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Due to their shared genetic history, antibodies from the same clonotype often bind to the same epitope. This knowledge is used in immune repertoire mining, where known binders are used to search bulk sequencing repertoires to identify new binders. However, current computational methods cannot identify epitope convergence between antibodies from different clonotypes, limiting the sequence diversity of antigen-specific antibodies that can be identified. We describe how the antibody binding site, the paratope, can be used to cluster antibodies with common antigen reactivity from different clonotypes. Our method, paratyping, uses the predicted paratope to identify these novel cross clonotype matches. We experimentally validated our predictions on a pertussis toxoid dataset. Our results show that even the simplest abstraction of the antibody binding site, using only the length of the loops involved and predicted binding residues, is sufficient to group antigen-specific antibodies and provide additional information to conventional clonotype analysis. Abbreviations: BCR: B-cell receptor; CDR: complementarity-determining region; PTx: pertussis toxoid

Plasma antimalarial Ab can mediate antiparasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IGs) or Abs with those expressed on B cells as part of the B cell receptor. We applied this strategy to define plasma IG and to determine variable (V) gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. Using proteomic tools coupled with bulk immunosequencing data, we determined human antigen-binding fragment [F(ab′)2] peptide sequences from plasma IG of adults who received 4 doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab′)2 peptides (Pfs25-IG) were aligned to cDNA sequences of IG heavy (IGH) chain complementarity determining region 3 from a data set generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by V heavy/V light chain sequencing of Pfs25-specific single B cells from 5 vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma Ab repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful for studying circulating Abs in response to other vaccines as well as those induced during infections or autoimmune disorders., An approach to characterize the human plasma antibody repertoire is applied to define plasma Ig and determine variable V gene usage after malaria vaccination.

The advent of next‐generation sequencing (NGS) now allows a detailed assessment of the adaptive immune system in health and disease. In particular, high‐throughput B‐cell receptor (BCR) repertoire sequencing provides detailed information about the functionality and abnormalities of the B‐cell system. However, it is mostly unknown how the BCR repertoire is altered in the context of primary immunodeficiencies (PID) and whether findings are consistent throughout phenotypes and genotypes. We have performed an extensive literature search of the published work on BCR repertoire sequencing in PID patients, including several forms of predominantly antibody disorders and combined immunodeficiencies. It is somewhat surprising that BCR repertoires, even from severe clinical phenotypes, often show only mild abnormalities and that diversity or immunoglobulin gene segment usage is generally preserved to some extent. Despite the great variety of wet laboratory and analytical methods that were used in the different studies, several findings are common to most investigated PIDs, such as the increased usage of gene segments that are associated with self‐reactivity. These findings suggest that BCR repertoire characteristics may be used to assess the functionality of the B‐cell compartment irrespective of the underlying defect. With the use of NGS approaches, there is now the opportunity to apply BCR repertoire sequencing to multiple patients and explore the PID BCR repertoire in more detail. Ultimately, using BCR repertoire sequencing in translational research could aid the management of PID patients by improving diagnosis, estimating functionality of the immune system and improving assessment of prognosis.

Repertori de cèl·lules B; SARS-CoV-2; Anticòs Repertorio de células B; SARS-CoV-2; Anticuerpo B-cell repertoire; SARS-CoV-2; Antibody 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. MR is supported by an Engineering and Physical Sciences Research Council (EPSRC) and Medical Research Council (MRC) grant (EP/L016044/1). AK is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/M011224/1).

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 B-cell receptor (BCR) 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 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 BCR repertoires and stress the importance of using well-selected, age-appropriate controls in BCR studies.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

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.

Key Points FAS-dependent apoptosis in Vδ1 T cells makes the latter possible culprits for the lymphadenopathy observed in patients with FAS mutations. Rapamycin and methylprednisolone resistance should prompt clinicians to look for Vδ1 T cell proliferation in ALPS-FAS patients.

High-throughput sequencing allows detailed study of the B cell receptor (BCR) repertoire post-immunization but it remains unclear to what extent the de novo identification of antigen-specific sequences from the total BCR repertoire is possible. A Hib-MenC-TT conjugate vaccine containing H. influenzae type b (Hib) and group C meningococcal (MenC) polysaccharides as well as tetanus toxoid (TT) was used to investigate the BCR repertoire of adult humans following immunization and test the hypothesis that public or convergent repertoire analysis could identify antigen specific sequences. A number of antigen-specific BCR sequences have previously been reported for Hib and TT which made a vaccine containing these 2 antigens an ideal immunological stimulus. Analysis of identical complementarity determining region (CDR)3 amino acid (AA) sequences that were shared by individuals in the post-vaccine repertoire identified a number of known Hib-specific sequences but only one previously described TT sequence. The extension of this analysis to non-identical but highly similar CDR3 AA sequences revealed a number of other TT-related sequences. The anti-Hib avidity index post-vaccination was strongly correlated with the relative frequency of Hib-specific sequences, indicating that the post-vaccination public BCR repertoire may be related to more conventional measures of immunogenicity correlating with disease protection. Analysis of public BCR repertoire provided evidence of convergent BCR evolution in individuals exposed to the same antigens. If this finding is confirmed, the public repertoire could be used for rapid and direct identification of protective antigen-specific BCR sequences from peripheral blood.

Next-generation sequencing was used to investigate the B cell receptor heavy chain transcript repertoire of different B cell subsets (naïve, marginal zone, IgM memory and IgG memory) at baseline, and of plasma cells 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 germ-line and complementarity-determining region 3 length) that were conserved between individuals. However, analyzing the complementarity-determining region 3 amino acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 plasma cells demonstrated nearly tenfold greater sequence sharing between individuals than the baseline subsets, consistent with the plasma cells being induced by the vaccine antigen, and sharing specificity for a more limited range of epitopes. By annotating plasma cell sequences based on IgG subclass usage and mutation, and also comparing them to the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. Plasma cells produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the plasma cells were predominantly IgG2, less mutated, and were equally likely to be related to marginal zone, 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.Immunology and Cell Biology accepted article preview online, 15 May 2015. doi:10.1038/icb.2015.57.

Advances in next-generation sequencing now allow characterization of the global B-cell receptor (BCR) heavy-chain repertoire at a level that reflects its huge diversity. This technology has provided great insight into the structure of the BCR repertoire and how it responds to specific antigen stimuli. There are numerous potential clinical and research applications of BCR repertoire sequencing, but a major hurdle in the realization of these applications is the identification of the antigen-specific sequences of interest from within the total repertoire. To deconvolute the antigen-specific sequences from total repertoire, either a source of antigen-enriched sequence data is required with which to annotate the total repertoire, or de novo annotation methods must be used based on preconceptions of the features of antigen-specific sequences and their behavior following antigen-specific immune stimulation. We present a review of how these different methods can be applied to identify antigen-specific BCR sequences from the total BCR repertoire.

Nearly all licensed vaccines have been developed to confer protection against infectious diseases by stimulating the production of antibodies by B cells, but the nature of a successful antibody response has been difficult to capture. Recent advances in next-generation sequencing (NGS) technology have allowed high-resolution characterization of the antibody repertoire, and of the changes that occur following vaccination. These approaches have yielded important insights into the B cell response, and have raised the possibility of using specific antibody sequences as measures of vaccine immunogenicity. Here, we review recent findings based on antibody repertoire sequencing, and discuss potential applications of these new technologies and of the analyses of the increasing volume of antibody sequence data in the context of vaccine development. © 2014 Elsevier Ltd.