O01.2\u2005Pyrococcus furiosus thioredoxin as a platform to express extracellular loops of treponema pallidum outer membrane proteins

Abstract

Background Development of a vaccine is a necessary step towards curtailing the global syphilis epidemic. Towards this end, our group has utilized bioinformatic and biophysical methods to characterize T. pallidum’s repertoire of outer membrane proteins (OMPs): the two ‘stand-alones’, BamA and LptD, and the T. pallidum repeat (Tpr), 8-stranded β-barrel, outer membrane factor (OMF) for efflux pump, and FadL paralogous families. We have hypothesized that antibodies against extracellular loops (ECLs) of OMPs are responsible for the opsonic and protective activities of immune rabbit serum (IRS). We used Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to present ECLs for assessment of reactivity with syphilitic sera and quantitation of circulating ECL-specific B-cells. To develop this system, we focused on BamA/ECL4 which we previously have shown is immunogenic and a target for opsonic antibodies. Methods PfTrx was engineered to express BamA-ECL4 (PfTrx-BamA/ECL4) with a N-terminal His6- and C-terminal Avi-tags for Ni-NTA purification and in vivo biotinylation, respectively. Reactivity of IRS and human syphilitic sera (HSS) with PfTrx-BamA/ECL4 was assessed by immunoblotting. To determine the accessibility of the BamA/ECL4 epitope, a pull-down assay was performed by incubating PfTrx-BamA/ECL4 or PfTrx with IgG from IRS or normal rabbit serum (NRS) immobilized on protein G agarose beads. BamA/ECL4-specific IgG+ B-cells were identified by flow cytometry using PfTrx-BamA/ECL4 conjugated with streptavidin (SP)-Alexa Fluor 647 and SP-Brilliant Violet-421. PfTrx was conjugated to SP-APC-Cy7 to exclude non-specific binding to scaffold. Results IRS and HSS recognized PfTrx-BamA/ECL4 but not PfTrx. PfTrx-BamA/ECL4 was pulled down by IgG from IRS but not NRS, while PfTrx was not pulled down by either. 1.66% of circulating IgG+ B-cells were PfTrx-BamA/ECL4-specific. Conclusion When used in conjunction with OMP structural models, PfTrx-ECL is a promising platform for identification of antigenic ECLs and isolation of ECL-specific B-cells for the generation of anti-ECL monoclonal antibodies with opsonic activity.