THE MAJOR ROLE OF JUNCTIONAL DIVERSITY IN THE HORSE ANTIBODY REPERTOIRE

The sequencing of the antibody repertoire (Rep-seq) revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis of diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-immunized horses using HTS technology. In this study, we obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 and 82% in IgM and IgG isotypes. We observed that besides horses antibody can use all of the functional IGHV genes, around 80% of their antibodies use only three IGHV gene segments, and around 55% use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horses antibodies is highly frequent, present in more than 90% of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45% of the antibody clones have more than ten nucleotides in both N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and in other species to better understand antibody repertoire characteristics.

[1]  Carolina Q. Sacramento,et al.  Polyclonal F(ab’)2 fragments of equine antibodies raised against the spike protein neutralize SARS-CoV-2 variants with high potency , 2021, iScience.

[2]  Carolina Q. Sacramento,et al.  Equine hyperimmune globulin raised against the SARS-CoV-2 spike glycoprotein has extremely high neutralizing titers , 2020 .

[3]  Narmada Thanki,et al.  CDD/SPARCLE: the conserved domain database in 2020 , 2019, Nucleic Acids Res..

[4]  James E. Crowe,et al.  High frequency of shared clonotypes in human B cell receptor repertoires , 2019, Nature.

[5]  F. Molina,et al.  Next‐generation sequencing reveals new insights about gene usage and CDR‐H3 composition in the horse antibody repertoire , 2019, Molecular immunology.

[6]  D. Burton,et al.  Commonality despite exceptional diversity in the baseline human antibody repertoire , 2018, Nature.

[7]  D. Wesemann,et al.  Analyzing Immunoglobulin Repertoires , 2018, Front. Immunol..

[8]  M. Pecaut,et al.  Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing , 2018, PloS one.

[9]  R. Suzuki,et al.  Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis , 2017, Front. Immunol..

[10]  G. Ippolito,et al.  Comparative analysis of the feline immunoglobulin repertoire. , 2017, Biologicals : journal of the International Association of Biological Standardization.

[11]  D. Merico,et al.  Preferential expression of IGHV and IGHD encoding antibodies with exceptionally long CDR3H and a rapid global shift in transcriptome characterizes development of bovine neonatal immunity , 2017, Developmental and comparative immunology.

[12]  A. Chao,et al.  iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers) , 2016 .

[13]  Patrice Duroux,et al.  IMGT/StatClonotype for Pairwise Evaluation and Visualization of NGS IG and TR IMGT Clonotype (AA) Diversity or Expression from IMGT/HighV-QUEST , 2016, Front. Immunol..

[14]  M. Delarue,et al.  Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination. , 2016, Structure.

[15]  Joseph Kaplinsky,et al.  Robust estimates of overall immune-repertoire diversity from high-throughput measurements on samples , 2016, Nature Communications.

[16]  U. Diesterbeck,et al.  Equine immunoglobulins and organization of immunoglobulin genes. , 2015, Developmental and comparative immunology.

[17]  Johannes Trück,et al.  In-Depth Assessment of Within-Individual and Inter-Individual Variation in the B Cell Receptor Repertoire , 2015, Front. Immunol..

[18]  R. Tallmadge,et al.  Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse. , 2014, Developmental and comparative immunology.

[19]  Z. Suo,et al.  N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis. , 2014, DNA repair.

[20]  X. He,et al.  Comparative analysis of human and mouse immunoglobulin variable heavy regions from IMGT/LIGM-DB with IMGT/HighV-QUEST , 2014, Theoretical Biology and Medical Modelling.

[21]  J. Galson,et al.  Studying the antibody repertoire after vaccination: practical applications. , 2014, Trends in immunology.

[22]  M. Niku,et al.  Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus , 2014, PloS one.

[23]  David A. Hafler,et al.  pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires , 2014, Bioinform..

[24]  G. Ippolito,et al.  Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice. , 2014, Molecular immunology.

[25]  S. Quake,et al.  The promise and challenge of high-throughput sequencing of the antibody repertoire , 2014, Nature Biotechnology.

[26]  J. Thurn,et al.  Decreased Mutation Frequencies among Immunoglobulin G Variable Region Genes during Viremic HIV-1 Infection , 2014, PloS one.

[27]  R. Tallmadge,et al.  Developmental progression of equine immunoglobulin heavy chain variable region diversity. , 2013, Developmental and comparative immunology.

[28]  Liming Ren,et al.  Immunoglobulin genes and diversity: what we have learned from domestic animals , 2012, Journal of Animal Science and Biotechnology.

