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Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies.

Several human B cell subpopulations are recognised 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.

Next-generation sequencing of immunoglobulin gene repertoires (Ig-seq) allows the investigation of large-scale antibody dynamics at a sequence level. However, structural information, a crucial descriptor of antibody binding capability, is not collected in Ig-seq protocols. Developing systematic relationships between the antibody sequence information gathered from Ig-seq and low-throughput techniques such as X-ray crystallography could radically improve our understanding of antibodies. The mapping of Ig-seq datasets to known antibody structures can indicate structurally, and perhaps functionally, uncharted areas. Furthermore, contrasting naïve and antigenically challenged datasets using structural antibody descriptors should provide insights into antibody maturation. As the number of antibody structures steadily increases and more and more Ig-seq datasets become available, the opportunities that arise from combining the two types of information increase as well. Here, we review how these data types enrich one another and show potential for advancing our knowledge of the immune system and improving antibody engineering.

Background Vaccines have greatly reduced the burden of infectious disease, ranking in their impact on global health second only after clean water. Most vaccines confer protection by the production of antibodies with binding affinity for the antigen, which is the main effector function of B cells. This results in short term changes in the B cell receptor (BCR) repertoire when an immune response is launched, and long term changes when immunity is conferred. Analysis of antibodies in serum is usually used to evaluate vaccine response, however this is limited and therefore the investigation of the BCR repertoire provides far more detail for the analysis of vaccine response. Results Here, we introduce a novel Bayesian model to describe the observed distribution of BCR sequences and the pattern of sharing across time and between individuals, with the goal to identify vaccine-specific BCRs. We use data from two studies to assess the model and estimate that we can identify vaccine-specific BCRs with 69% sensitivity. Conclusion Our results demonstrate that statistical modelling can capture patterns associated with vaccine response and identify vaccine specific B cells in a range of different data sets. Additionally, the B cells we identify as vaccine specific show greater levels of sequence similarity than expected, suggesting that there are additional signals of vaccine response, not currently considered, which could improve the identification of vaccine specific B cells.

Next‐generation sequencing was used to investigate the B‐cell receptor heavy chain transcript repertoire of different B‐cell subsets (naive, marginal zone (MZ), immunoglobulin M (IgM) memory and IgG memory) at baseline, and of plasma cells (PCs) 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein–polysaccharide conjugate vaccines. Baseline B‐cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germline and complementarity‐determining region 3 (CDR3) length) that were conserved between individuals. However, analyzing the CDR3 amino‐acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 PCs demonstrated nearly 10‐fold greater sequence sharing between individuals than the baseline subsets, consistent with the PCs being induced by the vaccine antigen and sharing specificity for a more limited range of epitopes. By annotating PC sequences based on IgG subclass usage and mutation, and also comparing them with the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. PCs produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the PCs were predominantly IgG2, less mutated and were equally likely to be related to MZ, IgM memory or IgG memory cells. High‐throughput B‐cell repertoire sequencing thus provides a unique insight into patterns of B‐cell activation not possible from more conventional measures of immunogenicity.

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species’ repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B cells are able to go through complete development.

High-throughput sequencing of the B cell receptor (BCR) repertoire can provide rapid characterization of the B cell response in a wide variety of applications in health, after vaccination and in infectious, inflammatory and immune-driven disease, and is starting to yield clinical applications. However, the interpretation of repertoire data is compromised by a lack of studies to assess the intra and inter-individual variation in the BCR repertoire over time in healthy individuals. We applied a standardized isotype-specific BCR repertoire deep sequencing protocol to a single highly sampled participant, and then evaluated the method in 9 further participants to comprehensively describe such variation. We assessed total repertoire metrics of mutation, diversity, VJ gene usage and isotype subclass usage as well as tracking specific BCR sequence clusters. There was good assay reproducibility (both in PCR amplification and biological replicates), but we detected striking fluctuations in the repertoire over time that we hypothesize may be due to subclinical immune activation. Repertoire properties were unique for each individual, which could partly be explained by a decrease in IgG2 with age, and genetic differences at the immunoglobulin locus. There was a small repertoire of public clusters (0.5, 0.3, and 1.4% of total IgA, IgG, and IgM clusters, respectively), which was enriched for expanded clusters containing sequences with suspected specificity toward antigens that should have been historically encountered by all participants through prior immunization or infection. We thus provide baseline BCR repertoire information that can be used to inform future study design, and aid in interpretation of results from these studies. Furthermore, our results indicate that BCR repertoire studies could be used to track changes in the public repertoire in and between populations that might relate to population immunity against infectious diseases, and identify the characteristics of inflammatory and immunological diseases.

Every human possesses millions of distinct antibodies. It is now possible to analyze this diversity next-generation sequencing of immunoglobulin genes (Ig-seq). This technique produces large volume sequence snapshots of B-cell receptors that are indicative of the antibody repertoire. In this paper, we enrich these large-scale sequence datasets with structural information. Enriching a sequence with its structural data allows better approximation of many vital features, such as its binding site and specificity. Here, we describe the structural annotation of antibodies pipeline that maps the outputs of large Ig-seq experiments to known antibody structures. We demonstrate the viability of our protocol on five separate Ig-seq datasets covering ca. 35 m unique amino acid sequences from ca. 600 individuals. Despite the great theoretical diversity of antibodies, we find that the majority of sequences coming from such studies can be reliably mapped to an existing structure.

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.