Establishment Of CV-A4 Actively Immunizing Murine Model And Evaluation Of Efficacy Of The Vaccine Candidate

Hand,foot,mouth diseases(HFMD)is a widespread disease worldwide.The main pathogens of HFMD are several viruses from enterovirus species A and B(A:Coxsackieviruses 2,4,5,6,7,9,10,16,EV-A71 and B:1,2,3,4,5,and Echoviruses).Since2012,HFMD caused by enteroviruses other than EV-A71 and CV-A16 has gradually increased in China,which resulted in more than 50%of incidence in Guangzhou,Beijing,Hunan,Shandong,Fuzhou and other provinces and cities,and 80%in some regions and years.With the launch of the EV-71 vaccine in 2016,severe and fatal cases have been significantly reduced,but the number of cases has not been significantly reduced.Therefore,in order to reduce the incidence of HFMD,it is very important to develop a multivalent HFMD vaccine.Since the first time isolation of CV-A4 virus,it has been circulating in many countries and regions around the world.There were two pandemics of CV-A4 in Taiwan,China.In2012-2013,CV-A4-associated HFMD accounted for 13.9%and is the fourth largest pathogen at Wuhan,Hubei.In Fujian Province,CV-A4 accounted for 6.8%of non-EV-A71 and non-CV-A16 pathogens from 2011 to 2015,ranking third.In 2012,Ningde,CV-A4 was the most important pathogen,accounting for 15.1%.In 2012,Yuyu County,Jiangsu Province,CV-A4-associated HFMD accounted for 16%,ranking second.Severe sympotoms caused by CV-A4appeared in Fujian,Yunnan,Guangzhou and other places.In addition,there was recombination between CV-A4 and other serotypes,which may lead to more severe outcomes of HFMD.Therefore,the development of a CV-A4 monovalent vaccine or a multivalent HFMD vaccine containing CV-A4 is very important for the prevention and control of HFMD.It is extremely urgent to establish mouse models of CV-A4 to study the pathogenesis of CV-A4,and to evaluate the efficacy of candidate vaccines in vivo.In order to prepare antibodies for characterization of CV-A4,the CV-A4 VP1 protein was expressed.CV-A4 VP1 and virus particles were used to immunize Japanese white rabbits.Rb-?-VP1 and Rb-?-virion with high specificity were obtained,providing reagents for quantitative analysis of antigens.In this study,CV-A4 RD cell isolates were alternately passaged in mouse brain and RD cells.After repeated alternate passages,mouse-adapted CV-A4-M14-RP3 was obtained and was lethal to 14-day-old Kunming mice through intraperitoneal(i.p.)and intracranial(i.c.)routes.Eight clones were selected for comparison of virulence in mice with the same infectious dose,and a virulent strain CV-A4-M14-613131 and a weak-virulent strain CV-A4-M14-623131 were screened out.There were eight nucleotide differences in the whole genome of the two strains,and 4 nucleotide differences resulted in amino acid changes,which might be related to virulence.These subsititutes and their locations are VP1-T2778G/D112E,3D-C5973G/I3M,3D-T7175A/W404R,3D-A7181G/K406R.The challenge virus stock was prepared by cell factory culture and the LD₅₀ was determined.In this study,CV-A4 was adapted to grow from RD cells to Vero cells by continuous passage at a high MOI,and clones suitable for vaccine candidates were selected.The complete genome sequence of the CV-A4 vaccine candidate strain was artificially synthesized in vitro,then inserted to the p BR322 plasmid,constructed the infectious c DNA clone of CV-A4.The virus was rescued in Vero cells,and the vaccine candidate strain without the genetic background of RD cells was successfully obtained.At the same time,the whole genome sequence analysis of parental RD isolates and Vero adapted strains was carried out.It was found that the synonymous nucleotide mutations between the RD isolate and the Vero-adapted strain were VP2-C1144T and 2C-C4783T;the nucleotides and amino acids(and positions)of the missense mutation were VP2-G1361A-A1362G/E137R,VP3-A2417G/I233V,VP1-G2930T/D164Y,VP1-A3354G/H305R and 3D-A7322C/M454L.These mutations might be related to cell tropism.The virus was cultured and the harvested virus solution was purified.The empty particles(EP)and full particles(FP)of CV-A4 were obtained.The morphology,purity and specificity of the purified virus particles were identified by TEM,SDS-PAGE and WB.The CV-A4 EP,CV-A4 FP,CV-A4 EP+FP(EP=19.23%,FP=80.77%)and CV-A4 VLP were used in vaccine formulation study(as antigen),conjugated with aluminum adjuvant(a final concentration 1.05 mg/m L).The groups of mice were immunized at the doses of 0.5?g,1.5?g or 4.5?g on 0 and 14 days,and the neutralizing antibody titers of serum were detected.All four vaccines induced the high levels of neutralizing antibodies.There was no significant difference in the immunogenicity of CV-A4 FP and CV-A4 EP+FP,which were superior to CV-A4 EP and CV-A4 VLP.The immune persistence of CV-A4 EP,CV-A4 FP,CV-A4EP+FP were long and better than CV-A4 VLP.Various antigens had obvious dose-dependent effects,and 1.5?g was a more appropriate dose.T cell mediated immune response was also determined.No significant differences in the CD4⁺and CD8⁺cell numbers in living cells were observed among PBS,PBS-Alum control groups and candidate vaccine group in the sera of mice.Howevere,the stastically significant differences in cytokines and chemokines produced by CD4⁺/CD8⁺cells were observed when immunized kidney cells were restimulated with antigens.The results demonstrated that the vaccine formulations stimulated T cell-medicated immune responcses.The significance of increased levels in immunized mice are discussed.After the candidate vaccine was inoculated,induced humoral immunity and cellular immunity(stimulated Th1 and Th2 immune response),activated B cells,and then secreted antibodies to stimulate T cell proliferation,and produced memory cells.Following re-stimulation,the memory cells would react quickly and secrete various inflammatory factors.In this study,the protective efficacy of the CV-A4 inactivated vaccine was evaluated.The results showed that the CV-A4 FP and CV-A4 EP+FP induced the production of high levels of neutralizing antibodies,which effectively protected the mice against the infection of CV-A4 virus,with a protection rate of 100%.While the protection rate of the vaccine prepared by CV-A4 VLP was 60%.The results of immunohistochemistry,histopathological section and viral load of various tissues and organs showed that the vaccine formulations(prepared by)of CV-A4 FP and CV-A4 EP+FP had better protective effect in mice than that of CV-A4 VLP.In conclusion,a CV-A4 vaccine candidate was developed and the safety,immunogenicity and efficacy of vaccine were evaluated in a newly established mouse model.Determinant mapping of cell tropism of vaccine strain and virulence of the mouse-adapted challenge strain were performed.The results indicated that the CV-A4 vaccine could be further developed into multivalent vaccines as one of antigen components.