Figure 7.

Vaccibodies targeting different APCs induce NA-specific immune responses and protection

(A) Vaccibodies are homodimers composed of two identical monomers, each having a targeting unit, a homodimerization unit, and an antigenic unit. The targeting units were scFvs binding to MHC class II (scFv^αMHCII, green) or CD11c (scFv^αCD11c, blue) or the chemokines mouse MIP-1α (light blue triangle) or mouse Xcl1 (Xcl1, green triangle). The targeting units are genetically linked to a homodimerization unit consisting of a shortened hinge (h1 and h4), ensuring covalent disulfide bonds between the two monomers (black lines), and C_H3 from human IgG3. The antigen was NA^PR8. scFv^αNIP was used as a non-targeted control (white). A plasmid encoding NA alone was also included. The names of the vaccines are indicated above. (B and C) HEK293E cells were transiently transfected with vaccine plasmids as indicated. (B) Vaccine proteins in supernatants were analyzed by ELISA. Antibodies used for coating and detection are indicated. Mean of technical triplicates ± SD are shown; results are representative of two independent experiments. (C) In western blot analysis, Vaccibody proteins were detected with anti-NA pAb under non-reducing conditions. Putative homodimers are indicated by an asterisk and are schematically depicted on the right. Monomers and dimers of NA alone are indicated by a triangle. Results are representative of two independent experiments. (D–F) BALB/c mice were immunized once i.m./EP with 10 μg of plasmids as indicated. n = 4 mice/group. (D) NA-specific IgG responses in vaccinated mice were measured by ELISA at the indicated time points. Mean titers ± SEM are shown. (E) IgG1/IgG2a ratios of anti-NA serum antibodies at week 5. Individual mice and mean ± SEM are indicated. (F) Mice were challenged with 5× LD₅₀ H1N1 PR8 virus 5 weeks after vaccination. Weight (left, mean ± SEM) and survival (right) were monitored for 12 days. Dead mice were assigned a weight of 75%. ∗p ≤ 0.05, ∗∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; two-way ANOVA (D, left, and F, weight curve), Kruskal-Wallis multiple-comparisons test with Dunn’s correction (D, right, and E), or Mantel Cox (F, survival). Statistical analysis compared NA with different targeting units with non-targeted control and NA alone (D) or across different targeting units (E). Non-significant values are not indicated.