Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 ofTreponema pallidumBamA (TP0326)

Abstract

ABSTRACTSyphilis, a sexually transmitted infection caused by the spirocheteTreponema pallidum(Tp), is resurging globally. Opsonic antibodies (Abs) targeting surface-exposed epitopes of the spirochete’s outer membrane proteins (OMPs) are believed to promote macrophage-mediated clearance of the bacterium during infection and are presumed to be key to vaccine development.Tp’s repertoire of outer membrane proteins includes BamA (β-barrelassemblymachinery subunitA/TP0326), the central component of the molecular machine that inserts newly exported OMP precursors into the OM lipid bilayer. BamA is a bipartite protein consisting of an 18-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptidetransport-associated) domains. Antisera directed against BamA ECL4 promote internalization ofTpby rabbit peritoneal macrophages. Herein, we employed a novel two-stage, phage display strategy, termed “Epivolve” (forepitopeevolution), to generate five site-directed murine monoclonal Abs (mAbs) targeting a centrally located peptide (S2) of BamA ECL4. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of BamA ECL4 displayed by aPyrococcus furiosusthioredoxin (PfTrx) scaffold (PfTrxBamA/ECL4). One mAb containing a unique amino acid sequence in both light and heavy chains showed activity in an opsonophagocytosis assay employing murine bone marrow-derived macrophages. Mice and rabbits hyperimmunized withPfTrxBamA/ECL4produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides including S2. In contrast, Abs generated duringTpinfection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve, which circumvents the natural immune response, can be utilized for the generation of mAbs that target subdominant opsonic epitopes in ECLs ofTpOMPs.