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Introduction: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been posing a severe threat to global public health. Although broadly neutralizing antibodies have been used to prevent or treat corona virus disease 2019 (COVID-19), new emerging variants have been proven resistant to these antibodies. Methods: In this study, we isolated receptor binding domain (RBD)-specific memory B cells using single-cell sorting method from two COVID-19 convalescents and expressed the antibody to test their neutralizing activity against diverse SARS-CoV-2 variants. Then, we resolved antibody-RBD complex structures of potent RBD-specific neutralizing antibodies by X-ray diffraction method. Finally, we analyzed the whole antibody repertoires of the two donors and studied the evolutionary pathway of potent neutralizing antibodies. Results and discussion: We identified three potent RBD-specific neutralizing antibodies (1D7, 3G10 and 3C11) from two COVID-19 convalescents that neutralized authentic SARS-CoV-2 WH-1 and Delta variant, and one of them, 1D7, presented broadly neutralizing activity against WH-1, Beta, Gamma, Delta and Omicron authentic viruses. The resolved antibody-RBD complex structures of two antibodies, 3G10 and 3C11, indicate that both of them interact with the external subdomain of the RBD and that they belong to the RBD-1 and RBD-4 communities, respectively. From the antibody repertoire analysis, we found that the CDR3 frequencies of the light chain, which shared high degrees of amino acid identity with these three antibodies, were higher than those of the heavy chain. This research will contribute to the development of RBD-specific antibody-based drugs and immunogens against multiple variants. [ABSTRACT FROM AUTHOR]

Summary: Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (VHs) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human VHs, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines.

Keywords: B-cell receptor repertoire; next generation sequencing; antibodyome; immunoglobulin; B-cell clonotypes; vaccination; immunogenetics; immunoinformatics EN B-cell receptor repertoire next generation sequencing antibodyome immunoglobulin B-cell clonotypes vaccination immunogenetics immunoinformatics 1 4 4 07/03/20 20200630 NES 200630 The next-generation sequencing (NGS) analysis of human antibody repertoires has enabled a heightened appreciation and comprehensive characterization of the B-cell receptor (BCR) landscape at an unprecedented resolution ([1]-[4]). These studies illustrate means by which to harness the power of NGS for antibody discovery, B-cell immunogenetics, and the evolution of affinity maturation, as well as the investigation of antibody lineages in HIV-1/SIV infections. Also, large-scale NGS analysis of individual antibodyome will lead to improved insights into overall diversity of the human antibody repertoire and B cell immunogenetics ([15]-[17]). [Extracted from the article]

In the decade since the human genome sequence was declared complete, the development of next-generation sequencing (NGS) or ‘deep’ sequencing to deliver cost-effective genomic sequencing has influenced advances beyond its primary application and changed the research landscape in many other areas. This review will survey recent applications of NGS which have broadened the understanding of natural antibody repertoires (the antibodyome) and how these evolve in response to viral infection. We will also report examples where deep sequencing of binding populations, derived from both natural and synthetic repertoires, have been used to benefit antibody engineering. This knowledge will ultimately lead to the design of more effective biological drugs and vaccines.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world's most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel's sifaka ( Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel's sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. [ABSTRACT FROM AUTHOR]

Abstract: The vast diversity of antibody repertoires is largely attributed to heavy chain (VH) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some VH families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines. [Copyright &y& Elsevier]

In the decade since the human genome sequence was declared complete, the development of next generation sequencing (NGS) or "deep" sequencing to deliver cost-effective genomic sequencing has influenced advances beyond its primary application and changed the research landscape in many other areas. This review will survey recent applications of NGS which have broadened the understanding of natural antibody repertoires (the "antibodyome") and howthese evolve in response to viral infection.We will also report examples where deep sequencing of binding populations, derived from both natural and synthetic repertoires, have been used to benefit antibody engineering.This knowledge will ultimately lead to the design of more effective biological drugs and vaccines. [ABSTRACT FROM AUTHOR]

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations. [ABSTRACT FROM AUTHOR]

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (V H s) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human V H s, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines. • NGS-based non-canonical cysteine landscape in human V H s • 1 to 8 non-canonical cysteines and up to 30% in long CDR-H3s • An array of potential disulfide motifs adds paratope diversity • Non-canonical cysteines in human V H s are reminiscent of lower animals Prabakaran and Chowdhury reveal the remarkable patterns of non-canonical cysteines in human antibody heavy chains (V H s) and their role in paratope diversification. These patterns mimic features observed separately in chicken, camel, llama, shark, and cow. These findings can help design and develop next-generation human antibodies and libraries. [ABSTRACT FROM AUTHOR]

Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.

Next-generation sequencing is making it possible to study the antibody repertoire of an organism in unprecedented detail, and, by so doing, to characterize its behavior in the response to infection and in pathological conditions such as autoimmunity and cancer. The polymorphic nature of the repertoire poses unique challenges that rule out the use of many commonly used NGS methods and require tradeoffs to be made when considering experimental design.We outline the main contexts in which antibody repertoire analysis has been used, and summarize the key tools that are available. The humoral immune response to vaccination has been a particular focus of repertoire analyses, and we review the key conclusions and methods used in these studies.