PilVax: A novel peptide antigen delivery system for the development of an effective vaccine against Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (pneumococcus) is a pervasive human pathogen responsible for significant global mortality and morbidity. In an attempt to reduce the infectious burden of this pathogen, multiple vaccines targeting the pneumococcal polysaccharide capsule have been developed. However, due to large variation in capsular structure between serotypes, these vaccines have had limited success. As such, there is substantial interest in developing a vaccine effective against all serotypes of S. pneumoniae by instead targeting conserved pneumococcal surface proteins. The aim of this project was to generate such a vaccine using the novel peptide antigen delivery system PilVax. PilVax is a mucosal vaccine technology where peptide antigens are engineered into the polymeric backbone of the pilus from Group A Streptococcus serotype M1 to improve their immunogenicity. The recombinant pilus is then expressed on the surface of the food-grade bacterium Lactococcus lactis to provide a safe, cheap and easy platform for vaccination. To achieve this, four peptides from the conserved pneumococcal surface protein PspA were engineered into the PilVax system. As the success of the PilVax system is based on pilus formation, the effect of peptide insertion on pilus assembly and expression were investigated by Western blotting and flow cytometry. While insertion of the four PspA peptides did not disrupt pilus assembly, it did significantly decrease pilus expression on the surface of L. lactis. Different insertion sites were trialled to improve pilus expression and the two best expressing PilVax constructs were used to intranasally immunise mice. Mice were also immunised with peptide fused to the mucosal adjuvant flagellin from Salmonella enterica serovar Typhimurium to compare the PilVax system to another peptide-based vaccine technology. However, immunisation with the PilVax constructs and the peptide-flagellin fusion proteins failed to generate substantial peptide-specific systemic or mucosal antibodies. For the PilVax constructs, this was likely because the dose of peptide was too low to stimulate a response due to the low level of pilus expression. As such, repeating the vaccination with a higher dose may generate a better response. For the flagellin fusion proteins, the low response was likely due to the fact the proteins were shown to be unstable and therefore degraded at some point during the immunisations. Again, repeating the vaccination and monitoring protein stability will likely generate a better response. Until these experiments are performed, the true efficacy of a PilVax-based vaccine against S. pneumoniae cannot be determined.