HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21(DE3)感受态细胞,经IPTG诱导,再进行SDS-PAGE,可得到有一条约34 kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带.该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示目的蛋白占整个菌体蛋白的36%以上,经Ni-柱纯化的E2蛋白纯度可达95%以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阴阳性血清,结果表明15份抗HCV阳性血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体.     

HCV E2区基因在真核表达系统中的表达

以原核表达载体pET-E2为模板,用PCR的方法重新扩增出带有适于真核表达载体多克隆位点的E2基因,PCR产物经纯化并双酶切后与相同处理的真核表达载体pSecTag2/Hygro连接并转化大肠杆菌DH5α感受态细胞,重组表达载体在大量扩增并纯化后再转染COS7细胞,收集的培养上清经过Ni-柱纯化,用ELISA进行血清检测显示,6份HCV阳性血清中有4份检出有E2抗体,而5份HCV阴性血清也有一份检出有E2抗体。     

HCV包膜蛋白E2的克隆、表达与检测

构建丙型肝炎HCV包膜蛋白糖蛋白的E2基因原核表达载体,获得大量重组HCVE2蛋白,进行E2蛋白的抗原性及潜在保护作用研究。通过RT-PCR从HCVRNA阳性血清标本中扩增出975bp(383～708)E2基因片段,PCR产物经EcoR I和Sall I双酶切后连接到经同样酶切的PET-41a原核表达载体上,转化到大肠杆菌BL21(DE3)菌株,经Amp筛选,得到阳性重组质粒PET41a-HCVE2菌株,并以IPTG诱导蛋白表达,SDS-PAGE鉴定,表达产物经固定化金属配体亲和层析纯化,用ELLSA方法检测生物学活性。结果表明,构建的HCVE2包膜蛋白基因片段原核表达质粒所表达产物主要以包涵体形式存在,表达的融合蛋白与HCV阳性血清具有较好的反应原性。以HCVE2融合蛋白检测患者阳性血清具有良好的抗原性,有望能提高HCV抗体检测试剂盒的检出率。     

羊抗人IgG的纯化及其在抗—HCV检测中的应用

单独或联合应用辛酸沉淀、饱和硫酸铵沉淀、阴离子交换等方法对羊抗人IgG进行纯化,对纯化前后抗体的纯度和免疫学活性进行比较,并与辣根过氧化物酶连接,作为二抗用于抗－HCV的ELISA检测。结果表明,不同方法纯化的抗体其纯度和免疫学活性具有一定程度的差别,其中经辛酸＋饱和硫酸铵沉淀纯化的抗体为最佳,凝胶扫描纯度为98．05％,比活性近1800,为纯化前的6．8倍。用痞根过氧化物酶标记后,作为酶标二抗检测HCV阴性和阳性标准血清各40份,阴性符合率为97．5％,阳性符合率为95％,可用于抗－HCV的ELISA检测。     

肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析

HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 ,评估其在临床肿瘤诊断中的作用     

丙型肝炎病毒分片段抗体检测蛋白质芯片质控参比品的建立及质量验证

建立能同时检测丙型肝炎病毒(HCV)嵌合抗体、核心抗体和NS3、NS4、NS5抗体的蛋白质芯片质控参比品,对质控合格的芯片进行质量验证。用3种HCV EIA试剂分别检测从3家医院收集的丙型肝炎病毒感染患血清及其他非HCV感染患血清,从3种EIA试剂同时阴性或阳性的血样中挑取阳性和阴性血清,然后用RNA hyb PCR试剂进行检测,从中再选取部分样本用RIBA3．0进行检测,确定HCV分片段抗体检测蛋白质芯片质控参比品。经质检合格的芯片用中国药品生物制品检定所的HCV参比品进行检定。通过490例临床标本的检测对芯片的质量进行进一步的验证。从收集的240份丙型肝炎病毒感染患血清及其他非HCV感染患血清筛选出30份血样(15份阳性,15份阴性)作为HCV分片段抗体检测蛋白芯片质控参比品。中国药品生物制品检定所的80份HCV参比品检定结果表明,混合抗体阳性检出率为39／40,阴性符合率为40／40,总符合率为98．7％;核心抗体阳性检出率为27／40,阴性符合率为40／40;NS3抗体阳性检出率为26／40,阴性符合率为39／40;NS4抗体阳性检出率为19／40,阴性符合率为40／40;NS5抗体阳性检出率2／40,阴性符合率为40／40。490例临床标本的检测结果表明,对于194例HCV阳性标本,蛋白质芯片混合抗体与ELISA的符合率达99．5％,分片段抗体符合率达97．4％,两种方法检测结果不符的标本经RIBA试剂确认,蛋白质芯片与RIBA试剂的符合率高度一致。对于296例各种HCV抗体阴性标本,蛋白质芯片检测结果全部为阴性。以上结果表明,制备的丙型肝炎病毒分片段抗体检测蛋白质芯片质控参比品可用于芯片生产的质量控制,经质控合格的芯片符合国家标准的要求,可用于临床检测。     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCV HVR1模拟表位融合蛋白.用Western blot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况.皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应.结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35).融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCV HVR1合成肽发生交叉反应.本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值.     

丙型肝炎病毒高变区1模拟表位的交叉反应性分析

研究丙型肝炎病毒(Hepatitis C virus,HCV)高变区1(Hypervariable region 1,HVR1)抗原表位的交叉反应性,获取高反应性的抗原表位.设计并合成5种HVR1模拟表位基因,构建编码HVR1模拟表位的表达载体,表达并纯化表位蛋白.ELISA法检测表位蛋白与35份HCV抗体阳性血清的交叉反应性.包装HCV假病毒(HCV pseudotype particles,HCVpp),评价表位蛋白免疫BALB/c鼠血清在假病毒感染Huh7.5细胞中的作用.结果表明,表达纯化的5种表位蛋白(P1、P2、P5、P6、P8)均可与HCV抗体阳性血清反应,阳性反应率分别为54.3%(P1)、62.9%(P2)、80%(P5)、68.6%(P6)、54.3%(P8).表位蛋白P6、P8免疫BALB/c鼠血清对HCV假病毒感染Huh7.5细胞具明显的抑制作用.结果提示,选取的HVR1模拟表位在HCV感染免疫与疫苗研制中可能具有潜在的价值.     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1 VLP两种抗原在检测宫颈癌抗 16L1或VLP抗体及在宫颈癌血清学诊断意义上的差别 ,应用PCR技术从宫颈癌组织的DNA中扩增出全长15 35bp的HPV16L1基因片段 ,克隆至 pUC18 T载体中 ,进行DNA测序鉴定。然后 ,将HPV16L1基因克隆至pGEX 2T表达载体中 ,并诱导表达HPV16L1融合蛋白 ,分子量为 83kD ,能被HPV16L1单克隆抗体所识别。经GST柱层析法纯化后 ,与重组腺病毒表达的HPV16L1 VLP分别经酶联免疫吸附 (ELISA)法检测 12份宫颈癌患者和 35份献血员血清。 12例宫颈癌血清标本中 ,抗HPV16L1蛋白的抗体阳性率为 7例 (占 5 8.3% ) ;抗HPV16L1 VLP的抗体阳性率为 8例 (占 6 6 .7% )。经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体IgG( )的 7份患者血清 ,利用HPV16L1 VLP试剂盒检测均阳性 ;经大肠杆菌表达的重组抗原检测为HPV16抗体IgG( )的 5份患者血清 ,利用HPV16L1 VLP试剂盒检测有 1份阳性。两者对HPV16抗体的阳性检出率并无显著差异 (P >0 .0 5 )。本实验结果说明HPV16与宫颈癌高度相关 ,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1 VLP重组抗原检测抗体的敏感性并不受影响。利用重组抗原HPV16L1对宫颈癌的抗体进行定性、定量分析有助于该疾病     

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl     

Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.     

Induction of Hepatitis C Virus E1 Envelope Protein-Specific Immune Response Can Be Enhanced by Mutation of N-Glycosylation Sites

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes.     

Intracellular delivery of serum-derived hepatitis C virus

A robust and reliable cell culture system for serum-derived HCV (HCVser) has not been established yet because of the presence of neutralizing antibody and tropism for infection. To overcome this obstacle, we employed a lipid-mediated protein intracellular delivery reagent (PIDR) that permits internalization of proteins into cells. Although entry of HCVcc was not enhanced by the treatment with PIDR, entry of HCVser into hepatoma cell lines (Huh7 and HepG2) and immortalized primary hepatocytes (Hc and HuS/E2) was significantly enhanced by the PIDR treatment. The entry of HCVser into Huh7 cells in the presence of PIDR was resistant to the neutralization by an anti-hCD81 antibody, suggesting that PIDR is capable of internalizing HCVser in a receptor-independent manner. Interestingly, the PIDR-mediated entry of HCVser and HCVcc was enhanced by the addition of sera from chronic hepatitis C patients but not from healthy donors. In addition, neutralization of HCVcc infection by anti-E2 antibody was canceled by the treatment with PIDR. In conclusion, the PIDR is a valuable tool to get over the obstacle of neutralizing antibodies to internalize HCV into cells and might be useful for the establishment of in vitro propagation HCVser.     

Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection

Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1–E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag–Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.     

Identification of a Domain Containing B-Cell Epitopes in Hepatitis C Virus E2 Glycoprotein by Using Mouse Monoclonal Antibodies

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.     

Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.     

Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells.

The putative envelope protein of hepatitis C virus (HCV) was expressed in insect cells by using a baculovirus expression vector and in monkey COS cells under the control of exogenous promoters. The expressed envelope proteins, identified by immunoblot analysis using sera from patients with chronic HCV infection, were a series of glycoproteins of 35 to 24 kDa (gp35-24) in insect cells and a single species of glycoprotein of 35 kDa (gp35) in monkey cells. The size difference of these proteins was due to the different degrees of glycosylation. The envelope proteins expressed in these cells were produced by common specific cleavage from the precursor protein, and cleavage positions of the envelope protein were mapped at about amino acids 190 and 380. The gp35-24 proteins expressed in insect cells were used for detection of antibody against HCV envelope protein in patient sera. The results showed that (i) the antibody is detected in 2 to 17% of various patients with hepatitis C, (ii) three patients were apparently cured after acquiring the antienvelope antibody, and (iii) in sera of patients with more than a 20-year history of infection, the antibody sometimes coexisted with HCV. These results suggest that the antienvelope antibody is neutralizing only in limited number of patients with hepatitis C.