Fig. 3.

Determination of ZIKV-80E NP-induced antibody titres in mice. (a) Schematic representation of the immunisation schedule. Mice (n = 14) were given i.p. injections on days 0, 14, and 28, and bled 10 days later for antibody titration. A single dose contained 20 µg NPs formulated with 500 µg alum. (b) Indirect ELISA titration curves obtained using pooled immune sera collected on day 38 using P. pastoris-produced purified 80E NPs of ZIKV (purple), DENV-1 (magenta), DENV-2 (green), DENV-3 (blue) and DENV-4 (black), as coating antigens. (c) Similar indirect ELISA titration curves of the same immune sera pool as in panel ‘b’, but using E. coli-expressed, purified EDIII (as MBP fusion) proteins, corresponding to the same set of five viruses listed above, as the coating antigens. Data in panels ‘b’ and ‘c’ are plotted as ELISA reactivity, in terms of absorbance at 450 nm (A₄₅₀), as a function of serum dilution. Each data point represents the average of duplicates. The ELISA titration curves shown depict one of two independent experiments. (d) Determination of ZIKV nAb titres in ZIKV-80E NP-immunised mice using FACS neutralisation assay. Serial dilutions of pooled heat-inactivated immune sera collected on day 38 were titrated against ZIKV MR766 (solid purple curve) and ZIKV PRVABC59 (dashed purple curve). In parallel, mock-immune serum was similarly titrated against the same two ZIKV strains (MR766: solid grey curve; PRVABC59: dashed grey curve). Data depict ZIKV infection (%) as a function of serum dilution. The horizontal dashed line denotes 50% ZIKV infection.