Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires

Bannoudi, Hanane El; Anquetil, Céline; Braunstein, Marc; Pond, Sergei L. Kosakovsky; Silverman, Gregg J.

doi:10.1007/978-1-4939-7180-0_5

Cited by 6 publications

(9 citation statements)

References 53 publications

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“…Human IgA mAbs have been isolated from memory B cell populations using recombinant methods [13,[21][22][23], and IgAs have been produced in plants [26,29,47,48] and mammalian cells [25,28,30,49]. In this study, we produced PV-neutralizing IgA mAbs using two strategies: (1) de novo IgA cloning from primary human B cells using an updated hybridoma method; and (2) isotype switching of existing IgG mAbs, with recombinant expression in transiently transfected HEK293F cells.…”

Section: Discussionmentioning

confidence: 99%

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Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

et al. 2020

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Poliovirus (PV)-specific intestinal IgAs are important for cessation of PV shedding in the gastrointestinal tract following an acute infection with wild type or vaccine-derived PV strains. We sought to produce IgA monoclonal antibodies (mAbs) with PV neutralizing activity. We first performed de novo IgA discovery from primary human B cells using a hybridoma method that allows assessment of mAb binding and expression on the hybridoma surface: On-Cell mAb Screening (OCMS™). Six IgA1 mAbs were cloned by this method; three potently neutralized type 3 Sabin and wt PV strains. The hybridoma mAbs were heterogeneous, expressed in monomeric, dimeric, and aberrant forms. We also used recombinant methods to convert two high-potency anti-PV IgG mAbs into dimeric IgA1 and IgA2 mAbs. Isotype switching did not substantially change their neutralization activities. To purify the recombinant mAbs, Protein L binding was used, and one of the mAbs required a single amino acid substitution in its κ LC in order to enable protein L binding. Lastly, we used OCMS to assess IgA expression on the surface of hybridomas and transiently transfected, adherent cells. These studies have generated potent anti-PV IgA mAbs, for use in animal models, as well as additional tools for the discovery and production of human IgA mAbs.

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Section: Discussionmentioning

confidence: 99%

“…PV-neutralizing mAb titers were determined in a microneutralization test [17,23]. The mAbs were diluted to 10 µg/mL in maintenance medium (DMEM with 2% FBS and 1% antibiotic/antimycotic solution; Thermo Fisher) and sterilized by filtration through Corning Spin-X columns (Thermo Fisher).…”

Section: Poliovirus Microneutralization Testmentioning

confidence: 99%

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

et al. 2020

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“…Adapting a validated previously reported protocol ( 44 ), we generated unbiased γ rearrangement transcript libraries from class-switched B cells from healthy adult blood donors. RNA was first isolated from PBMCs using the PAXgene Blood RNA Kit (PreAnalytiX).…”

Section: Methodsmentioning

confidence: 99%

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A

Radke

Hernández

et al. 2021

Front. Immunol.

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Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

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“…The immunized mouse was sacrificed, and splenocytes were isolated and fused with nonsecreting NS01 myeloma cells. Subcloned cell lines were then selected by screening supernatants for binding of the immunogen in an ELISA, and antibody gene sequences were determined as previously described (74).…”

Section: Methodsmentioning

confidence: 99%

Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes

et al. 2020

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Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

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Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires

Cited by 6 publications

References 53 publications

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A

Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes

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