International ImMunoGeneTics Information System Research Articles

Uncommon HLA Alleles Observed in a Population of Istanbul Province

Objective: New polymorphisms are formed in human leucocyte antigen (HLA) genes with point mutations, gene conversions, and duplication, and the diversity continues to increase. Various new HLA alleles have significant roles in transplantation, and epidemiologic and population studies. The aim of our study was to determine the status of HLA alleles in the Turkish population, which is uncommon, well-defined, and non-defined in the world population according to the international ImMunoGeneTics information system® (IMGT) database. Methods: We performed HLA-A, -B, -C, -DQB1, and DRB1 loci at the four-field resolution level, using Sanger- sequence-based typing (SBT) for 5592 healthy, unrelated bone marrow donor volunteers from Istanbul Province. The uncommon alleles were also confirmed using high-throughput next-generation sequencing (NGS). Results: Uncommon alleles were determined at five loci as follows: HLA-A*01:155, 02:66, 02:90, 02:110, 02:343, 03:82, 24:28, 24:146, 24:276, 24:356, 31:23,33:33, 68:38; HLA-B *07:240, 18:19, 35:193, 40:303, 51:69, 51:169; HLA-C*04:39, 06:40, 07:93, 12:149, 15:73; HLA-DRB1*11:149, 13:14:02 and HLA-DQB1*03:27. All alleles were arranged according to the common and well‐documented (CWD) 3.0.0 catalog. Conclusion: This is the first study to show uncommon alleles in our population. These reported data increase the knowledge of HLA polymorphisms in the Turkish population and provide a basis for further studies in population genetics. This information may also be useful in determining whether a matched, unrelated donor is unlikely to be found so that a mismatch strategy, an extended family search, or alternate therapy, can be pursued, thus saving time and cost for patients.

Treating paroxysmal nocturnal hemoglobinuria with humanized IgG 2/4 kappa antibody: understanding the ravulizumab structure

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease caused by somatic mutations in hematopoietic stem cells. As molecular studies are essential for a better understanding of new drugs, tools such as the international ImMunoGeneTics information system® are powerful approaches to better understanding the molecular conformations, arrangements, and orientations of two-dimensional (2D) structures of PNH treatments. This review aims to characterize the structure of Ravulizumab, describe its drug design process, and show its amino acid sequence through IGMT Colliers de Perles. Methods: The IMGT/mAb-DB interface was used to evaluate therapeutic antibodies and to clarify their immunological applications and pharmacological effects. Results: Ravulizumab binds to C5 and blocks its activation by complement pathway convertases, thereby preventing the release of the proinflammatory C5a and activation of the terminal pathway. The healthcare cost of PNH patients decreased because smaller doses of RAVUL could promote full complement blockage, maintaining the therapeutic effect. Choosing the epitope, hinge, and Fc region is crucial to balance efficacy and undesirable effects. The better intermolecular interactions of RAVUL with binding sites are due to its nanostructure design. Conclusion: These results bridge the gap between linear amino acid sequences and 3D structures. It may guide the development of new drugs for rare diseases.

IMGT/mAb-KG: the knowledge graph for therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) have demonstrated promising outcomes in diverse clinical indications, including but not limited to graft rejection, cancer, and autoimmune diseases lately.Recognizing the crucial need for the scientific community to quickly and easily access dependable information on monoclonal antibodies (mAbs), IMGT®, the international ImMunoGeneTics information system®, provides a unique and invaluable resource: IMGT/mAb-DB, a comprehensive database of therapeutic mAbs, accessible via a user-friendly web interface. However, this approach restricts more sophisticated queries and segregates information from other databases. To connect IMGT/mAb-DB with the rest of the IMGT databases, we created IMGT/mAb-KG, a knowledge graph for therapeutic monoclonal antibodies connected to IMGT structures and genomics databases. IMGT/mAb-KG is developed using the most effective methodologies and standards of semantic web and acquires data from IMGT/mAb-DB. Concerning interoperability, IMGT/mAb-KG reuses terms from biomedical resources and is connected to related resources. In February 2024, IMGT/mAb-KG, encompassing a total of 139,629 triplets, provides access to 1,489 mAbs, approximately 500 targets, and over 500 clinical indications. It offers detailed insights into the mechanisms of action of mAbs, their construction, and their various products and associated studies. Linked to other resources such as Thera-SAbDab (Therapeutic Structural Antibody Database), PharmGKB (a comprehensive resource curating knowledge on the impact of genetic variation on drug response), PubMed, and HGNC (HUGO Gene Nomenclature Committee), IMGT/mAb-KG is an essential resource for mAb development. A user-friendly web interface facilitates the exploration and analyse of the content of IMGT/mAb-KG.

