Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody

<div><p>Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.</p></div

Plaque reduction assay of viruses treated with oseltamivir acid.

<p>Plaque reduction assay of viruses treated with oseltamivir acid.</p

The G234E mutant HA of A/Perth/16/2009 binds and is blocked by 39.29 as well as the WT HA.

<p>(A) 293T cells expressing the WT or G234E A/Perth/16/2009 HA were incubated with a positive control antibody (left panel) or 39.29 (middle and right panels) at pH 7 (left and middle panels) or 4.8 (right panel). Flow cytometry profiles are shown. Mock, mock transfected cells. (B) Hela cells expressing the WT or G234E A/Perth/16/2009 HA were treated with trypsin to activate HA0 and then incubated with either 39.29 or a negative control antibody before pH drop to 5.4 to induce maximal cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. 39.29 was able to block the fusion mediated by the G234E mutant HA.</p

Differential membrane fusion properties of WT and mutant HAs.

<p>(A) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs were treated with trypsin to activate HA0 and then incubated with buffers at different pHs for 2 minutes to induce cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. Note the dramatic difference between the WT and mutant HAs at pH 5.8 and 5.9. (B) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs plus a tetracycline (Tet)-inducible luciferase protein were mixed with Hela Tet-On 3G cells expressing the WT or mutant HAs. Fusion was induced as in (A). After overnight culture, cells were lysed and incubated with a luminescent substrate of the luciferase. Luminescence signals were measured and normalized to the value at pH 5.5 for each HA. The percentages of fusion were plotted at various pH values and the data were fit with a nonlinear regression dose response curve. The assay was done in triplicate with data presented as Mean +/- SEM.</p

Effect of oseltamivir acid on A/Perth/16/2009 WT and mutant viruses.

<p>(A) Plaque reduction assay. MDCK cells in 6-well plates were infected with the WT or 39.29-resistant A/Perth/16/2009 viruses at 100 pfu/well for 1 hour. After removal of the virus inoculum, cells were overlaid with varying concentrations of oseltamivir acid in agarose. The numbers of plaques were counted for each virus and normalized to the number at the lowest oseltamivir acid concentration. The assay was done in triplicate with data presented as Mean +/- SEM. (B) Neuraminidase (NA) activity assay. Serial dilutions of the WT and 39.29-resistant viruses were incubated with a fluorescent NA substrate. NA activities as a function of the fluorescence intensities in relative fluorescence unit (RFU) were plotted on the y-axis versus the log10 virus dilutions on the x-axis. The assay was done in duplicate with data presented as Mean +/- SEM. (C) Virus dilutions with equal NA activities were incubated with varying concentrations of oseltamivir acid, followed by NA activity determination with the fluorescent NA substrate. The NA activities of each virus were normalized to the value at the lowest oseltamivir acid concentration. The assay was done in duplicate with data presented as Mean +/- SEM.</p

Summary of whole genome sequencing results of A/Perth/16/2009 WT and resistant viruses.

<p>Summary of whole genome sequencing results of A/Perth/16/2009 WT and resistant viruses.</p

Fusion kinetics of WT and mutant HAs and sensitivity to 39.29.

<p>(A) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs plus a tetracycline (Tet)-inducible luciferase protein were mixed with Hela Tet-On 3G cells expressing the WT or mutant HAs. Cells were treated with trypsin to activate HA0 and then incubated with a buffer of pH 5.7 for 20, 40, 60 or 120 seconds to induce cell-cell fusion. After overnight culture, cells were lysed and incubated with a luminescent substrate of the luciferase. Luminescence signals were measured and normalized to the largest value at 120 second for each HA. The percentages of fusion are shown as histograms at each time point. The assay was done in triplicate with data presented as Mean +/- SEM. Statistics were calculated between WT and each of the mutants using a multiple t test with the GraphPad Prism v.6.0 software (* P ≤ 0.05, indicating significant difference). (B) Hela cells expressing the WT or mutant A/Perth/16/2009 HAs were treated with trypsin to activate HA0 and then incubated with either 39.29 or a negative control antibody before pH drop to 5.5 to induce cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. 39.29 was able to block the fusion mediated by the WT HA but not the mutant HAs.</p

Binding affinities between antibodies and HAs at pH7 or pH4.8.

<p>Binding affinities between antibodies and HAs at pH7 or pH4.8.</p

Viruses resistant to mAb 39.29.

<p>(A) Micro-neutralization assay was performed on MDCK cells in 96-well plates. WT and mutant A/Perth/16/2009 viruses were incubated with serial dilutions of 39.29 ranging from 0.0032 to 250 μg/ml. Cells were incubated with the virus-antibody mixture for 16 hours prior to immuno-staining with anti-IAV NP and Hoechst 33342. The percentages of infected cells for each virus were normalized to the value at the lowest antibody concentration. The assay was done in triplicate with data presented as Mean +/- SEM (standard error of the mean). (B) The structure is generated from PDB ID, 4KVN with PYMOL. The stalk of A/Perth/16/2009 HA is in red, the light chain of 39.29 Fab is in blue, and the heavy chain is in green. Amino acid side chains for Asn32 (in CDR L1) and Asn93 (in CDR L3) of the light chain, and Asp391 and Gln387 of the HA are represented as sticks.</p

SdgB and SdgA sequentially modify the SDR-domain with GlcNAc moieties.

<p>(<b>A</b>) SdgB generates rF1 epitopes on SDR protein. A combination of MBP-SDR-His and SdgA or SdgB was co-expressed in <i>E. coli</i>, and cell lysates were immunoblotted with mAb rF1, or with mAb against unmodified SDR (9G4) or anti-His. (<b>B</b>) Cell-free system to reconstitute SDR glycosylation using purified components. Recombinant MBP-SDR-His was incubated with purified SdgA or SdgB, and in the presence or absence of UDP-GlcNAc; rF1 reactivity was induced only in the presence of SdgB and UDP-GlcNAc. (<b>C</b>) Final model for step-wise glycosylation of SDR proteins by SdgA and SdgB. First, SdgB appends GlcNAc moieties onto the SD-region on SDR proteins, followed by additional GlcNAc modification by SdgA. The epitope for mAb rF1 includes the SdgB-dependent GlcNAc moieties. (<b>D</b>) Mass spectrometry analysis to identify the SDR-sugar moieties using purified MBP-SDR-His expressed in <i>E. coli</i>. (Upper panel) Deconvoluted mass spectrum of purified MBP-SDR-His protein, showing the expected intact mass of 58719 Da. (Middle panel) MBP-SDR-His protein was treated with purified SdgB enzyme in the presence of UDP-GlcNAc for 2 h at 37°C. After incubation, the mass of the MBP-SDR-His protein showed several peaks, each peak being separated from the others by the mass of additional GlcNAc residues. (Bottom panel) The above-mentioned reaction mixture of MBP-SDR-His and SdgB (middle panel) was additionally treated with purified SdgA enzyme. After further incubation for 2 hrs at 37°C, up to an additional 47 GlcNAc groups were found to be added. Thus, most of the serines in the DSD motifs in MBP-SD can be modified with these disaccharide sugar moieties.</p