Fig. 4.

Immunological analyses of E1E2-Flag mouse sera, A. Immunization scheme. C57BL/6 mice were primed at day 0 with full length E1E2-Flag (gt1a, 1b or 2a), and boosted with the same antigen at weeks 2, 5 and 8. The terminal bleed was at week 10. B. Quantification of humoral responses induced by vaccination with E1E2-Flag proteins. Lectin-bound gt1a, 1b or 2a E1E2-Flag immunogens were probed with the corresponding homologous E1E2-Flag sera in three-step serial dilutions (starting from 1:500, up to 1:10⁶). Absorbance was measured at 450 nm. Each dot on the plot represents one animal (■ - 1a-Flag, ♦ - 1b-Flag, • - 2a-Flag). The antibody titer was estimated as the serum concentration, at which binding was 2 times higher than the pre-immune (preIM) control. The horizontal bars represent the median value for each group. C. Cross-reactivity of mice sera against selected HCV genotypes. Lectin-bound E1E2 complexes from 293T cell lysates, transfected with gt1a (the heterologous strain UKN 1a.12.18), gt2b (UKN 2b1.1), gt4 (UKN 4.21.16) or gt6 (UKN 6.5.34) were probed with each of the E1E2-Flag sera, followed by anti-mouse IgG–HRP conjugate and TMB substrate. Absorbance was measured at 450 nm. The anti-E2 AP33 mAb was used as a positive and the anti-Flag M2 and preimmune (preIM) serum as negative controls, respectively. Mock transfected 293T cell lysates (no env) were used for the estimation of background signal. Error bars represent standard deviations of the individual mouse responses.