Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus

A recombinant baculovirus expressing membrane bound form of hemagglutinin–neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263–368 and 538–609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.     

Characterization of immunodominant linear B-cell epitopes on the carboxy terminus of the rinderpest virus nucleocapsid protein

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N(1-525)), an amino-terminal construct (N(1-179)), and a carboxy-terminal construct (N(414-496)), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N(414-496) was much more antigenic than GST-N(1-179) when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues (440)VPQVRKETRASSR(452) (site 1), (479)PEADTDPL(486) (site 2), and (520)DKDLL(524) (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.     

Mapping of T-helper epitopes of Rinderpest virus hemagglutinin protein

Rinderpest virus (RPV) is a highly contagious and often fatal disease of domestic and wild ruminants, caused by rinderpest virus of the genus Morbillivirus under the family Paramyxoviridae. Hemagglutinin (H) and fusion (F) proteins of this enveloped virus confer protective immunity against experimental challenge with virulent rinderpest virus. We have earlier demonstrated that immunization with a single dose of recombinant extracellular baculovirus expressing H protein elicits H-specific humoral and lymphoproliferative responses in cattle. The lymphoproliferative responses are predominantly BoLA class II restricted. In this work, we have analyzed lymphoproliferative responses of peripheral lymphocytes from immunized cattle to truncated H protein fragments expressed in E. coli for locating domains harboring Th epitopes. One region (aa 113-182) recognized by immune T cells is conserved in the H protein of measles virus, which was earlier shown to contain a dominant Th epitope in mouse. Synthetic peptides within this region of measles virus H protein were used to identify a Th epitope conserved in the H protein of RPV virus (aa 123-137) in cattle. A second Th epitope located at the C-terminus of RPV-H was mapped to the region corresponding to aa 512-609 using truncated protein fragments expressed in E. coli. The C-terminal epitope (aa 575-583) was mapped using synthetic peptides corresponding to measles virus H as well as RPV-H protein.     

Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N_1-525), an amino-terminal construct (N_1-179), and a carboxy-terminal construct (N_414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N_414-496 was much more antigenic than GST-N_1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues ⁴⁴⁰VPQVRKETRASSR⁴⁵² (site 1), ⁴⁷⁹PEADTDPL⁴⁸⁶ (site 2), and ⁵²⁰DKDLL⁵²⁴ (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.     

Development of a novel rapid immunochromatographic test specific for the H5 influenza virus

Three anti-H5 influenza virus monoclonal antibody (mAb) clones, IFH5-26, IFH5-115 and IFH5-136, were obtained by immunising a BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs were found to recognise specifically the haemagglutinin (HA) epitope of the influenza H5 subtypes by western blotting with recombinant HAs; however, these mAbs have no neutralising activity for A/duck/Hokkaido/84/02 (H5N3) or A/Puerto Ric/8/34 (H1N1). Each epitope of these mAbs was a conformational epitope that was formed from the regions located between 46 to 60 amino acids (aa) and 312 to 322 aa for IFH5-115, from 101 to 113 aa and 268 to 273 aa for IFH5-136 and from 61 to 80 aa and 290 to 300 aa for IFH5-26. The epitopes were located in the loop regions between the receptor region and alpha-helix structure in haemagglutinin 1 (HA1). Influenza A virus H5-specific rapid immunochromatographic test kits were tested as solid phase antibody/alkaline phosphate-conjugated mAb in the following three combinations: IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115. In every combination, only influenza A H5 subtypes were detected. For effective clinical application, rapid dual discrimination immunochromatographic test kits in combination with H5 HA-specific mAb, IFA5-26 and IFA5-115 and the influenza A NP NP-specific mAb, FVA2-11, were developed. The dual discrimination immunochromatographic tests kits detected influenza A virus H5 subtypes as H5 line-positive and all influenza A subtypes as A line-positive simultaneously. The dual discrimination immunochromatographic test kits may be useful for discriminating highly pathogenic avian influenza A H5N1 viruses from seasonal influenza A virus, as well as for confirming influenza infection status in human, avian and mammalian hosts.     

Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus

Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.     

Development and application of monoclonal antibodies against avian influenza virus nucleoprotein

Rapid and accurate diagnosis of avian influenza (AI) infection is important for an understanding epidemiology. In order to develop rapid tests for AI antigen and antibody detection, two monoclonal antibodies (mAbs) against influenza nucleoprotein (NP) were produced. These mAbs are designated as F26-9 and F28-73 and able to recognize whole AI virus particles as well as the recombinant NP. Both of the mAbs were tested in a slot blot for their reactivity against 15 subtypes of influenza virus; F28-73 reacted with all tested 15 subtypes, while F26-9 failed to react with H13N6 and H15N8. The mAb binding epitopes were identified using truncated NP recombinant proteins and peptide array techniques. The mAb F26-9 reacted with NP-full, NP-1 (638bp), NP-2 (315bp), NP-4 (488bp), and NP-5 (400bp) in the Western blot. The peptide array results demonstrated that the mAb F26-9 reacted with NP peptides 15-17 corresponding to amino acids 71-96. The mAb F28-73 recognized the NP-full, -1 and -4 fragments, but failed bind to NP-2, -3, -5, and any peptides. This antibody-binding site is expected to be contained within 1-162 amino acids of AI NP, although the exact binding epitope could not be determined. The two mAbs showed reactivity with AI antigen in immunofluorescence, immunohistochemistry and immune plaque assays. Immune response of AI infected animals was determined using the mAb F28-73 in a cELISA. All tested chickens were positive at 11 days post-infection and remained positive until the end of the experiment on day 28 (>50% inhibition). The two mAbs with different specificities are appropriate for developing various tests for diagnosis of AI infection.     

The latex allergen hev B 5 is an antigen with repetitive murine B-cell epitopes

BACKGROUND: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. METHODS: The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. RESULTS: All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63-68), PAEGEK (120-125), and PAEEEK (126-131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K131 as critical aa in each epitope to change by site-directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that epitope 126-131 was the dominant epitope, but mutation of epitope 120-125 was also required to eliminate mAb reactivity to Hev b 5. The antibodies did not appear to recognize the epitope 63-68 in the recombinant fusion protein. CONCLUSIONS: We identified an immunodominant B-cell epitope in Hev b 5 that is repeated 3 times within the sequence, making Hev b 5 multivalent. Well-characterized monoclonals recognizing repeated epitopes would be a good choice for immunodetection of Hev b 5 in glove extracts where individual epitopes could get altered by the manufacturing process.     

以乙肝病毒核心蛋白为载体制备肠道病毒71 型非结构蛋白3D 单克隆抗体

目的：预测肠道病毒71型（EV71）非结构蛋白3D的表位,以HBc蛋白为载体展示多肽,制备并鉴定抗EV71-3D的特异性单克隆抗体（mAb）。方法：应用生物信息学方法分析预测出EV71-3D蛋白亲水性和免疫原性指数较高的多肽片段,并运用HBc颗粒型蛋白载体展示肽段,构建多肽融合蛋白,免疫BALB/c雌鼠,通过杂交瘤技术和亲和层析技术制备和纯化抗EV71-3D蛋白的特异性mAb,用间接ELISA、ELISPOT、IFA和IHC对mAb的性质进行初步鉴定。结果：构建表达分别嵌合3D蛋白34~43位氨基酸残基、 61~76位氨基酸残基、151~164位氨基酸残基的HBc重组蛋白,免疫并经过多轮克隆化筛选,获得抗EV71-3D单克隆抗体3E1,其亚类为IgG2a;免疫荧光试验、ELISPOT法和免疫组织化学染色结果显示其可与EV71特异性结合。结论：成功制备可特异性识别EV71的单克隆抗体3E1,为病毒的检测及进一步研究3D蛋白的功能奠定了基础,同时还验证了生物信息学技术与HBc颗粒型载体展示多肽技术相结合可快速高效地制备单克隆抗体。     

Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302

Abstract: To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA-DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B-lymphoblastoid cell lines (B-LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302-3, 0401-2, 0501-3, 0601-4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45–47 of the DQ β chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQP1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B–LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1 *0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39–46 of DR-and DQ- and 37–44 of DP p chains.     

