Identification des répertoires d'affinité des cellules sécrétrices d'anticorps

This research aimed to explore the complexities of antibody affinities of antibody-secreting cells (ASCs) in the context of both vaccination and autoimmune responses. The first aim was to characterize the ASC response to mRNA vaccination against SARS-CoV-2. The advent of novel mRNA vaccines to tackle the COVID-19 pandemic presented a unique opportunity to study a novel humoral B cell response post-immunization. We used a droplet microfluidic assay, DropMap, able to measure single cell antibody secretion and affinity towards antigen to characterize at high throughput the functional characteristics of ASCs in the weeks and months following mRNA vaccine administration, with a distinction made between individuals based on prior SARS-CoV-2 infection. Our characterization of the ASC response to mRNA vaccination against SARS-CoV-2 has provided valuable insights into how B cells respond to novel vaccines and affinity repertoires during human vaccination. The second aim was to measure the affinity repertoires of a non-natively secreting B cell population, memory B cells (Bmem), following mRNA vaccination against SARS-CoV-2. This involved characterizing the affinity repertoires of Bmem-derived antibodies towards numerous SARS-CoV-2 receptor-binding domain (RBD) variants by implementing a high throughput screening system based on biolayer interferometry (BLI) measurements. The third aim was to develop a novel technique (termed DropPick) for recovering cells of interest from DropMap. DropMap is a potent technique for direct identification of ASCs and affinity characterization. However, there is much more to be discerned from analyzing these cells of interest using additional methods, such as flow cytometry for phenotyping or B cell receptor sequencing for clonal analyses and re-expression. My thesis work has shown that after receiving the BNT162b2 vaccine targeting the Hu-1 SARS-CoV-2 Spike protein, high-affinity plasmablasts were initially induced targeting both the Hu-1 and Omicron Spike RBD variants but exhibited a rapid decline. Surprisingly, plasmablasts with low affinity consistently constituted more than 65% of the specific plasmablast response at all observed time points. Our high throughput BLI affinity measurements on Bmem antibodies from similarly vaccinated individuals found high affinity clones against several RBD variants with signs of affinity maturation; comparing results between variants allowed for key antibody binding epitopes to be determined. Following this cohort of Bmem antibodies also found that viral evolution to the Omicron RBD variant resulted in a large evasion of Bmems mutated towards Hu-1 RBD with high affinity, with especially high evasion of potently Hu-1 and Beta neutralizing antibodies. In parallel, I have developed a system to label cells of interest using photoactivatable markers within DropMap and the technical platform to achieve targeted cell photoactivation by targeted UV illumination. This involved the creation of automated programs for DropMap assay real-time analyses as well as macro control of microscope utilities to deliver discrete illumination to droplets. I have also shown the ability for this workflow to yield a clear population by flow cytometry with up to 70% efficiency, with the potential for sorting and future downstream analyses. These results will aid ongoing efforts to understand the role of affinity in ASC responses and their immune contexts. Our data provide broad affinity repertoires that provide valuable functional insights into B cell responses to SARS-CoV-2 and mRNA vaccination. The development of the DropPick platform has shown the potential to further leverage these studies to collect molecular data from specific functional subsets.Cette recherche à explorer les complexités des affinités d’anticorps des cellules sécrétant des anticorps (CSA). Le premier objectif était de caractériser la réponse de l’CSA à la vaccination par ARNm contre le SRAS-CoV-2. Les nouveaux vaccins à ARNm pour lutter contre la pandémie de COVID-19 a présenté une opportunité unique d’étudier une nouvelle réponse humorale des cellules B après l’immunisation. Nous avons utilisé un test microfluidique, DropMap, capable de mesurer la sécrétion d'anticorps et l'affinité envers l'antigène pour caractériser les caractéristiques des CSA dans les semaines et les mois suivant l'administration du vaccin à ARNm, avec une distinction faite entre les individus en fonction des antécédents du SRAS-CoV-2 infection. Notre caractérisation de la réponse des CSA à la vaccination par ARNm contre le SRAS-CoV-2 a fourni des informations précieuses sur la façon dont les cellules B répondent aux nouveaux vaccins et aux répertoires d'affinité lors de la vaccination humaine. Le deuxième objectif était de mesurer les répertoires d’affinité d’une population de cellules B non sécrétant de manière native, les cellules B mémoire (Bmem), après vaccination par ARNm contre le SRAS-CoV-2. Ceci impliquait de caractériser les répertoires d'affinité des anticorps dérivés de Bmem envers de nombreuses variantes du domaine de liaison au récepteur (RBD) du SRAS-CoV-2 en mettant en œuvre un système de criblage à haut débit basé sur des mesures d'interférométrie de biocouche. Le troisième objectif était de développer une nouvelle technique (appelée DropPick) pour récupérer des cellules d'intérêt à partir de DropMap. DropMap est une technique puissante pour l’identification directe des CSA et la caractérisation par affinité. Cependant, il reste beaucoup plus à discerner en analysant ces cellules d’intérêt à l’aide de méthodes supplémentaires, telles que la cytométrie en flux pour le phénotypage ou le séquençage des récepteurs des cellules B pour les analyses clonales et la réexpression. Mes travaux de thèse ont montré qu'après avoir reçu le vaccin BNT162b2, des CSA de haute affinité ont été initialement induits ciblant à la fois les variantes Hu-1 et Omicron RBD, mais ont présenté un déclin rapide. Les CSA de faible affinité représentaient systématiquement plus de 65% de la réponse spécifique des CSA à tous les moments observés. Nos mesures d’affinité sur les anticorps Bmem provenant d’individus vaccinés ont révélé des clones à haute affinité contre plusieurs variantes de RBD présentant de maturation d’affinité ; la comparaison des résultats entre les variantes a permis de déterminer les épitopes clés de liaison aux anticorps. Suite à cette cohorte d’anticorps Bmem, nous avons également découvert que l’évolution virale vers la variante Omicron RBD entraînait une évasion des Bmems mutés vers Hu-1 RBD avec une affinité élevée, avec une impacte élevée sur des anticorps neutralisants puissants Hu-1 et Beta. J'ai développé un système pour marquer les cellules d'intérêt avec des marqueurs photoactivables au sein de DropMap et de la plateforme pour réaliser une photoactivation cellulaire par illumination UV ciblée. Cela impliquait la création de programmes automatisés pour les analyses en temps réel du test DropMap ainsi que le contrôle macro des utilitaires du microscope pour fournir un éclairage discret aux gouttelettes. J'ai également montré la capacité de cette technique à produire une population par cytométrie avec une efficacité jusqu'à 70% avec un potentiel de tri et de futures analyses en aval. Ces résultats contribueront aux efforts en cours pour comprendre le rôle de l’affinité dans les réponses CSA. Nos larges répertoires d’affinité fournissent des informations fonctionnelles précieuses sur les réponses des cellules B à la vaccination contre le SRAS-CoV-2. La plateforme DropPick a montré le potentiel d’exploiter ces études pour collecter des données moléculaires provenant de sous-ensembles fonctionnels spécifiques

