Immunogenicity Assessment Of Functional Domains Of Group A Streptococcal C5a Peptidase And Their Application As Carrier Proteins For GAS Glycoconjugate Vaccine

Group A streptococcus(GAS)causes a variety of human diseases,such as mild pharyngitis,impetigo,severe toxic shock,rheumatic heart disease and acute glomerulonephritis.These fatal infections usually lead to high morbidity and mortality.Currently,the main treatment for GAS infection is antibiotic treatment.With the increasing usage of antibiotics,the drug-resistant strains have come to being correspondingly.The prevention and treatment of GAS infections has been becoming a great challenge.Therefore,the development of safe and effective vaccination strategies against GAS infectious diseases is still in urgent need.The development of GAS vaccines has been mainly focusing on protein-based vaccines.For instences,the 26-and 30-valent M protein-based vaccines have entered to the clinic.In addition,glycoconjugate vaccines prepared by coupling bacterial polysaccharides and protein carriers can improve the immunogenicity of carbohydrate antigens and effectively reduce disease infections.Therefore,GAS glycoconjugate vaccines have attracted more attention,and their immune protections have also been well identified in animal model.Although GAS vaccines have been well developed,there are still many shortcomings in these vaccines.For example,there are more than 80 serotypes of M protein in GAS;the GlcNAc side chain of GAS group A carbohydrate(GAC)may cross-react with the human tissues,and carrier-induced epitope suppression(CIES)caused by the same vector in vaccines,etc.Therefore,to overcome the the above-mentioned findings,we choosed the GAS highly conserved virulence factor C5a peptidase(ScpA)as the protein antigen,then recombined the different functional domains of ScpA and further explored their potential for subunit protein vaccine.Furthermore,we also screened the functional domain proteins with improved binding abilities to GAS as new carrier proteins to explore the potential of these proteins as new carriers for glycoconjugate vaccines.In addition,we synthesized GAC oligosaccharide derivatives containing rhamnose backbone without N-acetylglucosamine(GlcNAc)side chain,and then conjugated them to rsScpA193 carrier to prepare GAC oligosaccharide-rsScpA193 conjugates,which was expected to avoid the potential safety problem from the cross reaction with human tissue caused with GlcNAc side chain.Accordingly,this thesis mainly includes the following three parts:Part 1.Immunological assessment of different functional domains of ScpA as subunit protein vaccinesTo explore the possibility of different functional domains of ScpA as subunit vaccines,we obtained high purity and homogenous protein antigens of rsScpA193,Cat,Fn,PA,Fnl,Fn2,and Fn3,as well as wild-type rsScpA according to its crystal structure.To guarantee the biosafety of these protein antigens,the enzymatic activities of recombinant rsScpA193(His193Ala)and Cat proteins were first evaluated.The results of emzymatic assay indicated rsScpA 193 and Cat proteins were inactivated completely and could be used as potential antigens for immunological evaluation in this study.(1)We carried out systematic evaluation and comparison of the immunological characteristics of these recombinant proteins by immunizing the mice.The immunological results showed that,except for Fn3 protein,all other proteins elicited strong immunogenicity.Cat,Fn and Fnl proteins exhibited much stronger immunogenicity than the relative full-length rsScpA 193 protein.Likewise,all but Fn3 recombinant protein provoked high levels of IgG1,IgG2a,IgG2b antibody titers,which indicated the elicitation of T-cell mediated immune response.(2)We examined the cross-reactions of antisera of each protein toward the wild-type rsScpA.Except for antisera of Fn3 protein,all other antisera of proteins exhibited much better recognitions against rsScpA protein.Furthmore,Cat,Fn and Fnl proteins produced comparable level of positive control rsScpA 193 to cross-react with rsScpA protein.(3)We further verified antisera of each protein would really recognize the ScpA protein on GAS cells.FCM result indicated that these segmented proteins in mice could produce the functional antibodies that recognized ScpA on GAS and thus effectively bond to it.The antiserum of Fn and Fn2 protein displayed the comparative binding activity to GAS cells as compared to that of rsScpA193 serum.(4)ScpA are highly homologous in gene sequences in ScpB derived from Streptococcus agalactiae(GBS).Therefore,we further evaluated the recognization abilities of each antiserum toward GBS.As shown in experimental results,only the antisera of rsScpA193 protein exhibited the significant binding activity towards GBS cells.This result indicated that the relative full-length rsScpA193 protein might possess much more antigenic epitopes and similar spatial structure in comparison with ScpB.Collectively,our study disclosed that rsScpA193 protein could elicited effective antibodies to recognize and bind to both natural GAS and GBS cells,indicating that it might provide the broad spectrum of immunological protection against GAS and GBS infections.In conclusion,Fn,Fn2 and rsScpA193 might be promising target immunogens for subunit protein vaccine development.Part 2.Application of rsScpA193,Fn and Fn2 proteins as glycoconjugate vaccine carriersIn above study,we have proved the strong immunogenic properties and better binding abilities to GAS cells of Fn,Fn2 and rsScpA193 proteins.In this part,we further probed their potentiality as carrier proteins for glycoconjugate vaccine.