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

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. Keywords: B cells; diagnostics; human; immunoglobulin; immunology; inflammation; machine learning; prediction; repertoire

Structural diversity of B-cell receptor repertoires along the B-cell differentiation axis in humans and mice

Most current analysis tools for antibody next-generation sequencing data work with primary sequence descriptors, leaving accompanying structural information unharnessed. We have used novel rapid methods to structurally characterize the complementary-determining regions (CDRs) of more than 180 million human and mouse B-cell receptor (BCR) repertoire sequences. These structurally annotated CDRs provide unprecedented insights into both the structural predetermination and dynamics of the adaptive immune response. We show that B-cell types can be distinguished based solely on these structural properties. Antigen-unexperienced BCR repertoires use the highest number and diversity of CDR structures and these patterns of naïve repertoire paratope usage are highly conserved across subjects. In contrast, more differentiated B-cells are more personalized in terms of CDR structure usage. Our results establish the CDR structure differences in BCR repertoires and have applications for many fields including immunodiagnostics, phage display library generation, and “humanness” assessment of BCR repertoires from transgenic animals. The software tool for structural annotation of BCR repertoires, SAAB+, is available at https://github.com/oxpig/saab_plus

Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing

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

Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Patients Reveals Strong Convergent Immune Signatures.

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

Using antibody next generation sequencing data to aid antibody engineering

Future successful exploitation of antibodies as diagnostic and therapeutic agents will greatly benefit from an increased understanding of natural B-cell receptor (BCR) repertoire diversities. The advent of next-generation sequencing of immunoglobulin genes (Ig-seq) has made it possible to sequence large snapshots of BCR repertoires in a single experiment. In the results chapters of this thesis, we begin by describing a method (AntiBOdy Sequence Selector, “ABOSS”) for filtering BCR repertoire data, which considers the structural viability of each sequence and is orthogonal to all other current methods (Chapter 2). ABOSS leverages the presence/absence of a conserved disulphide bridge found in antibodies as a way of both identifying structurally viable BCR sequences and estimating the sequencing error rate. We show that this method is able to identify structurally impossible sequences missed by common error-correction methods. Next, we describe the development of Observed Antibody Space (OAS), the first resource that curates BCR sequences from publicly available studies. As of October 2020, OAS contains more than 1.9 billion sequences from 85 studies. In OAS, all BCR repertoire sequences are annotated and profiled for structural viability. We next describe the development of a novel method (SAAB+) to interrogate complete BCR repertoires at the structural level (Chapter 4). SAAB+ annotates large portions of BCR repertoires with three-dimensional information by mapping sequences to crystallographically solved antibody structures. By applying SAAB+ to BCR repertoires in OAS we, for the first time, document repertoire structural changes along the B-cell maturation axis in humans and mice. In the final experimental chapter, we describe our work in COVID-19 research where we have compared the structural and sequence diversities of SARS-CoV-2 BCR repertoires to healthy repertoires deposited in OAS. We also outline the development of the first organised database (CoV-AbDab) that curates all publicly available anti-SARS-CoV-2 antibodies in a standardised format. Finally, we discuss how recent developments in paired-chain Ig-seq platforms and deep learning algorithms could have a lasting impact on established Ig-seq analysis pipelines. We also outline how the tools described in this thesis can be combined with these field-disruptive technologies to advance our understanding of the immune system and improve computational antibody engineering

Public Baseline and shared response structures support the theory of antibody repertoire functional commonality.

The naïve antibody/B-cell receptor (BCR) repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger an effective antibody response to immunodominant epitopes. Sequence-based repertoire analysis has so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared ('public') antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we search for evidence of geometric similarity/convergence across human antibody repertoires. We first structurally profile naïve ('baseline') antibody diversity using snapshots from 41 unrelated individuals, predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples ('Public Baseline' structures). Our approach is the first computational method to find levels of BCR commonality commensurate with epitope immunodominance and could therefore be harnessed to find more genetically distant antibodies with same-epitope complementarity. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes ('Public Response' structures). We show that Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening

Structural diversity of B-cell receptor repertoires along the B-cell differentiation axis in humans and mice.

Most current analysis tools for antibody next-generation sequencing data work with primary sequence descriptors, leaving accompanying structural information unharnessed. We have used novel rapid methods to structurally characterize the complementary-determining regions (CDRs) of more than 180 million human and mouse B-cell receptor (BCR) repertoire sequences. These structurally annotated CDRs provide unprecedented insights into both the structural predetermination and dynamics of the adaptive immune response. We show that B-cell types can be distinguished based solely on these structural properties. Antigen-unexperienced BCR repertoires use the highest number and diversity of CDR structures and these patterns of naïve repertoire paratope usage are highly conserved across subjects. In contrast, more differentiated B-cells are more personalized in terms of CDR structure usage. Our results establish the CDR structure differences in BCR repertoires and have applications for many fields including immunodiagnostics, phage display library generation, and "humanness" assessment of BCR repertoires from transgenic animals. The software tool for structural annotation of BCR repertoires, SAAB+, is available at https://github.com/oxpig/saab_plus

Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Patients Reveals Strong Convergent Immune Signatures

Repertori de cèl·lules B; SARS-CoV-2; AnticòsRepertorio de células B; SARS-CoV-2; AnticuerpoB-cell repertoire; SARS-CoV-2; AntibodyDeep 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)