Study On The Immunogenicity And Protective Efficacy Of Hand Foot And Mouth Disease (EV71&CVA16) Virus-like Particle

HFMD（Hand, Foot and Mouth Disease） is a worldwide infectious disease, which mainly outbreaks in developing country（especially in Asia-pacific region）. The epidemiological investigation of mainland China showed that HFMD ranks the first among the C-class infectious diseases. Enterovirus71（EV71） and the Coxsackievirus A16（CVA16） were the major pathogens of HFMD, and the prevalent rate of which was more than 80%. However, until now there was no effective drug treatment of HFMD disease, so development of vaccine and vaccination was an important way to control the prevalence of HFMD.Based on the clinical trials of mainland China, the inactivated EV71 vaccine had a safe effect that could prevent over 90% of the EV71-associated HFMD and 80% of other EV71-associated diseases. The inactivated vaccine could induce the neutralizing antibodies carrying good broad-spectrum cross activity, but the serum of which did not neutralize the CVA16 and other HFMD virus, so development of the combined vaccine containing the EV71 and CVA16 was urgent particularly. Until now, the CVA16 inactivated vaccine was still under-clinical trials, which limits the application of the combined vaccines in the clinical trials.There was only one serotype of EV71 and CVA16 respectively. However, each viral strain contained multiple subtype-gene, and the cross activity of neutralizing antibody was different among these gene subtypes. So the key point to develop the effective HFMD vaccine preventing the HFMD caused by EV71 and CVA16, was to select vaccine strains with high coverage-rate of neutralizing antibody and good immunogenicity. However, until now, only the monovalent vaccine with broad spectrum neutralizing antibody was studied, there was no report about whether the cross neutralizing antibody activity was difference in combined vaccine vaccination. There was no detailed and comprehensive study about the immune-suppression and immune-interference between the two vaccines illustrated, and also whether the combined vaccine could prevent the infection of these two viruses. The investigation of HFMD vaccines mainly was focused on the inactivated vaccine and virus-like particles（VLP） vaccines. With the development of the genetic engineering technology and its unique natural advantages, VLP was widely studied. For the above problems, we have done a lot of comprehensive work and provided real and reliable animal experimental data. And we expect to solve current problems of the combined vaccines and develop an effective, safe, good cross activity combined vaccines（EV71 VLPs and CVA16 VLPs）.On the other hand, the dose effect relationship(ED₅₀) reflects the effectiveness of the vaccine and minimum-immune dose, which have the guiding significance to evaluate the vaccine in the preclinical trials and decide the dosage of vaccination in the clinical trials. The antigen ED₅₀ of EV71 inactivated vaccine had been evaluated in pre-clinical trials. However there was no detailed and reliable research data of the ED₅₀ of EV71 VLPs. So we did some research about this problem in this paper: the different dosage of EV71 VLPs purified by chromatographic and injected into mouse. Then we evaluated the ED₅₀ of both antigen and antibody. This work has a great significance for the EV71 VLPs study in the future.Part one: Study on the immunogenicity and protective efficacy of monovalent vaccine and combined vaccines.Firstly, based on the epidemiological investigation of mainland China.We chose the EV71 epidemic strains C4a（EU703814.1） and CVA16 epidemic strains B1a（AF177911.1） to produce the EV71 virus-like particles（VLPs） and CVA16 virus-like particles（VLPs） by utilizing the insect cell Baculovirus expression system. We chose the EV71 and CVA16 inactivated vaccines as the positive vaccines, and then produced monovalent vaccine and combined vaccines.Secondly, depending on the pseudovirus-detection platform in our laboratory, we evaluated the immunogenicity of both the monovalent vaccine and combined vaccines. The results showed that both the monovalent vaccine and combined vaccine could induce the strong neutralizing antibody titer; There was no significant difference between the VLPs monovalent vaccine and inactivated monovalent vaccine, the combined vaccine and monovalent vaccine in the neutralizing antibody titer; No immune-suppression and immune-interference existed in the combined vaccine; In this work, we firstly evaluated the broad spectrum neutralizing antibody of the combined vaccine.Thirdly, the protection evaluation was another important indicator for the vaccine effectiveness. The ICR mice（postnatal day 1） was used to establish the mouse model, which carried EV71 and CVA16 respectively. Then we evaluated the protection of the monovalent vaccine and combined vaccine. The results suggested that both the monovalent vaccine and combined vaccines showed the good protection, and the combined vaccines could also prevent the infection of the two viruses.Part two: Study on the ED₅₀ of both antigen and antibody of EV71 VLPs.In this part, we cooperated with National Institutes for Food and Drug Control（NIFDC） to study the ED₅₀ of the both antigen and antibody of the EV71 VLPs purified via the chromatography. The different dosage of EV71 VLPs were absorbed with the aluminum adjuvant and injected into the mouse, the neutralizing antibody of which showed the dose effect relationship. Through the newborn mice protection experiment, we calculated the ED₅₀ of the EV71 VLPs antigen（0.20μg/dose） based on survival rate of the newborn mice of the different dosage after injection. According to the neutralizing antibody titer of the different dosage challenge time, we calculated the neutralizing antibody titer（about 50 at this dosage）. At the same time, we calculated the ED₅₀ of antibody（about 54 and 24） via two kinds of methods to evaluate the newborn protection.In conclusion, in this study we used insect cell Baculovirus system to produce the EV71 VLPs and CVA16 VLPs, evaluated the neutralization antibody of monovalent vaccine and combined vaccine with the pseudovirus detection system and evaluated the broad spectrum of combined vaccine with all the genotypes pseudovirus. This work firstly had proved that there was no significant difference between combined vaccine and monovalent vaccine against the pseudovirus of the same subtype-gene in the neutralizing antibody titer. The vaccination of combined vaccine had no immunesuppression or immune-interference. The efficacy of monovalent vaccine and combined vaccine were evaluated via newborn mice model, and the two kinds of antibody protection methods demonstrated that the combined vaccine could prevent these two viruses’ attack. The evaluation of both the broad spectrum neutralizing antibody and the protection of newborn mice indicated that the combined vaccine provided the real and reliable data for future study and application of clinical trials. In addition, we firstly evaluated the ED₅₀ of the EV71 VLPs antigen and antibody purified via the chromatography, which paved the way for the use of EV71 VLPs in future study. Key Word:...