Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes
Identifieur interne : 000181 ( Istex/Corpus ); précédent : 000180; suivant : 000182Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes
Auteurs : D. M Theisen ; F. B Bouche ; K. C El Kasmi ; I. Von Der Ahe ; W. Ammerlaan ; S. Demotz ; C. P MullerSource :
- Journal of Immunological Methods [ 0022-1759 ] ; 2000.
English descriptors
- Teeft :
- Amino acids, Anking sequences, Antigenic, Apc, Bces, Brons, Cassette, Cell epitope, Cell epitopes, Cell line, Cell proliferation assay, Chimeric, Class peptides, Conformation, Different permutational, Epitope, Fournier, General rules, Helical, Helical conformation, Helix, Helix conformation, Hemagglutinin, Hemagglutinin protein, Immune, Immunogenicity, Immunol, Immunological, Immunological methods, Jung, Kasmi, Mabs, Mammalian cells, Maternal antibodies, Measles, Measles virus, Measles virus hemagglutinin protein, Molecular environment, Molecular weight, More antigenic, Multiple copies, Negative control, Neutralizing, Neutralizing antibodies, Peptide, Permutational, Plasmid, Polyepitope, Polyepitopes, Protective antibodies, Recombinant, Rotzschke, Schneider, Semliki forest virus replicon, Sequential, Sequential bces, Sequential epitopes, Stimulation index, Synthetic peptides, Tandem, Tandem tces, Tce, Theisen, Transfected cells, Transmembrane signal sequence, Vaccine, Virol, Western blot, Wiesmuller.
Abstract
Abstract: To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L4T4)2 and (L2T2)4] and the helix conformation in one [(H2T2)4] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L2T2)4, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L4T4)2, (L2T2)4, (H2T2)4] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.
Url:
DOI: 10.1016/S0022-1759(00)00197-6
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C El Kasmi</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Von Der Ahe, I" sort="Von Der Ahe, I" uniqKey="Von Der Ahe I" first="I" last="Von Der Ahe">I. Von Der Ahe</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Ammerlaan, W" sort="Ammerlaan, W" uniqKey="Ammerlaan W" first="W" last="Ammerlaan">W. Ammerlaan</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Demotz, S" sort="Demotz, S" uniqKey="Demotz S" first="S" last="Demotz">S. Demotz</name><affiliation><mods:affiliation>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</mods:affiliation></affiliation></author><author><name sortKey="Muller, C P" sort="Muller, C P" uniqKey="Muller C" first="C. P" last="Muller">C. P Muller</name><affiliation><mods:affiliation>E-mail: claude.muller@santel.lu</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:15E2F4832F500C24C588C078CCDE7CA309CBA52D</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0022-1759(00)00197-6</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000181</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000181</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title><author><name sortKey="Theisen, D M" sort="Theisen, D M" uniqKey="Theisen D" first="D. M" last="Theisen">D. M Theisen</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Bouche, F B" sort="Bouche, F B" uniqKey="Bouche F" first="F. B" last="Bouche">F. B Bouche</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="El Kasmi, K C" sort="El Kasmi, K C" uniqKey="El Kasmi K" first="K. C" last="El Kasmi">K. C El Kasmi</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Von Der Ahe, I" sort="Von Der Ahe, I" uniqKey="Von Der Ahe I" first="I" last="Von Der Ahe">I. Von Der Ahe</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Ammerlaan, W" sort="Ammerlaan, W" uniqKey="Ammerlaan W" first="W" last="Ammerlaan">W. Ammerlaan</name><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author><author><name sortKey="Demotz, S" sort="Demotz, S" uniqKey="Demotz S" first="S" last="Demotz">S. Demotz</name><affiliation><mods:affiliation>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</mods:affiliation></affiliation></author><author><name sortKey="Muller, C P" sort="Muller, C P" uniqKey="Muller C" first="C. P" last="Muller">C. P Muller</name><affiliation><mods:affiliation>E-mail: claude.muller@santel.lu</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Immunological Methods</title><title level="j" type="abbrev">JIM</title><idno type="ISSN">0022-1759</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">242</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="145">145</biblScope><biblScope unit="page" to="157">157</biblScope></imprint><idno type="ISSN">0022-1759</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-1759</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Amino