Single-Cell Technologies for the Study of Antibody-Secreting Cells

International audienceAntibody-secreting cells (ASC), plasmablasts and plasma cells, are terminally differentiated B cells responsible for large-scale production and secretion of antibodies. ASC are derived from activated B cells, which may differentiate extrafollicularly or form germinal center (GC) reactions within secondary lymphoid organs. ASC therefore consist of short-lived, poorly matured plasmablasts that generally secrete lower-affinity antibodies, or long-lived, highly matured plasma cells that generally secrete higher-affinity antibodies. The ASC population is responsible for producing an immediate humoral B cell response, the polyclonal antibody repertoire, as well as in parallel building effective humoral memory and immunity, or potentially driving pathology in the case of autoimmunity. ASC are phenotypically and transcriptionally distinct from other B cells and further distinguishable by morphology, varied lifespans, and anatomical localization. Single cell analyses are required to interrogate the functional and transcriptional diversity of ASC and their secreted antibody repertoire and understand the contribution of individual ASC responses to the polyclonal humoral response. Here we summarize the current and emerging functional and molecular techniques for high-throughput characterization of ASC with single cell resolution, including flow and mass cytometry, spot-based and microfluidic-based assays, focusing on functional approaches of the secreted antibodies: specificity, affinity, and secretion rate

Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics

International audienceSARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long-lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here we investigated qualitatively the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a unique droplet microfluidic and imaging approach, we analyzed >4,000 single IgG-secreting cells revealing high inter-individual variability in affinity for RBD with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented >65% of the plasmablast response at all timepoints. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols

High-affinity autoreactive plasma cells disseminate through multiple organs in patients with immune thrombocytopenic purpura

International audienceThe major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin αIIbβ3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies

mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants

International audienceIn addition to serum immunoglobulins, memory B cell (MBC) generation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is another layer of immune protection, but the quality of MBC responses in naive and coronavirus disease 2019 (COVID-19)-recovered individuals after vaccination remains ill defined. We studied longitudinal cohorts of naive and disease-recovered individuals for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of antibody repertoires, affinity, and neutralization against variants of concern (VOCs) using unbiased cultures of 2,452 MBCs. Upon boosting, the MBC pool of recovered individuals expanded selectively, matured further, and harbored potent neutralizers against VOCs. Although naive individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity toward multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs

Omicron BA.1 breakthrough infection drives long-term remodeling of the memory B cell repertoire in vaccinated individuals

Summary How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reaction, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies

SARS-CoV-2 Omicron BA.1 breakthrough infection drives late remodeling of the memory B cell repertoire in vaccinated individuals

International audienceHow infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant

International audienceThe SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals