Efficient generation of human IgA monoclonal antibodies
Introduction
Humoral immunity is essential to host protection against human infections, and relies on the phenomenal diversity of antibody molecules, which sense and inactivate pathogens. At mucosal surfaces, antibodies, and more specifically IgA immunoglobulins produced locally by sub-epithelial plasma cells, play a key role in the protection against toxins, bacteria, viruses and protozoa (Kaetzel, 2007). There are two IgA subclasses in humans, IgA1 and IgA2, which can exist as either monomers or as multimers made of IgA monomeric units polymerized by covalent bounds with the J-chain (principally dimers). IgAs are found in diverse mucosal secretions but are also abundant in serum (2–3 mg/ml) predominantly in the monomeric and IgA1 form (Kaetzel, 2007). On the other hand, mucosal IgAs are mainly present as “secretory IgA” (SIgAs) that are IgA polymers covalently associated with the “secretory component”, which corresponds to the extracellular domain of the polymeric immunoglobulin receptor (pIgR) cleaved away from the surface of epithelial cells (Kaetzel, 2007). Apart from their well-documented role in protecting epithelia from invading pathogens, SIgAs possess additional biological activities; they can act as anti-inflammatory and anti-allergenic molecules, maintain mucosal homeostasis, and regulate intestinal microbiota (Mantis et al., 2011). Surprisingly, the role of circulating serum IgAs is less clear, and should be explored more thoroughly.
In the past 15 years, the use of single cell antibody cloning techniques has enabled the rapid and efficient production of human monoclonal antibodies from different B-cell compartments (Sullivan et al., 2011, Tiller, 2011, Wilson and Andrews, 2012). The molecular and functional characterization of antibodies generated from selected single B-cell sub-populations (captured by flow cytometry or derived from screening of cultured B cells) has provided invaluable insight into the physiological and pathophysiological aspects of humoral immunity in humans (Wardemann and Nussenzweig, 2007, Meffre, 2011, Wilson and Andrews, 2012, Mouquet, 2014). Such studies have: (i) defined B-cell tolerance checkpoints at the transitions between different B-cell developmental stages (Wardemann et al., 2003, Tsuiji et al., 2006, Scheid et al., 2011); (ii) uncovered some of the tolerance mechanisms involved by studying these checkpoints in immunodeficient patients (Ng et al., 2004, Herve et al., 2005, Isnardi et al., 2008, Menard et al., 2011a); (iii) revealed defective tolerance checkpoints in autoimmune diseases (Samuels et al., 2005, Yurasov et al., 2005, Menard et al., 2011a, Menard et al., 2011b); (iv) dissected antibody responses to various pathogens (Wrammert et al., 2008, Scheid et al., 2009, Muellenbeck et al., 2013); (v) identified and characterized broadly neutralizing antiviral antibodies particularly, against HIV-1 (Mouquet, 2014). In fact, probing humoral immunity using single B-cell derived monoclonal antibodies, as both effectors of and surrogates for immune responses, has not only provided invaluable insight into the mechanisms that govern adaptive B-cell responses, but has also contributed to the development of therapeutic and/or vaccine strategies to fight human diseases. However, too few studies have investigated the role IgA-mediated humoral immunity in humans (Di Niro et al., 2010, Di Niro et al., 2012, Benckert et al., 2011), and in none of those, recombinant monoclonals were produced as native IgA antibodies, which definitively restricts certain molecular and functional characterizations.
In order to study IgA biology and IgA B-cell responses in physiological and pathophysiological conditions, we designed an expression cloning-based methodological approach to rapidly produce recombinant human IgA monoclonal antibodies. Expression vectors for human IgA1 and IgA2 compatible with the single B-cell antibody cloning method (Tiller et al., 2008) were created, and used to clone the immunoglobulin variable domain genes of a prototypic antigen-specific antibody, 10-1074, a neutralizing HIV-1 IgG1. Efficient production of purified monomeric and dimeric 10-1074 IgA molecules was made possible by combining a transient transfection-based eukaryotic expression system with fast protein liquid chromatography (FPLC) purification. Human IgA1 and IgA2 were FPLC-purified using a new affinity resin engrafted with anti-IgA chimeric Fabs. Finally, IgA monomers and dimers were separated by FPLC-based size exclusion chromatography.
Section snippets
Expression vectors and cloning of human IgA monoclonal antibodies
Total RNAs were previously extracted from human peripheral blood mononuclear cells (PBMCs) of a healthy donor (Scheid et al., 2011), using TRIzol® reagent (Life Technologies) following manufacturer's instructions. cDNAs were obtained by reverse transcription of RNAs primed with random hexamers (Roche) using SuperScript® III reverse transcriptase (Life technologies) according to the manufacturer's protocol. Primers used to amplify the Fc part of IgA1 and IgA2, and J chain gene products were as
Rapid expression cloning of human IgA monoclonal antibodies
To produce human IgA monoclonal antibodies, we first generated J chain-, Igα1- and Igα2-expression vectors by cloning for each specific product (J chain, IgA1 and IgA2 Fc regions), the corresponding nucleotide fragments were amplified from human PBMC cDNAs (Fig. 1A and B). Igα1- and Igα2-expression vectors were derived from an Igγ1-expression vector, allowing the same efficient and rapid cloning strategy using AgeI and SalI restriction sites as previously described (Tiller et al., 2008) (Fig. 1
Discussion
In the past decade, the development of methodologies for producing monoclonal antibodies from human B cells has allowed a rapid rise in the isolation and characterization of human antibodies (Tiller, 2011, Wilson and Andrews, 2012). Expression cloning of recombinant monoclonal antibodies from single B cells was originally developed 15 years ago to study level B-cell tolerance in humans at a molecular (Wardemann et al., 2003), and its breakage in autoimmune diseases (Yurasov et al., 2005). In the
Acknowledgments
We are grateful to Michel C. Nussenzweig (The Rockefeller University) for kindly providing us the cloning vectors for human immunoglobulins and 10-1074 IgG antibody, and to Caroline Eden (Icahn School of Medicine at Mount Sinai) for technical assistance, helpful comments and manuscript editing. We are very grateful to the ANRS (Agence Nationale de Recherche sur le Sida et les hépatites virales) to have supported the purchase of the FPLC instrument and the two PCR thermocyclers used in this
References (41)
- et al.
