Structure Basis And Neutralizing Mechanism Of A Novel Monoclonal Antibody Against EV71

Enterovirus 71(EV71), a member of the Enterovirus genus of the Picornaviridae, is the most frequently detected pathogen in hand–foot–mouth disease。Since its discovery in1969, enterovirus 71(EV71) has emerged several times as a serious worldwide health threat,especially in the Asian-Pacific region,causing Tens of millions of people infected by EV71 and tens of thousands of patients presenting with severe complication。China is a high-incidence area of hand, foot and mouth disease(HFMD).Since 2009, the number of deaths and the incidence of HFMD has been ranked first in the class C infectious disease for seven consecutive years. Presently,the phase III clinical trials of EV71 vaccine has been completed and approved to go to the public, but there is no effective healing strategies in severe cases,especially in fatal neurological disease.Therefore,it is extremely urgent to develop specific anti-virus drug。Virus particle,which has a diameter of approximately 300 ?,is composed of 60 copys each of VP1-VP4 and format icosahedron structure。Mature EV71 virus has four kind of capsid protein including VP1, VP2, VP3 and VP4。VP2, VP3 and most region of VP1 are located inside the virus capsid 。VP4 and VP1 N-terminal region are located outside the capsid. Several researchs suggest that the release of Sphingosine inside the hydrophobic pocket induce the "canyon" in the vicinity of the 5 axis region collapses to form a little hole,which causing virus conformational change during the EV71 uncoating process.A pair of reverse parallel α-helix in VP2 separate from each other to form a gateway which play an crucial role in virus genome release。During the uncoating process,VP1 N-terminal region,normally buried under the VP2 reverse parallel α-helix,move to the outside of virus and participate in the construction of virus nucleotide channel。 Therefore, screening antibodys targeting different epitope can help us further reveal the biological mechanisms of virus uncoating process。In this study, we obtained 15 Mabs against EV71 through immuning mice with EV71 particle and capsid proteins. Out of these 15 Mabs, six Mabs was demonstrated tohave EV71-neutralizing activity.Animal protection experiments showed that the protective rate of 6G5、12B6、2B6、2G12 can reach 100% in antibody dose of 10ug/g bodyweight。When the antibody dose decreased to 2ug/g bodyweight,6G5 and 12B6 protective rate can still respectively maintain 100% and 80%, which is higher than that of all other neutralizing antibodies currently reported.EV71 can cause aseptic meningitis,encephalitis,encephalomyelitis,acute flaccid paralysis and other serious neurological complications。By testing the protective effects of the 15 monoclonal antibodies on nerve cells,we found that,compared with RD cells,the protective effect of 2B6 increased significantly in the equal virus attacking titer。The reason of this appearence may be that EV71 has different mechanisms of invasion in muscle tissue and nervous system。Our study found that 6 EV71-neutralizing antibodies could be divided into 3 types according to the results of the protective effect in the in vitro assay,animal protection experiment and difference of recognizing epitope。Firstly,the epitopes of 2B6 and 2G12 was 215-KQEKD-219,which was located in the VP1-GH loop。Secondly,7G6 and 12H3 can recognize the VP1 N-terminal 4-VADVI-8。Thirdly,the recognizing site of 6G5 and12B6 is a conformational epitope。In order to identify this epitope,we solve three kind of complex between 6G5 Fab and different physiological cycle virus, including6G5Fab-mature virus, 6G5Fab-empty virus and 6G5Fab-shrink empty virus 。 Their resolution respectively reach 5.6?,4.9? and 5.0?。6G5 epitope is sited in the region between the gap of VP2 and VP3,which closed to 2-fold axis and 3-fold axis of virus particle, including 135-LDTKL-139, 223-GADG-226, 296-GAT-298 of VP2 and73-VSAQAGKGE-81,144-KDRA-147,209-PNTA-212 of VP3。All of these construct a sophisticated binding epitope comprising “notch” “knob” and “valley”。By mutating the critical amino acids of antibody variable region,we found six functional domains of 6G5 epitope are all involveing the interaction between 6G5 and EV71 virus。After analyzing the mechanism of three different type neutralizing antibody, we suggest that 2B6 binding decrease the flexibility of VP1-GHloop to stabilize the“canyon”area near the 5-fold axis,which restrain conformational change in virus uncoatingprocess.7G6 can handicap the construction of nucleotides channel。Interestingly,6G5 can play the function like“fake receptor”to mediate virus uncoating process earlier, which induce virus genome release and handicap the 135 S uncoating intermediate formation。These results indicate that the binding epitope of 6G5 is a new neutralizing epitope against EV71。Its neutralizing mechanism also provide some new approachs for healing strategy against virus and lay a solid foundation for the development of antibody drugs.