Reprogramming the antigen specificity of B cells using genome-editing technologies

  1. James E Voss  Is a corresponding author
  2. Alicia Gonzalez-Martin  Is a corresponding author
  3. Raiees Andrabi
  4. Roberta P Fuller
  5. Ben Murrell
  6. Laura E McCoy
  7. Katelyn Porter
  8. Deli Huang
  9. Wenjuan Li
  10. Devin Sok
  11. Khoa Le
  12. Bryan Briney
  13. Morgan Chateau
  14. Geoffrey Rogers
  15. Lars Hangartner
  16. Ann J Feeney
  17. David Nemazee
  18. Paula Cannon
  19. Dennis Burton  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Universidad Autónoma de Madrid (UAM), Spain
  3. Karolinska Institutet, United States
  4. University College London, United Kingdom
  5. University of Southern California, United States

Abstract

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope by FACS and sequenced mRNA from these cells showed PG9 HC expressed as several different isotypes after culture with CD40 ligand and IL-4.

Data availability

Next generation sequencing data from RT-PCR amplicons have been deposited at Dryad:DOI: https://doi.org/10.5061/dryad.45j0r70.Amplification free whole genome sequencing reads mapped to the human reference genome have been deposited to NCBI with BioSample accession numbers SAMN09404498 and SAMN09404497

The following data sets were generated

Article and author information

Author details

  1. James E Voss

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    jvoss@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4777-1596
  2. Alicia Gonzalez-Martin

    Department of Biochemistry, Universidad Autónoma de Madrid (UAM), Madrid, Spain
    For correspondence
    alicia.gonzalezm@uam.es
    Competing interests
    The authors declare that no competing interests exist.
  3. Raiees Andrabi

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Roberta P Fuller

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ben Murrell

    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Laura E McCoy

    Division of Infection and Immunity, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Katelyn Porter

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Deli Huang

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wenjuan Li

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Devin Sok

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Khoa Le

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Bryan Briney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Morgan Chateau

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Geoffrey Rogers

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Lars Hangartner

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Ann J Feeney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. David Nemazee

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Paula Cannon

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  19. Dennis Burton

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    burton@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (5R01DE025167-05)

  • Dennis Burton

Bill and Melinda Gates Foundation (OPP1183956)

  • James E Voss

Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades (RYC-2016-21155)

  • Alicia Gonzalez-Martin

Marie-Curie Fellowship (FP7-PEOPLE-2013-IOF)

  • Laura E McCoy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Voss et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 11,270
    views
  • 1,645
    downloads
  • 76
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. James E Voss
  2. Alicia Gonzalez-Martin
  3. Raiees Andrabi
  4. Roberta P Fuller
  5. Ben Murrell
  6. Laura E McCoy
  7. Katelyn Porter
  8. Deli Huang
  9. Wenjuan Li
  10. Devin Sok
  11. Khoa Le
  12. Bryan Briney
  13. Morgan Chateau
  14. Geoffrey Rogers
  15. Lars Hangartner
  16. Ann J Feeney
  17. David Nemazee
  18. Paula Cannon
  19. Dennis Burton
(2019)
Reprogramming the antigen specificity of B cells using genome-editing technologies
eLife 8:e42995.
https://doi.org/10.7554/eLife.42995

Share this article

https://doi.org/10.7554/eLife.42995

Further reading

    1. Immunology and Inflammation
    2. Microbiology and Infectious Disease
    Benita Martin-Castaño, Patricia Diez-Echave ... Julio Galvez
    Research Article

    Coronavirus disease 2019 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that displays great variability in clinical phenotype. Many factors have been described to be correlated with its severity, and microbiota could play a key role in the infection, progression, and outcome of the disease. SARS-CoV-2 infection has been associated with nasopharyngeal and gut dysbiosis and higher abundance of opportunistic pathogens. To identify new prognostic markers for the disease, a multicentre prospective observational cohort study was carried out in COVID-19 patients divided into three cohorts based on symptomatology: mild (n = 24), moderate (n = 51), and severe/critical (n = 31). Faecal and nasopharyngeal samples were taken, and the microbiota was analysed. Linear discriminant analysis identified Mycoplasma salivarium, Prevotella dentalis, and Haemophilus parainfluenzae as biomarkers of severe COVID-19 in nasopharyngeal microbiota, while Prevotella bivia and Prevotella timonensis were defined in faecal microbiota. Additionally, a connection between faecal and nasopharyngeal microbiota was identified, with a significant ratio between P. timonensis (faeces) and P. dentalis and M. salivarium (nasopharyngeal) abundances found in critically ill patients. This ratio could serve as a novel prognostic tool for identifying severe COVID-19 cases.

    1. Immunology and Inflammation
    2. Microbiology and Infectious Disease
    Yan Zhao, Hanshuo Zhu ... Li Sun
    Research Article

    Type III secretion system (T3SS) is a virulence apparatus existing in many bacterial pathogens. Structurally, T3SS consists of the base, needle, tip, and translocon. The NLRC4 inflammasome is the major receptor for T3SS needle and basal rod proteins. Whether other T3SS components are recognized by NLRC4 is unclear. In this study, using Edwardsiella tarda as a model intracellular pathogen, we examined T3SS−inflammasome interaction and its effect on cell death. E. tarda induced pyroptosis in a manner that required the bacterial translocon and the host inflammasome proteins of NLRC4, NLRP3, ASC, and caspase 1/4. The translocon protein EseB triggered NLRC4/NAIP-mediated pyroptosis by binding NAIP via its C-terminal region, particularly the terminal 6 residues (T6R). EseB homologs exist widely in T3SS-positive bacteria and share high identities in T6R. Like E. tarda EseB, all of the representatives of the EseB homologs exhibited T6R-dependent NLRC4 activation ability. Together these results revealed the function and molecular mechanism of EseB to induce host cell pyroptosis and suggested a highly conserved inflammasome-activation mechanism of T3SS translocon in bacterial pathogens.