acids</term><term>Anking sequences</term><term>Antigenic</term><term>Apc</term><term>Bces</term><term>Brons</term><term>Cassette</term><term>Cell epitope</term><term>Cell epitopes</term><term>Cell line</term><term>Cell proliferation assay</term><term>Chimeric</term><term>Class peptides</term><term>Conformation</term><term>Different permutational</term><term>Epitope</term><term>Fournier</term><term>General rules</term><term>Helical</term><term>Helical conformation</term><term>Helix</term><term>Helix conformation</term><term>Hemagglutinin</term><term>Hemagglutinin protein</term><term>Immune</term><term>Immunogenicity</term><term>Immunol</term><term>Immunological</term><term>Immunological methods</term><term>Jung</term><term>Kasmi</term><term>Mabs</term><term>Mammalian cells</term><term>Maternal antibodies</term><term>Measles</term><term>Measles virus</term><term>Measles virus hemagglutinin protein</term><term>Molecular environment</term><term>Molecular weight</term><term>More antigenic</term><term>Multiple copies</term><term>Negative control</term><term>Neutralizing</term><term>Neutralizing antibodies</term><term>Peptide</term><term>Permutational</term><term>Plasmid</term><term>Polyepitope</term><term>Polyepitopes</term><term>Protective antibodies</term><term>Recombinant</term><term>Rotzschke</term><term>Schneider</term><term>Semliki forest virus replicon</term><term>Sequential</term><term>Sequential bces</term><term>Sequential epitopes</term><term>Stimulation index</term><term>Synthetic peptides</term><term>Tandem</term><term>Tandem tces</term><term>Tce</term><term>Theisen</term><term>Transfected cells</term><term>Transmembrane signal sequence</term><term>Vaccine</term><term>Virol</term><term>Western blot</term><term>Wiesmuller</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L4T4)2 and (L2T2)4] and the helix conformation in one [(H2T2)4] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L2T2)4, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L4T4)2, (L2T2)4, (H2T2)4] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>epitope</json:string><json:string>bces</json:string><json:string>tce</json:string><json:string>peptide</json:string><json:string>theisen</json:string><json:string>kasmi</json:string><json:string>polyepitopes</json:string><json:string>recombinant</json:string><json:string>hemagglutinin</json:string><json:string>immunol</json:string><json:string>immunological</json:string><json:string>sequential</json:string><json:string>immunogenicity</json:string><json:string>immunological methods</json:string><json:string>vaccine</json:string><json:string>mabs</json:string><json:string>cassette</json:string><json:string>fournier</json:string><json:string>virol</json:string><json:string>jung</json:string><json:string>antigenic</json:string><json:string>helix</json:string><json:string>cell line</json:string><json:string>rotzschke</json:string><json:string>apc</json:string><json:string>schneider</json:string><json:string>permutational</json:string><json:string>neutralizing</json:string><json:string>plasmid</json:string><json:string>chimeric</json:string><json:string>wiesmuller</json:string><json:string>immune</json:string><json:string>brons</json:string><json:string>cell epitopes</json:string><json:string>polyepitope</json:string><json:string>helical</json:string><json:string>measles virus</json:string><json:string>tandem</json:string><json:string>cell epitope</json:string><json:string>hemagglutinin protein</json:string><json:string>synthetic peptides</json:string><json:string>molecular environment</json:string><json:string>transmembrane signal sequence</json:string><json:string>amino acids</json:string><json:string>conformation</json:string><json:string>cell proliferation assay</json:string><json:string>western blot</json:string><json:string>anking sequences</json:string><json:string>class peptides</json:string><json:string>more antigenic</json:string><json:string>measles virus hemagglutinin protein</json:string><json:string>measles</json:string><json:string>maternal antibodies</json:string><json:string>molecular weight</json:string><json:string>general rules</json:string><json:string>semliki forest virus replicon</json:string><json:string>sequential epitopes</json:string><json:string>mammalian cells</json:string><json:string>sequential bces</json:string><json:string>stimulation index</json:string><json:string>transfected cells</json:string><json:string>neutralizing antibodies</json:string><json:string>tandem tces</json:string><json:string>negative control</json:string><json:string>protective antibodies</json:string><json:string>helical