Establishment Of Specific Antigen Quantitative Assay And Study On Humoral Immunity Of Heat Resistant Vaccine Candidates Of Coxsackievirus A2

BackgroundHand,foot and mouth disease（HFMD）is the highest incidence of class C infectious diseases in China,which broke out repeatedly around the world.According to reports in literature,the pathogens of HFMD include Coxsackievirus A group 2,4,5,6,10,16 and EV-A71,etc.Since the launch of EV-A71 vaccine in 2015 in China,severe and fatal cases of HFMD have decreased significantly,but the number of HFMD cases has not decreased significantly.One reason is that the EV-A71 vaccine currently on the market does not prevent HFMD and related diseases caused by other enterovirus serotypes.The other is that the pathogen spectrum of HFMD has changed.In order to reduce the incidences of HFMD,it is necessary and urgent to develop HFMD multivalent vaccines.According to the epidemiological investigations by our laboratory,the proportion of HFMD caused by non-EV-A71 and non-CV-A16 enteroviruses,including CV-A2,is gradually increasing.Therefore,it is very important to develop HFMD multivalent vaccines including CV-A2 for the prevention and control of HFMD.At present,the technical route of HFMD multivalent vaccine development includes multivalent inactivated vaccine and VLPs vaccine.Except EV-A71 and CV-A16,other enterovirus serotypes for whole-virus multivalent,inactivated vaccines are difficult to be isolated in human vaccine cell matrix such as Vero cell,which makes it more difficult to the development of multivalent inactivated vaccines.The VLPs vaccine prepared by insect cell-baculovirus expression system has high expression level,safety profile and independence of virus isolation.However,humoral immunity of VLPs is lower than that of the whole virus inactivated vaccine,which is also a major challenge.It has been reported that enterovirus is sensitive to heat and temperature increasing can lead to conformation change of viral capsid.Heat resistant virus improves stability and humoral immunity of the vaccine candidate.Therefore,the heat resistant strain obtained by thermal screening might be a potent candidate vaccine strain of multivalent inactivated vaccine.CV-A2 VLPs can be prepared based on the sequence of heat resistant strain.ObjectiveOur laboratory has completed the screening of CV-A2 heat resistant strain in the early stage,and this strain is heat resistant.In this study,the humoral immunity of CV-A2 heat resistant strain was further studied.In order to study the humoral immunity of CV-A2 heat resistant strain,we selected the corresponding CV-A2 heat unresistant strain for comparison.Meanwhile,in order to provide a quality control method for the subsequent research and development of HFMD multivalent vaccine containing CV-A2,in this study a high-efficiency CV-A2 monoclonal antibody was prepared,and a CV-A2 ELISA antigen quantitative method was established and verified.MethodsThis study was divided into two parts.In the first part,CV-A2 whole virus particles were prepared in a 10-layer cell factory and the purified,inactivated antigen was finally obtained through formaldehyde inactivation.BALB/c mice were immunized with the inactivated antigen,and CV-A2 monoclonal antibody was prepared by conventional cell fusion technique,rapid neutralization test and ELISA method.The CV-A2 antigen detection method was established by using monoclonal antibody and the method was validated.The second part is the preparation and purification of EP and FP of CV-A2 heat resistant strain and heat unresistant strain by 10-layer cell factory.The antigens were identified by SDS-PAGE and TEM for further humoral immunity study.At the same time,CV-A2 heat resistant strain VLPs and CV-A2 heat unresistant VLPs were prepared and purified by insect cell-baculovirus expression system based on the gene sequences of heat resistant strain and heat unresistant strain.Mice were immunized with EP,FP or VLPs to study the humoral immunity of various virus particles of heat resistant strain and heat unresistant strain.Results1.CV-A2 monoclonal antibody with good specificity was successfully prepared,and the monoclonal antibody was used as the detecting antibody,and an ELISA was established for antigen detection and quantitation.The linear range of the detection method was determined,and its accuracy,precision,stability and specificity were verified.The method was used to determine the antigen content of CV-A2 virus particles during purification of CV-A2 virus particles used in multivalent vaccines.2.The results of humoral immunity study of CV-A2 heat resistant strain showed that the stability and immunity of EP and FP of the heat resistant strain was improved compared with the EP and FP of heat unresistant strain.The humoral immunity of heat resistant VLPs prepared by insect cell-baculovirus expression system was also stronger than that of heat unresistant VLPs.ConclusionsEstablished CV-A2 quantitative ELISA can be applied to antigen detection of samples at different stages in purification process of CV-A2 antigen,which provides a quantitative and quality control method for process development of multivalent HFMD vaccine containing CV-A2 component.In accordance with the results reported in literature,the humoral immunity of CV-A2 heat resistant strain was improved,and the humoral immunity of VLPs constructed from heat resistant strain genome sequence was also improved.