ED BIO SORBONNE PARIS CITE Proposition de sujet de thèse 2014-‐2015 Renseignements relatifs à l’Unité de Recherche : Label et intitulé : UMR 216 MERIT « Mère et enfant face aux infections tropicales » (IRD/UPD) Nom et prénom du Directeur : Philippe DELORON Téléphone : 01 53 73 96 57 ; Télécopie : 01 53 73 96 17 Courriel : [email protected] Renseignements relatifs à l’Equipe : Nom de l’Equipe d’Accueil : Immunologie Nom et prénom du responsable : Adrian LUTY Qualité du responsable : Directeur de Recherche DR2 IRD Téléphone : +229 64 80 99 96 ; Télécopie : +229 21 30 88 60 Courriel : [email protected] Renseignements relatifs au sujet de thèse : Nom et prénom du Directeur de thèse (HDR) : Florence MIGOT-‐NABIAS Qualité : Directrice de Recherche DR2 IRD Téléphone : 01 70 64 94 34 ; Télécopie : 01 53 73 96 17 Courriel : florence.migot-‐[email protected] Signature : Titre du sujet proposé : "Analyse du répertoire des immunoglobulines pour le diagnostic des infections parasitaires congénitales" Analysis of the immunoglobulin repertoire for the diagnosis of congenital parasitic infections Département :  Biologie Cellulaire et moléculaire, Physiologie et Physiopathologie  Immunologie  Développement Génétique Neurobiologie et Vieillissement  Infectiologie, Microbiologie Summary : The diagnosis of congenital infections in newborns is difficult when the methods of either antigen or immunoglobulin (Ig) detection are not reliable. It is the case of parasitic diseases such as congenital toxoplasmosis and congenital Chagas disease. The thesis program aims at establishing a new diagnostic tool, by means of the determination of the IgG repertoire from cord blood B lymphocytes after stimulation by specific antigens of Toxoplasma gondii or Trypanosoma cruzi. Proposition de sujet de thèse 2014-‐2015 Directeur de l'unité de recherche : Philippe DELORON Unité de recherche (et établissement de rattachement) : UMR 216 IRD/UPD Responsable de l'équipe d'accueil (EAD) : Adrian LUTY Directeur de thèse : Florence MIGOT-‐NABIAS Titre du sujet de thèse proposé : Analysis of the immunoglobulin repertoire for the diagnosis of congenital parasitic infections 5 mots clés : B lymphocyte ; Immunoglobulin (Ig) G ; neonate ; congenital toxoplasmosis ; congenital Chagas disease Candidat pressenti :  OUI  NON Contenu scientifique du programme de la thèse Context: the diagnosis of congenital infections in newborns is difficult when the methods of antigen detection are not reliable. It is the case of parasitic diseases such as congenital toxoplasmosis and congenital Chagas disease due to low parasite charges and limits of the microscopic or molecular methods employed for the identification of the causative Toxoplasma gondii and Trypanosoma cruzi pathogens1. On the other hand, serological methods are hampered either by an unsystematic presence of immunoglobulin (Ig) M signalling a recent infection or by the presence in the newborn’s serum, until six months of life, of shared maternal and intrinsic IgG. A new method is being validated in our laboratory, which exploits the individual amino acid polymorphism of the IgG heavy chain, in order to make a differential determination of pathogen-‐specific IgG from a mother and her newborn2. Another experimental approach would consist of the investigation of the IgG repertoire of cord blood B lymphocytes after specific stimulation. Objectives and feasibility: the thesis program will aim to determine the presence in the neonate of B lymphocytes primed in utero to produce pathogen-‐specific IgG in the context of congenital toxoplasmosis or Chagas disease. For this purpose, cellular and molecular techniques will be employed as well as bioinformatics for the data processing. The access to blood samples will be facilitated by collaborations already established with teams involved in the medical care of patients or in research projects. Proposed experiments: Selection of newborn B lymphocytes on parasite extracts: after their isolation from whole blood, part of mononuclear cells will be stimulated by a parasite extract and the other not, as a control. To optimize the protocols, experiments will first be performed with the blood of adults infected by T. gondii before moving to newborns and thereafter to patients (adults and then newborns) infected by T. cruzi. Sequencing of the immunoglobulin repertoire: mRNA will be retro-‐transcripted into cDNA which will be amplified using consensus primers anchored one in the constant CH1 region of the IgG heavy chain, and the other in the variable VH region. The amplified CDR3 region bears the immune signature consecutive to the specific recognition of an antigen. The diversity of the CDR3 lengths will thereafter be explored by sequencing3. Bioinformatics analysis: in the case of a naive repertoire, the B-‐cell population is polyclonal and the CDR3 lengths display a bell-‐shaped profile while an immune response generates an oligoclonal expansion of B lymphocytes with a more restricted profile of the CDR3 lengths. Sequence analysis through peak description will benefit from the International ImMunoGeneTics (IMGT ) database, a high quality integrated database specializing in immunoglobulin molecules4. References: 1. Carlier Y et al. (2012) Acta Trop 121(2):55-‐70. 2. Dechavanne C et al. (2012) PLoS ONE 7(9): e46097. 3. Boudinot P et al. (2008) Mol Immunol 45 :2437-‐45. 4. Alamyar E et al. (2014) Methods Mol Biol 1131:337-‐81. 