FIGURE 3.

Evaluation of heterosubtypic R1a-B6 reactivity in mouse sera 39 days after IM injection of 1.0 × 10¹¹ vg AAV encoding nanobodies. (A–C) Binding of R1a-B6 against different influenza subtypes was tested via ELISA. Plates were coated with 5 μg/mL influenza reference reagent (Supplementary Table 3) and the Absorbance at 450 nm was plotted against a serial dilution of recombinant nanobody for standard curve analysis. Recombinant nanobody concentration in serum corresponding to the sample binding activity was plotted. Samples were measured in duplicate. All values below the solid line represent no detectable binding activity. A/Texas/50/2012(H3N2) (TX/12) and B/Brisbane/60/2008 (B/BR/08) served as negative controls. (D) In vitro neutralization against different influenza pseudotypes was determined via a luciferase-reporter assay and given as IC₅₀ values (IC₅₀ is half maximal inhibitory concentration). All values above the dotted line indicate no neutralization. For all plots, n = 8 mice/group.