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1.
Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization. 总被引:3,自引:0,他引:3
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J C Siz J Cair M Medina D Zuidema C Abrams G J Belsham E Domingo J M Vlak 《Journal of virology》1994,68(7):4557-4564
A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens. 相似文献
2.
Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies. 总被引:1,自引:0,他引:1
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L Fiore B Ridolfi D Genovese G Buttinelli S Lucioli A Lahm F M Ruggeri 《Journal of virology》1997,71(9):6905-6912
Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level. 相似文献
3.
Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies 总被引:13,自引:0,他引:13
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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. 相似文献
4.
Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins. 总被引:17,自引:12,他引:5
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R B Roden E M Weissinger D W Henderson F Booy R Kirnbauer J F Mushinski D R Lowy J T Schiller 《Journal of virology》1994,68(11):7570-7574
We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface. 相似文献
5.
Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation 总被引:6,自引:0,他引:6
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The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody. 相似文献
6.
Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells. 相似文献
7.
Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans. 总被引:45,自引:35,他引:10
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We have used an indirect-capture enzyme-linked immunosorbent assay to quantitate the reactivity of sera from human immunodeficiency virus type 1 (HIV-1)-infected humans with native recombinant gp120 (HIV-1 IIIB or SF-2) or with the gp120 molecule (IIIB or SF-2) denatured by being boiled in the presence of dithiothreitol with or without sodium dodecyl sulfate. Denaturation of IIIB gp120 reduced the titers of sera from randomly selected donors by at least 100-fold, suggesting that the majority of cross-reactive anti-gp120 antibodies present are directed against discontinuous or otherwise conformationally sensitive epitopes. When SF-2 gp120 was used, four of eight serum samples reacted significantly with the denatured protein, albeit with ca. 3- to 50-fold reductions in titer. Only those sera reacting with denatured SF-2 gp120 bound significantly to solid-phase-adsorbed SF-2 V3 loop peptide, and none bound to IIIB V3 loop peptide. Almost all antibody binding to reduced SF-2 gp120 was blocked by preincubation with the SF-2 V3 loop peptide, as was about 50% of the binding to native SF-2 gp120. When sera from a laboratory worker or a chimpanzee infected with IIIB were tested, the pattern of reactivity was reversed, i.e., there was significant binding to reduced IIIB gp120, but not to reduced SF-2 gp120. Binding of these sera to reduced IIIB gp120 was 1 to 10% that to native IIIB gp120 and was substantially decreased by preincubation with IIIB (but not SF-2) V3 loop peptide. To analyze which discontinuous or conformational epitopes were predominant in HIV-1-positive sera, we prebound monoclonal antibodies (MAbs) to IIIB gp120 and then added alkaline phosphatase-labelled HIV-1-positive sera. MAbs (such as 15e) that recognize discontinuous epitopes and compete directly with CD4 reduced HIV-1-positive sera binding by about 50%, whereas neutralizing MAbs to the C4, V2, and V3 domains of gp120 were either not inhibitory or only weakly so. Thus, antibodies to the discontinuous CD4-binding site on gp120 are prevalent in HIV-1-positive sera, antibodies to linear epitopes are less common, most of the antibodies to linear epitopes are directed against the V3 region, and most cross-reactive antibodies are directed against discontinuous epitopes, including regions involved in CD4 binding. 相似文献
8.
A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1. 总被引:12,自引:1,他引:11
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C Wychowski S van der Werf O Siffert R Crainic P Bruneau M Girard 《The EMBO journal》1983,2(11):2019-2024
Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1. 相似文献
9.
Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins 总被引:8,自引:6,他引:8
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Andreas Gigler Simone Dorsch Andrea Hemauer Constance Williams Sonnie Kim Neal S. Young Susan Zolla-Pazner Hans Wolf Miroslaw K. Gorny Susanne Modrow 《Journal of virology》1999,73(3):1974-1979
Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development. 相似文献
10.
Maxime J. J. Fleury Antoine Touzé Marie‐Christine Maurel Thierry Moreau Pierre Coursaget 《Protein science : a publication of the Protein Society》2009,18(7):1425-1438
The aim of this study was to characterize the conformational neutralizing epitopes of the major capsid protein of human papillomavirus type 31. Analysis of the epitopes was performed by competitive epitope mapping using 15 anti‐HPV31 and by reactivity analysis using a HPV31 mutant with an insertion of a seven‐amino acid motif within the FG loop of the capsid protein. Fine mapping of neutralizing conformational epitopes on HPV L1 was analyzed by a new approach using a system displaying a combinatorial library of constrained peptides exposed on E. coli flagella. The findings demonstrate that the HPV31 FG loop is dense in neutralizing epitopes and suggest that HPV31 MAbs bind to overlapping but distinct epitopes on the central part of the FG loop, in agreement with the exposure of the FG loop on the surface of HPV VLPs, and thus confirming that neutralizing antibodies are mainly located on the tip of capsomeres. In addition, we identified a crossreacting and partially crossneutralizing conformational epitope on the relatively well conserved N‐terminal part of the FG loop. Moreover, our findings support the hypothesis that there is no correlation between neutralization and the ability of MAbs to inhibit VLP binding to heparan sulfate, and confirm that the blocking of virus attachment to the extracellular matrix is an important mechanism of neutralization. 相似文献
11.