conformation</json:string><json:string>different permutational</json:string><json:string>helix conformation</json:string><json:string>multiple copies</json:string></teeft></keywords><author><json:item><name>D.M Theisen</name><affiliations><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item><json:item><name>F.B Bouche</name><affiliations><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item><json:item><name>K.C El Kasmi</name><affiliations><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item><json:item><name>I von der Ahe</name><affiliations><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item><json:item><name>W Ammerlaan</name><affiliations><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item><json:item><name>S Demotz</name><affiliations><json:string>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</json:string></affiliations></json:item><json:item><name>C.P Muller</name><affiliations><json:string>E-mail: claude.muller@santel.lu</json:string><json:string>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Recombinant polyepitopes</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Measles virus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Sequential B cell epitopes</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>tt830–844, Hemagglutinin protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>APC, antigen presenting cell</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BCE, B cell epitope</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>H, helical epitope of MVH (aa236–255)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>L, loop epitope of MVH (aa386–400)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>mab, monoclonal antibody</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MV, measles virus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MVH, measles virus hemagglutinin protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MW, molecular weight</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TCE, T cell epitope</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>tt830–844, tetanus toxoid T cell epitope aa830–844</value></json:item></subject><arkIstex>ark:/67375/6H6-QQHDZG8L-Z</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L4T4)2 and (L2T2)4] and the helix conformation in one [(H2T2)4] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L2T2)4, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L4T4)2, (L2T2)4, (H2T2)4] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.</abstract><qualityIndicators><score>10</score><pdfWordCount>6278</pdfWordCount><pdfCharCount>38154</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>546 x 744 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>293</abstractWordCount><abstractCharCount>2042</abstractCharCount><keywordCount>14</keywordCount></qualityIndicators><title>Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title><pmid><json:string>10986397</json:string></pmid><pii><json:string>S0022-1759(00)00197-6</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Immunological Methods</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0022-1759</json:string></issn><pii><json:string>S0022-1759(00)X0097-X</json:string></pii><volume>242</volume><issue>1–2</issue><pages><first>145</first><last>157</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string></date><geogName><json:string>Semliki</json:string></geogName><orgName><json:string>Luxembourg, Luxembourg</json:string><json:string>Switzerland Received</json:string><json:string>University of Tubingen</json:string><json:string>Department of Immunology, Laboratoire National</json:string><json:string>Difco Laboratories, Detroit</json:string><json:string>University of Lausanne</json:string><json:string>Multisyntech, Bochum, Germany</json:string><json:string>EU Biotechnology Programme</json:string><json:string>Scripps Research Institute, La Jolla, CA, USA</json:string><json:string>WHO Collaborating Center for Measles</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>S. Demotz</json:string><json:string>F. Fack</json:string><json:string>P.O. Box</json:string><json:string>B. Chain</json:string><json:string>ELISA Peptides</json:string><json:string>C.P. Muller</json:string><json:string>W. Ammerlaan</json:string><json:string>S. Kreis</json:string><json:string>A. Lanzavecchia</json:string><json:string>F.B. Bouche</json:string><json:string>N. Kieffer</json:string><json:string>D.M. Theisen</json:string><json:string>Rotzschke</json:string><json:string>K.C. El Kasmi</json:string><json:string>Joseph