIU/ml,其中2例HBVDNA定量阳性者未出现“a”决定簇变异,4例HBVDNA定量阴性者中2例氨基酸发生变异(126,131),1例HBV DNA定量由阳性转为阴性者由无变异转为氨基酸126位点变异;9例肝移植术后痊愈患者HBsAb含量均高于150mIU/ml,HBV DNA定量均为阴性,未发现“a”决定簇变异;15例免疫失败儿童14例HBV DNA定性阳性,2例出现“a”决定簇145位点、1例126位点、1例134位点氨基酸变异。结论:部分HBsAg、HBsAb共存慢性乙肝患者、乙肝疫苗免疫失败儿童体内存在S基因“a”决定簇变异;“a”决定簇变异可改变HBsAg抗原性、逃避低含量抗HBs的中和作用;“a”决定簇变异对HBV的复制可能有一定影响;肝移值治愈乙肝患者未发现“a”决定簇变异。     

重组乙型肝炎病毒变异s基因的表达及其抗原性的分析

目的 构建乙型肝炎病毒（HBV）变异s基因真核表达载体。研究变异所致乙肝表面抗原（HBsAg)免疫学性状的改变。方法 利用Dra Ⅲ和XhoI双酶切含有突变位点的HBV DNA 1．2拷贝的质粒P．38Ⅱ，获得904bp带有突变点的HBV－S基因片段。将其置换含有HBV DNA（adr亚型）S和S2片段的真核表达载体pCMV-S2．S的相应片估，从而构建s基因nt587G→A突变的真核表达载体CMV-S2.S 145R；并转染人肝癌细胞系Hep G2，以获得分泌变异型HBsAg的细胞系。利用EIA及细胞免疫染色法，探讨变异抗原与抗－HBs结合力的变化。结果 构建了HBsAg的第145位甘氨酸－精氨酸突变体的真核表达载体。将其转染哺乳动物细胞后第3天，培养上清中用EIA检测用HBsAg呈阳性，A值随时间延长而上升，但变异型的A值明显低于野生型。变异型和野生型HBsAg细胞免疫化学检测均有部分细胞呈阳性，但差异不显著。结论 HBsAg第145位甘氨酸－精氨酸的突变体的真核表达载体产物具有良好的抗原性，能够与抗－HBs结合，但与野生型相比结合力明显降低。     

江西地区儿童感染的乙型肝炎病毒"a"抗原变异分析

目的:初步分析江西省乙型肝炎疫苗免疫儿童感染的乙型肝炎病毒(Hepatitis B Virus,HBV)"a"抗原决定簇的变异。方法:收集在江西省儿童医院就医的13 117名乙肝疫苗免疫儿童(7.39±3.66岁)血清标本,酶联免疫吸附方法检测HBV-M,抽提HBV表面抗原(HBsAg)阳性血清标本中的HBV DNA,扩增HBV S基因,PCR产物测序并与标准序列对比,分析"a"抗原决定簇的变异与血清型;利用在线Genotyping软件对儿童感染的HBV进行分型。同时用荧光定量PCR检测血清HBV DNA含量。结果:从13 117份血清样品中,检测出HBsAg阳性标本230份(1.75%),扩增HBV S基因并成功测序118份。检测出24份标本有"a"抗原决定簇变异(变异率20.34%),"a"抗原决定簇两茎环间的变异率和男女儿童感染的HBV"a"抗原决定簇的变异率无显著性差异。Q129H、G145A突变后,血清HBV DNA水平较未变异株降低(P<0.05)其他各组则无显著变化。118份测序标本利用Genotyping比对后,112份属于B型,其中adw血清型105份,ayw血清型7份;6份属于C型,均为adr血清型。结论:在江西省乙肝疫苗免疫儿童人群检测出"a"抗原决定簇变异的HBV感染,未发现有明显优势的变异株类型。HBV"a"抗原决定簇区突变对血清HBV DNA含量有一定的影响。     

乙肝疫苗免疫失败儿童病毒S基因"a"决定簇变异研究

目的 探讨江苏常州地区乙型肝炎疫苗免疫失败儿童病毒S基因“a”决定簇的变异情况。方法 对15例乙肝疫苗接种后血清表面抗原(HBsAg)阳性的儿童,采用聚合酶链反应方法(PCR)扩增其血清中HBV DNA S基因区。并对PCR产物直接标记测序。结果 15例HBsAg阳性儿童有14例血清HBV DNA阳性,其中有4例出现了S基因“a”决定簇的变异,变异率为28．6％,第126位的异亮氨酸（Ile)被苏氨酸(Thr)替代1例,第134位苯丙氨酸(Phe)被异亮氨酸替代1例。第145位甘氨酸(Gly)被丙氨酸(Pha)替代2例。结论乙型肝炎疫苗免疫失败儿童中存在s基因“a”抗原决定簇变异,江苏常州地区存在HBVS基因“a”决定簇变异的新类型。     

乙肝病毒核心区与"a"决定簇嵌合基因的体外表达及抗原性分析

目的评价乙肝病毒核心区与"a"决定簇嵌合基因表达产物的抗原性.方法应用基因工程技术将编码截短的乙型肝炎病毒核心抗原的主要免疫显性区替换为表面抗原"a"决定簇的基因片段,将嵌合基因装入原核表达载体pRESET-B内,在体外进行诱导表达,纯化目的蛋白并检测其抗原性.同时将嵌合基因亚克隆到真核表达载体pcDNA3内,瞬时转染 BHK细胞并采用间接免疫荧光法检测融合蛋白表达情况.结果诱导并纯化得到了分子量为28 ku的蛋白,经检测证实该蛋白具有一定的HBsAg抗原性和较弱的HBcAg抗原性.真核表达质粒可在 BHK细胞内表达,可检出良好的HBsAg抗原性及较弱的HBcAg抗原性.结论该嵌合质粒表达产物具有良好的HBsAg抗原性及较弱的HBcAg抗原性.     

Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity

Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity.     

Effect of variation in the common "a" determinant on the antigenicity of hepatitis B surface antigen

Antibody to the common "a" determinant of hepatitis B surface antigen (HBsAg) protects against infection with hepatitis B virus. A number of variant surface antigens with amino acid substitutions within the "a" determinant have been described in patients around the world. Both wild type and variant HBsAgs were expressed in the yeast Pichia pastoris and the antigens were semi-purified and quantitated. The effect on antigenicity of these changes was investigated in a quantitative fashion using four monoclonal antibodies known to bind to different epitopes within the common "a" determinant. The results suggest that amino acid substitution of T131I, K141E and G145R and insertion of 3 amino acids between residues 123 and 124 markedly affect the antigenic structure of HBsAg. These substitutions and insertions in the viral envelope may lead to evasion of the virus neutralizing antibody response and also to reduce efficiency of detection by immunoassays used for diagnosis and blood-bank screening.     

HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.     

48-mer synthetic peptide analogue of the hepatitis B virus "a" determinant induces an anti-HBs antibody response after a single injection

An extended (48 amino acid) synthetic peptide analogue of the hepatitus B virus (HBV) S protein (HBsAg) 'a' determinant has been produced by using 9-fluorenylmethoxycarbonyl (fmoc) chemistry and a low substitution polystyrene resin as the solid phase support. This peptide (S121/48) elicited a sustained anti-peptide antibody response in BALB/c (H-2(d)) mice when immunised with Freund's complete adjuvant (FCA). Cross-reactive, anti-HBs antibodies were induced, directed against a significant proportion of the conformationally restrained epitope repertoire on the native HBsAg particles. Similar responses were obtained by injection of guinea pigs, a species known both to be exquisitely sensitive to HBsAg and to produce a wide range of B cell responses to HBsAg antigens. Taken together, these data show for the first time, that a synthetic peptide mimicking conformational epitopes can be produced by chemical synthesis and can be used to induce significant titres of anti-HBs antibodies after a single injection. This immunogen has considerable potential for incorporation into novel delivery systems, e.g., microspheres, thus offering the potential of a controlled release, single dose hepatitis B vaccine.     

重组乙型肝炎病毒变异s基因疫苗诱导小鼠产生特异性体液免疫应答

目的：构建乙型肝炎病毒(HBV)变异s基因真核表达载体，检测其诱导小鼠产生特异性体液免疫应答。方法：利用限制性内切酶定位克隆构建s基因nt587 G→A的真核表达载体pcMV-S2．S 145R(PR).用其转染人肝癌细胞系Hep G2后，用EIA、EILISA及免疫细胞化学法，观察其抗原性。以重组变异型s基因真核表达载体(PR)和载体pcDNA3．0分别免疫C57BL／6小鼠各5只。每只小鼠各肌肉注射纯化质粒100μg.用ELISA法检测血清抗-HBs及抗-HBs2抗体的效价。结果：体外实验证实，变异型HBs矩可与抗-HBs结合；PR免疫小鼠可诱导其产生抗-HBs抗体及抗．HBs2抗体，但抗-HBs2抗体的出现早于抗-HBs抗体约1～2wk。结论：HBV变异s基因(nt587G→A)的真核表达载体的表达产物具有良好的抗原性，能够诱导C57BL／6小鼠产生体液免疫应答。     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

A novel hepatitis B virus variant S 129 (Gln-->Leu): lack of correlation between antigenicity and immunogenicity.

A point mutation has been detected in the "a" determinant of hepatitis B surface antigen (HBsAg) in an infant immunised with hepatitis B vaccine after exposure to hepatitis B virus (HBV). This A-to-T point mutation at nucleotide 540 resulted in a glutamine-to-leucine substitution at amino acid residue 129 (129L). The S gene fragment (nucleotide 58-1072) of this isolate was cloned and used to substitute the wild-type S gene in a plasmid (p3.8II), containing 1.2 copy of full-length HBV genome with expression and replication efficiency. This plasmid p3.8II-129L was used to transfect HepG2 cells. HBsAg expressed by p3.8II-129L showed higher binding efficiency compared with the original plasmid containing the wild-type clone. A panel of 24 anti-HBs monoclonal antibodies (MAbs) was used to characterise the binding efficiency of HBsAg expressed by p3.8II-129L. Eighteen showed higher binding to the antigen, whereas HBsAg expressed by p3.8II-145R gave a consistently lower absorbance or were negative. Surprisingly, when the immunogenicity of plasmid constructs was used for DNA immunisation in Balb/c mice, the anti-HBs response induced by p3.8II-129L was significantly lower than that of the wild-type p3.8II. The lack of correlation between the antigenicity profile (binding of expressed HBsAg to anti-HBs in vitro), and the immunogenicity (induction of anti-HBs by plasmid DNA in vivo) of HBsAg with leucine substitution at position 129 indicates that biological characteristics other than the binding efficiency of HBsAg to anti-HBs could occur in HBsAg variants. These different aspects of the biological characteristics of S mutants merit further investigation.