Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes.
Identifieur interne : 003667 ( Main/Merge ); précédent : 003666; suivant : 003668Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes.
Auteurs : D M Theisen [Luxembourg (pays)] ; 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
- KwdFr :
- Animaux, Antigènes viraux (), Antigènes viraux (biosynthèse), Antigènes viraux (génétique), Antigènes viraux (immunologie), Conformation des protéines, Cricetinae, Données de séquences moléculaires, Déterminants antigéniques des lymphocytes B (), Déterminants antigéniques des lymphocytes B (biosynthèse), Déterminants antigéniques des lymphocytes B (génétique), Déterminants antigéniques des lymphocytes B (immunologie), Déterminants antigéniques des lymphocytes T (), Déterminants antigéniques des lymphocytes T (biosynthèse), Déterminants antigéniques des lymphocytes T (génétique), Déterminants antigéniques des lymphocytes T (immunologie), Expression des gènes, Hémagglutinines virales (), Hémagglutinines virales (biosynthèse), Hémagglutinines virales (génétique), Hémagglutinines virales (immunologie), Lignée cellulaire, Peptides (), Peptides (génétique), Peptides (immunologie), Protéines de fusion recombinantes (), Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (immunologie), Séquence d'acides aminés.
- MESH :
- biosynthèse : Antigènes viraux, Déterminants antigéniques des lymphocytes B, Déterminants antigéniques des lymphocytes T, Hémagglutinines virales.
- génétique : Antigènes viraux, Déterminants antigéniques des lymphocytes B, Déterminants antigéniques des lymphocytes T, Hémagglutinines virales, Peptides, Protéines de fusion recombinantes.
- immunologie : Antigènes viraux, Déterminants antigéniques des lymphocytes B, Déterminants antigéniques des lymphocytes T, Hémagglutinines virales, Peptides, Protéines de fusion recombinantes.
- Animaux, Antigènes viraux, Conformation des protéines, Cricetinae, Données de séquences moléculaires, Déterminants antigéniques des lymphocytes B, Déterminants antigéniques des lymphocytes T, Expression des gènes, Hémagglutinines virales, Lignée cellulaire, Peptides, Protéines de fusion recombinantes, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Antigens, Viral (biosynthesis), Antigens, Viral (chemistry), Antigens, Viral (genetics), Antigens, Viral (immunology), Cell Line, Cricetinae, Epitopes, B-Lymphocyte (biosynthesis), Epitopes, B-Lymphocyte (chemistry), Epitopes, B-Lymphocyte (genetics), Epitopes, B-Lymphocyte (immunology), Epitopes, T-Lymphocyte (biosynthesis), Epitopes, T-Lymphocyte (chemistry), Epitopes, T-Lymphocyte (genetics), Epitopes, T-Lymphocyte (immunology), Gene Expression, Hemagglutinins, Viral (biosynthesis), Hemagglutinins, Viral (chemistry), Hemagglutinins, Viral (genetics), Hemagglutinins, Viral (immunology), Molecular Sequence Data, Peptides (chemistry), Peptides (genetics), Peptides (immunology), Protein Conformation, Recombinant Fusion Proteins (chemistry), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (immunology).
- MESH :
- chemical , biosynthesis : Antigens, Viral, Epitopes, B-Lymphocyte, Epitopes, T-Lymphocyte, Hemagglutinins, Viral.
- chemical , chemistry : Antigens, Viral, Epitopes, B-Lymphocyte, Epitopes, T-Lymphocyte, Hemagglutinins, Viral, Peptides, Recombinant Fusion Proteins.
- chemical , genetics : Antigens, Viral, Epitopes, B-Lymphocyte, Epitopes, T-Lymphocyte, Hemagglutinins, Viral, Peptides, Recombinant Fusion Proteins.
- chemical , immunology : Antigens, Viral, Epitopes, B-Lymphocyte, Epitopes, T-Lymphocyte, Hemagglutinins, Viral, Peptides, Recombinant Fusion Proteins.
- Amino Acid Sequence, Animals, Cell Line, Cricetinae, Gene Expression, Molecular Sequence Data, Protein Conformation.
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 [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(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 (L(2)T(2))(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 [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(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.