Delving into Molecular Pathways: Analyzing the Mechanisms of Action of Monoclonal Antibodies Integrated in IMGT/mAb-DB for Myasthenia Gravis.

Myasthenia Gravis (MG) is a rare autoimmune disease presenting with auto-antibodies that affect the neuromuscular junction. In addition to symptomatic treatment options, novel therapeutics include monoclonal antibodies (mAbs). IMGT®, the international ImMunoGeneTics information system®, extends the characterization of therapeutic antibodies with a systematic description of their mechanisms of action (MOA) and makes them available through its database for mAbs and fusion proteins, IMGT/mAb-DB. Using available literature data combined with amino acid sequence analyses from mAbs managed in IMGT/2Dstructure-DB, the IMGT® protein database, biocuration allowed us to define in a standardized way descriptions of MOAs of mAbs that target molecules towards MG treatment. New therapeutic targets include FcRn and molecules such as CD38, CD40, CD19, MS4A1, and interleukin-6 receptor. A standardized graphical representation of the MOAs of selected mAbs was created and integrated within IMGT/mAb-DB. The main mechanisms involved in these mAbs are either blocking or neutralizing. Therapies directed to B cell depletion and plasma cells have a blocking MOA with an immunosuppressant effect along with Fc-effector function (MS4A1, CD38) or FcγRIIb engager effect (CD19). Monoclonal antibodies targeting the complement also have a blocking MOA with a complement inhibitor effect, and treatments targeting T cells have a blocking MOA with an immunosuppressant effect (CD40) and Fc-effector function (IL6R). On the other hand, FcRn antagonists present a neutralizing MOA with an FcRn inhibitor effect. The MOA of each new mAb needs to be considered in association with the immunopathogenesis of each of the subtypes of MG in order to integrate the new mAbs as a viable and safe option in the therapy decision process. In IMGT/mAb-DB, mAbs for MG are characterized by their sequence, domains, and chains, and their MOA is described.

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Antibody Sequence and Structure Analyses Using IMGT®: 30 Years of Immunoinformatics.

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org , the global reference in immunogenetics and immunoinformatics, was created in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS) to manage the huge diversity of the antigen receptors, immunoglobulins (IG) or antibodies, and T cell receptors (TR) of the adaptive immune responses. The founding of IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® standardized analysis of the IG, TR, and major histocompatibility (MH) genes and proteins bridges the gap between sequences and three-dimensional (3D) structures, for all jawed vertebrates from fish to humans. This is achieved through the IMGT Scientific chart rules, based on the IMGT-ONTOLOGY axioms, and primarily CLASSIFICATION (IMGT gene and allele nomenclature) and NUMEROTATION (IMGT unique numbering and IMGT Colliers de Perles). IMGT® comprises seven databases (IMGT/LIGM-DB for nucleotide sequences, IMGT/GENE-DB for genes and alleles, etc.), 17 tools (IMGT/V-QUEST, IMGT/JunctionAnalysis, IMGT/HighV-QUEST for NGS, etc.), and more than 20,000 Web resources. In this chapter, the focus is on the tools for amino acid sequences per domain (IMGT/DomainGapAlign and IMGT/Collier-de-Perles), and on the databases for receptors (IMGT/2Dstructure-DB and IMGT/3D-structure-DB) described per receptor, chain, and domain and, for 3D, with contact analysis, paratope, and epitope. The IMGT/mAb-DB is the query interface for monoclonal antibodies (mAb), fusion proteins for immune applications (FPIA), composite proteins for clinical applications (CPCA), and related proteins of interest (RPI) with links to IMGT® 2D and 3D databases and to the World Health Organization (WHO) International Nonproprietary Names (INN) program lists. The chapter includes the human IG allotypes and antibody engineered variants for effector properties used in the description of therapeutical mAb.

Generation and Characterization of an Anti-diclazuril Monoclonal Antibody and Development of a Diagnostic Enzyme-Linked Immunosorbent Assay for Poultry.

