(P388) Development of an opsonic monoclonal antibody targeting extracellular loop 4 of Treponema pallidum BamA (TP0326)

Background: A vaccine will be critical to curtail the global syphilis epidemic. Generation of antibodies (Abs) targeting surface-exposed epitopes of the spirochete’s outer membrane proteins (OMPs) is believed to be the key to syphilis vaccine development. The T. pallidum (Tp) ‘OMPeome’ includes BamA (β-barrel assembly machinery subunit A), a bipartite protein consisting of an 18-stranded β-barrel with nine extracellular loops (ECLs) and a periplasmic arm containing five POTRA (polypeptide transport-associated) domains. Opsonic Ab activity is widely considered a correlate of protection. Antisera previously generated with a BamA/ECL4 peptide promoted Tp internalization by rabbit peritoneal macrophages. Herein we describe production and characterization of an opsonic BamA/ECL4 monoclonal Ab (mAb) and a novel murine macrophage opsonophagocytosis assay.

Methods: 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) (Figure). Additionally, antisera were generated by immunizing mice and rabbits with BamA/ECL4 displayed by a Pyrococcus furiosus thioredoxin (PfTrx) scaffold. MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A direct comparison of the murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse model. Sera from Tp-infected mice (MSS), rabbits (IRS) and syphilis patients were evaluated for BamA/ECL4-specific Abs using PfTrx^BamA/ECL4 and the overlapping BamA/ECL4 peptides in immunoblotting and ELISA assays.

Results: BamA/ECL4 mAbs, reactive against the S2 peptide, were produced by fusing five different scFvs to mouse or rabbit Fc-domains. Resulting mAbs recognized nanogram amounts of PfTrx^BamA/ECL4 and BamA β-barrel. Equilibrium dissociation constants were similar between species. Mouse mAb (IGX7141) and antisera opsonophagocytosis levels were comparable, while less robust than MSS. Uptake of Tp opsonized with rabbit antisera was similar to IRS. Abs generated during Tp infection poorly recognized BamA/ECL4 S2 and S3 peptides, whereas the polyclonal sera strongly recognized all three peptides.

Conclusion: “Epivolve” produced mAbs with opsonic activity against BamA/ECL4, a top tier 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 Tp infection.