Construction And Function Verified Of An Enterovirus EV71 Polypeptide Vaccine

BackgroundHand,foot and mouth disease(HFMD)is an infectious disease caused by enterovirus(EV)type 71,which is the most common type.As is still lacks effective therapeutic drugs,the only prescription made for treating HFMD should be based on patients’specific clinical symptoms.China is leading the development of HFMD vaccine.The EV71 HFMD vaccine developed in China is the only inactivated vaccine that can be used to prevent HFMD.Some immunostimulating complexes like adjuvant can help vaccines work better.As a non-specific immunopotentiator,adjuvants have been widely used in vaccines.In summary,in order to develop a vaccine that is more efficient and easier to prepare,we preliminary designed a novel HFM virus EV71 polypeptide vaccine and an adjuvant.PurposeTo investigate whether our self-designed EV71 polypeptide vaccine can stimulate the production of protective antibodies in mice.MethodFirst,we synthesized three peptide vaccines against EV71 virus which contained different T cell and B cell epitopes of EV71 virus.The three vaccines were named peptide1,peptide2,and peptide3 respectively.At the same time,we prepared a vaccine adjuvant containing Cp G ODN,named ZTS001,by the following methods:the following components were taken according to the volume percentage:0.5%Tween 80,0.5%Span85,4.3%peanut oil and 94.7%.The physiological saline was then mixed with a high-pressure homogenizer.Take an appropriate amount of ZTS001 in an EP tube,centrifuge at 5,000 rpm for 5 minutes,and observe whether the water and oil phase are separated after 24 hours.The HSP65-Her2 protein is a fusion protein of mycobacterium tuberculosis heat shock protein 65(HSP65)and the proto-oncogene human epidermal growth factor receptor-2(Her2).We mixed HSP65-Her2 with adjuvant ZTS001 in a volume ratio of 1:1,and then were inoculated at a concentration of 10μg into mice on days 0 and 7,respectively.Blood was collected 1 week after the second immunization.Antibody levels in serum were measured by ELISA to determine the immunogenicity of adjuvant ZTS001.The polypeptide vaccine was prepared at a concentration of 1 mg/ml,mixed with an adjuvant at a volume ratio of 1:1,and then were inoculated into mice on day 0 and day 7at a dose of 100μl(50μg).Mouse sera were collected on day 1 before immunization,on day7 after the first immunization,on week 1 and 6 after the second immunization,and the antibodies in serum were detected by ELISA.At the same time,we used flow cytometry to detect CD19~+and CD27~+B cells in the spleen.Results1.The self-administered vaccine adjuvant had good stability and produced higher antibody levels in mice;2.The self-designed EV71 peptide vaccine had good activity and could be used in combination with adjuvant to effectively induce antibodies against EV71 polypeptide in mice;3.Antibodies produced by the self-designed vaccine immunized mice could effectively identify EV71 inactivated virus;4.The self-designed EV71 peptide vaccine combined with adjuvant could efficiently stimulate CD19~+CD27~+B lymphocytes in the spleen.ConclusionThe EV71 polypeptide vaccine prepared by our research institute had good immunogenicity.The adjuvant could effectively improve the immunogenicity of the antigen.The vaccine injection could cause high antibody level of EV71,achieve preventive effect.