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B cell clonal expansion is vital for adaptive immunity. High-throughput B cell receptor (BCR) sequencing enables investigating this process, but requires computational inference to identify clonal relationships. This inference usually relies on only the BCR heavy chain, as most current protocols do not preserve heavy:light chain pairing. The extent to which paired light chains aids inference is unknown. Using human single-cell paired BCR datasets, we assessed the ability of heavy chain-based clonal clustering to identify clones. Of the expanded clones identified

The efficacy of the adaptive humoral immune response likely requires diverse, yet focused regional B cell antibody production throughout the body. Here we address, in the first study of its kind, the B cell repertoire in the bronchial mucosa, an important barrier to antigens inhaled from the atmosphere. To accomplish this, we have applied high-throughput Adaptive Immune Receptor Repertoire Sequencing (AIRR-Seq) to 10 bronchial biopsies from altogether four different sites in the right lungs from an asthmatic patient and a healthy subject. While the majority of identified B cell clones were restricted to a single site, many were disseminated in multiple sites. Members of a clone were shared more between adjacent biopsies than between distal biopsies, suggesting local mucosal migration and/or a homing mechanism for B cells through the blood or lymph. A smaller fraction of clones spanned the bronchial mucosa and peripheral blood, suggesting ongoing trafficking between these compartments. The bronchial mucosal B cell repertoire in the asthmatic patient was geographically more variable but less diverse compared to that of the healthy subject, suggesting an ongoing, antigen-driven humoral immune response in atopic asthma. Whether this is a feature of atopy or disease status remains to be clarified in future studies. We observed a subset of highly mutated and antigen-selected IgD-only cells in the bronchial mucosa. These cells were found in relative high abundance in the asthmatic individual but also, albeit at lower abundance, in the healthy subject. This novel finding merits further exploration using a larger cohort of subjects.

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones 1-4 . SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs) 5-9 . It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naïve B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs) 1-4 . Induction of the latter is a hallmark of durable immunity after vaccination 5 . SARS-CoV-2 mRNA vaccination induces a robust GC response in humans 6-8 , but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S + BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations., Competing Interests: Conflict of interest statement The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines which list Florian Krammer as co-inventor. Viviana Simon is also listed on the serological assay patent application as co-inventors. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. Florian Krammer has consulted for Merck and Pfizer (before 2020), and is currently consulting for Pfizer, Seqirus and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. Ali Ellebedy has consulted for InBios and Fimbrion Therapeutics (before 2021) and is currently a consultant for Mubadala Investment Company. The Ellebedy laboratory received funding under sponsored research agreements that are unrelated to the data presented in the current study from Emergent BioSolutions and from AbbVie.