(P387) Pyrococcus furiosus thioredoxin (PfTrx) scaffolds displaying extracellular loops of Treponema pallidum outer membrane proteins: “learning from nature” in the search for syphilis vaccine candidates

Background: The re-emergence of syphilis in the new millennium highlights the need for vaccine development to better understand the immune responses elicited by infection with Treponema pallidum (Tp). Limited studies have utilized the mouse for syphilis research; the rabbit remains the time-honored animal model. Ex vivo studies with immune rabbit serum (IRS) and peritoneal macrophages strongly support that an effective vaccine must be antibody (Ab) based. Structural models of Tp outer membrane proteins (OMPs) provide a ‘roadmap’ to identify potential vaccine candidates whose prime targets are extracellular loops (ECLs). Elucidating ECL-directed Ab responses in the animal models will inform vaccine development for humans.



Methods: Pyrococcus furiosus thioredoxin (PfTrx) was used as a scaffold to display individual ECLs from four Tp Nichols OMP families: four eight-stranded b-barrels (8SβB), five long-chain fatty acid transporters (FadLs), and four outer membrane factors (OMFs). Sera from five immune rabbits (immune rabbit sera, IRS) were tested for reactivity against 67 ECLs by immunoblotting and ELISA. Four cohorts of mice and rabbits were immunized with PfTrx^TPA/ECL antigens. The ECL-specific reactivities of the antisera were assessed using a second scaffold, transferrin binding protein B (TbpB) displaying the corresponding ECL. The mouse and rabbit opsonophagocytoisis assays were compared directly using the corresponding murine and rabbit antisera. Spirochete internalization was confirmed via confocal microscopy.

Results: ECLs from all OMFs reacted poorly with IRS. Only ECL4 and ECL3 of the 8SβBs TP0479 and TP0698, respectively, showed some reactivity. ECLs 2 and 4 of the FadLs TP0856 and TP0858, were the antigenic standouts (see figure). Mouse and rabbit antisera generated using PfTrx constructs containing ECLs 2 or 4 from TP0856 and TP0858 detected nanogram amounts of the corresponding ECLs displayed by TbpB. They also demonstrated strong ‘functional activity’ in opsonophagocytosis assays (see figure).



Conclusions: Our findings support the notion that antigenic analysis of ECLs can identify top-tier ECL vaccine candidates, in this case, ECLs 2 and 4 of TP0856 and TP0858. They also suggest that the murine opsonophagocytosis assay may be a viable alternative to the rabbit assay. Lastly, this ECL-based strategy can be utilized to identify OMPs vaccinogens of non-spirochetal diderm bacterial pathogens.