Trotter</json:string></persName><placeName><json:string>Bradford</json:string><json:string>Miami</json:string><json:string>Switzerland</json:string><json:string>Germany</json:string><json:string>UK</json:string><json:string>Austria</json:string><json:string>Immunogenicity</json:string><json:string>London</json:string><json:string>Luxembourg</json:string><json:string>USA</json:string><json:string>Basel</json:string><json:string>FL</json:string><json:string>Vinci</json:string><json:string>Belgium</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Hobman et al., 1997</json:string><json:string>Bouche et al., 1998</json:string><json:string>Ockenhouse et al., 1998</json:string><json:string>An and Whitton, 1997</json:string><json:string>Deroo et al., 1998</json:string><json:string>Leclerc et al., 1994</json:string><json:string>Ziegler et al., 1996</json:string><json:string>Rotzschke et al. (1997)</json:string><json:string>Falk et al., 1994</json:string><json:string>Kjerrulf et al., 1997</json:string><json:string>Zamorano et al., 1995</json:string><json:string>Niedermann et al., 1995</json:string><json:string>Shastri et al., 1995</json:string><json:string>Muller et al., 1995</json:string><json:string>Van Regenmortel, 1999</json:string><json:string>Montero et al., 1997</json:string><json:string>De Smet et al., 1994</json:string><json:string>Thomson et al., 1995</json:string><json:string>Charbit et al., 1997</json:string><json:string>El Kasmi et al., 1999, 2000</json:string><json:string>¨ Rotzschke and Falk, 1994</json:string><json:string>El Kasmi et al., 1999</json:string><json:string>Kropshofer et al., 1992</json:string><json:string>Ahlborg et al., 1994</json:string><json:string>Liljestrom and Garoff, 1991</json:string><json:string>Chicz et al., 1992</json:string><json:string>Fournier et al., 1997</json:string><json:string>Demotz et al., 1993</json:string><json:string>El Kasmi et al., 1998, 1999</json:string><json:string>Hanke et al., 1998</json:string><json:string>Muller et al., 1993</json:string><json:string>Whitton et al., 1993</json:string><json:string>El Kasmi et al., 1998</json:string><json:string>Wiesmuller et al., 1992</json:string><json:string>Fournier et al., 1996</json:string><json:string>Liljestrom and Garoff (1991)</json:string><json:string>Sambrook et al., 1989</json:string><json:string>Langedijk et al., 1997</json:string><json:string>Rotzschke et al., 1997</json:string><json:string>Chatterjee et al., 1995</json:string><json:string>Martineau et al., 1992</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-QQHDZG8L-Z</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - immunology</json:string><json:string>2 - biochemical research methods</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - clinical medicine</json:string><json:string>3 - immunology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Immunology</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Immunology and Allergy</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0022-1759(00)00197-6</json:string></doi><id>15E2F4832F500C24C588C078CCDE7CA309CBA52D</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2000 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2000</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Recombinant Technology</note><note type="content">Fig. 1: BHK-(L2T2)4 (A) or mock-transfected BHK-21 cells (B) were stained after ethanol fixation with BH195 (1:500) and a FITC-labelled second step antibody. Micrographs were taken with a DMRB fluorescence microscope (Leica, Germany) using a Fujicolor 36 CH 135, 400 ASA super G plus film (magnification 63×).</note><note type="content">Fig. 2: Western blot of two 16-mer constructs, (L4T4)2 (predicted MW 40 kDa) and (H2T2L2T2)2 (MW 42.7 kDa), and the corresponding 8-mers, L4T4 (MW 24.7 kDa) and H2T2L2T2 (MW 26 kDa). Mock-transfected BHK-21 and MV-hemagglutinin protein served as controls. Antibody BH195 and anti-TTB serum were used at a concentration of 1:1000.</note><note type="content">Fig. 3: Intracellular expression of polyepitopes in transfected BHK-21 cells. (A) BHK-(L4T4)2, -(L2T2)4 transfectants permeabilized with ‘Fix&Perm’ (An der Grub, Kaumberg, A) and stained with mabs (1:500). Shaded curves correspond to the transfectants stained with an irrelevant mab (BH47, 1:1000). Mock-transfectant (BHK-0) served as a negative control. (B) BHK-(H2T2)4 transfectants permeabilized with 90% ethanol and stained with mabs (1:500), anti-TTB peptide serum (1:1000), or anti-TB peptide serum (1:1000). Shaded curves correspond to an irrelevant mab (BH6, 1:500) or naive mouse serum (1:500). Irrelevant mabs were titrated on BHK-MVH cells (Bouche et al., 1998) and saturating concentrations were used. BHK-MVH cells also served as a positive antibody control (not shown).