Immunological Studies On Streptococcus Pneumoniae Serotype 3 Oligosaccharide-based Glycoconjugates And Construction Of A New Immunopotentiating Carrier Protein For Conjugate Vaccine

Lower respiratory tract infections(LRTIs)remained the most deadly communicable disease,causing 3.0 million deaths worldwide in 2016.Streptococcus pneumoniae(S.pneumoniae,Spn)is a bacterium that has been widely linked to LRTIs in infants,the elderly and immunocompromised individuals.Spn causes many pneumococcal diseases such as meningitis,bacteremia,pneumonia,acute otitis media and sinusitis,leading to a high rate of the morbidity and mortality.With the increase of antibiotic resistant Spn strains,such infectious dieases have been becoming a severe health issue.As a result,vaccination against bacterial infections including pneumococcal infections has become an attractive strategy.So far,all commercialized pneumococcal vaccines used the capsular polysaccharide(CPS)as the antigen,which were plain or conjugated to a carrier protein,such as anti-pneumonia 23-valent polysaccharide vaccine(PPV23)and glycoconjugate vaccines 7-valent polysaccharide conjugate vaccine(PCV7),10valent polysaccharide conjugate vaccine(PCV10)and 13-valent polysaccharide conjugate vaccine(PCV13).The inoculation of these vaccines has led to a drastically decrease in pneumonia cases in young children and adults.However,these vaccines have some pitfalls,such as hyporesponsiveness of S.pneumoniae serotype 3(ST3),restricted coverage,serotype replacement and carrier induced epitopic suppression(CIES).Therefore,more efficacious vaccines are urgently needed for Spn.To overcome the pitfalls of Spn vaccines,many endeavors have been done.For example,several semi-synthetic glycoconj ugates composed of synthetic oligosaccharide fragments of ST3 CPS have been investigated.Although studies on ST3 CPS oligosaccharides revealed that their chain length had a significant impact on the immunological activities,the optimal oligosaccharide antigens of ST3 CPS was still unknown.In addition,the development of Spn protein-based vaccines has been pursued for decades,but the immunogenicity for most of Spn proteins in humans has been proved to be lowefficacties.Therefore,utilization of self-contained antigen as a carrier protein in Spn conjugate vaccine to prevent serotype replacement and reduce selective pressure of Spn serotypes is a more powerful way to construct new Spn vaccine.In this thesis,three research contents for development of Spn vaccines are mianly included:(1)Semi-synthetic glycoconjugate vaccines to elicit T cell-mediated immune responses and protection against ST3.To develop more effective ST3 vaccines,the penta-,hexa-,hepta-,and octasaccharide fragments la-d of ST3 CPS were synthesized and covalently coupled with TT via the bifunctional glutaryl linker to give TT conjugates 2a-d.We performed a systematic study on ST3 oligosaccharide-TT conjugates 2a-d as potential ST3 vaccines.The immunological results revealed that conjugates 2a-d elicited strong carbohydrate-specific T-cell dependent immunity,which is desired for prophylactic vaccines.It was also demonstrated that immunizing mice 2 or 5 times with conjugate 2b and in combination with either Freund's adjuvant(FA)or Alum adjuvant(AL)gave similar immunological results.However,conjugates with different linkers showed slightly different activities,and 2b with the glutaryl linker was better than 4 with the squarate linker,which was consistent with literature results.Moreover,the antisera of 2a-d exhibited extensive cross-reactivities with all synthetic oligosaccharides,suggesting that the carbohydrate-specific antibodies elicited by 2a-d recognized a common core motif,which is an interesting finding worthy further investigation.Accordingly,it was anticipated that these antibodies should be able to recognize ST3 CPS on bacterial cells,which was eventually proved by the strong binding of the antisera with ST3 cells.More importantly,opsonophagocytic killing assay(OPA)results disclosed that all of the antisera obtained with 2a-d mediated strong bactericidal activities in vitro,indicating their potential protective function against ST3 infection.Both the immunological results and the binding assay and in vitro OPA results further showed that penta-and hexasaccharide conjugates 2a and 2b were significantly more potent vaccines than hepta-and octasaccharide conjugates 2c and 2d to induce very robust antibody responses that were highly effective to mediate opsonophagocytic killing of ST3 cell.This together with Seeberger and co-workers' finding that ST3 CPS tetrasaccharide exhibited better binding affinity than the shorter analogs to ST3-specific and protective monoclonal antibodies suggested that ST3 CPS penta-and hexasaccharides might be the optimal ST3 oligosaccharide antigens for vaccine development.Furthermore,in most assays,hexasaccharide conjugate 2b exhibited better properties and activities than pentasaccharide conjugate 2a.Therefore,2b was identified as the most promising ST3 vaccine candidate and was then subjected to in vivo evaluations using an established mouse model.It was ultimately proved that immunization with 2b could effectively protect mice from ST3-induced lung damages and infection and elongate animal survival.In-depth analysis of the induced the immune responses demonstrated that immunizing mice with conjugate 2b elicited long-term immunological memory.Therefore,2b should be useful for long-term protection.In vitro studies further revealed that a significant portion of primary CD4+T cells isolated from 2b-inoculated mice could be activated by ST3 CPS hexasaccharide and be stimulated to express IL-4,indicating their differentiation into Th2 cells.Th2 cells provided T cell help to B cells to evoke strong antibody responses and humoral immune responses responsible for the eradication of extracellular pathogens.According to the literature,a hexasaccharide hapten might be the desired fragment size for B cell receptor(BCR)recognition to facilitate B cell activation.Eventually,it is proposed that glycoconjugate 2b might mainly induce T cell-dependent humoral immune responses for the protection of mice against ST3 infection.