Neutralizing Effects And Mechanism Of Monoclonal Antibodies Against Enterovious71

Enterorirus71(Enterovirus71),one of the commom pathogen for hand-foot-mouthdisease(HFMD),can cause disorders with a wide range of clinical manifestations,includingfever,a serious of cutanaous,visceral diseases in faint and young children.Especially in theAsia-Pacific regions, enterovirus71infection has caused significant morbidityandmortality.From2008to2012, numerous outbreaks of HFMD occurred in mainland China,with over2,000fatal cases reported,which has become a serious public heathy problem.EV71virus is a non-enveloped, single positive-strandedRNA virus with a genome size ofapproximately7.4kb and belongs to Enterovirus species A within the Enterovirus genus ofthe Picornaviridae. The viral capsid,which has a diameter of approximately300, iscomposed of60copies each of VP1to VP4that are organized onto a quasi-T=3icosahedral lattice. The capsid proteins VP1, VP2, and VP3each possess a β-sandwichjelly roll fold and form the outer surface of the capsid shell, whereas VP4is situated insidethe shell.Nowdays, EV71infection prevention and control work still face many problems, itspathogenic mechanism and popμlar trend is not yet clear, neither clinically effectivevaccine to prevent infection, nor effective antiviral drugs used to treat, which makeEpidemic is hard to be suppressed for a long time. Aiming at the status quo, protectiveantibody-based therapyagainst EV71has been widely concerned. Although there areseveral neutralizing antibodies and a fewof neutralization epitopes, protection mechanismand structure foundation are still unknown.In this study, we intended to obtain neutralizing mAbs against EV71using hybridomatechnology, employing the method of immuning mice with EV71particle and cap sidproteins. Finally, we generated14mAbs against EV71. We design a series of experimentsto determine whether these mAbs can react with EV71and recombinant capsid proteins.As expected, all mAbs recognized EV71vaccine by ELISA. The neutralizing ability ofmAbs were then judged by cytopathic effects of human rhabdomyosarcoma cells (RD)infected with EV71.Four of these mAbs can effitively protect RD cells infected from EV71,mAb2G12and12B6coμld protect60%RD cells from cytopathic effectat theconcentration of100μg/ml.Protective effect of antibody on RD cell can be verified byvirus plaque assay.Western blotting have found that there are three mAbs not binding tothe recombinant VP1protein under denaturing conditions, suggesting that these three antibodies epitopes may be spatial. The remaining four antibodies can identify linearepitopes on VP1protein.To map the neutralizing epitope of mAb2G12, a standard phage-display techniquewas firstly carried out. Through analysis of phage-displayed peptide and sequencealignment, we found that mAb2G12specifically recognizes the215KQEKD219sequencewhich located on the GH loop of VP1,which is far away from the receptor bindingdomain.The synthesis of epitope peptides can be combinedwiththe correspondingmonoclonal antibody specificity ina concentration-dependentmannerand can competitivelyblock the binding of2G12with EV71.Researchs has shown VP1GH loop acts as an adaptor-sensor for the attachment ofcellular receptors, converting heterologous inputs to a generic uncoating mechanism.Neutralizing mAb2G12specifically recognizes the215KQEKD219sequence whichlocates on the GH loop of VP1. Next,we generate the extracellμlar domain of EV71receptor SCARB2with gene synthesis. We find that the extracellμlar domain ofSCARB2recognized EV71vaccine and VP1by ELISA,which is not affected by theconcentration of mAb2G12. In order to define the protection mechanism of2G12,we testrelative virus copies of RD cells using RT-PCR.The resμlts show that relative virus copiesdecreased with the rise of antibody concentration,which indicates that protectivemAb2G12could affect the virus uncoating rather than blocking the combination ofreceptor and virus.On the whole,we reckon that neutralizing antibody2G12could affectthe key functional domains of VP1GH loop,whose conformational changesinactiveenterovious uncoating process followed by closing extrusion of the viral genome into t hecytoplasm of the target cell,toprotect the host cell from infection.In summary, this study generated four neutralizing antibody againstEV71,mappedtheneutralizing epitope of mAb2G12and illustrate the neutralizationmechanisms of2G12from the perspective of virus uncoating.These findings help toelucidate the relationship between structure and functions of envelope protein, and providetheoretical foundations for further investigation of pathogenesis of EV71. Our findingprovides a new approach to studying the neutralization mechanisms of EV71antibody andmight be potential candidates for anti-EV71therapeutics.