부산대학교 도서관

Background: Recent increases in the global incidence of syphilis highlight the urgent need for a vaccine to prevent transmission of Treponema pallidum (Tp), the syphilis spirochete. An efficacious syphilis vaccine should elicit protective (i.e., opsonic) antibodies against the spirochete's limited repertoire of β- barrel-forming outer membrane proteins (OMPs). Highly promising Tp OMPeome targets include the 8- stranded β-barrels (Tp8SβB) paralogs (TP0126, TP0479, TP0698 and TP0733), which likely serve to import small molecules. Production of recombinant Tp8SbBs in a native conformation presents numerous challenges. To circumvent these difficulties, we grafted the extracellular loops (ECLs) from Tp8SbBs onto the well-characterized 8SbB for Escherichia coli OmpA, which has been characterized extensively at the atomic level. Methods: Crystal structure for OmpA-βB and Alphafold-models for all four Tp8SβBs were used for alignment. OmpA/Tp8SβB chimeras were engineered by replacing the ECLs from OmpA-βB with the predicted loops from each Tp8SbB. OmpA/Tp8SβB were over-expressed in E. coli as inclusion bodies and refolded by dilution in 1% LDAO. Proper refolding of recombinant chimeras was confirmed by demonstrating heat-modifiability. Antigenicity of all four OmpA/Tp8SβB chimeras was evaluated by immunoblot using immune rabbit sera (IRS) and human syphilitic sera (HSS) from patients in Colombia and Malawi. Sera from mice immunized with refolded chimeras were evaluated using recombinant Pyrococcus furiosus thioredoxin (PfTrx) containing single OMP ECLs. Results: Tp8SβBs models show high superposition scores when compared to the OmpA-βB, suggesting that the Tp8SbB ECLs are properly displayed in the OmpA/Tp8SβB scaffolds. High yields of refolded recombinant OmpA/Tp8SβBs were achieved and showed heat-modifiability, thus confirming their proper refolding into stable β-barrels. All four OmpA/Tp8SbB chimeras were recognized by HSS and/or IRS. Mice immunized with OmpA/Tp8SβB developed antibodies against individual Tp8SβB ECLs. Conclusions: Our results support the use of E. coli OmpA-βB as a highly versatile scaffold for presenting heterologous T. pallidum OMP ECLs. High yields of refolded OmpA/Tp8SβBs chimeras were easily achieved, enabling the generation of Tp8SβBs ECLspecific antisera. These antibodies will be investigated for their ability to promote opsonophagocytosis of T. pallidum by murine and rabbit macrophages and their ability to confer protection in the rabbit model of experimental syphilis. [ABSTRACT FROM AUTHOR]