Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

Abstract

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

Keywords: hemagglutinin; influenza; membrane fusion; neutralization stoichiometry; neutralizing antibody.

Fig. 1.

Experimental design and readouts. (A, Left) Transmission EM images of the two influenza A viruses, H1N1 (Top) and H3N2 (Bottom), depicting the high density of spike proteins present on the viral surface. (A, Middle and Left) Side and top views, respectively, showing a space-filling model of crF6261 bound to H1 [Top, Protein Data Bank (PDB) ID code 3GBN with epitope in red] and crF8020 bound to H3 (Bottom, PDB ID code 3SDY with epitope in yellow). These crystal structures highlight differences in proximity of the two epitopes to the viral membrane and of the HA–Fab angle upon binding. (B) Schematic depiction of experimental design. Alexa-488–labeled IgG (or Fab) are bound to R18-labeled influenza A viruses (magenta-edged sphere) immobilized on a glass-supported planar bilayer through interaction with glycophorin A; pH-sensitive fluorescein (pK_a 6.4) is also bound to the bilayer surface. Fluorescence is excited and detected via objective TIRF microscopy. Zoom: Acidification of the virus particles causes membrane fusion, resulting in escape of the R18 dye from the viral membrane into the target bilayer and producing a dequenching signal. (C, Left) False-color still frames from a fusion movie at time points before (Top) and 20 s after (Bottom) the pH drop. (Scale bar: 10 μm.) IgG/Fab (green spots, 50 nM incubation) and fluorescein (diffuse background) are visualized on the left, whereas the R18-labeled viruses (magenta) and their low pH-induced dequenching (white triangles) are simultaneously visualized on the right. (C, Right) Image montage of the virus highlighted by the yellow square, which is covered with a subinhibitory number of IgG molecules (green). Its fusion to the bilayer is seen as a flash of R18 intensity (magenta) followed by outward R18 diffusion. (Scale bar: 1 μm.) (D) Fluorescence time trajectory for the highlighted virus in C. Time t = 0 is set to loss of the fluorescein signal (dark green) upon arrival of the fusion-inducing pH 5.0 buffer. The time to hemifusion, t_hemi, occurs at t ≈ 30 s for this virus particle and is observed as the abrupt increase in R18 fluorescence (magenta). The virus-bound IgG/Fab fluorescence (light green) used for stoichiometry measurements is indicated by the box: 1 s after the pH drop and enclosing 3 s of fluorescence information.

Fig. 2.

Hemifusion inhibition and antibody stoichiometry. In A–C, IgG data are on the left-hand graphs (solid fit lines), Fab data are on the right (dashed fit lines); top rows are the H1N1 strain (blue) and bottom rows are H3N2 (black). Each data point represents a single experimental run (CR6261 IgG shown as diamonds, crF6261 Fab as upward triangles, CR8020 IgG as squares, and crF8020 as downward triangles); the best-fit lines are in blue or black and their 95% confidence bands are in light blue or gray. (A) Hemifusion efficiency decreases as the concentration of neutralizing IgG or Fab is increased. (B) The median number of neutralizing IgG or Fab bound to virions increases as the concentration used for incubation with virus increases. (C) Plot of hemifusion data (A) versus the number of IgG or Fab bound to the viral surface (B), allowing for the estimation of the number of IgG/Fab required for a given reduction in hemifusion efficiency. Fit lines used are the logistic function (A), hyperbolic function (B), and a combination of the two (C) (SI Materials and Methods). Fit lines in C are truncated at high coverage and correspond to the plateau values obtained in A and B at high IgG/Fab concentrations.

Fig. 3.

Hemifusion is delayed at higher IgG/Fab concentrations. Data are displayed as in Fig. 2 and are fit with a hyperbolic function having a constant offset (SI Materials and Methods); each data point is the geometric mean hemifusion time from a single experimental run. Fold increases in hemifusion times between zero and the highest IgG/Fab concentrations are listed in Table 1.

Fig. 4.

Cartoon illustrating inter-HA network disruption by IgG binding leading to fusion inhibition. Fusogenic HA at neutral pH in the prefusion conformation (dark blue triangles) initially have a network of connections (blue dashed lines) dictated by their spatial geometry relative to one another. Binding of IgG (black Y) neutralizes the HA (light gray triangles) by preventing their low pH-induced conformational changes and disrupts coordination with neighboring HA. Exposure to low pH conditions triggers HA to unfold (light blue triangles) and activates the inter-HA network (thick red lines) between neighboring, triggered HA. Continued low pH exposure causes HA inactivation by nonproductive refolding (light gray triangles), also removing inter-HA connections. Although productive fusion could arise at locations with a sufficiently high density of activated inter-HA connections, both IgG/Fab binding and low pH inactivation can inhibit accumulation of this density even in the presence of fusogenic HA. NA and M2 proteins are not depicted for clarity.