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Understanding the evolution of the B cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is fundamental to design the next generation of vaccines and therapeutics. We longitudinally analyze at the single-cell level almost 900 neutralizing human monoclonal antibodies (nAbs) isolated from vaccinated people and from individuals with hybrid and super hybrid immunity (SH), developed after three mRNA vaccine doses and two breakthrough infections. The most potent neutralization and Fc functions against highly mutated variants belong to the SH cohort. Repertoire analysis shows that the original Wuhan antigenic sin drives the convergent expansion of the same B cell germlines in vaccinated and SH cohorts. Only Omicron breakthrough infections expand previously unseen germ lines and generate broadly nAbs by restoring IGHV3-53/3-66 germ lines. Our analyses find that B cells initially expanded by the original antigenic sin continue to play a fundamental role in the evolution of the immune response toward an evolving virus. [Display omitted] • Omicron infection induces potent neutralization and Fc functions against variants • Neutralization relies on restored germ lines expanded by Wuhan antigenic sin (AS) • AS drives convergent antibody maturation in homologous and heterologous immunity • Omicron infection stretches the antibody repertoire to control SARS-CoV-2 variants Paciello et al. find that Omicron infection induces the most potent and broad antibody response to SARS-CoV-2 variants. This response relies on restored B cell germ lines expanded by the Wuhan antigenic sin, like IGHV3-53 and 3-66, and on new germ lines expanded to stretch the antibody repertoire to control SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure histories become increasingly complex through original and variant-adapted vaccines and infections with viral variants. Upon exposure to the highly altered Omicron spike glycoprotein, pre-immunized individuals predominantly mount recall responses of Wuhan-Hu-1 (wild-type)-imprinted memory B (B MEM) cells mostly targeting conserved non-neutralizing epitopes, leading to diminished Omicron neutralization. We investigated the impact of imprinting in individuals double/triple vaccinated with a wild-type-strain-based mRNA vaccine who, thereafter, had two consecutive exposures to Omicron BA.1 spike (breakthrough infection followed by BA.1-adapted vaccine). We found that depletion of conserved epitope-recognizing antibodies using a wild-type spike bait results in strongly diminished BA.1 neutralization. Furthermore, spike-specific B MEM cells recognizing conserved epitopes are much more prevalent than BA.1-specific B MEM cells. Our observations suggest that imprinted B MEM cell recall responses limit the induction of strain-specific responses even after two consecutive BA.1 spike exposures. Vaccine adaptation strategies need to consider that prior SARS-CoV-2 infections and vaccinations may cause persistent immune imprinting. [Display omitted] • Two exposures to Omicron BA.1 spike boost BA.1 neutralization in pre-vaccinated individuals • BA.1 neutralization is largely mediated by antibodies cross-recognizing wild-type SARS-CoV-2 • BA.1-specific neutralizing antibodies are detectable only in some individuals • Memory B cells recognizing conserved epitopes predominate over BA.1-specific B cells Muik et al. show that, in individuals pre-immunized with SARS-CoV-2 ancestral strain mRNA vaccines, cross-reactive humoral immunity targeting conserved epitopes remains dominant after two exposures to Omicron BA.1 spike. Their observations indicate that persistent immune imprinting may limit strain-specific naive B cell priming even after repeated variant spike exposure. [ABSTRACT FROM AUTHOR]

Memory B cells (MBCs) are essential for humoral immunological memory and can emerge during both the pre-germinal center (GC) and GC phases. However, the transcription regulators governing MBC development remain poorly understood. Here, we report that the transcription regulator Notch2 is highly expressed in MBCs and their precursors at the pre-GC stage and required for MBC development without influencing the fate of GC and plasma cells. Mechanistically, Notch2 signaling promotes the expression of complement receptor CD21 and augments B cell receptor (BCR) signaling. Reciprocally, BCR activation up-regulates Notch2 surface expression in activated B cells via a translation-dependent mechanism. Intriguingly, Notch2 is dispensable for GC-derived MBC formation. In summary, our findings establish Notch2 as a pivotal transcription regulator orchestrating MBC development through the reciprocal enforcement of BCR signaling during the pre-GC phase and suggest that the generation of GC-independent and -dependent MBCs is governed by distinct transcriptional mechanisms. [Display omitted] • Notch2 is required for MBC development at the pre-GC stage • The interplay of Notch2 and BCR signaling promotes MBC development • Notch2 is dispensable for GC-dependent MBC development Transcriptional regulation of MBC development remains poorly understood. Xu et al. report that the transcription regulator Notch2 guides activated B cells to form MBCs, but not GC B and plasma cells, through interplay with BCR signaling. Unexpectedly, the Notch2 signal is dispensable for the generation of GC-dependent MBCs. [ABSTRACT FROM AUTHOR]

