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Development and validation of a new analytical approach for the characterization of IgG N-glycans in COVID-19, sepsis and HIV infection

par Helali, Yosra
Président du jury Vermijlen, David
Promoteur Delporte, Cédric
Co-Promoteur Marchant, Arnaud
Publication Non publié, 2024-06-14

Thèse de doctorat

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Résumé :

N-glycosylation is one of the most prominent post-translational modifications (PTM) of glycoproteins. Antibodies are glycosylated proteins, and the alteration of this modification may influence its function, stability and half-life. Importantly, antibodies play a central role in humoral immunity against pathogens, particularly immunoglobulin G (IgG), which is a key effector of the humoral immune response.For this reason, the first part of this PhD work was dedicated to the development and validation of a reliable, sensitive and robust analytical method for the characterization of IgG N-glycans. To achieve this aim, procainamide was used for the labeling of the glycans. The analytical method is based on two steps: first an enrichment step using online solid phase extraction (SPE) with hydrophilic interaction liquid chromatography (HILIC) chemistry, then, a separation step using the same chemistry (HILIC) coupled to fluorescence (FLD) and mass spectrometry (MS) detectors Several parameters were optimized using an experimental design. After that, a validation process was performed, and repeatability, precision and stability were confirmed. NIST standard (reference material 8671, monoclonal antibodies), Trixuma© (biosimilar) and MabThera© (bio original) as well as commercial kits, were also used for the validation to compare our findings to the literature.After its validation, we decided to evaluate this method for the characterization of IgG N-glycans in patients with different diseases. Given the pandemic situation, the first studied disease was the COVID-19 with a group of patients suffering from severe COVID-19 and hospitalized in intensive care unit (ICU). Those patients were divided into two groups according to their clinical outcome (survival or death) and compared to healthy volunteers and ICU hospitalized patients suffering from sepsis. Our data indicated that a specific bisecting N-glycan, FA2B, is a potential biomarker of the mortality risk. The increase of FA2B was significant from the first day of admission in ICU. Our results are concordant with other studies indicating that an increase of total bisecting is associated with an increased affinity of IgG to the FcγRIII receptor, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and IgG pro-inflammatory effector functions in case of severe illness. Although, our study is the first to define a specific N-glycans as an indicator of poor prognosis.The second application of our method was the characterization of Fc IgG N-glycosylation in pregnant women living with Human Immunodeficiency virus (HIV) who started their antiviral therapy (ART) after conception (HIV+A) or before the conception (HIV+B) and uninfected pregnant women (HIV-) and their infants. Our results showed an increase in agalactosylation (absence of galactose residue) in HIV+A women as compared to HIV- and HIV+B women. This finding indicated that initiating ART before conception may correct alterations in IgG glycosylation profile induced by HIV infection. The different N-glycans profiles of maternal IgG could influence their transfer across the placenta and the effector functions in the newborn infant.