| Doctorant : |
Typhaine VIOLO
|
| Directeur de thèse : |
Cyrille GRANDJEAN ,
Directeur de recherche CNRS |
| co-directeur de thèse : | Jacques LEBRETON |
| Encadrant : |
Emilie CAMBERLEIN ,
Maître de conférences Université |
| Financement : |
Région Pays de la Loire |
| Date de la soutenance : |
mardi 03 mars 2020, 14h00 |
| Modalité : |
|
| Jury : |
- Président de jury : Gilles Truan, Directeur de Recherche CNRS, INSA de Toulouse
- Rapporteur : Gilles Truan, Directeur de Recherche CNRS, INSA de Toulouse
- Rapporteur : Laurence Mulard, Directrice de Recherche CNRS, Institut Pasteur
- Examinateur : Barbara Mouratou, Maître de conférences HDR, Université de Nantes
- Examinateur : Patrice Soumillon, Professeur, Université Catholique de Louvain, Belgique
- Directeur de thèse :
Cyrille GRANDJEAN ,
Directeur de recherche CNRS
- co-directeur de thèse : Jacques LEBRETON
- Encadrant :
Emilie CAMBERLEIN ,
Maître de conférences Université
- Invité : Elisabeth Ficko-Blean, Chargée de Recherche CNRS, Station Biologique de Roscoff
|
Au cours de cette thèse, nous avons mis en place une technologie d’incorporation d’acides aminés non naturels de manière site-spécifique au sein du laboratoire et l’avons appliqué à deux problématiques. Les vaccins glycoconjugués actuels ne couvrent que 13 des 97 sérotypes de Streptococcus pneumoniae. L’utilisation d’une protéine porteuse immunogène permet d’élargir la couverture sérotypique en couplant l’utilisation d’antigènes polysaccharidiques avec des antigènes protéiques. Cependant, les méthodes de conjugaison actuelles ne permettent pas l’étude de l’association optimale entre ces deux antigènes. Dans une première partie, nous avons généré différents vaccins glycoconjugués homogènes en incorporant la Propargyl-Lysine dans la protéine porteuse du pneumoncoque (mPsaA) afin de maîtriser la position ainsi que le nombre des haptens. Les lectines reconnaissent leur ligand saccharidique avec une très bonne spécificité mais avec une affinité généralement faible. Dans une seconde partie de la thèse, nous nous sommes intéressés à l’incorporation d’acides aminés non naturels permettant de former des liaisons covalentes avec les sucres et ainsi augmenter les forces d’interaction entre des lectines et leurs ligands. Nous avons tenté d’incorporer un premier acide aminé non naturel original sans succès. Mais deux autres ont pu être incorporés, dont l’un d’eux au sein de 2 cibles protéiques, en quantité suffisante pour réaliser des tests d’affinité. Cette thèse a donc permis la mise en place d’un nouvel axe technologique au sein du laboratoire et d’en explorer des applications potentielles.
Publications
2020
Violo, Typhaine; Dussouy, Christophe; Tellier, Charles; Grandjean, Cyrille; Camberlein, Emilie
Homogenous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes Article de journal
Dans: Journal of visualized experiments : JoVE, 2020.
@article{violo:hal-02990572,
title = {Homogenous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes},
author = {Typhaine Violo and Christophe Dussouy and Charles Tellier and Cyrille Grandjean and Emilie Camberlein},
url = {https://hal.archives-ouvertes.fr/hal-02990572},
doi = {10.3791/60821},
year = {2020},
date = {2020-01-01},
journal = {Journal of visualized experiments : JoVE},
publisher = {JoVE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2019
Pillot, Aline; Defontaine, Alain; Fateh, Amina; Lambert, Annie; Prasanna, Maruthi; Fanuel, Mathieu; Pipelier, Muriel; Csaba, Noemi; Violo, Typhaine; Camberlein, Emilie; Grandjean, Cyrille
Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation Article de journal
Dans: Frontiers in Chemistry, vol. 7, no. November, p. 1–9, 2019, ISSN: 22962646.
@article{Pillot2019,
title = {Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation},
author = {Aline Pillot and Alain Defontaine and Amina Fateh and Annie Lambert and Maruthi Prasanna and Mathieu Fanuel and Muriel Pipelier and Noemi Csaba and Typhaine Violo and Emilie Camberlein and Cyrille Grandjean},
doi = {10.3389/fchem.2019.00726},
issn = {22962646},
year = {2019},
date = {2019-01-01},
journal = {Frontiers in Chemistry},
volume = {7},
number = {November},
pages = {1--9},
abstract = {Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.
Lien
