ABSTRACT Syphilis, a sexually transmitted infection caused by the spirochete Treponema 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- b arrel a ssembly m achinery subunit A /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 ( po lypeptide tr ansport- a ssociated) domains. Antisera directed against BamA ECL4 promote internalization of Tp by rabbit peritoneal macrophages. Herein, we employed a novel two-stage, phage display strategy, termed “Epivolve” (for epi tope evol ution), 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 a Pyrococcus furiosus thioredoxin ( Pf Trx) scaffold ( Pf Trx BamA/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 with Pf Trx BamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides including S2. In contrast, Abs generated during Tp infection 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 of Tp OMPs.

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

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)