A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ∼82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (∼0.05%) and unswitched B cells (∼0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Abundance of B Cell Receptors Harboring Elongated Polytyrosine and Polyserine Rich Motifs within Their Heavy Chain CDR3 Distinguishes Catastrophic and Antiphospholipid Syndrome

ACKGROUND: Antiphospholipid syndrome (APS) is defined by arterial and/or venous thrombosis and/or pregnancy morbidity along with persistent circulating antiphospholipid antibodies (aPL). Central to APS pathogenesis are B cells that generate autoreactive antibodies, including anticardiolipin antibodies (aCL), and anti-β2-glycoprotein-I antibodies (anti-B2GPI). Several studies have implicated specific germline and/or antigen-driven somatic hypermutations within the complementarity determining regions (CDRs) of pathogenic antibodies in APS, as well as other thrombotic disorders. Nevertheless, the B cell repertoire in thrombotic APS remains poorly characterized. Recently, autoreactive and platelet activating pathogenic antibodies harboring elongated tyrosine rich CDR3 motifs were reported in patients with heparin-induced thrombocytopenia (HIT) (Zhu et.al, Blood 2019, 2020). Whether such antibodies are present in APS and their potential role, if so, has not been determined. APPROACH : We leveraged single cell immunoprofiling to characterize the presence of antibodies harboring elongated tyrosine rich CDR3 motifs in the circulating B cell repertoire obtained from peripheral blood mononuclear cells (PBMC) from patients with thrombotic (n=4) and catastrophic APS (n=3) as well as healthy individuals (n=3). We also correlated the abundance of these antibodies with criteria aPL and inflammatory cytokines in plasma. The modified Ham (mHam) test was used to assess functional complement activation in plasma in plasma from all of the subjects studied (Chaturvedi et al. Blood 2020). RESULTS : B cell clones containing elongated polytyrosine rich CDR3 motifs were most abundant in patients with a diagnosis of catastrophic APS (n=3) , and were markedly elevated in patients with APS (n=4) compared to healthy counterparts (n=3) (Table 1). Elongated polytyrosine rich CDR3 motifs from CAPS patients contained penta-tyrosine (Y5), hexa-tyrosine (Y6), hepta-tyrosine (Y7), and octa-tyrosine (Y8) motifs (table 2) and contained more tyrosine residues than the penta-tyrosines previously reported in HIT. In addition, all of the polytyrosine containing motifs CDR3 motifs were present on the heavy chain and was followed by MDVW motif of IGHJ6. The presence of B cell clones containing elongated polytyrosine rich CDR3 motifs exhibited significant positive correlations with complement activation measured by mHAM test (r=0.728, p=0.0032), criteria aPLs including anticardiolipin antibodies (aCL) and anti-β2-glycoprotein-I antibodies (anti-B2GPI), as well as plasma inflammatory cytokines; TNFα (r=0.901, p=1.5x10 -5), IL-23 (r=0.780, p=0.0016), IL-17C cytokines (r=0.729, p=0.0033), sICAM1 (r=0.740, p=0.0025), sVCAM1 (r=0.764, p=0.0015), (Figure 1). The presence of elongated polytyrosine rich CDR3 motifs was also associated with the increase abundance of polyserine rich CDR3 motifs that were present predominantly in patients with CAPS. CONCLUSION : These preliminary studies provide the first characterization of the prevalence of B cell clones containing elongated polytyrosine rich CDR3 motifs, previously reported in HIT, in the B cell repertoire of APS patients. Notably, the abundance of these B cell clones is markedly elevated in patients with CAPS and is associated with elevated levels of inflammatory cytokines TNFa, IL-23, IL-17C, sICAM1, and sVCAM1. Validation studies in larger cohorts from APS patients and other thrombotic disorders are ongoing. Figure 1 Figure 1. Disclosures Chaturvedi: Alexion: Other: Advisory board member; Dova: Other: Advisory board member; Sanofi Genzyme: Other: Advisory board member; Argenx: Other: Advisory board member; UCB: Other: Advisory board participation. Khorana: Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Anthos: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. McCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

Different B cell subpopulations show distinct patterns in their IgH repertoire metrics

Several human B-cell subpopulations are recognized in the peripheral blood, which play distinct roles in the humoral immune response. These cells undergo developmental and maturational changes involving VDJ recombination, somatic hypermutation and class switch recombination, altogether shaping their immunoglobulin heavy chain (IgH) repertoire. Here, we sequenced the IgH repertoire of naïve, marginal zone, switched and plasma cells from 10 healthy adults along with matched unsorted and in silico separated CD19+ bulk B cells. Using advanced bioinformatic analysis and machine learning, we show that sorted B cell subpopulations are characterised by distinct repertoire characteristics on both the individual sequence and the repertoire level. Sorted subpopulations shared similar repertoire characteristics with their corresponding in silico separated subsets. Furthermore, certain IgH repertoire characteristics correlated with the position of the constant region on the IgH locus. Overall, this study provides unprecedented insight over mechanisms of B cell repertoire control in peripherally circulating B cell subpopulations.

