Characterization And Preliminary Evaluation Of The Protective Efficacy Of Recombinant Double-layered Virus-like Particles Of Rotavirus Expressed In E.coli

Group A rotavirus is the leading cause of diarrhea among infants and young children,resulting in approximately 453,000 annual death and causing severe social and economical burden.Vaccine is important for control of rotavirus infection.Currently,two rotavirus vaccines,RotateqTM(Merck)and RotarixTM(GSK)have been approved by FDA.RotateqTM and RotarixTM have been licensed in many countries and have been shown to be efficacious in developed countries.However,the immunogenicity and efficacy of these vaccines was shown to be lower in certain low-income countries,where the rotavirus mortality is much higher than that in developed countries.Moreover,they were both live-attenuated vaccine that there are some potential problems in safety.Therefore,it is necessary to develop more effective and safer rotavirus vaccines,to decrease the rates of rotavirus-related morbidity and mortality worldwide.Virus-like particles was considered to be an excellent candidate for rotavirus vaccine,VP6,VP6-VLP and VP2/6-VLP expressed in eukaryotic system has been proved to be effective against rotavirus infection.In previous studies of our group,rotavirus VP2 and VP6 proteins have been succesfully expressed in Escherichia coli,and VP6-VLPs and dl2/6-VLPs were assembled from VP2 dimer and VP6 trimer.In this study,the antigenicity,stability,immunogenicity and immunoprotectivity of VP6,VP6-VLP and dl2/6-VLP were analyzed,to study the viability of them to be a rotavirus vaccine candidate.First,the antigenicity of VP6,VP6-VLP and dl2/6-VLP were studied by a panel of 15 anti-VP6 monoclonal antibodies.Most of the antibodies showed preference to dl2/6-VLP,indicating that the dl2/6-VLPs have better antigenicity.Second,the Tmi and cloud point of dl2/6-VLP were higher than that of VP6 and VP6-VLP;The binding capacity of dl2/6-VLPs to selected confirmational mAbs was not significantly decreased during a 48 hour heat stress,while the binding capacity of VP6 and VP6-VLP was significantly decreased.The above results indicated that dl2/6-VLPs were more stable,and dl2/6-VLPs were proved to be stable under physiological conditions.Further more,the immunogenicity and immunoprotectivity of the recombinant proteins were evaluated in a mouse maternal antibody model.There was no significant difference in the serum anti-VP6 antibody titer of the dams immunized with VP6,VP6-VLP and dl2/6-VLP.The diarrhea severity and pathogenic changes in intestine of pups in dl2/6-VLP immunized group were observed to be lighter than that in VP6 and VP6-VLP immunized group.Also,no virus shedding in dl2/6-VLP immunized group.Indicating that the efficacy of dl2/6-VLPs against rotavirus induced diarrhea and disease was higher than VP6 and VP6-VLP.The Anti-VP6 antibody titer stimulated by dl2/6-VLPs in aluminum adjuvant was significantly lower than that stimulated by dl2/6-VLP in Freund's adjuvant,but comparable to that stimulated by inactivated rotavirus.However,in a mouse maternal antibody model,the efficacy of dl2/6-VLP against rotavirus induced diarrhea was lower than that of inactivated rotavirus.The results suggested that when anti-VP6 antibody titer was high enough,it can provide enough protect,while when the antibody titer is low,neutralizing antibodies was important in the protection.Thus,the neutralizing antigens were also expressed to improve the protective efficacy of dl2/6-VLP.VP8 and VP8C(aa65-231)were succesfuly expressed in E.coli.The protective efficacy of recombinant VP8 was higher than that of VP8C,however,VP8 was prone to aggregate and degrade,while VP8C can form stable dimers.In addtion,a series of VP6 mutant was constructed by site-directed mutation.None of the VP6 mutant(D90A,R106A and D380A)can assemble into VP6-VLP,while the assembly efficiency of dl2/6-VLP was also decreased.Indicating that electrostatic interaction is the main interaction betweeen VP6 trimers,but the assembly of dl2/6-VLP mainly depend on the interaction between VP2 and VP6.In summary,the recombinant double-layered particle dl2/6-VLP,with higher antigenicity and thermal stability than that of VP6 trimer or VP6-VLP,was more viable to be a candidate antigen for rotavirus vaccine.But in aluminum adjuvant,the antigenicity of dl2/6-VLP is lower than that in Freund's adjuvant,though the anti-VP6 antibody was comparable to inactivated rotavirus in the same condition.Thus,much work is needed to improve its immunogenicity and protective efficacy in aluminum adjuvant;or it can be formulated with neutralizing antigens to improve the efficacy.Meanwhile,there was also much work to be done on VP8 was a viable candidate antigen for rotavirus vaccine.In conclusion,this study provide basis for rotavirus genetic vaccine,and will promote the development and application of genetic vaccines against rotavirus infection and disease.