tetano
Editor, Senior Moderator
J Virol. 2017 Dec 20. pii: JVI.02006-17. doi: 10.1128/JVI.02006-17. [Epub ahead of print]
[h=1]Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments.[/h] Williams JA[SUP]1[/SUP], Gui L[SUP]1,[/SUP][SUP]2[/SUP], Hom N[SUP]1[/SUP], Mileant A[SUP]1,[/SUP][SUP]2[/SUP], Lee KK[SUP]3,[/SUP][SUP]2,[/SUP][SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The neutralizing antibody (nAb) response against the influenza virus's hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency on inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor-binding pocket at HA's distal end, while FI6v3 binds primarily to the HA2 fusion subunit towards the base of the stalk. Surprisingly, HC19 inhibited HA's ability to induce lipid mixing by preventing structural rearrangement of HA under fusion activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding, but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to crosslink separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that inter-spike crosslinking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared down Fab-soluble HA structures.IMPORTANCEThe influenza virus's hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of Ab-mediated neutralization largely relies on high resolution characterization of antigen binding fragments (Fab) in complex with soluble, isolated antigen constructs by cryo-EM single particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well-characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occurs by multiple coexisting mechanisms and is largely dependent on the specific epitope that is targeted and is highly dependent on the bivalent nature of IgG molecules.
PMID: 29263254 DOI: 10.1128/JVI.02006-17
[h=1]Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments.[/h] Williams JA[SUP]1[/SUP], Gui L[SUP]1,[/SUP][SUP]2[/SUP], Hom N[SUP]1[/SUP], Mileant A[SUP]1,[/SUP][SUP]2[/SUP], Lee KK[SUP]3,[/SUP][SUP]2,[/SUP][SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The neutralizing antibody (nAb) response against the influenza virus's hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency on inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor-binding pocket at HA's distal end, while FI6v3 binds primarily to the HA2 fusion subunit towards the base of the stalk. Surprisingly, HC19 inhibited HA's ability to induce lipid mixing by preventing structural rearrangement of HA under fusion activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding, but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to crosslink separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that inter-spike crosslinking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared down Fab-soluble HA structures.IMPORTANCEThe influenza virus's hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of Ab-mediated neutralization largely relies on high resolution characterization of antigen binding fragments (Fab) in complex with soluble, isolated antigen constructs by cryo-EM single particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well-characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occurs by multiple coexisting mechanisms and is largely dependent on the specific epitope that is targeted and is highly dependent on the bivalent nature of IgG molecules.
PMID: 29263254 DOI: 10.1128/JVI.02006-17