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.influenza | neutralizing antibody | hemagglutinin | neutralization stoichiometry | membrane fusion

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

“…Strain-specific neutralization of G-specific antibodies correlates with epitope specificity. Antibody efficacy is determined by multiple variables, ranging from antibody and target glycosylation, binding on and off rate, the epitope, and the angle that the target is approached by the antibody (4,(22)(23)(24). Here, we hypothesized that for the G-specific antibodies, the neutralization potency may be determined by the binding site on the G protein.…”

“…Fc tail presentation could also play a role during other immune effector functions activated through bound antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of infected cells, as has been shown in influenza disease (24). Some of our antibodies were able to induce substantial ADCC (mainly epitopes B [AT34] and C [AT40 and AT50]), while others were more potent inducers of ADCP (mainly epitope D, AT32 and AT33).…”

Mechanisms of influenza viral membrane fusion

“…When the number of inhibited HAs is sufficiently large to prevent the formation of a fusogenic cluster, the particle will fail to achieve hemifusion altogether ( Figure 4G right and H rightmost). The presence of such time delay and concomitant reduction in fusion yield at intermediate inhibition levels was experimentally demonstrated [113].…”

“…Drugs targeting the conserved HA stem or extended intermediate seem particularly promising in finding a universal influenza therapy. A single-particle assay using antibodies and antibody fragments that bind the stem of the HA trimer in the prefusion state and prevent the low-pH conformational change showed the effect of inhibiting fusion-competent HAs one by one [113]. By counting the number of antibodies bound per individual virion and observing the resulting fusion yield, it was found that not all HA trimers need to be inhibitor-bound to fully abrogate fusogenicity, supporting the idea that hemifusion requires a network of HAs that can form a cluster ( Figure 4B left).…”

“…A parameter that sets the fraction of unproductive HAs in the population can be added to the model described above. In order to be able to quantitatively explain the inhibition levels measured in [113], a model including such unproductive HAs predicts that a large fraction of the HAs inherently is unproductive [114]. There are two major caveats, the first of which is that the contact patch size has not been experimentally measured making the relationship between experimental fusion data and modeling efforts somewhat uncertain [101,114].…”