Summary: Memory B cells seem to be more durable than antibodies and thus crucial for the long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here we investigate SARS-CoV-2 spike-specific memory B cells and their dependence on CD4+ T cell help in different settings of coronavirus disease 2019 (COVID-19). Compared with severely ill individuals, those who recovered from mild COVID-19 develop fewer but functionally superior spike-specific memory B cells. Generation and affinity maturation of these cells is best associated with IL-21+CD4+ T cells in recovered individuals and CD40L+CD4+ T cells in severely ill individuals. The increased activation and exhaustion of memory B cells observed during COVID-19 correlates with CD4+ T cell functions. Intriguingly, CD4+ T cells recognizing membrane protein show a stronger association with spike-specific memory B cells than those recognizing spike or nucleocapsid proteins. Overall, we identify CD4+ T cell subsets associated with the generation of B cell memory during SARS-CoV-2 infection.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria.

The memory B cell response consists of phenotypically distinct subsets that differ in their ability to respond upon antigen re-encounter. However, the pathways regulating the development and function of memory B cell subsets are poorly understood. Here, we show that CD62L and CD44 are progressively expressed on mouse memory B cells and identify transcriptionally and functionally distinct memory B cell subsets. Bcl6 is important in regulating memory B cell subset differentiation with overexpression of Bcl6 resulting in impaired CD62L + memory B cell development. Bcl6 regulates memory B cell subset development through control of a network of genes, including Bcl2 and Zeb2. Overexpression of Zeb2 impairs the development of CD62L + memory B cells. Importantly, CD62L is also differentially expressed on human memory B cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and identifies phenotypically distinct populations. Together, these data indicate that CD62L expression marks functionally distinct memory B cell subsets., Competing Interests: Declaration of interests The Ellebedy laboratory received funding from Emergent BioSolutions, AbbVie, and Moderna that is unrelated to the data presented in the current study. A.H.E. has received consulting and speaking fees from InBios International, Fimbrion Therapeutics, RGAX, Mubadala Investment Company, Moderna, Pfizer, GSK, Danaher, Third Rock Ventures, Goldman Sachs, and Morgan Stanley and is the founder of ImmuneBio Consulting. J.S.T. and A.H.E. are recipients of a licensing agreement with AbbVie that is unrelated to the data presented in the current study., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Germinal centers (GCs), sites of antibody affinity maturation, are organized into dark (DZ) and light (LZ) zones. Here, we show a B cell-intrinsic role for signal transducer and activator of transcription 3 (STAT3) in GC DZ and LZ organization. Altered zonal organization of STAT3-deficient GCs dampens development of long-lived plasma cells (LL-PCs) but increases memory B cells (MBCs). In an abundant antigenic environment, achieved here by prime-boost immunization, STAT3 is not required for GC initiation, maintenance, or proliferation but is important for sustaining GC zonal organization by regulating GC B cell recycling. Th cell-derived signals drive STAT3 tyrosine 705 and serine 727 phosphorylation in LZ B cells, regulating their recycling into the DZ. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses identified STAT3 regulated genes that are critical for LZ cell recycling and transiting through DZ proliferation and differentiation phases. Thus, STAT3 signaling in B cells controls GC zone organization and recycling, and GC egress of PCs, but negatively regulates MBC output. [Display omitted] • B cell STAT3 signaling controls germinal center dark and light zone organization • STAT3 signaling regulates GC output of plasma and memory B cells • Th cell-derived signals activate STAT3 in LZ B cells, facilitating their recycling • STAT3-regulated transcriptional program promotes LZ B cell recycling to the DZ The role of STAT3 in germinal center (GC) zone organization is unclear. Fike et al. describe a mechanism by which STAT3 signaling in B cells regulates GC dark and light zone organization, GC B cell recycling, and GC output of plasma and memory B cells. [ABSTRACT FROM AUTHOR]