(2)Protective immune response elicited by the chimeric protein YAPO and YAPL that are constructed by Spn surface protein Ply,PspA and PsaA.Many studies have been shown that fusion antigens or combination of several proteins are more powerful than single antigen.In order to obtain a more powerful Spn protein antigen,we construct two fusion proteins using different linkers.Firstly,according to the Spn protein virulence factors pneumolysin(Ply),Pneumococcal surface protein A(PspA)and Pneumococcal surface adhesin A(PsaA)of Spn,we constructed their relevant truncated protein rsPspA,PsaA and nontoxic rsPly.The immunological studies revealed that truncated protein rsPly,rsPspA and PsaA could elicit strong specific T-cell dependent immunity that are desired for prophylactic vaccines.Furthermore,the elicited antibodies could recognize S.pneumoniae serotype 3,4,5,6B,15B,19A and 23F(ST3,ST4,ST5,ST6B,ST15B,ST19A and ST23F,respectively)cells.Encouraged by the immunological results of truncated protein rsPly,rsPspA and PsaA,we constructed fusion protein YAPO and YAPL,and carried out a systematic study on the immunological characteristics.Immunological studies of fusion protein YAPO and YAPL revealed that fusion protein elicited strong specific T-cell dependent immunity,and corresponding antibodies induced by YAPO and YAPL had good recognition to wild type Ply,PspA and PsaA.Furthermore,antibodies elicited by fusion proteins could recognize various serotype Spn cells,and OPA studies demonstrated that antibodies could induce efficient protective to ST3 and ST6B bacterium in vitro.Moreover,in vivo evaluations using an established mouse model proved that immunization with YAPO and YAPL could effectively elongate animal survival in the condition of ST3 and ST6B infection.These results showed that YAPO and YAPL could provide broad-protection and they might be more potent glycoconjugate vaccine carrier protein than TT protein.(3)Protective immune response elicited by a semi-synthetic glycoconjugate vaccine containing a chimeric self-contained carrier protein and ST3 CPS oligosaccharide.To prevent serotype replacement and reduce selective pressure of Spn serotypes,we used self-contained antigen YAPO and YAPL as carrier protein in Spn glycoconjugate vaccine.To evaluate whether fusion protein could be really used as carrier protein of oligosaccharide,we conjugated ST3 pentasaccharide la with YAPO and YAPL to generat conjugate 5a and 5b,respectively.Immunological studies of conjugates 5a-b revealed that they elicited both strong protein-specific and pentasaccharide-specific Tcell dependent immunity.Moreover,TT conjugate 2a mainly elicited pentasaccharidespecific IgG1 antibody,whereas conjugate 5a-b induced specific IgG2b,suggesting a better binding to Fc receptors and correlated better with opsonophagocytosis than IgG1.Furthermore,antibodies induced by conjugates 5a and 5b could recognize all seven tested serotype Spn cells,while TT conjugate 2a only recognized ST3 cells.More significantly,OPA results showed conjugates 5a and 5b elicited highly efficient opsonophagocytic antibodies which could induce significant killing of the ST3,ST6B,ST19A and ST23F cells,whereas TT conjugate 2a only induced the killing of ST3 cells.All of these results suggested YAPO and YAPL were more potent carrier protein for Spn glycoconjugate vaccine than TT carrier protein.In depth analysis of the protection in vivo demonstrated the immune responses elicited by conjugate 5a could greatly reduce bacterial colonization in intraperitoneal and effective prolong survival time and survival rate in the case of ST3 or ST6B infection,whereas conjugate 2a could only protect mouse from ST3 infection.These results have ultimately proved that YAPO-pentasaccharide conjugate 5a could provide effective immunoprotective responses in mice more than conjugate corresponding serotype,further suggesting that YAPO was a more powerful carrier protein than TT protein for development of Spn glycoconjugate vaccine.In summary,our studies foucsed on the poteinal defects of PCV,and aimed to overcome the problems of hyporesponsiveness of ST3,restricted coverage,serotype replacement,and CIES.We preformed detailed immunological and structureimmunogenicity relationship studies of the TT conjugates 2a-d,and assessed the potential ability of YAPO and YAPL to be self-contained carrier protein of Spn oligosaccharide antigen.This research work should have a great significance on development of Spn glycoconjugate vaccine.First,the detailed immunological studies of the conjugates 2a-d promoted the structure-immunogenicity relationship study of ST3 CPS oligosaccharide-based glycoconjugates.Second,ST3 hexasaccharide was identified as the most promising oligosaccharide antigen for vaccine development,which might overcome current PCV problems such as hyporesponsiveness of ST3.Third,we constructed the chimeric proteins of YAPO and YAPL,and demonstrated their protection against Spns,which could possibely promote the development of Spn protein vaccines.Eventually,YAPO and YAPL are poved to be better carrier proteins for Spn vaccine than TT protein,which could provide broader range of protection against Spn infection.Our studies provide a new self-contained carrier protein for Spn conjugate vaccine and might overcome the problems of restricted coverage,serotype replacement and CIES.Most importantly,all of our results obtained above can promote the development of new PCV.