Background: Pseudomonas aeruginosa continues to be a significant opportunistic pathogen, with growing antibiotic resistance and no available vaccine. Previous work showed that the P. aeruginosa type III secretion system (T3SS) protein, PopB, elicits a protective Th17 response after intranasal immunization using the Th17 adjuvant curdlan, but the protection is of low potency. In those studies, PopB was co-purified with its chaperone PcrH. In the current studies, we tested whether fusing or mixing PopB with PcrV, a T3SS protein known to elicit protective antibodies, would enhance protective efficacy. We also hypothesized that adding a Th17 antigen (PopB) to PcrV would improve mucosal IgA responses to PcrV.

Methods: Fusion proteins to the avidin homolog rhizavidin (Rhavi) were constructed to achieve multimeric antigen presentation as dimers and also for future coupling to biotinylated polysaccharides. A fusion protein of Rhavi to full-length PcrV and PopB (denoted RVB) was purified from E. coli using a pACYCDuet-1 vector co-expressing His-PcrH, where untagged RVB bound to His-PcrH was pulled down by nickel affinity resin, followed by ion exchange chromatography and then negative affinity chromatography with nickel resin using elution with 0.1% LDAO to dissociate RVB from His-PcrH. Recombinant His-tagged Rhavi-PcrV, PcrV, and PopB/PcrH were prepared by published methods. Mice were immunized intranasally using curdlan as adjuvant. IgA titers in bronchoalveolar lavage fluid (BALF) were assayed by ELISA, and Th17 responses were assessed by measuring IL-17 (ELISA) in supernatants of splenocytes stimulated with antigens in vitro. Protective efficacy was evaluated in a murine acute pneumonia model using P. aeruginosa strain N13.

Results: Our results show significantly higher IgA titers to PcrV in BALF of mice immunized with RVB when compared to mice immunized with either PcrV or Rhavi-PcrV, where IgA titers were undetectable. Th17 responses to PopB were significantly higher in RVB-immunized mice compared to mice immunized with PopB/PcrH. In the P. aeruginosa acute pneumonia model, RVB-immunized mice displayed 100% survival, whereas PcrV- and Rhavi-PcrV-immunized mice had approximately 60% survival (logrank P=0.06 compared to RVB), all significantly higher than curdlan-immunized mice (0% survival).

Conclusion: These results suggest that integrating PopB with PcrV into the same rhizavidin fusion protein elicits improved IgA titers to PcrV while maintaining a robust Th17 response to PopB, ultimately conferring broad and potent protection against P. aeruginosa lung infection.