Figure legends
FIGURE 1 Expression of the HA protein in baculovirus expression
system. (a) Detection of HA expression in sf9 cells inoculated with the
recombinant baculovirus using indirect immunofluorescence assay. Scale
bar, 200 µm. (b) SDS-PAGE analysis of the purified HA protein. M,
marker; lane 1, elution with 30 mM imidazole; lane 2, elution with 60 mM
imidazole; lane 3, elution with 250 mM imidazole. (c) Detection of HA
expression using Western blot under non-reducing conditions.
FIGURE 2 Epitope mapping of the mAbs using
peptide-based ELISA. Reactivity of 1A1 1D7 (a), 1A12 2A4 (b), 2E3 3D8
(c) and 5D3 1B5 (d) with 19 potential linear B cell epitopes in HA was
determined using peptide-based ELISA. The HA protein and bovine serum
albumin (BSA) was used as the positive and negative control
respectively. (e) The overlap motif between peptide 12 and 13 was
truncated into three overlapping short peptides. (f) The peptide 19 was
truncated into five overlapping short peptides. Reactivity of 1A1 1D7
(g), 1A12 2A4 (h), 2E3 1D8 (i) and 5D3 1B5 (j) with the truncated
peptides was determined using peptide-ELISA. The BSA was used as the
negative control.
FIGURE 3 Reactivity of the mAbs with H7N9 virus determined by
IFA. MDCK cells were infected with H7N9 GD15 strain at a MOI of 0.1.
After 30 h, the cells were fixed and incubated with anti-H7N9 mAbs
(1:500) as the primary antibodies. Goat anti-mouse IgG Alexa 488 was
used as the secondary antibody. Non-infected cells that were treated in
a same manner were used as the mock control. Scale bar, 200 µm.
FIGURE 4 Identification of the immunodominant epitopes in HA
using competition ELISA. (a) Reactivity of chicken NDV-H7N9 immune sera
with potential B cell epitopes. (b) Reactivity of mouse anti-H7N9 sera
with potential B cell epitopes. (c) Competition between non-neutralizing
mAbs and chicken NDV-H7N9 immune sera. (d) Competition between the mAbs
(4B7 4D5 and 1B10 1D1) and chicken NDV-H7N9 sera. (e) Competition of 4B7
4D5 and 1B10 1D1 with chicken rGD15 immune sera. (f) Competition between
4B7 4D5 and mouse anti-H7N9 sera. Adj, adjuvanted; Non-adj,
non-adjuvanted. (g, h, i) Area under curve (AUC) was calculated based on
the curves of percentage of inhibition (PI) and PI AUC values were
analyzed using one-way ANOVA. *, p < 0.05; **, p< 0.01; ns, non-significant.
FIGURE 5 Conformational analysis of the epitopes in HA and
cross-reactivity of the stalk-reactive mAbs. (a) Localization of the
identified epitopes were illustrated in the HA monomer. (b) Sequence
variation analysis of the epitopes recognized by the mAbs. The HA
sequences of H1-H17 subtypes were aligned using BioEdit software and
variation of the epitopes was illustrated using Weblogo. The size of
letters correlates with the conservation of the amino acid. (c)
Reactivity of the mAbs with different subtypes. The HA proteins
belonging to group 1 (H1N1, H5N1 and H9N2) and group 2 (H7N7, H7N9 and
H15N8) were used. The bovine serum albumin (BSA) was used as the
negative control.