</note><note type="content">Fig. 4: Stimulation of the human T cell line TCL-tt830 by antigen-pulsed APCs. APCs, pulsed with L8 or H4L4, served as negative controls. Data are shown as the stimulation index (S.I.) based on the mean (100 c.p.m.) of triplicates of BHK-0 pulsed APCs.</note><note type="content">Fig. 5: Reactivity of pooled sera induced by L-containing constructs with the biotinylated reporter peptide MVH386-400. Anti-BHK-0 serum served as a negative control. The insert shows the cross-reactivity of anti-(H2T2L2T2)2 serum (1:100) with MV-superinfected EBV-transformed human B cell line (WMPT). Naive or irrelevant sera on MV-positive cells served as negative controls (shaded curve).</note><note type="content">Table 1: Intracellular detection of polyepitope constructs</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title><author xml:id="author-0000"><persName><forename type="first">D.M</forename><surname>Theisen</surname></persName><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><author xml:id="author-0001"><persName><forename type="first">F.B</forename><surname>Bouche</surname></persName><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><author xml:id="author-0002"><persName><forename type="first">K.C</forename><surname>El Kasmi</surname></persName><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><author xml:id="author-0003"><persName><forename type="first">I</forename><surname>von der Ahe</surname></persName><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><author xml:id="author-0004"><persName><forename type="first">W</forename><surname>Ammerlaan</surname></persName><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><author xml:id="author-0005"><persName><forename type="first">S</forename><surname>Demotz</surname></persName><affiliation>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</affiliation></author><author xml:id="author-0006"><persName><forename type="first">C.P</forename><surname>Muller</surname></persName><email>claude.muller@santel.lu</email><note type="correspondence"><p>Corresponding author. Department of Immunology, Laboratoire National de Santé, P.O. Box 1102, L-1011 Luxembourg, Luxembourg. Tel.: +352-490-604; fax: +352-490-686</p></note><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation></author><idno type="istex">15E2F4832F500C24C588C078CCDE7CA309CBA52D</idno><idno type="ark">ark:/67375/6H6-QQHDZG8L-Z</idno><idno type="DOI">10.1016/S0022-1759(00)00197-6</idno><idno type="PII">S0022-1759(00)00197-6</idno></analytic><monogr><title level="j">Journal of Immunological Methods</title><title level="j" type="abbrev">JIM</title><idno type="pISSN">0022-1759</idno><idno type="PII">S0022-1759(00)X0097-X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">242</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="145">145</biblScope><biblScope unit="page" to="157">157</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L4T4)2 and (L2T2)4] and the helix conformation in one [(H2T2)4] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L2T2)4, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L4T4)2, (L2T2)4, (H2T2)4] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Recombinant polyepitopes</term></item><item><term>Measles virus</term></item><item><term>Sequential B cell epitopes</term></item><item><term>tt830–844, Hemagglutinin protein</term></item></list></keywords></textClass><textClass><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>APC, antigen presenting cell</term></item><item><term>BCE, B cell epitope</term></item><item><term>H, helical epitope of MVH (aa236–255)</term></item><item><term>L, loop epitope of MVH (aa386–400)</term></item><item><term>mab, monoclonal antibody</term></item><item><term>MV, measles virus</term></item><item><term>MVH, measles virus hemagglutinin protein</term></item><item><term>MW, molecular weight</term></item><item><term>TCE, T cell epitope</term></item><item><term>tt830–844, tetanus toxoid T cell epitope aa830–844</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-02-13">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>JIM</jid><aid>8612</aid><ce:pii>S0022-1759(00)00197-6</ce:pii><ce:doi>10.1016/S0022-1759(00)00197-6</ce:doi><ce:copyright type="full-transfer" year="2000">Elsevier Science B.V.