Localization And Structural Character Of Conformational-dependant Neutralizing Epitopes Of Enterovirus 71 And Coxsackievirus A16

HFMD is the highest number of infections and mortality in Class C infectious diseases seriously endanger the life health of our children.CA16 and EV71 are the major pathogen of HMFD.So the primary task nowadays is to develop CA16 and EV71 bivalent vaccine to control epidemics.CA16 and EV71 both belong to A serotype of human enterovirus,highly homologous in gene and similar in viral structural.But the ability of eliciting specific neutralizing antibody of formalin-inactivated CA16 are obviously lower than EV71,while the P-particles of CA16 even failed to induce neutralization antibody.The phenomenon reminder that the structure feature of neutralizing epitopes between CA16 and EV71 may be different,as the potent and important cross-genotype neutralization epiopes are conforamtional.However,due to the lack of research on the EV71 and CA16 conformational epitopes,the phenomenon above can not get a good explanation,which obstacle to the development of vaccines.Firstly.We estabished a blocking methodology to evaluate the immune advantage degree of Site A?Site B?Site C conformational neutralizing epitopes of Jiangsu-C4 pre-identified in the early research.As all the antibody binding to Site B advantage to block the immuned serum of EV71,we identified Site B is an immunodominance epitope.Then we selected two escape virus mutating VP3-S64 to I64 or VP3-P59 to L59 as antigens immuning Balb/c and evluated the ability of neutralization to different genotypes of EV71.Finding that the serum immuned by the escape virus mutating VP3-S64 to I64 could no neutralizing some genotype of EV71 as well as the serum immuned by wild of EV71.Reminding that VP3-S64 is a key amino acid for viurs to induce a broad spectrum neutralization antibody,and hint that conformational neutralization epitope Site B is important to the antigenicity of EV71.Second.According to phylogeny tree,we selected a viral strain 190-CA16 as the immunogen for CA16-specific neutralizing antibody preparation,and constructed an CA16-sepcific NT-ab library containing 41 clones.Further,we established anti-CA16 neutralizing antibody panel containing 22 clones according to the neutralizing potent against to 9 natural isolates of 2 different genogypes of CA16.Moving forward,we constructed the methodology of escape mutants to identify the epitope of anti-CA16 antibody using relate experience applied in EV71.Using the methodology we gained a library of neutralizing escape mutants and identifide the key bind site of 22 antibodies.Locating the binding site's spatial distribution resulting from homology modeling study,we identified two major conformational neutralizing epitope region of CA16:Site ?(VP1-E213,L215,A217,N218,L220,D292,K293;VP3-K59,N141)and Site p(VP1-N282;VP2-K69,S70,V159,S230;VP3-V75,S77,K78).Thirdly.We analysised the differences of the spatial structure of comformational neutralization epitopes between CA16 and EV71.Founding that the spatial structure of Site a and Site ? of CA16 changed significantly between solid and empty virus particles,while only small changes occurring on the spatial structure of Site A?Site B and Site C of EV71.Suggesting that the comformational neutralization epitopes of EV71 are affected by lower levels of the damage to the viral particle structure than CA16.May be that's the reason why formalin-inactivated EV71 P-particles and R-particles could induce strong EV71-specific neutralizing antibody responses while the CA16 P-particles could no.These results illustrate the localization,composition and structural characteristics of conformational neutralizing epitopes between CA16 and EV71 and provide an important theoretical basis for vaccine development.