An anti-diclazuril monoclonal antibody (mAb) was developed for use in enzyme-linked immunosorbent assay (ELISA)-based detection of diclazuril with high sensitivity and specificity, which can be used to measure anti-coccidial drug residues. The anti-diclazuril mAb had a half-maximal inhibitory concentration of 0.449–0.517 ng/mL. The mAb cross-reactivity with toltrazuril, toltrazuril 18 sulfone, clozaril, monesin, madurmycin, and salinomycin was very minimal (< 0.1%). The detection limit of the ELISA using this mAb was 0.10 ng/mL and the sensitivity was 0.05 ng/mL. A standard curve generated in the range of 0.05–16.2 ng/mL had a linear correlation coefficient value of ≥ 0.99. The average recoveries of diclazuril from chicken and duck samples ranged from 85.0 to 102.5%.Intra- and inter-assay coefficients of variation ranged from 5.9 to 8.5% and 9.2 to 12.6%, respectively. Using the International Immunogenetics Information System®, the VH domain of the mAb was found to be encoded by an IGHV3 family gene and had the following complementarity determining region (CDR) sequences: GFTFSRY (CDR1), SRGGS (CDR2), and GDDNYAFAY (CDR3). The VL domain was encoded by an IGKV1 family gene and had the following CDR sequences: KSSQSLLNSRTRKNYLA (CDR1), WASTRES (CDR2), and KQSYNLHT (CDR3). This study provides a method to generate anti-diclazuril mAbs and determine their variable region sequences. The diagnostic ELISA developed using this mAb may drive additional studies on the monitoring and detection of food and veterinary drug residues.

IMGT®Homo sapiens IG and TR Loci, Gene Order, CNV and Haplotypes: New Concepts as a Paradigm for Jawed Vertebrates Genome Assemblies.

IMGT®, the international ImMunoGeneTics information system®, created in 1989, by Marie-Paule Lefranc (Université de Montpellier and CNRS), marked the advent of immunoinformatics, a new science which emerged at the interface between immunogenetics and bioinformatics for the study of the adaptive immune responses. IMGT® is based on a standardized nomenclature of the immunoglobulin (IG) and T cell receptor (TR) genes and alleles from fish to humans and on the IMGT unique numbering for the variable (V) and constant (C) domains of the immunoglobulin superfamily (IgSF) of vertebrates and invertebrates, and for the groove (G) domain of the major histocompatibility (MH) and MH superfamily (MhSF) proteins. IMGT® comprises 7 databases, 17 tools and more than 25,000 pages of web resources for sequences, genes and structures, based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts. IMGT® reference directories are used for the analysis of the NGS high-throughput expressed IG and TR repertoires (natural, synthetic and/or bioengineered) and for bridging sequences, two-dimensional (2D) and three-dimensional (3D) structures. This manuscript focuses on the IMGT® Homo sapiens IG and TR loci, gene order, copy number variation (CNV) and haplotypes new concepts, as a paradigm for jawed vertebrates genome assemblies.

IMGT® Immunoinformatics Tools for Standardized V-DOMAIN Analysis.

The variable domains (V-DOMAIN) of the antigen receptors, immunoglobulins (IG) or antibodies and Tcell receptors (TR), which specifically recognize the antigens show a huge diversity in their sequences. This diversity results from the complex mechanisms involved in the synthesis of these domains at the DNA level (rearrangements of the variable (V), diversity (D), and joining (J) genes; N-diversity; and, for the IG, somatic hypermutations). The recognition of V, D, and J as "genes" and their entry in databases mark the creation of IMGT by Marie-Paule Lefranc, and the origin of immunoinformatics in 1989. For 30years, IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org , has implemented databases and developed tools for IG and TR immunoinformatics, based on the IMGT Scientific chart rules and IMGT-ONTOLOGY concepts and axioms, and more particularly, the princeps ones: IMGT genes and alleles (CLASSIFICATION axiom) and the IMGT unique numbering and IMGTCollierdePerles (NUMEROTATION axiom). This chapter describes the online tools for the characterization and annotation of the expressed V-DOMAIN sequences: (a) IMGT/V-QUEST analyzes in detail IG and TR rearranged nucleotide sequences, (b) IMGT/HighV-QUEST is its high throughput version, which includes a module for the identification of IMGT clonotypes and generates immunoprofiles of expressed V, D, and J genes and alleles, (c) IMGT/StatClonotype performs the pairwise comparison of IMGT/HighV-QUEST immunoprofiles, (d) IMGT/DomainGapAlign analyzes amino acid sequences and is frequently used in antibody engineering and humanization, and (e) IMGT/Collier-de-Perles provides two-dimensional (2D) graphical representations of V-DOMAIN, bridging the gap between sequences and 3D structures. These IMGT® tools are widely used in repertoire analyses of the adaptive immune responses in normal and pathological situations and in the design of engineered IG and TR for therapeutic applications.

