Study On Prophylactic Recombinant Genetic Engineering Vaccine Against Hand Foot And Mouth Disease

Human enterovirus71and coxsackievirus A16（CA16）have been identified asthe two major etiological agents of hand, foot and mouth disease（HFMD）. Largeoutbreaks of HFMD have recently been reported in the Asia-Pacific region, which isbecoming a common acute viral disease in these areas and posing a serious healththreat to children. While HFMD is usually mild and self-limiting, it may lead tosevere neurological complications and even death. So far, prevention of hand foot andmouth disease vaccine is not yet available.EV71is a non-enveloped RNA virus of the Picornaviridae family. The virion isaround30nm in diameter. The icosahedral capsid is composed of60sets structuralprotein（sVP1to VP4）. It has been shown that VP1-3form a pseudo T=3icosahedralcapsid that are located on the surface of viral capsid. VP4is located inside, which isapproximately70amino acids in length and is myristoylated at the N terminus.Crystallographic analysis showed that the mature EV71virus is structurally similar toother enteroviruses.In the present study, the gene of peptide VP4-N20（N terminal residues1-20ofEV71VP4of genotype C4）was fused with HBcAg-encoding gene and subcloned intothe vector pET-22b（+）. HBcAg protein （HBc-N149）and a fusion protein（HBc-N149-VP4-N20） were expressed in E.coli, respectively. The efficientexpression of both proteins was demonstrated by Western-blot after IPTG induction.They were further purified using Ni Sepharose column. Electron microscopy analysisshowed that both HBc-N149and HBc-N149-VP4-N20proteins were able toefficiently form particles with the size around25-30nm. The results suggest that thechimeric HBcAg protein harboring N-terminal20amino acids of EV71VP4stillkeeps the same physical characteristic as the original HBcAg particles.To determine whether chimeric HBcAg particles were capable of elicitinganti-VP4-N20antibody, female BALB/c mice were immunized i.m. with eitherpurified HBc-N149-VP4-N20or HBc-N149. Mice immunized with PBS were used asnegative controls. The immunized animals were bled for the serological analysis. Ourresults indicated that chimeric particles were able to induce anti-VP4-N20immuneresponses.To evaluate whether the chimeric HBcAg particles could induce neutralizingantibodies against EV71, sera from immunized mice were tested for the ability toneutralize live EV71in vitro. The sera from the group immunized withHBc-N149-VP4-N20were able to neutralize EV71. Compare to chimeric HBcAgparticles, HBcAg particles failed to induce neutralizing antibody responses against EV71. Our results suggest that immunization of chimeric HBcAg particles containingVP4-N20epitope can elicit neutralizing antibody responses against EV71.To investigate whether anti-HBc-N149-VP4-N20sera conferred protection tomice, In vivo challenge experiments were performed. EV71BrCr-TR strain was usedfor viral challenge because of its high virulence in neonatal mice. Groups ofone-day-old BALB/c suckling mice were inoculated intraperitoneally（i.p.）with thevirus-sera（anti-HBc-N149-VP4-N20sera and anti-HBc-N149sera）or virus-PBSmixture. After7days, the mice receiving the mixture of EV71with PBS oranti-HBc-N149sera started to show disease symptoms, such as reduced mobility,limb weakness, limb paralysis, and death; the survival rates were20%and40%forthe PBS and anti-HBc-N149sera recipient groups, respectively, at the end of the16-day period. In contrast,90%of mice treated with the mixture ofanti-HBc-N149-VP4-N20sera remained healthy and survived throughout the course.Our results suggested that the immune sera elicited by chimeric HBcAg particlesHBc-N149-VP4-N20conferred protection to neonatal mice against lethal EV71challenge.We further investigated the most immunologically essential sequence of thepeptide by epitope mapping experiments to find high efficacy and minimal peptidesequence required for the neutralizing antibody induction. A panel of peptidescorresponding to the N-and C-terminal truncations of VP4-N20peptide was used forepitope mapping. Once si（xN-terminal）or ten（C-terminal）residues were clipped fromeither end of the inoculation peptide, the polyclonal antibodies raised againstVP4-N20peptide were no longer able to bind. One interpretation of these results isthat there is an essential “core” of the peptide that does not tolerate truncation. Thissuggests that this peptide can elicit a pan-serotypic immune response once the rightsegment of VP4is identified.In the present study, the peptide consisting of N terminal residues1-20of EV71VP4of genotype C4was fused to HbcAg and expressed in E. coli. The resultingfusion proteins were able to spontaneously assemble into chimeric VLPs, whichelicited virus-neutralizing antibody response. We further identified a linearneutralizing epitope in the N-terminus of EV71VP4by epitope mapping experiments.Our results suggest that chimeric HBcAg particles carrying a neutralizing epitope ofEV71VP4could be a promising vaccine candidate and provide novel strategy againstEV71infection.