The Study Of Recombinant Pneumococcal Protein-Based Vaccine

Streptococcus pneumoniae a common pathogenic bacterium which can cause many diseases such as bacteremic pneumonia,bacteremia,meningitis,nasosinusitis and otitis media.It is one of the predominant pathogen threatening human health,especially in children,elderly and immunocompromised patient.Streptococcus pneumoniae is one of the most common pathogenic bacterium in developing countries where pneumonia remains the leading cause of mortality for children and elderly due to lower economic and health conditions.It is estimated that in 2000 alone,pneumococcal diseases caused about one million deaths in children younger than 5 years old,accounting for 11%of total death in this age group.The main treatment of pneumococcal diseases is antibiotics.However,antibiotic resistance has emerged due to the widespread use and misuse,which negatively influences the therapeutical effect of antibiotics.In such case,vaccine is a good method to control infectious diseases compared with antibiotics.At present,there are two kinds of pneumococcal vaccines available clinically,which are both based on capsular polysaccharides.The 23-valent polysaccharide vaccine induces T.cell-independent immune response,which could protect host from infection.However,T cell-independent antigen has no immunological memory and thus cannot induce the second immune response when exposed the antigen again.The problem can be solved by the pneumococcal conjugated vaccine,and this pneumococcal vaccine becomes T cell-dependent antigen after capsular polysaccharides conjugated with protein.However,the pneumococcal conjugated vaccine needs complex processing technology leading to high price and only limited number of serotypes included in the vaccine formulation.Due to the drawbacks of pneumococcal polysaccharide vaccine and pneumococcal conjugated vaccine,it is necessary to develop a protein-based vaccine as an alternative with broad serotype coverage,good immunogenicity and low cost,and thus have improved effectiveness.New generation of pneumococcal protein-based vaccines has been studied for decades with some promising results.Many pneumococcal protein candidates have been found by classical procedures,such as pneumococcal surface protein A(PspA)and other choline-binding proteins,pneumolysin(Ply),pneumococcal surface adhesin A(PsaA).Since the publication of the pneumococcus genome in 2001,more novel pneumococcal proteins for vaccine candidate have emerged.However,most of the potential candidates have not progressed past the preclinical trials.Some candidates have progressed into clinical trials in humans,but most of them still showed low immunogenicity in humans.Therefore,the key to success of pneumococcal protein-based vaccine should lie in high immunogenicity and broad coverage of serotypes.In this research,we summarized the experience of former studies and designed two types of pneumococcal protein vaccine,one for systemic immunization and another for nasal mucosal immunization.We expect that nasal mucosal immunization which simulates natural route with respect to immune activation has better protection at the site of infection.We chose Bacterium-like particles(BLP)as mucosal adjuvant,which is one of potent mucosal adjuvants.BLP also acts as carrier displaying antigens on the surface of itself.According to the results,the mucosal administration of BLP-based vaccine not only elicited a strong and robust systemic immune response,but also raised strong local immune response in the local mucosal layer.This research comprises of the following two parts;Part one:systemic immunization(subcutaneous immunizaiton)Firstly,according to former reports,we indentified two pneumococcal proteins(PsaA and PspA)as vaccine candidates.They are both the virulence factors and surface proteins of Streptococcus pneumoniae.Because of the structural variability of PspA protein,we designed two kinds of proteins based on the family difference of PspA,PsaA-PspA fusion protein and PspA4,in order to broaden the coverage of protection.We aimed to confer protection against more than 95%of Streptococcus pneumoniae strains bearing heterologous PspAs by combinating immunization of PsaA-PspA and PspA4.Secondly,the DNA encoding of PsaA-PspA and PspA4 were designed and synthesized after genetic code optimization adapting to Escherichia coli.We successfully constructed the prokaryotic expression plasmids of PsaA-PspA and PspA4.We then purified and identified both of the two proteins.Third,we evaluated the immunogenicity and immune-protective effect in mice challenging model.Either using single or combination of the two proteins produced good immunogenicity and conferred protection against fatal challenge by Streptococcus pneumoniae strains bearing different PspAs.We proved that antibodies against PsaA-PspA and PspA4 had broad immune-reactivity to Streptococcus pneumoniae strains by Western-blot method and immune-fluorescence technique.Part two:intranasal mucosal immunization(BLP-based vaccine)Firstly,we successfully applied new type of mucosal adjuvant(BLP)to pneumococcal protein vaccine and assessed the possibility of production of this vaccine.Secondly,we designed PspA2-PA,PspA4-PA and Plym2-PA fusion proteins.We successfully constructed the prokaryotic expressive plasmids and expressed the three proteins in Escherichia coli.We also produced BLP successfully.Third,we produced three kinds of mucosal vaccine antigens,PspA2-PA-BLP,PspA4-PA-BLP and Plym2-PA-BLP.The BLP-based vaccine didn't affect the immune-reactivity after the antigen binding with BLP.Fourth,we evaluated the immunogenicity and immune-protective effect of the three BLP-based vaccine antigens in mice challenging model.The intranasal mucosal administration of BLP-based vaccine elicited a strong and robust systemic immune response,as well as raised strong local immune response in the local mucosal layer.It could confer protection against fatal challenge with Streptococcus pneumoniae.In conclusion,we successfully designed,generated and evaluated two kinds of pneumococcal protein vaccines,one for systemic immunization including PsaA-PspA and PspA4,another for mucosal immunization of BLP-based vaccine.Both of the two vaccines had good immunogenicity and could protect mice against fatal challenge with Streptococcus pneumoniae bearing different PspAs.Administrating the two vaccines could provide around 100%survival rates for some challenge Streptococcus pneumoniae strains.Compared with the 23-valent polysaccharide vaccine,the two protein vaccines had a better protection effects.Therefore,both of the two protein vaccines exhibit promising prospects to become the new generation of pneumococcal vaccine.The innovation of this paper as bellows:First,we construct fusion protein including PsaA part and PspA part for the first time,we also constructed PspA4 protein in order to increase broad coverage of protection,the efficacy of the PsaA-PspA fusion protein and PspA4 protein as systemic immunization antigens,in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in animal models;Second,we investigate the BLP as a new platform to display PspA and Ply for pneumococcal vaccine development for the first time,the efficacy of the BLP based vaccine as mucosal immunization antigens,in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in animal model.