Summary Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium- antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria. [ABSTRACT FROM AUTHOR]

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development. [Display omitted] • More than 100 antibodies from two older adult donors are expressed and characterized • Potent neutralization is not biased toward prefusion-specific antigenic sites • Two prefusion antigenic sites at the trimer interface are identified • Structural basis for neutralization of hMPV F by two prefusion-specific antibodies Human metapneumovirus (hMPV) infections can lead to severe outcomes for the elderly. Rush et al. use engineered hMPV fusion proteins to isolate and characterize antibodies generated from natural infection of two older adult donors. Epitope mapping and structural studies provide insights useful for the rational design of an hMPV vaccine. [ABSTRACT FROM AUTHOR]

Influenza infection imparts an age-related increase in mortality and morbidity. The most effective countermeasure is vaccination; however, vaccines offer modest protection in older adults. To investigate how aging impacts the memory B cell response, we track hemagglutinin-specific B cells by indexed flow sorting and single-cell RNA sequencing (scRNA-seq) in 20 healthy adults that were administered the trivalent influenza vaccine. We demonstrate age-related skewing in the memory B cell compartment 6 weeks after vaccination, with younger adults developing hemagglutinin-specific memory B cells with an FcRL5 + "atypical" phenotype, showing evidence of somatic hypermutation and positive selection, which happened to a lesser extent in older persons. We use publicly available scRNA-seq from paired human lymph node and blood samples to corroborate that FcRL5 + atypical memory B cells can derive from germinal center (GC) precursors. Together, this study shows that the aged human GC reaction and memory B cell response following vaccination is defective., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Memory B cells seem to be more durable than antibodies and thus crucial for the long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here we investigate SARS-CoV-2 spike-specific memory B cells and their dependence on CD4+ T cell help in different settings of coronavirus disease 2019 (COVID-19). Compared with severely ill individuals, those who recovered from mild COVID-19 develop fewer but functionally superior spike-specific memory B cells. Generation and affinity maturation of these cells is best associated with IL-21+CD4+ T cells in recovered individuals and CD40L+CD4+ T cells in severely ill individuals. The increased activation and exhaustion of memory B cells observed during COVID-19 correlates with CD4+ T cell functions. Intriguingly, CD4+ T cells recognizing membrane protein show a stronger association with spike-specific memory B cells than those recognizing spike or nucleocapsid proteins. Overall, we identify CD4+ T cell subsets associated with the generation of B cell memory during SARS-CoV-2 infection. [Display omitted] • Mild COVID-19 induces fewer but functionally superior B cells than severe disease • B cells are associated with different CD4+ T cell functions in both disease settings • Severe COVID-19 causes excessive activation and exhaustion of B cells • Membrane-specific CD4+ T cells are strongly associated with spike-specific B cells Pušnik et al. report that individuals who recovered from mild COVID-19 develop fewer but functionally superior SARS-CoV-2 spike-specific memory B cells than individuals with severe disease. The development and activation of those cells are associated with distinct CD4+ T cell functions in both clinical outcomes of COVID-19. [ABSTRACT FROM AUTHOR]

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity. [Display omitted] • Integrated, single-cell RNA- and BCR-seq in three organs after influenza infection • Switched, mutated memory B cells are continuously produced from germinal centers • Lung memory B cells originate from lymphoid organs and assume residency phenotype • Memory B cells are derived from both low- and high-affinity GC precursors Combining single-cell RNA and BCR sequencing, Mathew et al. report that, after influenza infection, memory B cells (Bmems) are stochastically selected from both low- and high-affinity germinal center (GC) precursors. They reveal that lung Bmems acquire tissue-residency signatures and continuously differentiate from the spleen and mediastinal lymph nodes GCs. [ABSTRACT FROM AUTHOR]

