Alsharifi, MohammedBrazel, ErinPaton, JamesGates, Chloe2024-10-042024-10-042024https://hdl.handle.net/2440/142701Streptococcus pneumoniae is the world’s foremost respiratory bacterial pathogen and the largest global killer of children under the age of five. Responsible for up to two million deaths annually, the high genetic diversity of the pneumococcus has complicated universal vaccine development. Current vaccination strategies target the dominant virulence factor, the capsular polysaccharide. These vaccines have been highly effective at reducing the incidence of disease caused by vaccine serotypes, however, global use has inadvertently led to the increase of disease by non-vaccine included serotypes. Therefore, development of serotype-independent vaccines is a key requirement for the control of pneumococcal infections and invasive disease. Our lab has previously described a new, gamma-irradiated pneumococcal vaccine, termed Gamma-PN2. This vaccine consists of an unencapsulated whole cell antigen that is inactivated using gamma-irradiation and has been shown to provide antibody-mediated serotype-independent protection. For clinical advancement, this gamma-irradiated vaccine was compared to those inactivated using heat, ethanol, or formalin. Structural integrity and aggregation analyses demonstrated that Gamma-PN2 exhibited comparable cellular morphology to live bacteria, in contrast to varying degrees of damage and aggregation observed for other inactivated material. Intramuscular vaccination of mice showed that vaccination with Gamma-PN2 or heat-inactivated PN2 (Heat-PN2) induced pneumococcal-specific IgG but with significantly different profiles of IgG subclasses. High IgG1 levels were induced after vaccination with Heat-PN2, while Gamma-PN2 biased the response towards IgG2. In addition, vaccination of animals with Gamma-PN2 was associated with enhanced recognition of a wider array of pneumococcal proteins compared to other inactivated vaccines. This study also describes the effect of lytic proteins on vaccine thermostability during manufacturing. In this study, the vaccine strain, termed GPN-002, showed poor thermostability throughout down-stream processing during manufacturing. Attenuation of the lytic lysozyme, LytC, rescued thermostability during manufacturing, suggesting that lytic activity by this enzyme led to lysis of the vaccine strain at ambient temperatures. Importantly, deletion of lytC was also associated with enhanced vaccine-mediated immunogenicity. Immunisation in mice with this vaccine, termed Gamma-PN3, was associated with enhanced pneumococcal-specific antibody responses. This was characterised by greater IgG binding to encapsulated pneumococci and enhanced protein recognition compared to other vaccine candidates. Along with IgG responses, cell-mediated responses have been reported in their role in clearing infecting pneumococci, with emphasis on IL-17+ cells in the mucosa. This study also characterised the cell-mediated response induced after vaccination with Gamma-PN3 and the effect of these responses on IgG subclass profiles. Importantly, vaccination with Gamma-PN3 significantly enhanced IgG2 antibody responses and IFN-g+ and GMCSF+ Th1 cell production. In addition, neutralisation of IFN-g during vaccination demonstrated a significant reduction in IgG2 responses, highlighting the direct relationship between vaccine-mediated humoral and cell-mediated immunity. This study was also the first to show that IL-17+ responses had no impact on vaccine-induced IgG subclass responses. Overall, these findings illustrate significant progress in the generation and understanding of a gamma-irradiated whole-cell pneumococcal vaccine for advancement of clinical development.envaccinespneumococcusimmune responseantibodiesserotype-independentT-cellsThesis