Epitopic characterization of native bovine beta-lactoglobulin

Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.     

应用噬菌体展示技术筛选、鉴定抗汉滩病毒单克隆抗体的模拟表位

目的 :应用噬菌体 12肽文库筛选抗汉滩病毒单抗 (mAb)F3、B11的模拟配体肽 ,并对其免疫学特性进行初步分析。方法 :以纯化的mAb为筛选配基 ,进行噬菌体肽库的生物亲和淘选。用ELISA法鉴定筛选克隆的结合活性 ,对阳性克隆进行序列测定和分析。用动物免疫试验初步分析噬菌体颗粒展示的抗原肽的免疫特性。结果 :通过 3～ 4轮生物淘选 ,ELISA显示筛选到的多数噬菌体克隆均可与mAb特异性结合 ;与mAbF3结合的阳性克隆的氨基酸序列高度一致 ,均为 MHGP TKNQMWHT ,与HTNV/SEOVM蛋白G2区第 75 0～ 75 9位氨基酸具有较高的同源性 ;而mAbB11特异性的结合肽在氨基酸水平上表现出一定的多态性 ,其序列的基序尚未能确定 ;动物免疫试验表明 ,噬菌体肽抗原具有良好的免疫反应性和免疫原性 ,是天然病毒抗原较好的免疫原模拟物。结论 :获得了具有良好结合活性的模拟表位肽 ,为基于表位的HFRSV多肽疫苗及DNA疫苗的研制奠定了基础。     

Evaluation of neutralizing efficacy of monoclonal antibodies specific for 2009 pandemic H1N1 influenza A virus in vitro and in vivo

Pandemic influenza A virus (H1N1) 2009 poses a serious public-health challenge worldwide. To characterize the neutralizing epitopes of this virus, we generated a panel of eight monoclonal antibodies (mAbs) against the HA of the A/California/07/2009 virus. The antibodies were specific for the 2009 pdm H1N1 HA, as the antibodies displayed HA-specific ELISA, hemagglutination inhibition (HAI) and neutralization activity. One mAb (mAb12) showed significantly higher HAI and neutralizing titers than the other mAbs. We mapped the antigenic epitopes of the HA by characterizing escape mutants of a 2009 H1N1 vaccine strain (NYMC X-179A). The amino acid changes suggested that these eight mAbs recognized HA antigenic epitopes located in the Sa, Sb, Ca1 and Ca2 sites. Passive immunization with mAbs showed that mAb12 displayed more efficient neutralizing activity in vivo than the other mAbs. mAb12 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge in mice. In addition, a single injection of 10 mg/kg mAb12 outperformed a 5-day course of treatment with oseltamivir (10 mg/kg/day by gavage) with respect to both prophylaxis and treatment of lethal viral infection. Taken together, our results showed that mouse-origin mAbs displayed neutralizing effectiveness in vitro and in vivo. One mAb in particular (mAb12) recognized an epitope within the Sb site and demonstrated outstanding neutralizing effectiveness.     

Mapping of B cell epitopes in measles virus nucleocapsid protein

Summary. The B-cell response against measles nucleoprotein (MeN) plays an important role in the control of measles infection. However, the data on B cell epitopes of MeN are still limited. The objective of this study was to identify B cell epitopes in MeN using monoclonal and polyclonal antibodies raised against recombinant yeast-expressed MeN (rMeN) as well as human sera from measles-positive individuals. After immunization of mice, 15 monoclonal antibodies (mAbs) against rMeN were generated. The B cell epitopes were localized using recombinant overlapping MeN fragments, PepScan analysis, and competitive ELISA. The epitopes of 14 mAbs were mapped within the C-terminus of MeN between amino acids (aa) 419 and 525. Four mAbs recognized a linear epitope located within a sequence of aa 440–448. Competitive ELISA revealed a cluster of conformational mAb epitopes. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals. Thus, the majority of B cell epitopes are located at the C-terminal domain of MeN. These findings provide new data on the antigenic structure of MeN and are in agreement with recent experimental evidence indicating that the C-terminal domain of MeN is well accessible on the surface of nucleocapsid-like structures.     

