Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

Immunochemical properties of short recombinant polypeptides of proteine of, the West Nile virus bearing epitopes of domains I and II

Fragments cDNA (nt 935-1475, 1091-1310, 935-1193) encoding N-terminal part of protein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human were obtained and cloned. Recombinant polypeptides of glycoprotein E (E1-86, E53-126, E1-180) of the WNV with corresponding amino acid sequence to the cloned fragments of cDNA and modeling the epitopes of domains I and II of surface glycoprotein E were purified by affinity chromatography. Twelve types of monoclonal antibodies (MAbs) created in our laboratory against recombinant polypeptide E1-180 interact with glycoprotein E of the WNV as results of Western blot and ELISA that is demonstrating an similarity of chemical structure of short recombinant polypeptides and corresponding amino acid sequence regions of WNV protein E. Analysis of interactions of MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus let us reveal no less than six epitopes within domains I and II of glycoprotein E of the WNV. No less than seven types of MAbs to 86-126 aa region of the domain II were found where located peptide providing fusion of virus--cell membranes (98-110 aa). The epitope for anti-receptor MAbs 10H10 within 53-86 aa region of domain II of protein E of the WNV was mapped and it shows that the fusion peptide and co-receptor of protein E for cellular laminin-binding protein (LBP) are spatial nearness. X-ray model of protein E let us suppose that bc-loop (73-89 aa) of domain II interacts with LBP and together with cd-loop (fusion peptide) determines an initial stages of penetration virions into cell.     

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.     

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.     

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108–119 (domain A), a subtype I/IV-specific epitope at amino acids 211–226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231–246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131–146 and 241–256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.     

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.     

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.     

Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains.

We have analyzed a panel of eight murine monoclonal antibodies (MAbs) that depend on the V2 domain for binding to human immunodeficiency virus type 1 (HIV-1) gp120. Each MAb is sensitive to amino acid changes within V2, and some are affected by substitutions elsewhere. With one exception, the MAbs were not reactive with peptides from the V2 region, or only poorly so. Hence their ability to bind recombinant strain IIIB gp120 depended on the preservation of native structure. Three MAbs cross-reacted with strain RF gp120, but only one cross-reacted with MN gp120, and none bound SF-2 gp120. Four MAbs neutralized HIV-1 IIIB with various potencies, and the one able to bind MN gp120 neutralized that virus. Peptide serology indicated that antibodies cross-reactive with the HxB2 V1 and V2 regions are rarely present in HIV-1-positive sera, but the relatively conserved segment between the V1 and V2 loops was recognized by antibodies in a significant fraction of sera. Antibodies able to block the binding of V2 MAbs to IIIB or MN gp120 rarely exist in sera from HIV-1-infected humans; more common in these sera are antibodies that enhance the binding of V2 MAbs to gp120. This enhancement effect of HIV-1-positive sera can be mimicked by several human MAbs to different discontinuous gp120 epitopes. Soluble CD4 enhanced binding of one V2 MAb to oligomeric gp120 but not to monomeric gp120, perhaps by inducing conformational changes in the oligomer.     

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.     

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.     

Peptide immunogen mimicry of putative E1 glycoprotein-specific epitopes in hepatitis C virus.

Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B hepatitis with serious consequences that may lead to hepatocellular carcinoma. The putative envelope glycoproteins (E1 and E2) of HCV probably play a role in the pathophysiology of the virus. In order to map the immunodominant domains of the E1 glycoprotein, two epitopes from amino acid residues 210 to 223 (P1) and 315 to 327 (P2) were predicted from the HCV sequence. Immunization of mice with the synthetic peptides conjugated to bovine serum albumin induced an antibody response, and the antisera immunoprecipitated the E1 glycoprotein (approximately 33 kDa) of HCV expressed by recombinant vaccinia virus. A panel of HCV-infected human sera was also tested with the synthetic peptides by enzyme-linked immunosorbent assay for epitope-specific responses. Of 38 infected serum samples, 35 (92.1%) demonstrated a spectrum of reactivity to the P2 peptide. On the other hand, only 17 of 38 (44.7%) serum samples were reactive to the P1 peptide. Strains of HCV exhibit a striking genomic diversity. The predicted P1 epitope showed localization in the sequence-variable region, and the P2 epitope localized in a highly conserved domain. Results from this study suggest that the E1 glycoprotein of HCV contains at least two potential antigenic epitopes. Synthetic peptides corresponding to these epitopes and antisera to these peptides may serve as the monospecific immunological reagents to further determine the role of E1 glycoprotein in HCV infection.     

