Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes
Identifieur interne : 000091 ( PascalFrancis/Corpus ); précédent : 000090; suivant : 000092Differential 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.
Descripteurs français
- Pascal (Inist)
English descriptors
- KwdEn :
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.
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Format Inist (serveur)
NO : | PASCAL 01-0262336 INIST |
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ET : | Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes |
AU : | THEISEN (D. M.); BOUCHE (F. B.); EL KASMI (K. C.); VON DER AHE (I.); AMMERLAAN (W.); DEMOTZ (S.); MULLER (C. P.) |
AF : | Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102/1011 Luxembourg/Luxembourg (1 aut., 2 aut., 3 aut., 4 aut., 5 aut., 7 aut.); Fakultät für Biologie, University of Tübingen/Tübingen/Allemagne (1 aut.); Service de Génétique Appliquée, Université Libre de Bruxelles/Nivelles/Belgique (2 aut.); Medizinische Fakultät, University of Tübingen/Tübingen/Allemagne (3 aut., 4 aut., 7 aut.); Institut de Biochemie, University of Lausanne/Epalinges/Suisse (6 aut.) |
DT : | Publication en série; Niveau analytique |
SO : | Journal of immunological methods; ISSN 0022-1759; Coden JIMMBG; Pays-Bas; Da. 2000; Vol. 242; No. 1-2; Pp. 145-157; Bibl. 1 p.1/2 |
LA : | Anglais |
EA : | 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. |
CC : | 002A05C10 |
FD : | Lymphocyte T; Lymphocyte B; Déterminant antigénique; Virus rougeole; Antigène; Chimère; Protéine; Hémagglutinine; Structure hélice; Antigénicité |
FG : | Morbillivirus; Paramyxovirinae; Paramyxoviridae; Mononegavirales; Virus; Glycoprotéine |
ED : | T-Lymphocyte; B-Lymphocyte; Antigenic determinant; Measles virus; Antigen; Chimera; Protein; Hemagglutinin; Helical structure; Antigenicity |
EG : | Morbillivirus; Paramyxovirinae; Paramyxoviridae; Mononegavirales; Virus; Glycoprotein |
SD : | Linfocito T; Linfocito B; Determinante antigénico; Measles virus; Antígeno; Quimera; Proteína; Hemoaglutinina; Estructura helicoidal; Antigenicidad |
LO : | INIST-15654.354000090666720140 |
ID : | 01-0262336 |
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Pascal:01-0262336Le document en format XML
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<series><title level="j" type="main">Journal of immunological methods</title>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antigen</term>
<term>Antigenic determinant</term>
<term>Antigenicity</term>
<term>B-Lymphocyte</term>
<term>Chimera</term>
<term>Helical structure</term>
<term>Hemagglutinin</term>
<term>Measles virus</term>
<term>Protein</term>
<term>T-Lymphocyte</term>
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<term>Lymphocyte B</term>
<term>Déterminant antigénique</term>
<term>Virus rougeole</term>
<term>Antigène</term>
<term>Chimère</term>
<term>Protéine</term>
<term>Hémagglutinine</term>
<term>Structure hélice</term>
<term>Antigénicité</term>
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<front><div type="abstract" xml:lang="en">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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
and (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] and the helix conformation in one [(H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
, (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
, (H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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>
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<fA11 i1="05" i2="1"><s1>AMMERLAAN (W.)</s1>