A virus binding assay for studying the antigenic landscape on intact, native, primary human immunodeficiency virus-type 1

This protocol describes a simple assay that can be used to study the nature of exposure of antigenic epitopes and antigenic relatedness of different intact, native HIV-1 strains. The assay is based on the principle that mAbs coated on microtiter wells bind to epitopes on the surface of intact, native virions. The bound virion is then lysed to release p24, which is then quantitated (pg/ml) to give a measure of the amount of virion bound to the mAb. High p24 levels released after lysis correlate with high level capture of virions by mAbs, and as such, reflect good exposure of the epitope on the virion. Likewise, binding patterns of a specific mAb with different virus strains reveal information on their antigenic relatedness. In establishing this assay, the nature of exposure of antigenic epitopes and the antigenic relatedness of six intact, native HIV-1 virions of clades A, B, C, D, F and G were examined using anti-HIV-1 mAbs directed at epitopes in the V2, V3, CD4bd and C5 of gp120, and in clusters I and II of the gp41 region. Analysis of the binding data shows that mAbs directed at epitopes in the V3, C5 and gp41 Cluster I region bound best to the viruses examined, suggesting that these are the regions most exposed and conserved on intact, native HIV-1 virions of different clades. Epitopes in the V2 and CD4bd of gp120, and in gp41 cluster II, are not exposed on intact, native virions.     

Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus

Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/H?lln?s, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/H?lln?s and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.     

Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.     

Antigenic and immunogenic investigation of B-cell epitopes in the nucleocapsid protein of peste des petits ruminants virus

Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.     

The Leu477 and Leu613 of ORF2-Encoded Protein Are Critical in Forming Neutralization Antigenic Epitope of Hepatitis E Virus Genotype 4

Hepatitis E virus （HEV） genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody （mAb） IG10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids （aa） of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and/or C-terminal truncated and 6 site-directed mutated p166Chn proteins were prepared. Only those N- or C-terminal truncated proteins containing the region 477-613 aa could react with the mAb 1G10, suggesting the neutralization epitope of HEV genotype 4 is located between aa477 and aa613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both terminal truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neutralization epitope to the immune system. Substituting Leu477 and/or Leu613 with the polar, uncharged threonine （Thr） caused 〉 50% reduction of the mutants＇ immunoreactivity to IG10, whereas replacement by hydrophobic phenylalanine （Phe） made little impact on the immunoreactivity, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggested Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4. Cellular ＆ Molecular Immunology. 2008;5（6）：447-456.     

Characterization of monoclonal antibodies against the nonstructural 5A protein of hepatitis C virus

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a multifunctional protein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here, we produced monoclonal antibodies (mAbs) directed against the HCV NS5A protein. The N-terminal epitope was mapped to amino acids 60–80 of the NS5A protein, and the epitope in the middle region was mapped to amino acids 221–236. Because these epitopes overlap with binding regions of human vesicle-associated membrane-protein-associated protein (hVAP)-B and TNF-receptor-associated factor 2 (TRAF2), respectively, we investigated these mAbs for their potential capacity to inhibit viral and cellular interactions. We found that NS5A and hVAP-B interaction was abolished by mAb E5D3, and NS5A and TRAF2 interaction was inhibited by mAb C6D4. Since hVAP-B is necessary for HCV replication and TRAF2 is the major transducer in TNF signaling cascades, these data may provide further insights into the mechanisms underlying HCV replication and viral modulation of host signal transduction.