Study On The Immunoprotective Effect Of EV71-CA16 Combined Vaccination

Objective:To prepare the EV71-CA16 bivalent inactivated vaccine and evaluate its protective efficacy in vivo. To establish a neonatal mouse model via passive transfusion intraperitoneally of sensitized T for evaluating the relationship between celluar immunity and protective efficacy. Methods:The EV71-KM and CA16-KM strain was propagated in Vero cells. After harvested, the virus was concentrated by ultrafilter, followed by purified using sucrose gradient ultracentrifugation, subsequently treated with β-propiolactone to inactivate the virus to prepare as antigen. The inactivated virus was mixed with the equal volume of aluminum adjuvant (1mg/ml) to prepare the inactivated vaccine. The 8-week female mice were divided into two groups and inoculated intramuscularly on days 0 and 14 with inactivated vaccine, while PBS was used as control. They were allowed to mate with the male mice at 1h after the first injection. After delivery, we collected serum samples from female and neonatal mice to measure the mean neutralization titres. The neonatal mice not more than 24h were challenged with the viruses at 100 LDso/mouse, survival rates and clinical manifestation were monitored for 21 days post-challenge. One-day ICR mice inoculated intraperitoneally on days 0 and 14 with CA16 inactivated vaccine. Seven days after the second immunization splenocytes, T cell, B cell,CD4+T cell, CD8+T cell, Memory T cell, Effector T cell were isolated from CA16-immunized and CA16-unimmunized mice and intraperitoneally transfused into neonatal mouse.Two hours after transfusion, the neonatal mouse were challenged with CA16-CC at 100LD50 through the intracranial injection, survival rates and clinical manifestation were monitored for 21 days post-challenge. Results:The mean neutralization titres in different groups were all negative. Inactivated vaccine of EV71-KM and CA16-KM can protect neonatal mice from lethal challge, but there was no immune interference exists between the two immunogens. In bivalent inactivated vaccine (256EU EV71+32EU CA16) group the survival rates of mice after challenged with EV71-CC, CA16-CC, EV71-CC and CA16-CC was 100%,0%,0% respectively. In bivalent inactivated vaccine (256EU EV71+320EU CA16) group the survival rates of mice after challenged with EV71-CC, CA16-CC, EV71-CC and CA16-CC was 100%, 67%,30% respectively. In bivalent inactivated vaccine (256EU EV71+960 EU CA16) group the survival rates of mice after challenged with EV71-CC, CA16-CC, EV71-CC and CA16-CC was 100%,100%,70% respectively. The survival rates of mice in the group of transfused splenocytes (2×107/mice), T cell (1×107/mice), B cell (1×107/mice), CD4+T cell (5×106/mice), CD8+T cell (5×106/mice), Memory T cell (2×105/mice), Effector T cell (2×105/mice) was 100%,60%,56%,0%,0%,0%,0% respectively. Conclusion:The EV71-CA16 bivalent inactivated vaccine immune animals can induced immune response against the two virus. In the different groups EV71 256EU can protect neonatal mice from the challenge of two viruses completely, the protective effect of CA16 along with the increase of the dose. Sensitized splenocytes can protect neonatal mice from the challenge of CA16-CC completely. Sensitized T cell, B cell of can protect neonatal mice from the challenge of CA16-CC partly.