Fundamental Research For Vaccines And Monoclonal Antibodies Against Enterovirus D68

Enterovirus EVD68,a member of species D of enterovirus,belongs to picornaviridae.In recent years,the outbreak of enterovirus infection worldwide has aroused public attentions.EVD68 virus has been one of the focus of epidimic surveillance in many countries around the world.Currently,no effective preventive vaccines and therapeutic drugs commercially available against for EVD68 infection.In the first part of this study,we illustrate an EVD68 vaccine based on inactivated virus.The virus was inoculated to the Vero cells grown on the surface of cytodex-1 microcarriers(5 g/L)in 500 ml spinner flasks with a multiplicity of infection(MOI)of 1/5000.On the fifth day post-infection the virus supernatant was harvested,with the viral titer up to 108.15 TCID50/ml.The virus was then inactivated with ?-propiolactone(BPL)and purified by PEG precipitation and sucrose gradient ultracentrifugation.The highly pure viral particles were obtained and analyzed: three major viral proteins(VP1,VP2 and VP3)were observed by SDS-PAGE and western blot.Electron microscopy analysis also revealed 30 nm spherical particles of EVD68.The inactivated and purified EVD68 vaccine candidate formulated with alum adjuvant was found to induce EVD68-specific serum antibody responses in mice and the antisera strongly neutralized both the prototype(Fermon)and circulating strains of EVD68 in vitro.In this part we developed an EVD68 vaccine based on inactivated virus,which may offer an alternative way for fighting against epidemic EVD68 virus.In the second part,we studied the potential of VLP as candidate vaccine against EV68 infection.We successfully generated EV68 VLPs in insect cells by co-expressing the P1 precursor and 3CD protease of EVD68 delivered by a recombinant baculovirus.Biochemical and electron microscopic morphology analyses showed that EVD68 VLPs were composed of three capsid proteins VP0,VP1,and VP3,the products of precursor P1 cleavage,and their appearance showed spherical particles with diameter ~30 nm.EVD68 VLPs immunized mouse produced serum antibodies displayed broad and potent neutralizing activities against protype and clinical isolates of EVD68 virus in vitro.In vivo protect studies,antibodies induced by maternal immunization with these VLPs provided complete protection when challenged with lethal EVD68 in suckling mice.And also,the passive transfer of anti-VLP sera provided full protection of neonatal recipient mice against lethal EVD68 infection.Together,this part results provide us the recombinant VLP-based EVD68 vaccine as a promising vaccine candidate.According to the first two parts of studies on EVD68 vaccine,neutralizing antibodies play an important role when host fighting against virus infection.Therefore,we further studied the neutralizing antibodies in immunized mouse for a deep insight on virus and host interactions.In the third part,we immunized mouse with inactivated vaccines and VLP vaccines and 9 neutralizing antibodies were acquired from these mice by using hybridoma technology.After isotype identification and neutralization assay against different strains of EVD68,we generated a serious of antibody escaping isolates.Then we get the neutralization panels of antibodies against all escaping isolates.Consequently,the MAbs and escaping isolates were classed into three different groups in Fermon and US/MO/14-18947 respectively according to the similarity of their neutralizing features.After sequencing the capsid protein of all escaping isolates,a series of amino acid substitutions were unveiled.When all the substitutions were displayed on the surface of the virus crystal or cryo-electron microscope high-resolution structures,we found these amino acid substitutions distribute in some highly exposed surface loops like VP1 BC loop(amino acids 1081,1082,1085,1087),VP1 C terminal(1285,1293),VP2 EF loop(2137,2139,2142)and ?B(2070),VP3 BC loop(3079)and EF loop(3144).When viewed from the structure of whole virus structure,we found these continuous or discontinuous substitutions concentrated in four areas of the capsid respectively.All together,we identified four functionally independent neutralizing antigen sites I,II,III and IV.Among them,site I located close to the five-fold axis,site II and III flanked on both sides of the south rim of the canyon respectively,and site IV sited in the middle of the two fold axis and the three fold.All these information provide us a map of EVD68 neutralizing antigen sites on the virus surface,which will help us understand antibodies and EVD68 virus interaction.Deepen our understanding about the virus mutation and diversity of EVD68 epidemic strains.