Development of an immunocontraceptive vaccine: development of immunological tests for the vulpine model and of DNA vaccines coding for the sperm antigens fSP8 and fSP13
Développement d'un vaccin immunocontraceptif : mise au point de tests immunologiques dans le modèle vulpin et développement de vaccins ADN avec les antigènes spermatiques fSP13 et fSP8
Résumé
The initial aim of the work presented in this thesis was to develop the immunological tools necessary to investigate the immune response in the fox (Vulpes vulpes). After assuring the quality and efficiency of density gradient centrifugation for purifying PBMCs, and having selected suitable antibodies recognising the different classes of vulpine immunoglobulins, ovalbumin and cholera toxinB were used as an antigenic model to develop and test the techniques necessary for investigating the vulpine immune response. Immunoglobulin subtype specifie ELISA were developed to measure the antigen specifie humoral response for IgG, IgM, and IgA in both serum samples and vaginal swabs. The ELISPOT antigen secreting cell assay, allowing the observation of IgG secreting precursor cells from both splenocytes and PBMCs in response to an antigenic stimulus is described here. Cytokines, playing a major role in the orientation oft he immune response were also investigated. The ORFs for 4 vulpine cytokines IL2, IL6, IL10, and IFNy have been cloned and their sequence reported here.
Primers have been designed, and PCR reactions optimised to allow the analysis oftheir expression by quantitative RT-PCR after in vitro re-stimulation of vulpine PBMCs. Subsequently, several different DNA vaccines have been constructed using the commercial pVAXl coding for the testes specifie spenn antigens tSP8, and fSP13 identified in our laboratory. Using successive PCR reactions several elements were added to the coding ORF, allowing the unidirectional insertion (different restricton sites at each extremity), a better protein expression (inclusion of Kozak sequences), and the purification and detection of the protein produced (poly-His
tag). After insertion of the different constructs into the pVAX1 vector their functionality was tested in vitro. The expression off SP8, and fSP13, after transfection into MDCK cells was observed.
Le but de ce travail était tout d'abord, la mise au point d'outils immunologiques permettant l'analyse de la réponse immune humorale et cellulaire chez le renard (Vulpes vulpes). Après s'être assuré de la bonne qualité des PBMCs obtenus par gradient de centrifugation, et avoir sélectionné des anticorps reconnaissant les différentes classes d'immunoglobuline de renard, le modèle antigénique Ovalbumine et Choléra toxine B a été utilisé pour mettre au point les techniques ELISA et ELISPOT. Quatre cytokines vulpines Il2, Il6, Il10 et INFy ont été clonées et séquencées, et une technique d'analyse de leur expression par RT-PCR quantitative, après re-stimulation antigénique in vitro de PBMCs vulpins a été mise au point. Par la suite, différents vaccins ADN utilisant le vecteur vaccinaI commercial pVAX1 et les antigènes spermatiques spécifiques du testicule fSP13 et fSP8 identifiés au laboratoire ont été construits. La fonctionnalité de celles-ci a tout d'abord été testée in vitro pour l'expression de fSP 13 et fSP8 après transfection de cellules MDCK.