Immunochemical Properties of Recombinant Polypeptides Mimicking Domains I and II of West Nile Virus Glycoprotein E

Complementary DNA fragments (nucleotides 935–1475, 1091–1310, and 935–1193) encoding the N-terminal portion of glycoprotein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were cloned. Recombinant polypeptides of glycoprotein E (E_1–180, E_53–126, and E_1–86) of the WNV having amino acid sequences corresponding to the cloned cDNA fragments and mimicking the main functional regions of domains I and II of surface glycoprotein E were purified by affinity chromatography. According to ELISA and Western blotting, 12 types of monoclonal antibodies (MAbs) raised in our laboratory against recombinant polypeptide E_1–180 recognized the WNV glycoprotein E. This is indicative of similarity between the antigenic structures of the short recombinant polypeptides and corresponding regions of the glycoprotein. Analysis of interactions of the MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus revealed at least six epitopes within domains I and II of the WNV protein E. We found at least seven MAb types against the region between amino acid residues (aa) 86 and 126 of domain II, which contains the peptide responsible for fusion of the virus and cell membranes (residues 98–110). The epitope for antireceptor MAbs 10H10 was mapped within the 53–86 aa region of domain II of WNV protein E, which is evidence for the spatial proximity of the fusion peptide and the coreceptor of protein E (residues 53–86) for cellular laminin-binding protein (LBP). The X-ray pattern of protein E suggests that the bc loop (residues 73–89) of domain II interacts with LBP and, together with the cd loop (fusion peptide), determines the initial stages of flavivirus penetration into the cell.     

Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ～100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.     

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.     

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.     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoVN蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Westernblotting和间接免疫荧光方法分别检测了4株抗SARS-CoVN蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoVN蛋白对上述4种单克隆抗体的识别表位进行了初步定位。结果显示:(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoVN蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoVN蛋白的aa30-60,1-1C2针对的抗原表位则位于SARS-CoVN蛋白的aa170-184。这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料。     

Epitope mapping of rat cytochrome P450 2B1 using glutathione S-transferase-2B1 fusion constructs

We have previously produced 12 monoclonal antibodies (MAbs) against rat cytochrome P450(CYP)2B1, and 8 of these inhibit CYP2B1 catalytic activity to varying extents. Using competitive binding studies we showed that this collection of 12 MAbs recognize at least 6 spatially distinct epitopes. The complete coding sequence of CYP2B1 DNA in plasmid pSR-P450 was inserted into a glutathione S-transferase (GST) expression vector pGEX-1lambdaT so that it was in frame with the GST gene. Expression of GST-CYP2B1 was detected with most of the MAbs in Western blots except those which were conformation-specific. Fourteen different constructs were then made using PCR with oligonucleotide primers having EcoRI sites at their ends and were introduced into the GST expression vector at the EcoRI site. Each fusion construct was expressed in Escherichia coli, subjected to SDS-PAGE, blotted, and probed individually with each MAb. MAbs, which inhibited catalytic activity and were mutually competitive in binding to CYP2B1 (viz. BEA33, BE44, BE45, and BE28), recognized several fusion constructs and by deduction recognized amino acids 250-261 in CYP2B1. Other antibodies inhibiting catalytic activity recognized amino acids 262-272 (BEF29) and 306-491 (BE46, B50, and BE49) on CYP2B1. Non-inhibitory MAbs BE26 and BE32 were mapped to region 380-398 in CYP2B1. It was interesting to note that MAbs BEA33 and BE26, which also recognize spatially distinct epitopes on human CYP2E1 but not rat CYP2E1, had corresponding regions of high homology in human CYP2E1 but not rat CYP2E1. Identifying the epitopes recognized by this collection of MAbs will add to our understanding the sequences that may be important for producing inhibitory and specific antibodies to closely related antigens.     

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.     

Immunochemical properties of West Nile virus protein prM and protein M C-end

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.     

猪瘟病毒糖蛋白E^rns中和表位的鉴定和比较

猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣ｂｉｎｇｅｎ毒株Ｅｒｎｓ糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位～ 35 6位或 348位～ 35 0位、384位～ 386及 32 2位～ 32 3位、380位～ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异