Novel strategies to prevent and treat experimental pneumococcal disease

With over 90 different serotypes of Streptococcus pneumoniae unevenly distributed around the world, current vaccine formulations vary significantly in their ability to protect against invasive pneumococcal disease (IPD). The adult pneumococcal vaccine is composed of capsular polysaccharide from 23 of the most prevalent disease causing serotypes. Purely polysaccharide vaccines are unable to protect those most at risk; infants under the age of two. This has led to the development of a paediatric conjugate vaccine, composed of capsular polysaccharide from seven of the most common disease causing serotypes, each chemically conjugated to a carrier protein. Although efficacious at protecting the target age group from disease caused by homologous serotypes, it fails to protect against the non-vaccine serotypes. Serotype specific vaccination is a short-term solution to pneumococcal disease. As the most common serotypes are eradicated by vaccination, previously less common serotypes fill the vacant niche and cause serotype replacement disease. A solution to this problem would be the development of pneumococcal vaccines containing antigens that elicit non-serotype specific protection. Pneumolysin, the pore-forming toxin produced by S. pneumoniae, may play a role in future pneumococcal vaccine. It is a major virulence factor produced by all invasive isolates and has previously been demonstrated to confer non- serotype specific protection. In this thesis, pneumolysin retained the ability to bind to cell membranes and form pores even when other antigens were fused genetically to the N terminus. Pneumolysin performed as a highly immunogenic mucosal adjuvant, with substantial mucosal and systemic immune responses to the fused antigen, when nanograms quantities when applied to the mucosal surface of the nasopharynx. A fusion between pneumococcal surface adhesin A (PsaA) and pneumolysin (PLY) was created to investigate potential protection conferred by the antigen specific response. Vaccination of Balb/c and MF1 mice with PsaAPLY conferred no protection against challenge with virulent S. pneumoniae TIGR4. The toxicity of pneumolysin is problematic and existing pneumolysin mutants possess residual cytotoxicity. By ablating the toxicity of pneumolysin with formalin it permits its use in parental vaccines. The pneumococcal histidinetriad proteins (Pht) are a recently identified family of surface exposed proteins that have homologues in other Streptococcus species and are therefore novel potential vaccine candidates. In vivo models of disease require the sacrifice of a large number of animals at time points to investigate the impact of vaccine or pharmaceuticals on disease progression. Real-time photonic imaging of bioluminescent bacteria offers significant advantages over conventional methods for monitoring and combating bacterial disease in animals. Not only does this approach reduce the time and costs associated with such experiments, but also it considerably reduces the number of animals used. Furthermore, because bioluminescent imaging allows the same group of animals to be monitored over time, animal-to-animal variations are overcome by including the zero time point as an internal control. Models of pneumococcal pneumonia were established in MF1 mice. Newly established bioluminescent models were then used to investigate the impact of vaccination with the paediatric pneumococcal conjugate vaccine as proof of principle