Impact of Host Immune Status on Discordant Anti-SARS-CoV-2 Circulating B Cell Frequencies and Antibody Levels

Individuals with pre-existing chronic systemic low-grade inflammation are prone to develop severe COVID-19 and stronger anti-SARS-CoV-2 antibody responses. Whether this phenomenon reflects a differential expansion of antiviral B cells or a failure to regulate antibody synthesis remains unknown. Here, we compared the antiviral B cell repertoire of convalescent healthcare personnel to that of hospitalized patients with pre-existing comorbidities. Out of 277,500 immortalized B cell clones, antiviral B cell frequencies were determined by indirect immunofluorescence screening on SARS-CoV-2 infected cells. Surprisingly, frequencies of SARS-CoV-2 specific clones from the two groups were not statistically different, despite higher antibody levels in hospitalized patients. Moreover, functional analyses revealed that several B cell clones from healthcare personnel with low antibody levels had neutralizing properties. This study reveals for the first time a key qualitative defect of antibody synthesis in severe patients and calls for caution regarding estimated protective immunity based only on circulating antiviral antibodies.

A multi-objective based clustering for identifying clonally-related sequences from high-throughput B cell repertoire data

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clones. A high number of clones in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity within clones can be related to antigen's selective pressure. Identifying clones is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial clones and iteratively applies two objective functions that optimize cohesion and separation within clones simultaneously. We show that under simulations with varied mutation rates, our method greatly improves clonal grouping as compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools. The method based on multi-objective clustering can accurately identify clone members, has fewer parameter settings and presents the lowest running time among existing tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context to unravel the mechanisms involved in the development and evolution of B cell malignancies.

Different B cell subpopulations show distinct patterns in their IgH repertoire metrics

Background: Several human B-cell subpopulations are recognized in the peripheral blood, which play distinct roles in the humoral immune response. These cells undergo developmental and maturational changes involving VDJ recombination, somatic hypermutation and class switch recombination, altogether shaping their immunoglobulin heavy chain (IgH) repertoire. Methods: Here, we sequenced the IgH repertoire of naïve, marginal zone, switched and plasma cells from 10 healthy adults along with matched unsorted and in silico separated CD19+ bulk B cells. We used advanced bioinformatic analysis and machine learning to thoroughly examine and compare these repertoires. Results: We show that sorted B cell subpopulations are characterised by distinct repertoire characteristics on both the individual sequence and the repertoire level. Sorted subpopulations shared similar repertoire characteristics with their corresponding in silico separated subsets. Furthermore, certain IgH repertoire characteristics correlated with the position of the constant region on the IgH locus. Conclusion: Overall, this study provides unprecedented insight over mechanisms of B cell repertoire control in peripherally circulating B cell subpopulations.

A third dose of inactivated vaccine augments the potency, breadth, and duration of anamnestic responses against SARS-CoV-2

Emergence of variants of concern (VOC) with altered antigenic structures and waning humoral immunity to SARS-CoV-2 are harbingers of a long pandemic. Administration of a third dose of an inactivated virus vaccine can boost the immune response. Here, we have dissected the immunogenic profiles of antibodies from 3-dose vaccinees, 2-dose vaccinees and convalescents. Better neutralization breadth to VOCs, expeditious recall and long-lasting humoral response bolster 3-dose vaccinees in warding off COVID-19. Analysis of 171 complex structures of SARS-CoV-2 neutralizing antibodies identified structure-activity correlates, revealing ultrapotent, VOCs-refractory and broad-spectrum antigenic patches. Construction of immunogenic and mutational heat maps revealed a direct relationship between "hot" immunogenic sites and areas with high mutation frequencies. Ongoing antibody somatic mutation, memory B cell clonal turnover and antibody composition changes in B cell repertoire driven by prolonged and repeated antigen stimulation confer development of monoclonal antibodies with enhanced neutralizing potency and breadth. Our findings rationalize the use of 3-dose immunization regimens for inactivated vaccines.