N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with changes in antibody recognition of the V1/V2 region of gp120. 总被引:3,自引:1,他引:2
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The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although NB-DNJ appears to inhibit HIV entry at the level of post-CD4 binding (P.B. Fischer, M. Collin, G.B. Karlsson, W. James, T.D. Butters, S.J. Davis, S. Gordon, R.A. Dwek, and F.M. Platt, J. Virol. 69:5791-5797, 1995), the exact mechanism of action remains to be established. In this study we have examined the effect of NB-DNJ on the structure of recombinant gp120 (rgpl20), expressed in CHO cells, by using a panel of 40 monoclonal antibodies. The levels of binding of antibodies to rgp120 produced in the presence [rgpl20(+)] and absence [rgpl20(-)] of NB-DNJ were compared by enzyme-linked immunosorbent assay and surface plasmon resonance (BIAcore; Pharmacia). The results showed an increase in the binding to rgp120(+) of antibodies directed against the C1 and C2 regions and a decrease in the binding of antibodies directed against the V1/V2 loops compared with antibody binding to rgpl20(-). A decrease in the binding to rgpl20(+) of antibodies directed against discontinuous epitopes was also observed. No differences were seen in the binding of antibodies directed against the crown of the V3 loop and the C4 region of gp120. Treatment of rgpl20 with alpha-glucosidases I and II had no effect on the differential binding observed, whereas treatment with sialidase abolished the differences seen in the binding of antibodies directed against the C1 and C2 regions of gp120. In addition to these findings, rgpl20(+) showed increased sensitivity to proteases released by CHO cells during expression, as well as to exogenous thrombin. Taken together, the data presented in this paper suggest that production of gp120 in the presence of NB-DNJ affects the conformation of the Vl/V2 loops of gpl20, as well as the overall charge of the C1 and C2 regions. These effects may play a role in the previously described NB-DNJ-mediated inhibition of HIV entry at the level of post-CD4 binding. 相似文献
12.
Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action. 总被引:16,自引:16,他引:0
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Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope. 相似文献
13.
Group A rotaviruses are classified into serotypes, based on the reactivity pattern of neutralizing antibodies to VP4 and VP7, as well as into subgroups (SGs), based on non-neutralizing antibodies directed against VP6. The inner capsid protein (VP2) has also been described as a SG antigen; however, little is known regarding the molecular determinants of VP2 SG specificity. In this study, we characterize VP2 SGs by correlating genetic markers with the immunoreactivity of the SG-specific monoclonal antibody (YO-60). Our results show that VP2 proteins similar in sequence to that of the prototypic human strain Wa are recognized by YO-60, classifying them as VP2 SG-II. In contrast, proteins not bound by YO-60 are similar to those of human strains DS-1 or AU-1 and represent VP2 SG-I. Using a mutagenesis approach, we identified residues that determine recognition by either YO-60 or the group A-specific VP2 monoclonal antibody (6E8). We found that YO-60 binds to a conformationally dependent epitope that includes Wa VP2 residue M328. The epitope for 6E8 is also contingent upon VP2 conformation and resides within a single region of the protein (Wa VP2 residues A440 to T530). Using a high-resolution structure of bovine rotavirus double-layered particles, we predicted these epitopes to be spatially distinct from each other and located on opposite surfaces of VP2. This study reveals the extent of genetic variation among group A rotavirus VP2 proteins and illuminates the molecular basis for a previously described SG specificity associated with the rotavirus inner capsid protein. 相似文献
14.
Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2 总被引:13,自引:0,他引:13
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Lekcharoensuk P Morozov I Paul PS Thangthumniyom N Wajjawalku W Meng XJ 《Journal of virology》2004,78(15):8135-8145
Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein. 相似文献
15.
Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease. 总被引:1,自引:0,他引:1
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Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection. 相似文献
16.
For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies. 相似文献
17.
Warrington KH Gorbatyuk OS Harrison JK Opie SR Zolotukhin S Muzyczka N 《Journal of virology》2004,78(12):6595-6609
Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands. 相似文献
18.
Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation 总被引:4,自引:0,他引:4
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Bloom ME Best SM Hayes SF Wells RD Wolfinbarger JB McKenna R Agbandje-McKenna M 《Journal of virology》2001,75(22):11116-11127
Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines. 相似文献
19.
Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus 总被引:1,自引:0,他引:1
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Letzel T Coulibaly F Rey FA Delmas B Jagt E van Loon AA Mundt E 《Journal of virology》2007,81(23):12827-12835
Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the United States exhibit antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse-genetics approach to introduce selected amino acid changes-individually or in combination-into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 amino acids selected by sequence comparison and their locations on loops P(BC) and P(HI) at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivities of the mutants generated were assessed using a panel of five monoclonal antibodies (MAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five MAbs analyzed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display identical reactivity patterns but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the P(BC) loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains. 相似文献
20.
Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly. 相似文献