Vaccine-Induced Antibody Responses Against CHIKV and ZIKV

Groups of male or female (as indicated) 6–8-week-old C57BL/6J (ARC, Canning Vale, WA, Australia) or IFNAR^−/− mice^{27 (link)} were vaccinated intramuscularly (50 μl into both quadriceps femoris muscles) with SCV-CHIK, SCV-ZIKA/CHIK or SCV-cont (a control SCV vaccine encoding dsRed^{2 (link)}) in 10 mM Tris HCl pH 8. Serum antibody responses were determined by standard ELISA using (i) whole inactivated CHIKV as antigen as described^{62 (link)} and (ii) whole-ZIKV_MR766 virus preparations purified from infected Vero cell supernatants by 8% polyethylene glycol precipitation and ultracentrifugation through a 20% sucrose cushion. CHIKV neutralization titers against the Reunion Island isolate (LR2006-OPY1) of CHIKV were determined as described^{63 (link)}. As ZIKV_Natal plaques poorly, ZIKV neutralizing antibody titers were determined (against ZIKV_Natal and ZIKV_MR766) by incubating dilutions of heat-inactivated mouse serum (in duplicate) with 100 CCID₅₀ of virus for 3 h before adding Vero E6 cells (Sigma, ECACC Vero C1008) (10⁴/well of a 96-well plate). After 5 days cells were fixed, stained with crystal violet and the reciprocal 50% neutralizing titers determined.