Identification of pneumococcal vaccine antigens using Reverse Vaccinology 2.0

Abstract

The phenomenon of serotype replacement and the increase in antibiotic resistance among serotype replacement strains highlight the need for new pneumococcal vaccines with a broader coverage against Streptococcus pneumoniae strains. Because pneumococci express a large number of capsular polysaccharides, vaccine antigen discovery is now focused on the search for conserved protein antigens. To identify novel vaccine candidate antigens, this study utilized an approach that involves the expression and cloning of fully human monoclonal antibodies (hmAbs). This thesis describes the successful use of the Reverse Vaccinology 2.0 approach to identify novel functional pneumococcal antigens. Three different assays using S. pneumoniae were assessed. The first assay targeted the use of plasmablasts cells, from which one pneumococcal specific hmAb was mono-cloned - SMP13-D9K. The second assay, the immunoglobulin capture assay (ICA) had been previously developed using single viral vaccine antigens to enhance the efficiency of antigen-specific hmAb cloning but was optimised in this project for use with whole cell S. penumoniae. Eighteen out of 22 hmAbs (~82%), cloned from individual plasmablasts, were specific for disparate pneumococcal polysaccharide epitopes, however no anti-protein hmAbs were cloned using the ICA. Finally, the third assay targeted the used of enriched memory B cells from healthy volunteers, from which one pneumococcal specific hmAbs was mono-cloned -MBC-E12K. Furthermore, the protein antigens recognised by P13-D9K and MBC-E12K were assessed as a potential component in a pneumococcal vaccine. Results showed that P13-D9K and MBC-E12K were highly cross-reactive recognising 23 serotypes, and both showing functional, i.e., opsonophagocytic activity in vitro against strains of serotypes 4, 6B, 14 and 19A. Passive transfer of D9K and E12K antibody resulted in 50% and 30% of mice clearing infection, respectively, while a synergistic effect was seen when the mixed hmAbs were transferred with a 12.5% higher percentage of clearance (62.5%). Reactivity of the bactericidal hmAbs was found against a single ca. 63 kDa protein in western blots. Finally, the results presented in this thesis provide a proof-of-principle for the use of Reverse Vaccinology 2.0 as a powerful tool in the search for novel pneumococcal vaccine candidate antigens.