Serum-free production and purification of chimeric IgA antibodies
J. Immunol. Methods
(2009) - et al.
IRAK-4- and MyD88-dependent pathways are essential for the removal of developing autoreactive B cells in humans
Immunity
(2008) - et al.
Targeting B cell responses in universal influenza vaccine design
Trends Immunol.
(2011) - et al.
Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells
Mol. Biotechnol.
(2008) - et al.
Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut
Mucosal Immunol.
(2011) - et al.
Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography
Methods
(2014) Antibody B cell responses in HIV-1 infection
Trends Immunol.
(2014)- et al.
Recombinant IgA production: single step affinity purification using camelid ligands and product characterization
J. Immunol. Methods
(2012) Single B cell antibody technologies
N. Biotechnol.
(2011)- et al.
Autoreactivity in human IgG + memory B cells
Immunity
(2007)
Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning
J. Immunol. Methods
Cloning and expression of murine Ig genes from single B cells
J. Immunol. Methods
B-cell self-tolerance in humans
Adv. Immunol.
Large-scale transfection of mammalian cells
Methods Mol. Biol.
The majority of intestinal IgA + and IgG + plasmablasts in the human gut are antigen-specific
J. Clin. Invest.
A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine
Proc. Natl. Acad. Sci. U. S. A.
Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa
J. Immunol.
High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions
Nat. Med.
Glycosylation and Fc receptors
Curr. Top. Microbiol. Immunol.
Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity
J. Clin. Invest.
Cited by (43)
Anti-V1/V3-glycan broadly HIV-1 neutralizing antibodies in a post-treatment controller
2023, Cell Host and MicrobeInduced antigen-binding polyreactivity in human serum IgA
2022, ImmunobiologyCitation Excerpt :Variable regions were cloned in IgA1 expression vectors by the In-Fusion technology (Takara) as per manufacturer's protocol. The expression vectors were kindly provided by Dr. Hugo Mouquet (Institute Pasteur, Paris France) (Lorin and Mouquet, 2015). For each antibody, expression vectors for the heavy and light chain were co-transfected in the ExpiCHO cell line (Thermo Fisher Scientific, USA) using the manufacturer's protocol.
Fusogenicity and neutralization sensitivity of the SARS-CoV-2 Delta sublineage AY.4.2
2022, eBioMedicineCitation Excerpt :Human anti-SARS-CoV2 mAbs were cloned from S-specific blood memory B cells of Covid19 convalescents (Planchais et al, manuscript in preparation). Recombinant human IgG1 mAbs were produced by co‐transfection of Freestyle 293‐F suspension cells (Thermo Fisher Scientific) as previously described36 and purified by affinity chromatography using protein G sepharose 4 fast flow beads (GE Healthcare). Antibodies were validated by flow cytometry, by measuring their ability to bind spike expressing cells and not control cells.
Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models
2021, Cell Host and MicrobeTLR9 signalling in HCV-associated atypical memory B cells triggers Th1 and rheumatoid factor autoantibody responses
2019, Journal of HepatologyCitation Excerpt :The IgM- and IgM Fab-expression vectors (Igµ and Igµ-Fab, respectively) were generated from the original IgG1-expression vector20 by substituting the DNA sequence coding for the constant region of IgG1 by the one of the IgM CH1 followed by an hexahistine tag or of the full IgM (synthetic DNA fragment, GenScript HK Limited).18,20 Purified digested PCR products were cloned into expressing vectors, and then used to co-transfect FreeStyle™ 293-F cells as previously described.18,19,20,23 For recombinant IgM production, the co-transfection of Expi293 cells with IgM and IgL (1 µg/ml) and J-chain (2.5 µg/ml) expression vectors was performed using the Expi293™ expression system (Thermo Fisher Scientific).
HIV-1 Envelope Recognition by Polyreactive and Cross-Reactive Intestinal B Cells
2019, Cell ReportsCitation Excerpt :Purified digested PCR products were cloned into human Igγ1-, Igα1-, Igα2-, Igκ- or Igλ-expressing vectors as previously described (Lorin and Mouquet, 2015; Tiller et al., 2008). Recombinant antibodies were produced by transient co-transfection of Freestyle 293-F suspension cells (Thermo Fisher Scientific) using PEI-precipitation method as previously described (Lorin and Mouquet, 2015; Tiller et al., 2008). Recombinant human antibodies and serum IgG from HIV-1 donors were purified by batch/gravity-flow affinity chromatography using peptide M-coupled agarose (Invivogen, San Diego, CA) and protein G Sepharose 4 fast flow beads (GE Healthcare, Chicago, IL) for IgAs and IgGs, respectively.