[29]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[30]  C. Nusbaum,et al.  High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans , 2011, PloS one.

[31]  Liming Ren,et al.  A comprehensive analysis of germline and expressed immunoglobulin repertoire in the horse. , 2010, Developmental and comparative immunology.

[32]  M. Ares,et al.  Purification of RNA using TRIzol (TRI reagent). , 2010, Cold Spring Harbor protocols.

[33]  Jan Berka,et al.  Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire , 2009, Proceedings of the National Academy of Sciences.

[34]  I. Kacskovics,et al.  Porcine IgG: structure, genetics, and evolution , 2009, Immunogenetics.

[35]  U. Stenzel,et al.  PatMaN: rapid alignment of short sequences to large databases , 2008, Bioinform..

[36]  Kevin A. Fiala,et al.  Up-regulation of the Fidelity of Human DNA Polymerase λ by Its Non-enzymatic Proline-rich Domain* , 2006, Journal of Biological Chemistry.

[37]  J. C. Almagro,et al.  Analysis of the horse VH repertoire and comparison with the human IGHV germline genes, and sheep, cattle and pig VH sequences , 2006 .

[38]  J. Spencer,et al.  Hypermutation at A-T Base Pairs: The A Nucleotide Replacement Spectrum Is Affected by Adjacent Nucleotides and There Is No Reverse Complementarity of Sequences Flanking Mutated A and T Nucleotides12 , 2005, The Journal of Immunology.

[39]  G. Crooks,et al.  WebLogo: a sequence logo generator. , 2004, Genome research.

[40]  Jishan Sun,et al.  Antibody Repertoire Development in Fetal and Neonatal Piglets. VI. B Cell Lymphogenesis Occurs at Multiple Sites with Differences in the Frequency of In-frame Rearrangements1 , 2003, The Journal of Immunology.

[41]  G. Ippolito,et al.  Constraints on the Hydropathicity and Sequence Composition of HCDR3 are Conserved Across Evolution , 2002 .

[42]  C. Papanicolaou,et al.  Crystal structures of a template‐independent DNA polymerase: murine terminal deoxynucleotidyltransferase , 2002, The EMBO journal.

[43]  R. Tarone,et al.  Third complementarity‐determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non‐mutated genes , 2001, Immunology.

[44]  J. Lang,et al.  Safety and immunogenicity of a new equine tetanus immunoglobulin associated with tetanus-diphtheria vaccine. , 2000, The American journal of tropical medicine and hygiene.

[45]  A. Bogan,et al.  Anatomy of hot spots in protein interfaces. , 1998, Journal of molecular biology.

[46]  A. Casadevall,et al.  Serum Therapy for Tuberculosis Revisited: Reappraisal of the Role of Antibody-Mediated Immunity againstMycobacterium tuberculosis , 1998, Clinical Microbiology Reviews.

[47]  Jean-Claude Weill,et al.  Somatic hyperconversion diversifies the single VH gene of the chicken with a high incidence in the D region , 1989, Cell.

[48]  F. Burnet.  A modification of jerne's theory of antibody production using the concept of clonal selection , 1976, CA: a cancer journal for clinicians.

[49]  J. T. Curtis,et al.  An Ordination of the Upland Forest Communities of Southern Wisconsin , 1957 .

[50]  H. Moore,et al.  THE PRODUCTION OF ANTIPNEUMOCOCCIC SERUM , 1917, The Journal of experimental medicine.

[51]  L. Bukata,et al.  Development of a hyperimmune equine serum therapy for COVID-19 in Argentina. , 2020, Medicina.

[52]  Patrice Duroux,et al.  IMGT/HIGHV-QUEST: THE IMGT® WEB PORTAL FOR IMMUNOGLOBULIN (IG) OR ANTIBODY AND T CELL RECEPTOR (TR) ANALYSIS FROM NGS HIGH THROUGHPUT AND DEEP SEQUENCING , 2012 .

[53]  R. Schelonka,et al.  Genetic control of DH reading frame and its effect on B-cell development and antigen-specifc antibody production. , 2010, Critical reviews in immunology.

[54]  Mehdi Yousfi Monod,et al.  IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs , 2004, ISMB/ECCB.

[55]  V. Giudicelli,et al.  IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. , 2003, Developmental and comparative immunology.

[56]  C. G. Anderson.  The distribution of diphtheria antitoxin in pepsin-digested horse antiserum. , 1955, The Biochemical journal.