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Lymphocytes B et immunoglobulines néonatales dans un contexte d'infection parasitaire congénitale : stratégies méthodologiques de caractérisation

Abstract : In the first months of life, the newborn is protected from infections by the maternal immunoglobulins (Ig) G, which are transferred during pregnancy, and are present in his serum together with his neo-synthesized IgG.The distinction in neonatal serum between maternal and fetal IgG is difficult to implement, but could be very useful for diagnosing congenital infections early, particularly in the case of parasitic infections. For this purpose, our team has established a methodology based on mass spectrometry that exploits individual peptide polymorphisms located on the CH2 and CH3-CHS domains from the constant IgG heavy chain. In this work, we propose to validate this approach by molecular biology. The specific amplification and sequencing of the CH2 and CH3-CHS constant domains of the 4 total IgG subclasses allowed i/to validate the bottom-up mass spectrometry approach and ii/to highlight new nucleotide polymorphisms causing or not an amino acid change. This approach requires an exclusive purification of pathogen-specific IgG, which can be circumvented using another cell-based approach, based on the specific IgG secreted by the newborn B lymphocytes (Ly). Thus, the individual and antigenic specificities of Ig are reconciled. To do this, another development of my work consisted in the adaptation of the ELISPOT technique (Enzyme-Linked ImmunoSpot), in the context of the toxoplasmosis, caused by Toxoplasma gondii parasite, and responsible with the Chagas disease of the most cases of congenital infections of parasitic origin. Developments were made with mononuclear cells from adult volunteers seronegative and seropositive for toxoplasmosis, which led us to select a parasitic T. gondii type I lysate as multi-epitopes antigenic candidate compared to the specific recombinant protein SAG1 (Surface Antigen 1), a membrane protein representative of the parasite. The investigation of other parameters is necessary to complete the ELISPOT adaptation in the specific context of congenital parasitic infection. These include assessing i/the suitability of the test in the case of a recent infection with T. gondii, using B Ly from seroconverting adults and/or neonates with congenital toxoplasmosis, and ii/the test ubiquity, by studying its ability to reveal with the same efficiency the IgG secreted by B Ly from individuals infected with toxoplasm strains circulating in different geographical areas. To make this last ELISPOT adaptation phase possible, the implementation of field studies was essential in order to constitute a bio-bank resulting from toxoplasmosis follow-ups of pregnant women and their newborns at childbirth: a first study was conducted for 3 months in 2018 in the CHU maternity in Cotonou, Benin; also, a clinical trial has been in progress for 18 months since June 2018 in 3 AP-HP hospitals maternities, in Ile de France. In addition, a retrospective sero-epidemiological study of toxoplasmosis in about 1000 pregnant women in southern Benin, was conducted, using plasma samples collected 2008-2010 in our unit. This will document for the first time toxoplasmosis seroprevalence on a wide effective of pregnant women in Benin (53%) as well as the rate of toxoplasmic seroconversion during pregnancy (ongoing). In addition to the stated objectives, all of this work contributes to better documenting the exploration of the fetal immune system.
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Magalie Dambrun Dit Tambrun. Lymphocytes B et immunoglobulines néonatales dans un contexte d'infection parasitaire congénitale : stratégies méthodologiques de caractérisation. Immunologie. Université Sorbonne Paris Cité, 2018. Français. ⟨NNT : 2018USPCB118⟩. ⟨tel-02517078⟩

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