Engineering a novel IgG-like bispecific antibody against enterovirus A71

Figures

• 

  Fig. 1. Design and generation of anti-EVA71 antibodies. (A) Bivalent anti-EVA71 sdAb-F1, anti-EVA71 E18 IgG, and anti-EVA71 IgG-like bispecific antibody E18-F1. (B) Recombinant proteins.

• 

  Fig. 2. Characterization of recombinant proteins. Anti-EVA71 antibodies were electrophoresed on SDS-PAGE in nonreducing conditions (DTT (−)) and reducing conditions (DTT (+)). SDS-PAGE analysis of Protein A purified anti-EVA71 sdAb-F1 (A), anti-EVA71 E18 IgG (B), and anti-EVA71 E18-F1 BiSAb (C).

• 

  Fig. 3. Analyses of binding of E18 or E18-F1 to EVA71. (A) Indirect ELISA was performed by coating ELISA plate wells with formalin-inactivated EVA71. Various concentrations of E18-F1 or E18 were added in duplicates. The antibodies were detected by HRP assay and the data are presented as average OD450 value. (B) Effects of E18 on E18-F1 binding to EVA71. Various concentrations of E18 IgG, as a binding competitor, were premixed with 100 nM E18-F1 BiSAb for competitive binding assay. A different secondary antibody, as compared to the one used in Fig. 3(A), was employed in this assay.

• 

  Fig. 4. Evaluation the antiviral activity of E18 IgG and E18-F1 BiSAb by plaque reduction assay. (A) Plaque assays were performed with agarose overlay and incubated for 2 days. Plaque formation assay shows the reduction of plaque generation with a dose dependent response. (B) Histogram shows that E18 IgG and E18-F1 BiSAb are able to inhibit infection by EVA71 after normalization to untreated control. Results are expressed as mean ± SD from a representative experiment performed in triplicate. (C) The inhibitory activity of E18 IgG and E18-F1 BiSAb at different concentrations was calculated.

• 

  Fig. 5. Prophylactic efficacy of E18-F1 BiSAb in hSCARB2 transgenic mice. Kaplan–Meier survival curve (A) and clinical scores (B) of hSCARB2 transgenic mice administered with E18-F1 BiSAb one day before EVA71 infection at lethal dose. The total number (N) of mice per group is shown. Logrank test was used to analyze the statistic difference of survival rate. One-way ANOVA with the Kruskal-Wallis test was used to analyze the statistic difference of the individual groups with disease score. The symbols * and ** are used to indicate P values < 0.05 and < 0.01, respectively.