DOI: 10.1016/s0022-1759(00)00197-6
PubMed: 10986397
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<author><name sortKey="Theisen, D M" sort="Theisen, D M" uniqKey="Theisen D" first="D M" last="Theisen">D M Theisen</name>
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<author><name sortKey="Bouche, F B" sort="Bouche, F B" uniqKey="Bouche F" first="F B" last="Bouche">F B Bouche</name>
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<series><title level="j">Journal of immunological methods</title>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Antigens, Viral (biosynthesis)</term>
<term>Antigens, Viral (chemistry)</term>
<term>Antigens, Viral (genetics)</term>
<term>Antigens, Viral (immunology)</term>
<term>Cell Line</term>
<term>Cricetinae</term>
<term>Epitopes, B-Lymphocyte (biosynthesis)</term>
<term>Epitopes, B-Lymphocyte (chemistry)</term>
<term>Epitopes, B-Lymphocyte (genetics)</term>
<term>Epitopes, B-Lymphocyte (immunology)</term>
<term>Epitopes, T-Lymphocyte (biosynthesis)</term>
<term>Epitopes, T-Lymphocyte (chemistry)</term>
<term>Epitopes, T-Lymphocyte (genetics)</term>
<term>Epitopes, T-Lymphocyte (immunology)</term>
<term>Gene Expression</term>
<term>Hemagglutinins, Viral (biosynthesis)</term>
<term>Hemagglutinins, Viral (chemistry)</term>
<term>Hemagglutinins, Viral (genetics)</term>
<term>Hemagglutinins, Viral (immunology)</term>
<term>Molecular Sequence Data</term>
<term>Peptides (chemistry)</term>
<term>Peptides (genetics)</term>
<term>Peptides (immunology)</term>
<term>Protein Conformation</term>
<term>Recombinant Fusion Proteins (chemistry)</term>
<term>Recombinant Fusion Proteins (genetics)</term>
<term>Recombinant Fusion Proteins (immunology)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term>
<term>Antigènes viraux ()</term>
<term>Antigènes viraux (biosynthèse)</term>
<term>Antigènes viraux (génétique)</term>
<term>Antigènes viraux (immunologie)</term>
<term>Conformation des protéines</term>
<term>Cricetinae</term>
<term>Données de séquences moléculaires</term>
<term>Déterminants antigéniques des lymphocytes B ()</term>
<term>Déterminants antigéniques des lymphocytes B (biosynthèse)</term>
<term>Déterminants antigéniques des lymphocytes B (génétique)</term>
<term>Déterminants antigéniques des lymphocytes B (immunologie)</term>
<term>Déterminants antigéniques des lymphocytes T ()</term>
<term>Déterminants antigéniques des lymphocytes T (biosynthèse)</term>
<term>Déterminants antigéniques des lymphocytes T (génétique)</term>
<term>Déterminants antigéniques des lymphocytes T (immunologie)</term>
<term>Expression des gènes</term>
<term>Hémagglutinines virales ()</term>
<term>Hémagglutinines virales (biosynthèse)</term>
<term>Hémagglutinines virales (génétique)</term>
<term>Hémagglutinines virales (immunologie)</term>
<term>Lignée cellulaire</term>
<term>Peptides ()</term>
<term>Peptides (génétique)</term>
<term>Peptides (immunologie)</term>
<term>Protéines de fusion recombinantes ()</term>
<term>Protéines de fusion recombinantes (génétique)</term>
<term>Protéines de fusion recombinantes (immunologie)</term>
<term>Séquence d'acides aminés</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
<term>Epitopes, T-Lymphocyte</term>
<term>Hemagglutinins, Viral</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
<term>Epitopes, T-Lymphocyte</term>
<term>Hemagglutinins, Viral</term>
<term>Peptides</term>
<term>Recombinant Fusion Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
<term>Epitopes, T-Lymphocyte</term>
<term>Hemagglutinins, Viral</term>
<term>Peptides</term>
<term>Recombinant Fusion Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
<term>Epitopes, T-Lymphocyte</term>
<term>Hemagglutinins, Viral</term>
<term>Peptides</term>
<term>Recombinant Fusion Proteins</term>
</keywords>
<keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Antigènes viraux</term>
<term>Déterminants antigéniques des lymphocytes B</term>
<term>Déterminants antigéniques des lymphocytes T</term>
<term>Hémagglutinines virales</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Antigènes viraux</term>
<term>Déterminants antigéniques des lymphocytes B</term>
<term>Déterminants antigéniques des lymphocytes T</term>
<term>Hémagglutinines virales</term>
<term>Peptides</term>
<term>Protéines de fusion recombinantes</term>
</keywords>
<keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Antigènes viraux</term>
<term>Déterminants antigéniques des lymphocytes B</term>
<term>Déterminants antigéniques des lymphocytes T</term>
<term>Hémagglutinines virales</term>
<term>Peptides</term>
<term>Protéines de fusion recombinantes</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Cell Line</term>
<term>Cricetinae</term>
<term>Gene Expression</term>
<term>Molecular Sequence Data</term>
<term>Protein Conformation</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Animaux</term>
<term>Antigènes viraux</term>
<term>Conformation des protéines</term>
<term>Cricetinae</term>
<term>Données de séquences moléculaires</term>
<term>Déterminants antigéniques des lymphocytes B</term>
<term>Déterminants antigéniques des lymphocytes T</term>
<term>Expression des gènes</term>
<term>Hémagglutinines virales</term>
<term>Lignée cellulaire</term>
<term>Peptides</term>
<term>Protéines de fusion recombinantes</term>
<term>Séquence d'acides aminés</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(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(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 (L(2)T(2))(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 [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(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>
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