Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa

Abstract

The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor-binding domain of the main structural subunit of the pilus, PilA. The receptor-binding domain is a 14-residue disulfide loop at the C-terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide-conjugate to a protein subunit immunogen to determine which was superior for use in an anti-pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor-binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor-binding domain was used to characterize receptor-binding domain-specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide-conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti-peptide sera for the intact pilin receptor-binding domain were significantly higher than affinities of anti-pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide-conjugate over a subunit pilin protein for an anti-pilus vaccine.

Figure 1

Panel (A) Ribbon diagram showing the structure of PAK monomeric pilin (residues 29−144, PDB ID: 1DZO). The receptor binding domain is highlighted in magenta, showing the disulfide bond between residues 129 and 142 in green. Panel (B) Comparison of the conformation of the PAK strain receptor binding domain (RBD, residues 128−144) as part of the pilin protein (cyan) and as a free peptide in solution (magenta, PDB ID: 1NIL). The two chains are aligned to the type II beta-turn (residues 139−142). The disulfide bond is yellow.

Figure 2

Amino acid sequence alignment of truncated PAK pilin (29−144), the scrambled PAK pilin construct (29−144) with a scrambled receptor-binding domain, and PAK peptide (128−144) used in immunizations and binding studies. A disulfide bond is present between cysteine residues 129 and 142.

Figure 3

Indirect ELISA using antisera from mice immunized with either PAK pilin (residues 29−144) (A and C) or PAK peptide (residues 128−144)-KLH conjugate (B and D) binding to a microtiter plate coated with PAK peptide-BSA conjugate (A and B) or PAK pilin (C and D). Each curve corresponds to the titration curve from one mouse.

Figure 4

Western blot demonstrating recognition of PAK pilin with a scrambled receptor binding domain 128−144 (RBD) protein by pilin-specific polyclonal sera, but not by a RBD-specific antibody. Purified GST, PAK pilin with a scrambled RBD, and PAK pilin were blotted with either polyclonal mouse anti-PAK pilin antisera or an anti-PAK (128−144) monoclonal antibody. The presence of GST was demonstrated by SDS-PAGE (not shown). The two bands observed in the scrambled pilin-GST and PAK pilin blots represent the monomer and dimer.

Figure 5

Indirect ELISA binding curves of antisera raised to the PAK pilin protein demonstrating inhibition of binding by the PAK pilin protein with a scrambled RBD. Panel (A) Mouse 7. Panel (B) Mouse 12. Panel (C) Mouse 15. In panels (A–C), closed circles show serum dilutions without addition of PAK pilin protein with a scrambled RBD and open triangles show serum dilution PAK pilin with a scrambled RBD binding to PAK pilin. Panel (D) Mouse 8, closed circles show serum dilution only, open diamonds show serum with 20 μm PAK peptide (128−144), triangles show serum plus PAK pilin with scrambled RBD, closed squares show serum with PAK pilin with scrambled RBD and 20 μm PAK peptide.

Figure 6

Competitive ELISA inhibition curves showing competition between PAK pilin (A and B) or PAK peptide (C and D) in solution and a PAK pilin-coated surface using anti-pilin sera (A and C) and anti-peptide sera (B and D). A fixed dilution of scrambled pilin was included in the competition experiments using anti-pilin sera (A and C) at a concentration high enough to eliminate all antibody binding to pilin other than the receptor binding domain. The N^α-acetylated synthetic peptide was used in the competition experiments.