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.     

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.     

Immunochemical properties of the West Nile virus prM protein and the C-terminal fragment of the M protein

The cDNA fragments 466–966 and 878–1088 coding for the precursor M (prM) protein and polypeptide M_31–75-E_1–30 of the Russian strain LEIV-Vlg99-27889-human of the West Nile virus (WNV) were synthesized and cloned. The corresponding prM and M_31–75-E_1–30 recombinant polypeptides were purified by affinity chromatography. The prM polypeptide interacted with a polyclonal serum against WNV in ELISA and immunoblotting, demonstrating the immunochemical similarity of the recombinant polypeptide and the native WNV prM protein. Six species-specific monoclonal antibodies (mAbs) against the prM recombinant polypeptide recognized at least four epitopes on the recombinant polypeptides. In addition, mAb 7D11 displayed a virus-neutralizing activity. The patterns of mAb interactions with the prM, M_31–75-E_1–30, E_1–180, and E_260–466 recombinant polypeptides revealed cross-reacting epitopes in regions 260–466 of the E protein and 31–75 of the M_31–75-E_1–30 polypeptide and the WNV prM protein. A spatial model revealed structural similarity of the C-terminal regions of the E and M proteins of WNV, supporting the results of immunochemical experiments. Based on virus neutralization by mAb 7D11, which recognized an epitope mapping to region 31–75 of the WNV M protein, an important function in virus penetration into the cell was assumed for the C-terminal region of the M protein.     

Characterization of glycoprotein E C-End of West Nile virus and evaluation of its interaction force with αVβ3 integrin as putative cellular receptor

Recombinant polypeptide containing the 260–466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E_260–466) was constructed. Immunochemical similarity between the E_260–466 and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E_260–466. Polypeptide E_260–466 induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E_260–466 with one of the proposed cell receptors of WNV that average E_260–466-αVβ3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and αVβ3 integrin) during virus entry.     

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.     

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.     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.     

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     

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.     

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.     

Crystal structure of the West Nile virus envelope glycoprotein

The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-A crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel alpha-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.     

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.     

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.     

Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection

The domain III of the West Nile virus (WNV) envelope glycoprotein (E) was shown to serve as virus attachment domain to the cellular receptor, and neutralizing Abs have been mapped to this specific domain. In this study, domain III of the WNV E protein (WNV E DIII) was expressed as a recombinant protein and its potential as a subunit vaccine candidate was evaluated in BALB/C mice. Immunization of WNV E DIII protein with oligodeoxynucleotides (CpG-DNA) adjuvant by i.p. injection was conducted over a period of 3 wk. The immunized mice generated high titer of WNV-neutralizing Abs. Murine Ab against WNV E DIII protein was also capable of neutralizing Japanese encephalitis virus. The IgG isotypes generated were predominantly IgG2a in the murine sera against the recombinant protein. Splenocyte cultures from the mice coadministrated with WNV E DIII protein and CpG secreted large amounts of IFN-gamma and IL-2 and showed proliferation of T cells in the presence of WNV E DIII protein. Overall, this study highlighted that recombinant WNV E DIII protein delivered in combination with CpG adjuvant to mice generated a Th1 immune response type against WNV and can serve as a potential vaccine to prevent WNV infection.     

Cloning and sequencing of the bovine adenovirus type 2 early region 4

Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characterisitics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5–100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14 kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3–6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.     

Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.     

A fusion-loop antibody enhances the infectious properties of immature flavivirus particles

Flavivirus-infected cells secrete a mixture of mature, partially immature, and fully immature particles into the extracellular space. Although mature virions are highly infectious, prM-containing fully immature virions are noninfectious largely because the prM protein inhibits the cell attachment and fusogenic properties of the virus. If, however, cell attachment and entry are facilitated by anti-prM antibodies, immature flavivirus becomes infectious after efficient processing of the prM protein by the endosomal protease furin. A recent study demonstrated that E53, a cross-reactive monoclonal antibody (MAb) that engages the highly conserved fusion-loop peptide within the flavivirus envelope glycoprotein, preferentially binds to immature flavivirus particles. We investigated here the infectious potential of fully immature West Nile virus (WNV) and dengue virus (DENV) particles opsonized with E53 MAb and observed that, like anti-prM antibodies, this anti-E antibody also has the capacity to render fully immature flaviviruses infectious. E53-mediated enhancement of both immature WNV and DENV depended on efficient cell entry and the enzymatic activity of the endosomal furin. Furthermore, we also observed that E53-opsonized immature DENV particles but not WNV particles required a more acidic pH for efficient cleavage of prM by furin, adding greater complexity to the dynamics of antibody-mediated infection of immature flavivirus virions.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

戊型肝炎病毒细胞吸附模型的建立及病毒吸附区域初步研究

E.coli中表达的HEV衣壳蛋白片段P239(aa368~606)形成的类病毒颗粒与戊肝患者恢复期血清及中和单抗具有良好的反应性,较好地模拟了天然HEV病毒颗粒的表面空间结构。利用P239吸附HepG2细胞的模型来模拟HEV对宿主细胞的吸附,多株中和单抗对吸附的阻断验证了吸附的特异性。P239与多株传代细胞系的吸附结果则表明了这种特异性吸附的细胞选择性。对阻断P239吸附的线性单抗进行定位,初步确定了P239与细胞相互作用的区域:ORF2上的aa423~443很可能和病毒上的细胞膜受体结合部位非常靠近,或可能直接参与构成了病毒与细胞特异性识别的表位。此本研究为进一步研究HEV与宿主细胞的相互作用提供一定的线索。     

Immunochemical and single molecule force spectroscopy studies of specific interaction between the laminin binding protein and the West Nile virus surface glycoprotein E domain II

ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.