title: Natural immunity to pneumococcal Pilus-1 RrgA and RrgB antigens, and its relationship with pneumococcal carriage
Creators: name: Ahmed, Muhammad Shamsher
Publisher: name: University of Liverpool Repository
Year: 2014

description: Streptococcus pneumoniae (pneumococcus) is a leading cause of childhood morbidity and mortality around the world. The available polysaccharide-based vaccines are limited by either low efficacy in young children or narrow serotype coverage. Recent research has focused on developing protein vaccines. Pneumococcal pilus-1 proteins play an important role in pneumococcal adherence to host respiratory epithelium and subsequent bacterial colonization or invasion; and may therefore be an effective vaccine candidate. This PhD project investigated natural immunity in humans to pneumococcal pilus-1 proteins and its association with nasopharyngeal carriage of pneumococcus. Nasopharyngeal carriage of pneumococcus was analysed by bacteriological culture of nasal swabs on blood agar. Pneumococci were identified based on their colony morphology and optochin sensitivity; and further confirmed by PCR detection of pneumolysin gene in the isolates. Pneumococcal carriage was found to be common in young children, which gradually decreased with advancing age. PCR detection of pilus islet 1 (PI-1) gene revealed that percentage of pilus-1 positivity was low among these carriage isolates. This low prevalence may be associated with the recent introduction of pneumococcal conjugate vaccination, which covers the common piliated serotypes. ELISA based measurement of serum and salivary antibodies to pilus-1 proteins detected significant antibody levels to both RrgA and RrgB, presumably developed as a part of natural immune response in children and adults. An age-dependent increase in serum antibody levels to both RrgA and RrgB was also observed, and anti-RrgA appeared to develop earlier in childhood than anti-RrgB. Moreover, higher levels of antibody, especially anti-RrgA were found in children who were culture-negative than in those who were culture-positive for pneumococcus. It suggests that these naturally developed antibodies may contribute to the protection against pneumococcal carriage in humans. Using an in vitro model of human NALT, the study revealed that adenotonsillar tissues are important induction sites for immune response to these antigens, by priming B cell memory. The induction of antibody secreting cells in NALT was enumerated by ELISpot assay; and the antibody production was measured by antigen-specific ELISA. In vitro stimulation with a wild type (TIGR4) pneumococcal culture supernatant (containing both RrgA and RrgB proteins) induced a significant memory B cell and antibody response in adenotonsillar cells. Current carriage in vivo enhanced the memory B cell response and antibody production. Flowcytometric analysis of CFSE labelled adenotonsillar MNC suggested that pneumococcal pilus-1 proteins RrgA and RrgB were capable of stimulating CD4+ T cell proliferative response in human NALT. Stimulation with pneumococcal CCS induced Th17 related cytokines production in both human adenotonsillar MNC and PBMC culture supernatant. Importantly, this in vitro production of Th17 cytokines after stimulation with TIGR4wt CCS was significantly higher than that of CCS derived from its isogenic RrgA-/- and RrgB-/- mutants, indicating a contribution from both of these proteins. The ability of these pilus-1 proteins to stimulate CD4+ T cell proliferation and Th17 response may contribute to the natural immunity to pneumococcus in humans. This study has revealed that both of these pilus-1 proteins, RrgA and RrgB are immunogenic and are capable of priming for memory B and T cell response in human NALT. These findings aid to our understanding on the naturally developed immune response to pilus-1 proteins, and may inform future vaccination strategy with intranasal immunisation containing protein antigens against pneumococcal infection.