Establishment Of An Active Immunization-challenge Mice Model For CV-A10 And Evaluation Of Efficacy In Vivo Of Candidate Vaccines

Hand-foot-and-mouth disease（HFMD）is an infectious disease caused by multiple human enteroviruses（HEVs）and is the most common disease occurred in infants and young children under the age of five,and occasionally in adolescents and adults.Typical clinical symptoms of HFMD are fever,rash,herpes or maculopapular rash on the hands,feet and buttocks,aseptic meningitis,acute flaccid paralysis,and even death.Since May 2,2008,HFMD,as the 38th infectious disease,has been included in the management of category C statutory reporting infectious diseases in China’s Infectious Disease Prevention and Control Law.According to the National Center for Disease Control（CDC）,from 2006 to2019,a total of 22,715,730 cases of HFMD have been reported in China,of which the proportion of deaths was 3,396,and the mortality rate was approximately0.015%.Since 2015,the mortality rate of HFMD has begun to decrease significantly,but the incidence rate remains high.With the launch of the EV-A71 inactivated vaccine in 2016,the proportion of cases infected by EV-A71 is decreasing.According to the analysis of the epidemiological investigations at the multiple areas in Hubei province and Jiangsu province from 2016 to 2019,CV-A6,CV-A10 and CV-A16 have replaced EV-A71 and become the main pathogens of HFMD in these places.In order to better prevent the outbreak of HFMD,it is urgent to develop a multivalent vaccine for HFMD,including EV-A71,CV-A6,CV-A10 and CV-A16.At present,there have been a large number of reports in literature on mice active or passive immunization-challenge models for efficacy evaluation of CV-A6,CV-A16,and EV-A71 vaccine candidates.The age of mice susceptible to human enterovirus infection is only 3 days old.The immune system of one-to three-day-old mice is not fully developed,resulting in a lack of interval time between two doses of vaccinations,which cannot be used for a reasonable efficacy evaluation of vaccines.Therefore,an active immunization-challenge model based on two-week-old mice is urgently needed,which can completely simulate the process of vaccination and prevention of infectious diseases in mice following priming,boosting and challenging or simulate the immune response and disease prevention process of susceptible people after exposure to the same pathogen,making a more reliable assessment of the protective efficacy and tolerability of candidate vaccines,which can provide efficacy reference data for clinical research of candidate vaccines.This kind animal model makes a comprehensive evaluation of the CV-A10 monovalent vaccine candidate was conducted by establishing an active immunization-challenge model with a challenge virus that can kill 14-day-old mice.In this study,CV-A10-L12 virus was cultured by a 10-layer Vero cell factory,concentrated by ultrafiltration,and purified by ultracentrifugation through a sucrose cushion and Cs Cl isodensity density gradient.The purified CV-A10-L12 empty particles（EP）and full particles（FP）,and the EP-to-FP ratio of CV-A10 under natural conditions（about 4:6）were characterized and analyzed.The CV-A10 vaccine candidate was prepared with EP and FP（Mixed particles,MP）at the natural ratio of4:6 and Al（OH）₃ adjuvant at a concentration of 0.18 mg/dose.The particle integrity of EP,FP and MP before and after formaldehyde inactivation were observed by TEM.The content and purity of EP,FP and MP were analyzed by SDS-PAGE and Western Blot.The results showed that the CV-A10 vaccine candidate had a higher purity of proximately 92%.Next,the CV-A10 vaccine candidate was used to immunize 6～8-week-old Balb/c mice,and the humoral and cellular immunity caused by the CV-A10 vaccine candidate was studied.Immunogenicity can be an important parameter of vaccine efficacy.The results showed that the neutralization antibody（Nt Ab）conversion rates of the CV-A10 vaccine candidate after priming and booster immunization was 60%and 100%,respectively,and the Nt Ab level reached to 48.By studying the expression of cytokines such as TNF-α,IFN-γ,IL-2,IL-6 and other cytokines in mice spleen lymphocytes on the 14th day after restimulation of peripheral lymphocyte with peptides derived from P1 region.It was found that the inactivated CV-A10 vaccine candidate adjuvanted with Al（OH）₃ could induce cellular immunity.Further,the epitopes of T cellular immunity caused by CV-A10 were mapped by designing a set of overlapped polypeptides covering all structural proteins in the CV-A10 P1 region.Also,the study showed that a polypeptide at the CV-A10 VP1 or VP3 C terminus caused a strong T cell stimulation response.This provides important information and strategies for the development of CV-A10 novel peptide vaccines and other recombinant protein vaccines.Our laboratory successfully obtained a strain of CV-A10-M14 that can kill 14-day-old mice through mice brain-adapted inoculation.In this study,by culturing the CV-A10 virus in a 10-layer RD cell factory at MOI=0.01 and concentrating viruses35-fold by ultrafiltration concentration,a challenge strain causing death of 100%infected mice was successfully obtained,denoted as CV-A10-M14,and the LD₅₀assay was performed.An active immunization-challenge model of mice was established with the virulent CV-A10-M14.Then the CV-A10 vaccine at a concentration of 0.5μg or 2μg was formulated with Al（OH）₃ adjuvant and injected intraperitoneally,and the primary or boosting immunization was carried out at 3 or 9days of age,respectively.The lethal dose of CV-A10-M14 was innoculated at 14days of age.After the challenge,the body weight changes,clinical scores and survival rates of the mice were monitored,and the serum and tissue of each group of mice were collected at the time of death of the immunized Al（OH）₃ adjuvant control groups for serum neutralization titer determination and tissue viral load measurement.Studies have shown that the CV-A10 vaccine candidate had a good tolerability and protective efficacy,with a protection rate of 90%for the CV-A10 vaccine candidate in the 0.5μg dose group and 100%for the 2μg dose group.Through the pathological and immunohistochemical analysis of sections of mice tissues and organs,the results provided important evidences that the less pathogenic changes and lower virus protein detection of the immunized mice following virus challenge compared with the Al（OH）₃ control mice.The results further provided strong support data for the enhanced virus clearance ability of the immunized mice and protection mechanism of CV-A10 vaccine candidate.