(1)We synthesized GAC trisaccharide as the model antigen,and conjugated with Fn,Fn2 and rsScpA193 and CRM197 proteins via di(N-succinimidyl)glutarate(DSG)as bifuntional linker,resulting in the glycoconjugates of Fn-Tri,Fn2-Tri,rsScpA193-Tri and CRM197-Tri.And CRM197-Tri conjugate was used as positive control.The trisaccharide loadings of these synthetic glycoconjugates were all in the desired range of 5～10%for glycoconjugate vaccines.(2)We immunized mice with each conjugate,and all immunized protein conjugates provoked significantly high titers of trisaccharide-specific IgG1,IgG2a and IgG2b antibodies.This observation revealed the stimulation of T cell-mediated immune responses.Compared to CRM197 protein,Fn and Fn2 proteins as carriers possessed the comparative capability whilst the relative full-length rsScpA193 protein shows better ability on conversion of nonimmunogenic trisaccharide into T-cell dependent antigen after their conjugation.Our results suggest that Fn,Fn2 and rsScpA193 protein might be promising carrier proteins for development of glycoconjugate vaccines.(3)We evaluated the effect of these glycoconjugates on the corresponding carrier protein.The results depicted that glycoconjugates after conjugated with GAC trisaccharide might enhance the immunogenicity of the GAC oligosaccharide and carrier rsScpA93 protein.There are no significant effect on Fn and Fn2 proteins.(4)We further verified antisera of these glycoconjugates would recognize the GAS cells.All antisera of these glycoconjugates could effectively recognize and bind GAS cells,and the antiserum of rsScpA193-tri performed best recognition ability than other glycoconjugates.We also further evaluated the recognization abilities of each antiserum toward GBS cells.The similar results were disclosed that all antisera of these glycoconjugates could effectively recognize GBS cells.The antiserum of rsScpA193-tri displayed significantly stronger binding activity than those of Fn-Tri and Fn2-Tri serum.We speculate that ScpB may contained some or most of the effective and similar epitopes of ScpA carrier protein and similar epitopes of GAC trisaccharide existing in GBS cells wall polysaccharide.Collectively,our immunological studies demonstrated that Fn,Fn2 and rsScpA193 proteins,especially for rsScpA193 protein,had great potential application as carrier molecules for glycoconjugate vaccine development when compared to the commonly CRM 197 carrier protein.Part 3.Synthesis of GAC oligosaccharide-rsScpA193 glycoconjugatesIn order to avoid tissue-cross-reactive epitopes by GlcNAc side chain of GAC during vaccine development,we synthesized GAC oligosaccahrides with polyrhamnose core backbone,which lacked the potentially autoimmune GlcNAc epitope.We choosed rsScpA193 that had good immunological activity and extensive binding activity to GAS and GBS cells as the carrier protein to prepare a series of GAC related oligosaccharide-rsScpA193 conjugates.Firstly,we synthesized several rhamnose monomers and important disaccharide module GA-16 and GA-18.We adopt efficient synthesis strategy to synthesis the target molecules,including tri-,tet-,pentaand hexasaccharides.Thereafter,GAC oligosaccharides were coupled with rsScpA193 and BSA via the bifunctional glutarate linker to generate GAC oligosaccharidersScpA193 conjugates and their BSA conjugates.The successful preparation of these oligosaccharide conjugates has laided foundation for further exploring immunological properities of new GAC oligosaccharide conjugate vaccines.In summary,this thesis focused on the following issues,such as the numerous serotypes in GAS,the potential cross-reactions with human tissues,and the CIES effect,etc.,to develp safe and effetious GAS vaccines.We carried out the studies of immunogenicity assessment of different functional domains of ScpA and their application as carrier protein for glycoconjugate vaccines.The important significances of this dissertation are:(1)We first segmented and expressed the different functional domains of ScpA and carried out immunological evaluation.We demonstrated that Fn,Fn2 and rsScpA193 proteins showed great immunogenicity and better recognition of GAS cells,which revealed their potential as subunit protein vaccines.(2)We chosed Fn,Fn2 and rsScpA193 proteins as carriers to construct GAC trisaccharide conjugates.Immunological study of these glycoconjugates revealed that Fn,Fn2 and rsScpA193 proteins exhibited the camparable and even better ability with CRM 197 protein carrier on promotion of the immune response of oligosaccharides.All these results indicated that Fn,Fn2 and rsScpA193 were potential new carrier candidates for glycoconjugate vaccine vaccines.(3)We synthesized GAC oligosaccharide antigens basedpolyrhamnose backbone,and the rsScpA193 as carrier to prepare a series of GAC oligosaccharide-rsScpA193 protein conjugates.We designed these conjugates without GlcNAc side chain to avoid tissue cross-reactive epitopes caused by the GlcNAc side chain of GAC during vaccine development.