The Optimization And Broad Spectrum Evalution Of Pneumococcal PspA Protein-based Vaccine

Strptococcus pneumoniae,（S.pneumoniae）,was separated in1881 for the first time,is gram-positive bacterium located on the nasopharynx,causing pneumonia,is a major bacte-rial pathogen in young children and the elderly.More than 11%of the deaths under 5 were related to infection of S.pneumoniae.Polysaccharides（PS）are the major virulence factor which is outside the bacteria to protect it away from the complement deposition.There has been found 96 serotypes strain,over 30 can cause diseases.All currently commercial available pneumococcal vaccines are designed based on the serotype-specific polysaccharide capsule of the bacterium,mainly including the polysac-charide vaccine and the conjugate vaccine.The polysaccharide can’t induce a T cell-dependent immune response,so its formulations fail to protect the major risk group,young children under 2.While the conjugate vaccines（PCV）have been shown to be safe,immunogenic and induce a T cell-dependent immune response in children 2 years of age,they are based on limited types of polysaccharides.Moreover,they are associated with serotype replacement and restricted coverage and are too expensive for use in the parts of the world with the greatest need,unless heavily subsided.Therefore,development of new kind of S.pneumonia vaccine is critical and significant.Much research effort focus on pneumococcal factors to find candidate proteins that to be included in future protein based vaccines.Target is to develop a protein-based pneumococ-cal vaccine that confers serotype-independent protection in all age groups.PspA,a pneumococci virulence factor and a choline-binding protein.It inhibits other-wise spontaneous classical complement activation on the pneumococcal surface.It has five major domains:signal peptide,an alpha-helical amino-terminal domain（N-terminal）,which exhibits a pattern of sequence variation that was used to classify PspA molecules in-to clades;a proline-rich region（PRR）,a choline-binding domain to anchor the protein across the cell wall and C-terminal.Antibodies generated against PspA are highly cross-reactive and cross-protective.The major cross-protective epitopes are located in the N-terminal alpha-helical sequence of PspA,especially the first and last 100 amino acids.This area called clade defining region,CDR,divides PspA variants into three families,which are further sub-divided into six clades:family 1（clades 1 and 2）,family 2（clades 3,4 and 5）and family 3（clade 6）.Since over 98%of strains express family 1 or 2 PspA.PspA has immunogenicity,it can induce antibody in mice,which could recognize the pneumococci and promote the C3 deposition.Immunizations of recombinant PspA frag-ments have also been shown to induce protective immune responses against pneumococcal colonization,against lobar pneumonia and invasive infection.In animal models,PspA showed cross-protective with strains in other clades.It can also provide protection when mice were immunized with human serum through passive immunization.It has been shown that selection of appropriate PspA molecules as immunogens and the use of optimal adju-vants can offer increased coverage.Several preclinical trials utilizing live vectors,such as Salmonella and WP,enhanced the protection when against with pneumococcal infection.Due to the variability of PspA,the PspA-based vaccine should contain at least PspA of two strains,to against all the pneumococcal infection.Moreover,our group has developed mucosal vaccines with BLPs as adjuvants.Bacte-rium-like particles（BLPs）are based on non-genetically modified gram-positive bacteria and can be used to potentially enhance mucosal vaccines.They consist of non-living bacte-rial-shaped delivery particles with adjuvant properties,which can easily be loaded with an-tigens containing a cell-wall binding domain,called protein anchor（PA）.The BLPs are made from acid-pretreated Lactococcus lactis bacteria,with their original size and struc-tures of about 1μm retained,and are thus ideally sized for uptake by the M-cells on the mucosal surface.The PA domain is composed of three LysM motifs of about 45 amino ac-ids,separated by spacer regions,and can be added to antigens as recombinant fusion pro-tein,which are covalent binding with BLPs and stable.Mucosal vaccination with BLPs are safe and repeatable,don’t induce the immune tolerance in animal model.According to the situations,our lab developed two kinds of PspA-based pneumococcal vaccine,a systemic vaccine with Al（OH）₃ as adjuvants and a mucosal vaccine with BLPs.The antigen mixture of mucosal vaccine has completed.So in this study,we will continue the research.There are four major parts in the research.In the first part,the antigen mixtures of sys-temic vaccine were compared in groups,with PBS as negative control and PPV23 as posi-tive control,antibody titers were measured.Then challenged with two strains from different families.The protections helped us to choose three better groups to enter following re-search.In the second part,two types of vaccines were generated,a systemic vaccine,includ-ing the fusion protein PsaA-PspA23 and single protein PspA4 as antigens,and a mucosal vaccine,using fusion proteins of PspA2-PA and PspA4-PA antigens attached to BLP.We compared antibody types and levels,bacterial colonization and the survival rate conferred by these vaccines after challenge with two strains from different families.Through these methods,the systemic vaccine should be the better choice.In the third part,we compared the inoculation modes,the subcutaneous（s.c.）and the intramuscular（i.m.）.We measured the antibodies last for 6 months and the cytokines.The decrease of bacteria loads and survival rate were also considered.The final choice is s.c.In the fourth part,we aimed to evaluate the protective properties of the bivalent sys-temic PspA vaccine.With 10%Al（OH）₃ as an adjuvant,PsaA-PspA23 5ug+PspA4 20ug as antigens,mice were immunized subcutaneously three times every two weeks,and serum was collected to measure the bacteria-binding antibodies.The mice were then challenged intranasally with different PspA clades to measure the decrease of bacteria loads and sur-vival rate.All 25 strains covered by PPV23 were texted with a complement killing method in vitro.The innovation of this paper as bellows:Through several rounds of animal experi-ments,optimized the association of the antigens（PsaA-PspA23 5mg+PspA4 20mg）,the immunization route（systemic vaccine S.C.）,established the animal models,evaluated the broad-spectrum of the vaccine in vivo and in vitro.At the same time,we compared the protective properties of two kinds of vaccine against pneumococcal infection;lay a founda-tion for the strategy of combined immunization.