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

<p>(<b>A</b>) 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>B</b> and <b>C</b>) Stills of movies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s007" target="_blank">Movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s008" target="_blank">S2</a>) showing the joint and directed motion of R18-labeled A/Aichi/2/1968-X31 (H3N2) (red) and AF647-labeled CR8020 (green) (<b>B</b>), and R18-labeled A/Puerto Rico/8/1934 (H1N1) virus (red) and AF647-labeled CR6261 (green) (<b>C</b>), along <i>TubulinTracker</i>-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s007" target="_blank">movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s008" target="_blank">S2</a>. (<b>D</b>) A/Aichi/2/1968-X31 (H3N2) virus was pre-incubated with AF647-labeled CR8020 (green) before addition to live MDCK cells labeled with <i>LysoTracker</i> (magenta) and imaged when virus-antibody complexes reached the perinuclear region. Arrows indicate co-localization of virus-antibody complexes with low-pH vesicles (white). (<b>E</b>) As in (<b>D</b>), except that here A/Puerto Rico/8/1934 (H1N1) virus and AF647-labeled CR6261 were used. (<b>F</b>) 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. (<b>G</b>) 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). (<b>H</b>) 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 (<b>I</b>). Examples of progeny cells are indicated with numbers. Scale bars B–E equal 10 µm, F–I equal 25 µm.