</ce:copyright></item-info><head><ce:dochead><ce:textfn>Recombinant Technology</ce:textfn></ce:dochead><ce:title>Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</ce:title><ce:author-group><ce:author><ce:given-name>D.M</ce:given-name><ce:surname>Theisen</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>F.B</ce:given-name><ce:surname>Bouche</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF3"><ce:sup>c</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>K.C</ce:given-name><ce:surname>El Kasmi</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF4"><ce:sup>d</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>I</ce:given-name><ce:surname>von der Ahe</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF4"><ce:sup>d</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>W</ce:given-name><ce:surname>Ammerlaan</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>S</ce:given-name><ce:surname>Demotz</ce:surname><ce:cross-ref refid="AFF5"><ce:sup>e</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>C.P</ce:given-name><ce:surname>Muller</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF4"><ce:sup>d</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>claude.muller@santel.lu</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Fakultät für Biologie, University of Tübingen, Tübingen, Germany</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>Service de Génétique Appliquée, Université Libre de Bruxelles, Nivelles, Belgium</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>d</ce:label><ce:textfn>Medizinische Fakultät, University of Tübingen, Tübingen, Germany</ce:textfn></ce:affiliation><ce:affiliation id="AFF5"><ce:label>e</ce:label><ce:textfn>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Department of Immunology, Laboratoire National de Santé, P.O. Box 1102, L-1011 Luxembourg, Luxembourg. Tel.: +352-490-604; fax: +352-490-686</ce:text></ce:correspondence></ce:author-group><ce:date-received day="8" month="9" year="1999"></ce:date-received><ce:date-revised day="13" month="2" year="2000"></ce:date-revised><ce:date-accepted day="7" month="4" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L<ce:inf>4</ce:inf>T<ce:inf>4</ce:inf>)<ce:inf>2</ce:inf> and (L<ce:inf>2</ce:inf>T<ce:inf>2</ce:inf>)<ce:inf>4</ce:inf>] and the helix conformation in one [(H<ce:inf>2</ce:inf>T<ce:inf>2</ce:inf>)<ce:inf>4</ce:inf>] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L<ce:inf>2</ce:inf>T<ce:inf>2</ce:inf>)<ce:inf>4</ce:inf>, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L<ce:inf>4</ce:inf>T<ce:inf>4</ce:inf>)<ce:inf>2</ce:inf>, (L<ce:inf>2</ce:inf>T<ce:inf>2</ce:inf>)<ce:inf>4</ce:inf>, (H<ce:inf>2</ce:inf>T<ce:inf>2</ce:inf>)<ce:inf>4</ce:inf>] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Recombinant polyepitopes</ce:text></ce:keyword><ce:keyword><ce:text>Measles virus</ce:text></ce:keyword><ce:keyword><ce:text>Sequential B cell epitopes</ce:text></ce:keyword><ce:keyword><ce:text>tt830–844, Hemagglutinin protein</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>APC, antigen presenting cell</ce:text></ce:keyword><ce:keyword><ce:text>BCE, B cell epitope</ce:text></ce:keyword><ce:keyword><ce:text>H, helical epitope of MVH (aa236–255)</ce:text></ce:keyword><ce:keyword><ce:text>L, loop epitope of MVH (aa386–400)</ce:text></ce:keyword><ce:keyword><ce:text>mab, monoclonal antibody</ce:text></ce:keyword><ce:keyword><ce:text>MV, measles virus</ce:text></ce:keyword><ce:keyword><ce:text>MVH, measles virus hemagglutinin protein</ce:text></ce:keyword><ce:keyword><ce:text>MW, molecular weight</ce:text></ce:keyword><ce:keyword><ce:text>TCE, T cell epitope</ce:text></ce:keyword><ce:keyword><ce:text>tt830–844, tetanus toxoid T cell epitope aa830–844</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</title></titleInfo><name type="personal"><namePart type="given">D.M</namePart><namePart type="family">Theisen</namePart><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">F.B</namePart><namePart type="family">Bouche</namePart><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">K.C</namePart><namePart type="family">El Kasmi</namePart><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">I</namePart><namePart type="family">von der Ahe</namePart><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">W</namePart><namePart type="family">Ammerlaan</namePart><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">S</namePart><namePart type="family">Demotz</namePart><affiliation>Institut de Biochemie, University of Lausanne, Epalinges, Switzerland</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C.P</namePart><namePart type="family">Muller</namePart><affiliation>E-mail: claude.muller@santel.lu</affiliation><affiliation>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102, L-1011 Luxembourg, Luxembourg</affiliation><description>Corresponding author. Department of Immunology, Laboratoire National de Santé, P.O. Box 1102, L-1011 Luxembourg, Luxembourg. Tel.: +352-490-604; fax: +352-490-686</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">2000-02-13</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5–45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830–844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L4T4)2 and (L2T2)4] and the helix conformation in one [(H2T2)4] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L2T2)4, were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L4T4)2, (L2T2)4, (H2T2)4] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.