Recombinant Human Anti-IAV HA Antibody (39.29) (CAT#: PABL-204)

Figure 1 Differential binding properties of the mutants to 39.29.

(A) 293T cells expressing the WT or mutant A/Perth/16/2009 HAs 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) Immunogold EM images of the WT and mutant A/Perth/16/2009 viruses bound by a positive control antibody (top panel with 6 nm gold conjugate) or 39.29 (middle and bottom panels, showing filamentous and spherical particles respectively, with 10 nm gold conjugate). Scale bar is 50 nm. Note the loss of binding of 39.29 to Q387 particles.

Chai, N., Swem, L. R., Reichelt, M., Chen-Harris, H., Luis, E., Park, S.,... & McBride, J. (2016). Two escape mechanisms of influenza A virus to a broadly neutralizing stalk-binding antibody. PLoS pathogens, 12(6), e1005702.

Figure 2 Fusion kinetics of WT and mutant HAs and sensitivity to 39.29.

(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.

Chai, N., Swem, L. R., Reichelt, M., Chen-Harris, H., Luis, E., Park, S.,... & McBride, J. (2016). Two escape mechanisms of influenza A virus to a broadly neutralizing stalk-binding antibody. PLoS pathogens, 12(6), e1005702.

Figure 3 39.39 blocks the low-pH induced conformational change of the WT HA but not the mutant HAs.

293T cells expressing the Q387K, D391Y, F175Y/D391G or WT A/Perth/16/2009 HA were collected before (Stage 1) or after (Stage 2) trypsin treatment to activate HA0. A fraction of trypsin-treated cells were incubated with 39.29 and then subjected to a pH4.8 buffer to induce HA conformational change (Stage 3). Cells at each stage were tested for 39.29 binding by flow cytometry. The mean fluorescence intensities were normalized to the WT and the percentages of binding are shown as histograms. The possible HA conformations at each stage are depicted above the binding data. HA1 is in blue and HA2 is in red. Mock, mock transfected cells.

Chai, N., Swem, L. R., Reichelt, M., Chen-Harris, H., Luis, E., Park, S.,... & McBride, J. (2016). Two escape mechanisms of influenza A virus to a broadly neutralizing stalk-binding antibody. PLoS pathogens, 12(6), e1005702.

Figure 4 Binding and fusion properties of the Q386K, D390Y and D390G mutant HAs of A/California/7/2009.

The Q387K, D391Y and D391G mutations of the A/Perth/16/2009 resistant viruses were introduced into the corresponding residues of the A/California/7/2009 HA to generate the Q386K, D390Y and D390G mutant HAs. (A) 293T cells expressing the WT or mutant A/California/7/2009 HAs 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 mutant A/California/7/2009 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. (C) Hela cells expressing the WT or mutant A/California/7/2009 HAs were treated with trypsin to activate HA0 and then incubated with either 39.29 or a negative control antibody before pH drop to 4.8 to induce cell-cell fusion. After overnight culture, representative images were obtained under a phase contrast microscope. 39.29 was able to block fusion mediated by either the WT or the mutant HAs.

Chai, N., Swem, L. R., Reichelt, M., Chen-Harris, H., Luis, E., Park, S.,... & McBride, J. (2016). Two escape mechanisms of influenza A virus to a broadly neutralizing stalk-binding antibody. PLoS pathogens, 12(6), e1005702.

Figure 5 Reduced viral fitness of the A/Perth/16/2009 resistant viruses.

(A) A multiple sequence alignment of 11,981 HA amino acid sequences from human and zoonotic isolates of the H1–16 subtypes was used to assess the genetic diversity at positions corresponding to H3 HA 387 and 391. The results are shown as pie charts. (B) In vitro fitness. MDCK cells were infected with the WT or 39.29-resistant A/Perth/16/2009 viruses at MOI 0.01. Released viral genome in the supernatant was quantitated daily by qPCR of the viral M1 Matrix gene. Genome copy numbers per 50 μl of supernatant are shown as histograms. Statistics were calculated between WT and each of the mutant viruses using a multiple t test with the Prism 6.0 software. (C) In vivo fitness. DBA/2J mice were infected with same dose of WT or 39.29-resistant A/Perth/16/2009 viruses. At 6 hr, 24 hr, 48 hr and 72 hr post-infection, lung homogenates were prepared and viral titers in the homogenates were determined on MDCK cells. Each group at each time point contained 5 mice. Lung titers were presented as Mean +/- SEM. Statistics were calculated between WT and each of the mutant viruses using a multiple t test with the Prism 6.0 software.

Chai, N., Swem, L. R., Reichelt, M., Chen-Harris, H., Luis, E., Park, S.,... & McBride, J. (2016). Two escape mechanisms of influenza A virus to a broadly neutralizing stalk-binding antibody. PLoS pathogens, 12(6), e1005702.