| Hand,Foot and Mouth Disease(HFMD)is a global acute infectious disease that mainly affects children under 5 years old,especially in the Asia-Pacific region.At present,it has been identified that there are more than 20 kinds of enterovirus infection that can cause HFMD.In addition to enterovirus 71(EV71)and coxsackievirus A16(CVA16),the prevalence of coxsackievirus A6(CVA6)has increased significantly in recent years and has become the dominant pathogen of HFMD in many regions.However,there is no approved vaccines or specific therapeutic drugs for CVA6.Therefore,based on previous work,the study of neutralizing antibodies against CVA6 was carried out by biochemistry,cell biology and other techniques.In this study,specific neutralizing antibodies against CVA6 were screened and their biochemical properties were systematically identified in vitro.Firstly,the identity analysis of CVA6 strains in our laboratory was carried out based on the viral capsid protein VP1 gene sequence,and the representative strains used in subsequent experiments were identified as the epidemic strain 00141(E genotype)and the prototype strain Gdula(A genotype).Then,CVA6 virus particles with high purity and concentration were obtained by using the established methods of purification of CVA6,which were used for mice immunity.In this study,12 CVA6-neutralizing-mAbs were obtained through hybridioma technique and screened by a rapid and high-throughput neutralization test in vitro.One of the antibodies,4E9,not only had a high efficacy of broad-spectrum neutralization,but also had a good interaction with CVA6 virus.In this study,we further optimized the previously established mouse infection model of CVA6,determined the optimal challenge dose of different subtypes of CVA6,and evaluated the antiviral ability of 4E9 in vivo systematically.First,mice were treated with a single injection of 4E9 at different doses on the first day after infection.The results showed that the mice treated with 1 μg/g were 100%resistant to the lethal attack of strain 00141.The protective rate of 10 μg/g treatment group against strain Gdula was 100%,and that of 1 μg/g treatment group against strain Gdula was as high as 80%.This result indicated that the neutralizing antibody 4E9 had a good broad-spectrum antiviral activity against CVA6 infection in vivo.Further,the results of different treatment time and successive treatment strategies for 4E9 after challenge strain 00141 showed that the protective rate of mice treated with a single injection of 4E9 on the second day after challenge achieved 80%and three consecutive injections of 4E9 from the second day after challenge could protect mice from the lethal attack of CVA6,which further confirmed 4E9 has a good potential for the application of therapeutic drugsIn summary,this research has successfully obtained and screened 12 CVA6specific monoclonal neutralizing antibodies,of which antibody 4E9 has high-efficiency broad-spectrum neutralizing activity.The potential of antibody 4E9 as a therapeutic agent for CVA6 was verified by using the optimized CVA6 animal infection model.This study sets a foundation for the method of quality control for vaccine,the design of specific therapeutic drugs and potential broad-spectrum epitopes. |
