Study On Broad-spectrum Protein Vaccines Against Streptococcus Pneumoniae And Staphylococcus Aureus

Pneumonia is a common disease in young children,the elderly and populations with underlying diseases.In developing countries,pneumonia is the most important cause in the death of young children.Lots of pathogens cloud cause pneumonia.Streptococcus pneumoniae and Staphylococcus aureus are two of the most important pathogens.Streptococcus pneumoniae causes significant morbidity and mortality throughout the world and is responsible for approximately 1.6 million deaths and at least 1.2 million infant deaths worldwide each year.Staphylococcus aureus is the most commonly isolated bacterial pathogen.Staphylococcus aureus causes many skin and soft tissue infections and invasive diseases,such as sepsis,pneumonia and osteomyelitis.Antibiotics are often prescribed for the treatment of infection caused by Streptococcus pneumoniae and Staphylococcus aureus.In recent years,using antibiotics to control the infections has become increasingly difficult because of widespread and progressive antimicrobial resistance.A preventive staphylococcal vaccine would be highly desirable.Several candidate vaccines against Staphylococcus aureus have been proposed and tested,but a safe and effective vaccine is still not available.There are two pneumococcal vaccines on sale.The first is 23-valent pneumococcal vaccine.It have not been found to induce immunological memory nor to be immunogenic in children<2 years of age.The second is the seven-valent pneumococcal conjugate vaccine(PCV7).Moreover,since each PS-protein conjugate is prepared separately,increasing the number of serotypes in the vaccine formulation increases the cost of preparation,quality control and hence the price of the vaccine.Thus,research efforts are now being directed toward recombinant protein vaccines.Although they invade the epithelial cells during the process of infection,Streptococcus pneumoniae and Staphylococcus aureus are not considered as intracellular pathogens.Prevention of disease caused by Streptococcus pneumoniae and Staphylococcus aureus mainly relies on the production of antibodies that either neutralize the activity of virulence factors or function as opsonin to enhance the clearance of infecting cells.In this respect,surface proteins and virulence factors are the potential vaccine candidates.Several strategies have been employed to identify these candidates,including Pneumolysin(Ply),Pneumococcal surface adhesin A(PsaA),Protein Required for Cell Wall Separation of Group B Streptococcus(PcsB),Serine/Threonine protein Kinase(StkP),?-hemolysin(Hla)and Iron responsive surface determinant B(IsdB).This study contains four parts.Firstly,the candidates should be the the virulence factors or surface proteins in Streptococcus pneumoniae or Staphylococcus aureus.We identified four highly conserved proteins(Ply,PsaA,PcsB and StkP)of Streptococcus pneumoniae and two highly conserved proteins(Hla and IsdB)as vaccine antigens.Secondly,design of the candidates.Because of the toxicity of Ply and Hla in their native forms,a candidate vaccine would ideally be a molecule that lacks cytotoxic activity.We developed a novel highly detoxified Ply mutant,designated PlyM2,by engineering two key mutations(C428G and W433F)into wild-type Ply(PlyWT)and a highly detoxified Hla mutant,designated HlaM3 5,by engineering one key mutation(H35L)into wild-type Hla(HlaWT).We designed the truncated protein of PcsB,StkP and IsdB to be highly expressed in the syetem of E.coli.Third,the gene sequences of the candidates were designed and synthesis after the Escherichia coli codon optimization.We constructed the prokaryotic expression plasmids of Ply,PsaA,PcsB,StkP,Hla and IsdB successfully.The proteins were effectively purificated and identified.Fourthly,we evaluated the possibility that immunization with the single protein or the combine of them would confer additive protection against invasive challenge by Streptococcus pneumoniae or Staphylococcus aureus.Inoculation of mice with single protein of Streptococcus pneumoniae could elicit a broad-spectrum protective immune response against infection by three serotypes of Streptococcus pneumoniae.Inoculation of mice with the combine of Hla and IsdB could elicit a protective immune response against infection by Staphylococcus aureus.In conclusion,we generated Ply,PsaA,PcsB,StkP as vaccine candidates against Streptococcus pneumoniae and Hla,IsdB as vaccine candidates against Staphylococcus aureus.We evaluated the immune responses and protective efficacy of the candidates,when they used alone or combine.Our results therefore support these proteins as new protein antigens for inclusion in the development of an effective vaccine against Streptococcus pneumoniae or Staphylococcus aureus.