IMGT/3Dstructure-DB: T-Cell Receptor TR Paratope and Peptide/Major Histocompatibility pMH Contact Sites and Epitope.

T-cell receptors (TR), the antigen receptors of T cells, specifically recognize peptides presented by the major histocompatibility (MH) proteins, as peptide/MH (pMH), on the cell surface. The structure characterization of the trimolecular TR/pMH complexes is crucial to the fields of immunology, vaccination, and immunotherapy. IMGT/3Dstructure-DB is the three-dimensional (3-D) structure database of IMGT®, the international ImMunoGenetics information system®. By its creation, IMGT® marks the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. The IMGT® immunoglobulin (IG) and TR gene and allele nomenclature (CLASSIFICATION axiom) and the IMGT unique numbering and IMGT/Collier-de-Perles (NUMEROTATION axiom) are the two founding breakthroughs of immunoinformatics. IMGT-ONTOLOGY concepts and IMGT Scientific chart rules generated from these axioms allowed IMGT® bridging genes, structures, and functions. IMGT/3Dstructure-DB contains 3-D structures of IG or antibodies, TR and MH proteins of the adaptive immune responses of jawed vertebrates (gnathostomata), IG or TR complexes with antigens (IG/Ag, TR/pMH), related proteins of the immune system of any species belonging to the IG and MH superfamilies, and fusion proteins for immune applications. The focus of this chapter is on the TR V domains and MH G domains and the contact analysis comparison in TR/pMH interactions. Standardized molecular characterization includes "IMGT pMH contact sites" for peptide and MH groove interactions and "IMGT paratopes and epitopes" for TR/pMH complexes. Data are available in the IMGT/3Dstructure database, at the IMGT Home page http://www.imgt.org .

IMGT® databases, related tools and web resources through three main axes of research and development.

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/, is at the forefront of the immunogenetics and immunoinformatics fields with more than 30 years of experience. IMGT® makes available databases and tools to the scientific community pertaining to the adaptive immune response, based on the IMGT-ONTOLOGY. We focus on the recent features of the IMGT® databases, tools, reference directories and web resources, within the three main axes of IMGT® research and development. Axis I consists in understanding the adaptive immune response, by deciphering the identification and characterization of the immunoglobulin (IG) and T cell receptor (TR) genes in jawed vertebrates. It is the starting point of the two other axes, namely the analysis and exploration of the expressed IG and TR repertoires based on comparison with IMGT reference directories in normal and pathological situations (Axis II) and the analysis of amino acid changes and functions of 2D and 3D structures of antibody and TR engineering (Axis III).

TR Locus Annotation and Characteristics of Rhinolophus ferrumequinum.

T-cell antigen receptors (TRs) in vertebrates can be divided into αβ or γδ, encoded by TRA/D, TRG, or TRB loci. TRs play a central role in mammal cellular immunity, which occurs by rearrangement of V, D, J, and C genes in the loci. The bat is the only mammal with flying ability and is considered the main host of zoonotic viruses, an important public health concern. However, at present, little is known about the composition of bat TR genes. Based on the whole genome sequence of the greater horseshoe bat (Rhinolophus ferrumequinum) and referring to the TR/IG annotation rules formulated by the international ImMunoGeneTics information system (IMGT), we present a complete annotation of TRA/D, TRG, and TRB loci of R. ferrumequinum. A total of 128 V segments, three D segments, 85 J segments, and 6 C segments were annotated and compared with other known mammalian data. The characteristics of the TR locus and germline genes of R. ferrumequinum are analyzed.

Antibody upstream sequence diversity and its biological implications revealed by repertoire sequencing

The sequence upstream of the antibody variable region (antibody upstream sequence [AUS]) consists of a 5′ untranslated region (5′ UTR) and a preceding leader region. The sequence variations in AUS affect antibody engineering and PCR based antibody quantification and may also be implicated in mRNA transcription and translation. However, the diversity of AUSs remains elusive. Using 5′ rapid amplification of cDNA ends and high-throughput antibody repertoire sequencing technique, we acquired full-length AUSs for human, rhesus macaque, cynomolgus macaque, mouse, and rat. We designed a bioinformatics pipeline and identified 3307 unique AUSs, corresponding to 3026 and 1457 unique sequences for 5′ UTR and leader region, respectively. Comparative analysis indicated that 928 (63.69%) leader sequences are novel relative to those recorded in the international ImMunoGeneTics information system. Evolutionarily, leader sequences are more conserved than 5′ UTR and seem to coevolve with their downstream V genes. Besides, single-nucleotide polymorphisms are position dependent for leader regions and may contribute to the functional reversal of the downstream V genes. Finally, the AUGs in AUSs were found to have little impact on gene expression. Taken together, our findings can facilitate primer design for capturing antibodies efficiently and provide a valuable resource for antibody engineering and molecule-level antibody studies.

