Maturation and persistence of the anti-SARS-CoV-2 memory B cell response.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

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

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 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.

The 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.

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

In 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.

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

SARS-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 qualitatively investigated 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 (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual 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 more than 65% of the plasmablast response at all time points. 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.

The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. Eighty percent of patients with ITP possess anti-integrin αIIbß3 IgG autoantibodies that cause 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 approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbß3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-αIIbß3 IgG-SCs 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.

Rituximab-resistant splenic memory B cells and newly engaged naive B cells fuel relapses in patients with immune thrombocytopenia.

Rituximab (RTX), an antibody targeting CD20, is widely used as a first-line therapeutic strategy in B cell-mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Here, we characterize the cellular basis responsible for disease relapse in secondary lymphoid organs in humans, taking advantage of the opportunity offered by therapeutic splenectomy in patients with relapsing immune thrombocytopenia. By analyzing the B and plasma cell immunoglobulin gene repertoire at bulk and antigen-specific single-cell level, we demonstrate that relapses are associated with two responses coexisting in germinal centers and involving preexisting mutated memory B cells that survived RTX treatment and naive B cells generated upon reconstitution of the B cell compartment. To identify distinctive characteristics of the memory B cells that escaped RTX-mediated depletion, we analyzed RTX refractory patients who did not respond to treatment at the time of B cell depletion. We identified, by single-cell RNA sequencing (scRNA-seq) analysis, a population of quiescent splenic memory B cells that present a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell-specific factors and expression of prosurvival genes. Our results clearly demonstrate that these RTX-resistant autoreactive memory B cells reactivate as RTX is cleared and give rise to plasma cells and further germinal center reactions. Their continued surface expression of CD19 makes them efficient targets for current anti-CD19 therapies. This study thus identifies a pathogenic contributor to autoimmune diseases that can be targeted by available therapeutic agents.