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.     

Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.     

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     

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.     

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.     

Immunogenicity of a Cholera Toxin B Subunit <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Fimbrial Antigen Fusion Protein Expressed in <Emphasis Type="Italic">E. coli</Emphasis>

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.     

Development of HIV/AIDS vaccine using chimeric gag-env virus-like particles

We attempted to develop a candidate HIV/AIDS vaccine, by using unprocessed HIV-2 gag pr45 precursor protein. We found that a 45 kDa unprocessed HIV-2 gag precursor protein (pr45), with a deletion of a portion of the viral protease, assembles as virus-like particles (VLP). We mapped the functional domain of HIV-2 gag VLP formation in order to find the minimum length of gag protein to form VLP. A series of deletion mutants was constructed by sequentially removing the C-terminal region of HIV-2 gag precursor protein and expressed truncated genes in Spodoptera frugiperda (SF) cells by infecting recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not affect VLP formation. There is a proline-rich region at the amino acid positions 373 to 377 of HIV-2 gag, and replacement of these proline residues by site-directed mutagenesis completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein, and zinc finger domains, are dispensable for gag VLP assembly, but the presence of at least one of the three prolines at amino acid positions 373, 375 or 377 of HIV-2NIH-Z is required for VLP formation. Animals immunized with these gag particles produced high titer antibodies and Western blot analyses showed that anti-gag pr45 rabbit sera react with p17, p24 and p55 gag proteins of HIV-1. We then constructed chimeric gag genes, which carry the hypervariable V3 region of HIV-1 gp120, because the V3 loop is known to interact with chemokine receptor as a coreceptor, and known to induce the major neutralizing antibodies and stimulate the cytoxic T lymphocyte responses in humans and mice. We expressed chimeric fusion protein of HIV-2 gag with 3 tandem copies of consensus V3 domain that were derived from 245 different isolates of HIV-1. In addition, we also constructed and expressed chimeric fusion protein that contains HIV-2 gag with V3 domains of HIV-1IIIB, HIV-1MN, HIV-1SF2 and HIV-1RF. The chimeric gag-env particles had a spherical morphology, and the size was slightly larger than that of a gag particle. Immunoprecipitation and Western blot analyses show that these chimeric proteins were recognized by HIV-1 positive human sera and antisera raised against V3 peptides, as well as by rabbit anti-gp120 serum. We obtained virus neutralizing antibodies in rabbits by immunizing these gag-env VLPs. In addition, we found that gag-env chimeric VLPs induce a strong CTL activity against V3 peptide-treated target cells. Our results indicate that V3 peptides from all major clades of HIV-1 carried by HIV-2 gag can be used as a potential HIV/AIDS vaccine.     

Characterisation of bacterially expressed structural protein E2 of hepatitis C virus

The E2 glycoprotein is a structural component of the hepatitis C virus (HCV) virion. It interacts with putative cellular receptors, elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti-HCV vaccine. Despite this, relatively little is known about this protein. Previous studies have focused on the antigenic and functional analysis of the glycosylated forms. This report describes expression of the ectodomain of E2 (recE2) in Escherichia coli cells, its purification, and initial characterisation of its structural and functional properties. It is demonstrated that the purified protein forms small soluble aggregates, which retain functional characteristics of its native counterpart, i.e., it interacts with a putative cellular receptor, CD81, and is recognised by both conformation-dependent and -independent anti-E2 monoclonal antibodies.     

CD81-dependent binding of hepatitis C virus E1E2 heterodimers

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.     

Multimeric HCV E2 protein obtained from <Emphasis Type="Italic">pichia pastoris</Emphasis> cells induces a strong immune response in mice

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2₆₈₀) was produced in Pichia pastoris cells. E2₆₈₀, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulide-linked multimers. Both monomeric and oligomeric forms of E2₆₈₀ were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCV-positive patients were reactive against E2₆₈₀. By immunizing E2₆₈₀ in BALB/c mice, both a specific cellular immune response and anti-E2₆₈₀ IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2₆₈₀ could be useful to successfully induce strong anti-HCV immunity.     

兔抗五种鱼免疫球蛋白抗体的制备和HRP标记

目的制备兔抗鱼类免疫球蛋白抗体并进行辣根过氧化酶标记,为鱼类血清学检测系统的建立提供工具。方法利用proteinA亲和层析的方法纯化鱼血清免疫球蛋白,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗鱼免疫球蛋白的抗体,采用简易过碘酸钠法对纯化的兔抗鱼免疫球蛋白的抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting来考察标记抗体与其他常见鱼类血清蛋白间的交叉反应。结果纯化了鳙、鲤、乌鳢、黄鳝、鲈五种鱼血清免疫球蛋白,免疫双扩散法测定兔抗这五种鱼免疫球蛋白的抗血清效价均达到1∶32,并对纯化的兔抗鳙、鲤、乌鳢、黄鳝、鲈五种鱼类免疫球蛋白的抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶10000左右,Western blots显示标记抗体与部分其他鱼类免疫球蛋白之间存在不同程度的交叉反应。结论成功制备了HRP标记的兔抗鱼类免疫球蛋白抗体,为鱼类血清学检测体系的建立提供了工具。     

