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. 2013 Dec 11;8(12):e80034. doi: 10.1371/journal.pone.0080034

Figure 1. Stem-binding bnAbs are internalized into live cells in complex with viral particles, reach late endosomes, and prevent infection.

Figure 1

(A) Experimental layout. Fluorescently labeled viruses and antibodies were pre-incubated and subsequently added to live cells and tracked. Whether or not cells were eventually infected was determined by staining for influenza NP after tracking individual cells for 15 hours. (B and C) Stills of movies (Movies S1 and S2) showing the joint and directed motion of R18-labeled A/Aichi/2/1968-X31 (H3N2) (red) and AF647-labeled CR8020 (green) (B), and R18-labeled A/Puerto Rico/8/1934 (H1N1) virus (red) and AF647-labeled CR6261 (green) (C), along TubulinTracker-stained microtubules (white) of live MDCK cells (nucleus, blue) approximately 30 minutes after addition of the pre-incubated virus-antibody mixtures. Dashed lines outline the trajectories of the virus-antibody complexes (red triangles) as seen in movies S1 and S2. (D) A/Aichi/2/1968-X31 (H3N2) virus was pre-incubated with AF647-labeled CR8020 (green) before addition to live MDCK cells labeled with LysoTracker (magenta) and imaged when virus-antibody complexes reached the perinuclear region. Arrows indicate co-localization of virus-antibody complexes with low-pH vesicles (white). (E) As in (D), except that here A/Puerto Rico/8/1934 (H1N1) virus and AF647-labeled CR6261 were used. (F) R18-labeled A/Aichi/2/1968-X31 (H3N2) virus (red) was incubated with AF647-labeled CR8020 (green) before addition to live MDCK cells expressing a GFP-cell tracer (grey cell outline). Virus-antibody complexes (co-localization shown in yellow, compare also split channels in the inset) were detected in live cells 30 minutes after inoculation. (G) To determine whether internalized virus-antibody complexes prevent infection, the fate of individual cells was assessed by tracking them over night (imaged in 30 min intervals). 15 hours post-incubation (hpi) the same cells (including their progeny) were fixed and stained for expression of influenza nuclear protein (NP, blue). (H) Incubation of R18-labeled A/Aichi/2/1968-X31 (H3N2) virus (red) with non-binding AF647-labeled CR6261 did not result in internalization of antibody. Only viral particles were detected in live cells 30 minutes after addition of the virus-antibody mixture and infection was not prevented, as demonstrated by the expression of NP (blue) in these same cells 15 hours later (I). Examples of progeny cells are indicated with numbers. Scale bars B–E equal 10 µm, F–I equal 25 µm.