Memory B cells seem to be more durable than antibodies and thus crucial for the long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here we investigate SARS-CoV-2 spike-specific memory B cells and their dependence on CD4 + T cell help in different settings of coronavirus disease 2019 (COVID-19). Compared with severely ill individuals, those who recovered from mild COVID-19 develop fewer but functionally superior spike-specific memory B cells. Generation and affinity maturation of these cells is best associated with IL-21 + CD4 + T cells in recovered individuals and CD40L + CD4 + T cells in severely ill individuals. The increased activation and exhaustion of memory B cells observed during COVID-19 correlates with CD4 + T cell functions. Intriguingly, CD4 + T cells recognizing membrane protein show a stronger association with spike-specific memory B cells than those recognizing spike or nucleocapsid proteins. Overall, we identify CD4 + T cell subsets associated with the generation of B cell memory during SARS-CoV-2 infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change. • CD27dull and CD27bright MBCs share their VH repertoire but have different functions • CD27dull MBCs are the long-lived substrate of selected and specific CD27bright MBCs • The interplay between CD27dull and CD27bright MBCs preserves B cell memory • In pregnancy, MBCs decline, but persisting CD27dull MBCs re-expand after delivery Grimsholm et al. show that CD27dull and CD27bright represent sequential MBC developmental stages. T cell- and germinal center (GC)-independent CD27dull MBCs are the plastic source of strongly selected and GC-dependent CD27bright MBCs. CD27dull MBCs, able to expand and differentiate in response to change, ensure stability and flexibility of human B cell memory. [ABSTRACT FROM AUTHOR]

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27 dull and CD27 bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27 dull and CD27 bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27 dull into the CD27 bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27 dull MBCs transit to the more differentiated CD27 bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27 dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27 dull MBCs that expand and differentiate in response to change., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Broadly neutralizing antibodies are crucial for the control of many life-threatening viral infections like HIV, influenza, or hepatitis. Their induction is a prime goal in vaccine research. Using computer simulations, we identify strategies to promote the generation of broadly neutralizing antibodies in natural germinal center (GC) reactions. The simulations predict a feedback loop based on antibodies and memory B cells from previous GC reactions that promotes GCs to focus on new epitopes. Memory-derived or injected antibodies specific for immunodominant epitopes control epitope availability, suppress the participation of memory B cells in the GC reaction, and allow for the evolution of other B cells to affinity mature for hidden or rare epitopes. This defines a natural selection mechanism for GC B cells to concentrate on new epitopes rather than refine affinity to already-covered epitopes. This principle can be used for the design and testing of future therapies and vaccination protocols. • Injected or pre-exisiting antibodies mask immunodominant epitopes in germinal centers • Epitope masking can shift the focus of germinal centers to less-accessible epitopes • Memory B cells are less competitive in germinal centers with masked epitopes • Memory-derived antibodies promote natural affinity maturation to other epitopes Antibodies specific for low-accessible pathogen epitopes are crucial for the control of life-threatening infections. Meyer-Hermann shows with computer simulations that memory-derived antibodies mask immunodominant epitopes, suppress participation of memory B cells in germinal centers, and promote affinity maturation to less-accessible epitopes. Vaccination with antibodies can induce the same effect. [ABSTRACT FROM AUTHOR]

Pemphigus vulgaris (PV) is an autoimmune disease characterized by blistering sores on skin and mucosal membranes, caused by autoantibodies primarily targeting the cellular adhesion protein, desmoglein-3 (Dsg3). To better understand how Dsg3-specific autoantibodies develop and cause disease in humans, we performed a cross-sectional study of PV patients before and after treatment to track relevant cellular responses underlying disease pathogenesis, and we provide an in-depth analysis of two patients by generating a panel of mAbs from single Dsg3-specific memory B cells (MBCs). Additionally, we analyzed a paired sample from one patient collected 15-months prior to disease diagnosis. We find that Dsg3-specific MBCs have an activated phenotype and show signs of ongoing affinity maturation and clonal selection. Monoclonal antibodies (mAbs) with pathogenic activity primarily target epitopes in the extracellular domains EC1 and EC2 of Dsg3, though they can also bind to the EC4 domain. Combining antibodies targeting different epitopes synergistically enhances in vitro pathogenicity. • Single-cell analysis of autoantigen-specific memory B cells in pemphigus vulgaris • Memory B cells undergo extensive affinity maturation and acquire pathogenic activity • Memory B cell-derived autoantibodies are limited in epitope specificity and repertoire • Multiple antibodies can work synergistically to enhance pathogenic activity Cho et al. use single-cell sorting methods to detect and characterize autoantigen-specific memory B cells before and during the development of pemphigus vulgaris disease. They find that memory B cells undergo ongoing affinity maturation to generate a limited repertoire or pathogenic antibodies, which work synergistically to enhance overall pathogenic activity. [ABSTRACT FROM AUTHOR]