</abstract><note type="content">Section title: Recombinant Technology</note><note type="content">Fig. 1: BHK-(L2T2)4 (A) or mock-transfected BHK-21 cells (B) were stained after ethanol fixation with BH195 (1:500) and a FITC-labelled second step antibody. Micrographs were taken with a DMRB fluorescence microscope (Leica, Germany) using a Fujicolor 36 CH 135, 400 ASA super G plus film (magnification 63×).</note><note type="content">Fig. 2: Western blot of two 16-mer constructs, (L4T4)2 (predicted MW 40 kDa) and (H2T2L2T2)2 (MW 42.7 kDa), and the corresponding 8-mers, L4T4 (MW 24.7 kDa) and H2T2L2T2 (MW 26 kDa). Mock-transfected BHK-21 and MV-hemagglutinin protein served as controls. Antibody BH195 and anti-TTB serum were used at a concentration of 1:1000.</note><note type="content">Fig. 3: Intracellular expression of polyepitopes in transfected BHK-21 cells. (A) BHK-(L4T4)2, -(L2T2)4 transfectants permeabilized with ‘Fix&Perm’ (An der Grub, Kaumberg, A) and stained with mabs (1:500). Shaded curves correspond to the transfectants stained with an irrelevant mab (BH47, 1:1000). Mock-transfectant (BHK-0) served as a negative control. (B) BHK-(H2T2)4 transfectants permeabilized with 90% ethanol and stained with mabs (1:500), anti-TTB peptide serum (1:1000), or anti-TB peptide serum (1:1000). Shaded curves correspond to an irrelevant mab (BH6, 1:500) or naive mouse serum (1:500). Irrelevant mabs were titrated on BHK-MVH cells (Bouche et al., 1998) and saturating concentrations were used. BHK-MVH cells also served as a positive antibody control (not shown).</note><note type="content">Fig. 4: Stimulation of the human T cell line TCL-tt830 by antigen-pulsed APCs. APCs, pulsed with L8 or H4L4, served as negative controls. Data are shown as the stimulation index (S.I.) based on the mean (100 c.p.m.) of triplicates of BHK-0 pulsed APCs.</note><note type="content">Fig. 5: Reactivity of pooled sera induced by L-containing constructs with the biotinylated reporter peptide MVH386-400. Anti-BHK-0 serum served as a negative control. The insert shows the cross-reactivity of anti-(H2T2L2T2)2 serum (1:100) with MV-superinfected EBV-transformed human B cell line (WMPT). Naive or irrelevant sera on MV-positive cells served as negative controls (shaded curve).</note><note type="content">Table 1: Intracellular detection of polyepitope constructs</note><subject><genre>Keywords</genre><topic>Recombinant polyepitopes</topic><topic>Measles virus</topic><topic>Sequential B cell epitopes</topic><topic>tt830–844, Hemagglutinin protein</topic></subject><subject><genre>Abbreviations</genre><topic>APC, antigen presenting cell</topic><topic>BCE, B cell epitope</topic><topic>H, helical epitope of MVH (aa236–255)</topic><topic>L, loop epitope of MVH (aa386–400)</topic><topic>mab, monoclonal antibody</topic><topic>MV, measles virus</topic><topic>MVH, measles virus hemagglutinin protein</topic><topic>MW, molecular weight</topic><topic>TCE, T cell epitope</topic><topic>tt830–844, tetanus toxoid T cell epitope aa830–844</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Immunological Methods</title></titleInfo><titleInfo type="abbreviated"><title>JIM</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0022-1759</identifier><identifier type="PII">S0022-1759(00)X0097-X</identifier><part><date>2000</date><detail type="volume"><number>242</number><caption>vol.</caption></detail><detail type="issue"><number>1–2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>210</end></extent><extent unit="pages"><start>145</start><end>157</end></extent></part></relatedItem><identifier type="istex">15E2F4832F500C24C588C078CCDE7CA309CBA52D</identifier><identifier type="ark">ark:/67375/6H6-QQHDZG8L-Z</identifier><identifier type="DOI">10.1016/S0022-1759(00)00197-6</identifier><identifier type="PII">S0022-1759(00)00197-6</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©2000</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-QQHDZG8L-Z/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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