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.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1. Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.     

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.     

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.     

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。     

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.