AUTHOR=Ferguson Mary R. , Delgado Kristina N. , McBride Shannon , Orbe Isabel C. , La Vake Carson J. , Caimano Melissa J. , Mendez Qiana , Moraes Trevor F. , Schryvers Anthony B. , Moody M. Anthony , Radolf Justin D. , Weiner Michael P. , Hawley Kelly L. 

TITLE=Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1222267

DOI=10.3389/fimmu.2023.1222267

ISSN=1664-3224

ABSTRACT=<sec><title>Introduction</title><p>Syphilis, a sexually transmitted infection caused by the spirochete <italic>Treponema pallidum</italic> (<italic>Tp</italic>), is resurging globally. <italic>Tp</italic>’s repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of <italic>Tp</italic> by rabbit peritoneal macrophages.</p></sec><sec><title>Methods</title><p>Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed “Epivolve” (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a <italic>Pyrococcus furiosus</italic> thioredoxin scaffold (<italic>Pf</italic>Trx<sup>BamA/ECL4</sup>). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (<italic>e.g.</italic>, opsonization) and validate the mouse assay. Sera from <italic>Tp</italic>-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using <italic>Pf</italic>Trx<sup>BamA/ECL4</sup> and overlapping ECL4 peptides in immunoblotting and ELISA assays.</p></sec><sec><title>Results</title><p>Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of <italic>Pf</italic>Trx<sup>BamA/ECL4</sup>. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with <italic>Pf</italic>Trx<sup>BamA/ECL4</sup> produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during <italic>Tp</italic> infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.</p></sec><sec><title>Discussion</title><p>Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by <italic>Tp</italic> infection.</p></sec>