A fusion protein based pneumococcal vaccine

The Gram-positive bacterium Streptococcus pneumoniae(pneumococcus) is a major human pathogen, causing Otitis media, pneumonia, bacteraemia and meningitis worldwide. There are over 90 different serotypes of pneumococcus and current pneumococcal vaccines are somewhat limited in their protection against pneumococcal diseases. Currently available vaccines are based on the capsular polysaccharide. The 23-valent polysaccharide (PSP) vaccine can protect most of adults and children older than five, but it cannot protect children under age 2, immunodeficiency patients and cannot protect the elderly very well. The polysaccharide/ protein conjugated vaccines which are complementary to the 23-valent PS vaccine have been introduced. The PSP conjugate vaccine is highly immunogenic and protects children under the age of 2 and immunodeficiency patients against invasive pneumococcal diseases, but its efficacy is threatened by strain replacement and serotype switching since it only protects against the limited number of serotypes contained within the vaccine. There is therefore a need to develop improved vaccines against pneumococcal diseases. Pneumolysin (PLY), a cholesterol dependent cytolysin and an important virulence factor of pneumococcus, can act as a powerful mucosal adjuvant to induce both systemic and mucosal immunity to proteins genetically fused to PLY. Which regions of PLY are required for the novel adjuvant activity is not currently known. The model antigen eGFP was fused to domain 1-3 of PLY (D123PLY) and domain 4 of PLY (D4PLY) by ligation-dependent DNA recombinant technology during my master project. Balb/c mice were intranasally immunised with purified eGFPD4PLY or eGFPD123PLY fusion protein and immune response were then monitored by enzyme linked immunosorbent assay (ELISA). The ELISA data shows truncated PLY lost the adjuvant property and this adjuvant property was restored in the presence of free PLY or A6PLY when antigen was fused with truncated PLY. The aim of the current work is to determine whether a fusion protein based pneumococcal vaccine can be developed to provide protection against three pneumococcal strains in animal models of colonisation and invasive diseases. For this purpose, pneumococcal virulence factors, PsaA, PspA, PspC and PhtD were genetically fused to PLY or Δ6PLY separately by In-fusion cloning technology. Δ6PLY is a toxoid that lacks haemolytic activity but retains the immunogenic and adjuvant activity of PLY. MF1 mice were then vaccinated either intranasally or subcutanously by purified fusion protein antigens and immune responses were also monitored by ELISA; vaccinated mice were finally challenged intranasally by three strains of pneumococcus and monitored for colonisation. The immunity elicited by pneumococcal antigens fused to PLY/Δ6 PLY is protective in a colonisation model