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<fA14 i1="05"><s1>Institut de Biochemie, University of Lausanne</s1>
<s2>Epalinges</s2>
<s3>CHE</s3>
<sZ>6 aut.</sZ>
</fA14>
<fA20><s1>145-157</s1>
</fA20>
<fA21><s1>2000</s1>
</fA21>
<fA23 i1="01"><s0>ENG</s0>
</fA23>
<fA43 i1="01"><s1>INIST</s1>
<s2>15654</s2>
<s5>354000090666720140</s5>
</fA43>
<fA44><s0>0000</s0>
<s1>© 2001 INIST-CNRS. All rights reserved.</s1>
</fA44>
<fA45><s0>1 p.1/2</s0>
</fA45>
<fA47 i1="01" i2="1"><s0>01-0262336</s0>
</fA47>
<fA60><s1>P</s1>
</fA60>
<fA61><s0>A</s0>
</fA61>
<fA64 i1="01" i2="1"><s0>Journal of immunological methods</s0>
</fA64>
<fA66 i1="01"><s0>NLD</s0>
</fA66>
<fC01 i1="01" l="ENG"><s0>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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
and (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] and the helix conformation in one [(H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
, (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
, (H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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.</s0>
</fC01>
<fC02 i1="01" i2="X"><s0>002A05C10</s0>
</fC02>
<fC03 i1="01" i2="X" l="FRE"><s0>Lymphocyte T</s0>
<s5>01</s5>
</fC03>
<fC03 i1="01" i2="X" l="ENG"><s0>T-Lymphocyte</s0>
<s5>01</s5>
<s6>«T»-Lymphocyte</s6>
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<fC03 i1="01" i2="X" l="SPA"><s0>Linfocito T</s0>
<s5>01</s5>
</fC03>
<fC03 i1="02" i2="X" l="FRE"><s0>Lymphocyte B</s0>
<s5>02</s5>
</fC03>
<fC03 i1="02" i2="X" l="ENG"><s0>B-Lymphocyte</s0>
<s5>02</s5>
<s6>«B»-Lymphocyte</s6>
</fC03>
<fC03 i1="02" i2="X" l="SPA"><s0>Linfocito B</s0>
<s5>02</s5>
</fC03>
<fC03 i1="03" i2="X" l="FRE"><s0>Déterminant antigénique</s0>
<s5>03</s5>
</fC03>
<fC03 i1="03" i2="X" l="ENG"><s0>Antigenic determinant</s0>
<s5>03</s5>
</fC03>
<fC03 i1="03" i2="X" l="SPA"><s0>Determinante antigénico</s0>
<s5>03</s5>
</fC03>
<fC03 i1="04" i2="X" l="FRE"><s0>Virus rougeole</s0>
<s2>NW</s2>
<s5>04</s5>
</fC03>
<fC03 i1="04" i2="X" l="ENG"><s0>Measles virus</s0>
<s2>NW</s2>
<s5>04</s5>
</fC03>
<fC03 i1="04" i2="X" l="SPA"><s0>Measles virus</s0>
<s2>NW</s2>
<s5>04</s5>
</fC03>
<fC03 i1="05" i2="X" l="FRE"><s0>Antigène</s0>
<s5>05</s5>
</fC03>
<fC03 i1="05" i2="X" l="ENG"><s0>Antigen</s0>
<s5>05</s5>
</fC03>
<fC03 i1="05" i2="X" l="SPA"><s0>Antígeno</s0>
<s5>05</s5>
</fC03>
<fC03 i1="06" i2="X" l="FRE"><s0>Chimère</s0>
<s5>06</s5>
</fC03>
<fC03 i1="06" i2="X" l="ENG"><s0>Chimera</s0>
<s5>06</s5>
</fC03>
<fC03 i1="06" i2="X" l="SPA"><s0>Quimera</s0>
<s5>06</s5>
</fC03>
<fC03 i1="07" i2="X" l="FRE"><s0>Protéine</s0>
<s5>07</s5>
</fC03>
<fC03 i1="07" i2="X" l="ENG"><s0>Protein</s0>
<s5>07</s5>
</fC03>
<fC03 i1="07" i2="X" l="SPA"><s0>Proteína</s0>
<s5>07</s5>
</fC03>
<fC03 i1="08" i2="X" l="FRE"><s0>Hémagglutinine</s0>
<s5>08</s5>
</fC03>
<fC03 i1="08" i2="X" l="ENG"><s0>Hemagglutinin</s0>
<s5>08</s5>
</fC03>
<fC03 i1="08" i2="X" l="SPA"><s0>Hemoaglutinina</s0>
<s5>08</s5>
</fC03>
<fC03 i1="09" i2="X" l="FRE"><s0>Structure hélice</s0>
<s5>09</s5>
</fC03>
<fC03 i1="09" i2="X" l="ENG"><s0>Helical structure</s0>
<s5>09</s5>
</fC03>
<fC03 i1="09" i2="X" l="SPA"><s0>Estructura helicoidal</s0>
<s5>09</s5>
</fC03>
<fC03 i1="10" i2="X" l="FRE"><s0>Antigénicité</s0>
<s5>10</s5>
</fC03>
<fC03 i1="10" i2="X" l="ENG"><s0>Antigenicity</s0>
<s5>10</s5>
</fC03>
<fC03 i1="10" i2="X" l="SPA"><s0>Antigenicidad</s0>
<s5>10</s5>
</fC03>
