The Study Of Antiviral Mechanism Of Cross-neutralizing Monoclonal Antibodies Against Rotavirus VP4 Proteins

Rotavirus is the leading cause of diarrhea in children under 5 years old worldwide,and vaccination is an effective strategy to prevent and control rotavirus infection.There is substantial evidence for heterotypic protection after natural infection of rotavirus or vaccination,while the underlying mechanisms remain unclear.Cross-neutralizing antibodies stimulated by VP4 protein play a key role in mediating cross-protective immunity.Comprehensive analysis of neutralizing epitopes and mechanism of crossneutralizing antibodies will provide a theroretical foundation for the protective mechanisms conferred by vaccine and promote the development of broad-spectrum rotavirus vaccines.Currently,only a few numbers of VP4 specific cross-neutralizing antibodes have been reported,and the underlying mechanism of neutralization is poorly understood.In our previous studies,the truncated VP4*protein(aa26-476)has been proven to be highly immunogenic and can stimulate cross-neutralizing antibodies.The purpose of this study was to develop VP4-specific cross-neutralizing mAbs and to explore the antigen-binding sites and antiviral mechanism through a variety of technologies such as escape mutations,site-direated mutagenesis,and structural elucidation of rotavirus-neutralizing antibody immune-complexes.Furthermore,we hope to elucidate the mechanism of the cross-neutralizing and provided a basis for the development of highly effective broad-spectrum rotavirus vaccine.To achieve these purposes,the current study is structured into three parts.Firstly,a panel of monoclonal antibodies(mAbs)against VP4 protein was generated.In total,50 mAbs were generated and tentatively characterized,including subtype,binding activity,genotype specificity,neutralizing activity and so on.35 of the 50 mAbs were neutralizing mAbs,among which nine mAbs(11A8,5G8,7H13,7H11,2C1,2A12,7C7,9C4 and 15D9)could neutralize all three P-genotype rotaviruses.In part two of this study,9 cross-neutralizing antibodies were further evaluated for neutralizing activity against different rotavirus strains,and 7H11 and 7H13 exhibited broad-spectrum neutralizing activity against all the rotavirus strains tested.The neutralizing epitopes and the mechanism of neutralization of the 7 representative mAbs were further investigated.The key amino acids recognized by these mAbs were obtained by peptide scanning or escape mutation experiments.It was found that the epitopes recognized by 3 mAbs located in the N-terminus of VP8 and the epitopes recognized by the remaining 4 mAbs located in the antigenic domain of VP5.From the life cycle of rotavirus infection,the possible inhibition mechanism of these crossneutralizing mAbs including preventing VP4 protein proteolysis,blocking viral adsorption or blocking infection after adsorption were studied.Our results showed that cross-neutralizing mAbs against VP4 could inhibit rotavirus infection via several mechanisms.4 mAbs 7H11,7H13,2A12 and 2C1 primarily blocking absorption of the virus to the cell,while the remaining 3 mAbs 7C7,9C4 and 15D9 inhibiting rotavirus infection after viral adsorption.In addition,we report for the first time that 3 mAbs(2A12,2C1 and 15D9)could inhibit viral infection by blocking proteolytic cleavage of VP4 protein.Finally,we used Cryo-EM and 3D image reconstruction methods to investigate the structure of the rotavirus particle in complex with the Fab portion of cross-neutralizing antibody 7H13.The amino acids(N268,D270,S284,K290,Y332,N374,F418 and R467)were found to be critical residues determining the binding activity of 7H13.Unexpectedly,we found that 7H13 could inhibit rotavirus infection by antibodyinduced conformational changes,which was also proved by biochemical experiments.Overall,a panel of mAbs was generated against recombinant VP4*protein by immunizing mice for the first time.The neutralizing epitopes and neutralizing mechanisms of cross-neutralizing mAbs were comprehensively investigated.In addition,we first report the high-resolution Cryo-EM sturcures of SA11:7H13 immunecomplex,and reveal details about the neutralizing epitopes and molecular determinants in SA11 related to virus neutralization.These findings can provide an important basis for the development of rotavirus vaccine or antivral treatment,as well as the mechanisms of virus infection and vaccine-elicited cross-protection.