| Hand,foot and mouth disease is a Class C infectious disease,required legally for case report,that is caused by multiple enterovirus serotypes.HFMD occurs mostly in children under 5 years of age,and current research indicates that the main pathogens causing the disease are EV71,CVA6,CVA10,CVA16 and CVA2[1,2].The capsid structure of CVA2 is symmetrical icosahedron,which is composed of four structural proteins（VP1,VP2,VP3,VP4）,and the binding epitope located in VP1 can be specifically recognized by cell receptors[3].At present,there is a lack of research on the function of CVA2 protein of CVA2.Based on other enterovirusstudies,it is speculated that CVA2 VP1,VP2 and VP3 may contain epitopes that can stimulate the immune system to produce antiviral neutralizing antibodies.Genefragments of CVA2-VP1 and CVA2-VP3 were inserted into pGEX-6p-1 vector,respectively.The GST-VP1 and GST-VP3 fusion proteins were expressed in E.coli and purified for immunizing rabbit.The rabbit antisra were obtained andcharacterized by using PAGE,WB,IF assays.In the development of vaccines,there are two very important technical hurdles:one is the selection of candidate vaccine strains;the other is the establishment ofanimal models to evaluate the protective efficacy of candidate vaccines.This research is a sub-project of the multivalent HFMD vaccine research and development project.The main purpose is to establish a CVA2 active immune protection model and toconduct preliminary screening of candidate vaccine strains.Animal models are the most effective tool for evaluating the efficacy ofcandidate vaccines.Among them,the protective efficacy of EV71,CVA16,CVA6,CVA10 whole virus inactivated vaccines and corresponding virus-like particles has been reported using mouse infection models[4-5].In order to obtain vaccine strains with high titer and high virion yield for production of inactivated,whole virusvaccines,four RD-cell strains of CVA2-1580,CVA2-1730,CVA2-1881 and CVA2-2703 were adapted to grow in Vero cell which is cell line permitted for humanvaccine production.After 11 generations of cultivation,CVA2-1730 with a higher virus titer was selected as the candidate vaccine strain for 10-layer cell factorycultivation,and a certain amount of viral protein FP and EP were obtained for studies of immunogenicity of the candidate vaccine.During the vaccination of the targeted population,the vaccinated usually needs to receive two immunizations to generate a high-level immune response and immune persistence.In animal model for efficacy evaluation,there is an interval between the priming and boosting immunization and between boosting immunization andchallenge to accumulate protective antibody.So obviously,it is necessary to obtain a challenge strain that can cause disease or death of adult animals.In the case of mouse model,minimum 14 days old mice are needed for priming,boosting and challenging.In this study,in order to obtain a virulent strain that can be used to perform a lethal challenge,14-day-old Kunming mice are needed to carry out an immunization andprotection schedule.CVA2-1388 was adaptively passaged in Kunming mice at route of intracranial infection,and attempts were made to increase the virus titer andvirulence（grouth of challenge strain in RD cell subculture）for challenge at high doses.Different routes of inoculation were also tried.Finally,A CVA2 virus seed bank capable of stably killing 14-day-old Kunming mice was generated.In the end,this study will use the virus species that can persistantly kill 14-day-old Kunming mice as the challenge virus,and use the whole virus-inactivated CVA2-1730-R11V11 FP as the antigen to immunize the mice,and successfully establish the CVA2 active immune protection model. |
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