<fC07 i1="01" i2="X" l="FRE"><s0>Morbillivirus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="ENG"><s0>Morbillivirus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="SPA"><s0>Morbillivirus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="FRE"><s0>Paramyxovirinae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="ENG"><s0>Paramyxovirinae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="SPA"><s0>Paramyxovirinae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="FRE"><s0>Paramyxoviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="ENG"><s0>Paramyxoviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="SPA"><s0>Paramyxoviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="04" i2="X" l="FRE"><s0>Mononegavirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="04" i2="X" l="ENG"><s0>Mononegavirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="04" i2="X" l="SPA"><s0>Mononegavirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="05" i2="X" l="FRE"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="05" i2="X" l="ENG"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="05" i2="X" l="SPA"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="06" i2="X" l="FRE"><s0>Glycoprotéine</s0>
<s5>28</s5>
</fC07>
<fC07 i1="06" i2="X" l="ENG"><s0>Glycoprotein</s0>
<s5>28</s5>
</fC07>
<fC07 i1="06" i2="X" l="SPA"><s0>Glicoproteína</s0>
<s5>28</s5>
</fC07>
<fN21><s1>183</s1>
</fN21>
</pA>
</standard>
<server><NO>PASCAL 01-0262336 INIST</NO>
<ET>Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes</ET>
<AU>THEISEN (D. M.); BOUCHE (F. B.); EL KASMI (K. C.); VON DER AHE (I.); AMMERLAAN (W.); DEMOTZ (S.); MULLER (C. P.)</AU>
<AF>Department of Immunology and WHO Collaborating Center for Measles, Laboratoire National de Santé, B.P. 1102/1011 Luxembourg/Luxembourg (1 aut., 2 aut., 3 aut., 4 aut., 5 aut., 7 aut.); Fakultät für Biologie, University of Tübingen/Tübingen/Allemagne (1 aut.); Service de Génétique Appliquée, Université Libre de Bruxelles/Nivelles/Belgique (2 aut.); Medizinische Fakultät, University of Tübingen/Tübingen/Allemagne (3 aut., 4 aut., 7 aut.); Institut de Biochemie, University of Lausanne/Epalinges/Suisse (6 aut.)</AF>
<DT>Publication en série; Niveau analytique</DT>
<SO>Journal of immunological methods; ISSN 0022-1759; Coden JIMMBG; Pays-Bas; Da. 2000; Vol. 242; No. 1-2; Pp. 145-157; Bibl. 1 p.1/2</SO>
<LA>Anglais</LA>
<EA>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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
and (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] and the helix conformation in one [(H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
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<sub>4</sub>
T<sub>4</sub>
)<sub>2</sub>
, (L<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
, (H<sub>2</sub>
T<sub>2</sub>
)<sub>4</sub>
] 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.</EA>
<CC>002A05C10</CC>
<FD>Lymphocyte T; Lymphocyte B; Déterminant antigénique; Virus rougeole; Antigène; Chimère; Protéine; Hémagglutinine; Structure hélice; Antigénicité</FD>
<FG>Morbillivirus; Paramyxovirinae; Paramyxoviridae; Mononegavirales; Virus; Glycoprotéine</FG>
<ED>T-Lymphocyte; B-Lymphocyte; Antigenic determinant; Measles virus; Antigen; Chimera; Protein; Hemagglutinin; Helical structure; Antigenicity</ED>
<EG>Morbillivirus; Paramyxovirinae; Paramyxoviridae; Mononegavirales; Virus; Glycoprotein</EG>
<SD>Linfocito T; Linfocito B; Determinante antigénico; Measles virus; Antígeno; Quimera; Proteína; Hemoaglutinina; Estructura helicoidal; Antigenicidad</SD>
<LO>INIST-15654.354000090666720140</LO>
<ID>01-0262336</ID>
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