Evaluation of the potency of the anti-idiotypic antibody Ab2/3H6 mimicking gp41 as an HIV-1 vaccine in a rabbit prime/boost study

The HIV-1 envelope protein harbors several conserved epitopes that are recognized by broadly neutralizing antibodies. One of these neutralizing sites, the MPER region of gp41, is targeted by one of the most potent and broadly neutralizing monoclonal antibody, 2F5. Different vaccination strategies and a lot of efforts have been undertaken to induce MPER neutralizing antibodies but little success has been achieved so far. We tried to consider the alternative anti-idiotypic vaccination approach for induction of 2F5-like antibodies. The previously developed and characterized anti-idiotypic antibody Ab2/3H6 was expressed as antibody fragment fusion protein with C-terminally attached immune-modulators and used for immunization of rabbits to induce antibodies specific for HIV-1. Only those rabbits immunized with immunogens fused with the immune-modulators developed HIV-1 specific antibodies. Anti-anti-idiotypic antibodies were affinity purified using a two-step affinity purification protocol which revealed that only little amount of the total rabbit IgG fraction contained HIV-1 specific antibodies. The characterization of the induced anti-anti-idiotypic antibodies showed specificity for the linear epitope of 2F5 GGGELDKWASL and the HIV-1 envelope protein gp140. Despite specificity for the linear epitope and the truncated HIV-1 envelope protein these antibodies were not able to exhibit virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine.     

Definition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies

Development of a successful hepatitis C virus (HCV) vaccine requires the definition of neutralization epitopes that are conserved among different HCV genotypes. Five human monoclonal antibodies (HMAbs) are described that cross-compete with other antibodies to a cluster of overlapping epitopes, previously designated domain B. Each HMAb broadly neutralizes retroviral pseudotype particles expressing HCV E1 and E2 glycoproteins, as well as the infectious chimeric genotype 1a and genotype 2a viruses. Alanine substitutions of residues within a region of E2 involved in binding to CD81 showed that critical E2 contact residues involved in the binding of representative antibodies are identical to those involved in the binding of E2 to CD81.     

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.     

重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的：表达纯化黄热病毒（YFV）囊膜蛋白（E蛋白）结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒（JEV）感染的可能。方法：扩增YFVE蛋白结构域Ⅲ（YFDⅢ）的cDNA片段333bp,将其连接到原核表达载体pET-32a（＋）中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta（DE3）,IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果：在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论：重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。     

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.     

HIV-2 gag-gp105嵌合基因DNA疫苗对小鼠的免疫原性研究

利用真核表达载体 pVAX1 构建 HIV-2 gag-gp105 嵌合基因的重组质粒 pVAX1gag-gp105,将其转入 BHK21细胞中,利用间接免疫荧光方法检测其表达情况。进一步分别将核酸疫苗质粒 pVAX1gag-gp105、对照组质粒pVAX1 和 PBS 溶液经肌肉注射免疫 BALB/c 小鼠,检测免疫小鼠脾 CD4 、CD8 T 细胞亚群的数量,脾特异性 CTL杀伤活性和血清抗体滴度。结果显示,重组核酸疫苗质粒 pVAX1gag-gp105 疫组小鼠脾 CD4 、CD8 T 细胞亚群的数值均比对照组高( P<0.01),脾特异性 CTL 杀伤活性与对照组相比差异极显著(P<0.01),血清抗体滴度显著高于对照组(P<0.01) 。以上结果表明,HIV-2 gag-gp105 嵌合基因 DNA 疫苗对 BALB/c 小鼠具有良好的体液和细胞免疫原性。     

嵌合表皮生长因子疫苗E5T-mSEA的设计及抑瘤活性检测

表皮生长因子受体(Epidermal growth factor receptor,EGFR)及其配体在多种肿瘤细胞中高表达,免疫注射表皮生长因子(Epidermal growth factor,EGF)-载体蛋白所产生的抗体能够干扰EGFR与EGF的相互作用,进而抑制肿瘤生长。本实验室设计了EGF和TGFα的嵌合配体E5T,并将具有免疫增强作用的葡萄球菌肠毒素A(Staphylococcal enterotoxin A,SEA)与之融合。融合蛋白在大肠杆菌内表达后,通过金属螯合亲和层析,得到了纯度较高的E5T-mSEA融合蛋白。将E5T-mSEA免疫小鼠,能够产生高滴度的抗体,该抗体能够同时识别EGF和TGFα。将抗E5T-mSEA免疫血清与肿瘤细胞共培养,在1:10的稀释度下能显著抑制EGFR阳性肿瘤细胞的生长,而对EGFR阴性肿瘤细胞293T的生长没有明显影响。     

Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.     

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.