Immunoinformatics taught to middle and high school pupils in IMGT®

IMGT®, the international ImMunoGeneTics information system® (http://www.imgt.org), is the global reference in immunogenetics and immunoinformatics. In 2019, for the first time, two pupils were welcomed within the team and introduced to immunoinformatics thank to the «Apprentis Chercheurs » programme founded by the association «l’Arbre des Connaissances ». This article describes the method and pedagogical approaches used, and demonstrates that immunoinformatics can be successfully taught to pupils of middle and high school.

Immunoinformatics taught to middle and high school pupils in IMGT®

IMGT®, the international ImMunoGeneTics information system® (http://www.imgt.org), is the global reference in immunogenetics and immunoinformatics. In 2019, for the first time, two pupils were welcomed within the team and introduced to immunoinformatics thank to the «Apprentis Chercheurs » programme founded by the association «l’Arbre des Connaissances ». This article describes the method and pedagogical approaches used, and demonstrates that immunoinformatics can be successfully taught to pupils of middle and high school.

IMGT® and 30 Years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.

At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. Under these HGM auspices, IMGT®, the international ImMunoGeneTics information system®, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties.

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC—focusing, to begin with, on human IGHV genes—with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

Single B cell sorting-based amplification and functional identification of HIV-1 monoclonal antibody genes

Objective To amplify and identify monoclonal antibody genes from HIV-1-infected patients. Methods Single cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay. Results The heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml. Conclusion In this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification. Key words: Single B cell sorting; Antibody gene; Sequence analysis; Binding ability; Neutralizing activity

VDJ Gene Usage among B-Cell Receptors in ABO-incompatible Kidney Transplantation Determined by RNA-seq Transcriptomic Analysis

Background Studies on B-cell subtypes and V(D)J gene usage of B-cell receptors in kidney transplants are scarce. This study aimed to investigate V(D)J gene segment usage in ABO-incompatible (ABOi) kidney transplant (KT) patients compared to that in ABO-compatible (ABOc) KT patients. Methods We selected 16 ABOi KT patients with accommodation (ABOiA), 6 ABOc stable KT patients (ABOcS), and 6 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10, whose graft tissue samples had been stored in the biorepository between 2010 and 2014. Complete transcriptomes of graft tissues were sequenced and analyzed through RNA sequencing (RNA-seq). The international ImMunoGeneTics information system (IMGT®) was used for in-depth comparison of V(D)J gene segment usage. Results The mean age of the 28 KT recipients was 43.3 ± 12.8 years, and 53.6% were male. By family, IGHV3, IGHJ4, IGLV2, and IGLJ3 gene segments were most frequently used in all groups, and their usage was not statistically different among the three patient groups. While IGKV3 was most frequently used in both the ABOiA and ABOiR groups, IGKV1 was most commonly used in the ABOcS group. In addition, while IGKJ1 was most commonly used in the ABOiA and ABOcS groups, IGKJ4 was most frequently used in the ABOiR group. According to individual gene segments, IGHV4-34 and IGHV4-30-2 were more commonly used in the ABOiR group than in the ABOiA group, and IGHV6-1 was more commonly used in the ABOcS group than in the ABOiR group. IGLV7-43 was more commonly used in the ABOcS group than in the ABOi group. However, technical variability, small sample size, and potential confounding effects of Rituximab or HLA mismatching are limitations of our study. Conclusions Our findings suggest that RNA-seq transcriptomic analyses can provide information on the V(D)J gene usage of B-cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT. Astellas Pharma Korea, Inc. The Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (grant NRF-2015M3A9D3051413).

Coupling of Single Molecule, Long Read Sequencing with IMGT/HighV-QUEST Analysis Expedites Identification of SIV gp140-Specific Antibodies from scFv Phage Display Libraries.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system® (IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9–25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.