Multimerization And Protective Efficacy Of Rotavirus VP4 Truncated Proteins

Rotavirus is a major pathogen that causes diarrhea in infants and young children under 5 years of age worldwide,with 40 to 60 million deaths worldwide due to rotavirus infection.Vaccine is the most effective way to prevent rotavirus infections.Two rotavirus vaccines(Rotateq and Rotarix)are now available worldwide,and more than 70 countries have included rotavirus vaccines in immunization programs.With the promotion of rotavirus vaccines,the number of rotavirus-induced deaths become from 40-60 million to 20 million or so.However,the current rotavirus vaccine has significant regional differences in efficacy,and could induce intussusception and other safety issues,so a more safe and effective study of rotavirus subunit vaccine is important for further reducing rotavirus-induced morbidity and mortality.VP4,as a spike protein,mediates rotavirus adsorption and cellular entry process.VP4 antibody can block the two processes,rotavirus genetic engineering subunit vaccine is an important candidate antigen.In the previous study,VP4 truncated protein with N-terminal amino acid at position 26 and C-terminal amino acid at position 476 was expressed in soluble status in Escherichia coli,and its immunogenicity with aluminum adjuvant is significantly higher than VP8-1(aa26-231).In this study,we investigated the reasons for the effect of 26-476 protein immunization,evaluated the formation conditions and properties of 26-476 polymers preliminarily,and introduced the multimerization to other genotype VP4 protein.In order to obtain high immunoprotective VP4 recombinant protein with uniformity,stability,immunogenicity to be the rotavirus candidate vaccine.In this study,we first explored the immunogenicity of 26-476 with different adjuvants,endotoxin and disulfide bond.It was found that immune adjuvant and endotoxin had some influence on the immunogenicity of protein,but not major factors.26-476 could become polymer because of the formation of disulfide bonds,which significantly enhance the immunogenicity and immunoprotection.Then we investigated the formation conditions of the 26-476 polymer.It was confirmed that the salt ion concentration was directly related to the formation of the 26-476 polymer,whereas pH was a key factor in the size of the polymer.The morphological size and thermal stability of different polymers were analyzed by TEM,AUC,HPLC and DSC.It was confirmed that the 26-476 polymer was uniform in size and in thermal stability but irregular in morphology.And the antigenicity,immunogenicity,immuno-neutralizing activity of the polymer were investigated.It was found that the polymers formed with TB8.0 and TB8.8 maintained the integrity of the surface epitope.Its three properties have been strengthened.Finally,we introduced the multimerization to other genotypic strains,including human strains,and found that the formation conditions for VP4 truncated protein polymers of different genotypes is versatile.In conclusion,this study obtained homogeneous polymer of VP4 truncated protein,the polymer size uniformity,well thermal stability,compared with the trimer which has a higher antigenicity,immunogenicity and immunity neutralizing activity.It provides a great idea for the VP4 gene engineering subunit vaccine development.