Fig 1.
The eight representative strains were selected by pie chart analysis and subjected for antigenic landscaping using cross-HAI assay. Based on sequence comparison and antigenic landscaping data, critical mutation was identified and verified its role by site-directed mutation. HAI: hemagglutination inhibitaion, ROC: receiver operating characteristic curve, VLPs: Virus Like Particles.
Fig 2.
Frequencies of influenza HA clusters in 2000 and 2018.
Total of 3633 Influenza HA sequences uploaded between 2000 and 2018 in GISAID databases were aligned to understand how the H7Nx viruses evolved. Due to the overwhelming number of Anhui/13-like viruses during Asian H7N9 epidemics, the pie chart analysis was separately conducted on sequences isolated before and after 2013 Asian H7N9 epidemics (A and B). The non-Asian H7N9 sequences isolated after 2013–2018 were further analyzed as a separate pie (C). The aligned sequences were clustered by 3% amino acid similarity and dissected into each pie. The viruses to represent each pie were chosen from WHO candidate vaccine viruses.
Table 1.
Selected panel strains.
Fig 3.
Phylogenetic relations among selected H7 strains.
The phylogram based on HA1 amino acid sequences (H7 HA20-300) was constructed by Neighbor-Joining method with the boot-strap resampling (100 replicates) using the Geneious software (Auckland, New Zealand). The horizontal branch lengths are proportional to the number of nucleotide changes. (A) Phylogeny of all H7 HA1 amino acid sequences Red: Asian H7N9s isolated between 2013–2020, Orange: Eurasian H7NXs isolated between 2000–2012, Cyan: Ohio/03 H7N3-like cluster, Magenta: New York/02-like cluster (B) Selected panel strains Phylogenetic trees based on HA1 amino acid sequences of selected H7 panel strains.
Table 2.
Putative antigenic sites of selected panel strains.
Fig 4.
HAI antibody titers against Anhui/13 H7 HA in mouse sera after immunization with various H7 HAs.
Serum samples collected at week 8 were tested for the HAI antibody response specific to A/Anhui/1/2013 H7 virus like particles (VLPs) and A/Anhui/1/2013 H7N9 virus (A and B, respectively. Individual titer was plotted and the mean value was presented as bars. Dotted line represents the lower detection limit (10 HAI units). *p<0.05, **p<0.01.
Fig 5.
Protection against stringent H7N9 challenge.
C57BL/6 mice (8 mice/group) vaccinated with H7 VLPs at week 0 and 4 were intranasally infected with the A/Anhui/1/2013 H7N9) virus. Mice were monitored daily for weight loss (A and B, respectively) and viral lung titers in selected mice at day 4 post infection (C). Weight loss and lung viral titer was presented as mean± standard deviation (A and C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 6.
Cross-reactiveness among H7 panel strains.
The week 8 sera was tested for the cross-reactivity to H7 VLPs expressing HA from all eight panel strains. Individual titer was plotted. Interquartile range, median, minimum and maximum values were presented as box, middle line, upper and lower whiskers, respectively. Dotted line indicates the cut-off for the protection (80 HAI unit). *p<0.05, **p<0.01.
Fig 7.
Alanine to threonine mutation at HA169 resulted in significant antigenic change in Anhui/13 H7 HA.
(A) Mutagenesis to Anhui/13 H7 HA Site directed mutation was conducted on plasmid expressing wildtype (WT) Anhui/13 HA. The mutation is expected to result in alanine to threonine substitution at HA169 (H7 numbering), primer F = forward primer, primer R = reverse primer (B) Change of cross-reactiveness by the mutagenesis The plasmid with mutation was expressed as virus-like particle (VLP) tested for the reactivity to anti-H7 panel sera. Individual HAI titer was plotted. The box indicates the mean± standard deviation. *p<0.05, **<0.1, dotted line: protection cut-off (80 HAI unit). (C) Predictive location of mutation on the HA trimer The trimeric structure of Anhui/13 H7 HA was generated using the 3D-JIGSAW algorithm, and renderings were performed using MacPyMol. The trimeric structure was based on the structure on protein data base (PDB number = 4N5J). The putative antigenic site B and mutation site (H7 HA169) was shown in blue and red, respectively. Dashed circle indicates receptor binding site.
Fig 8.
Immunization with Anhui/13 A169T H7 VLP.
C57BL/6 mice (8 mice) was vaccinated with Anhui/169T H7 VLPs at week 0 and 4. Vaccinated mice were bled and the serum was tested for the antigenic breath across panel H7 strains (A). At week 8, vaccinated mice were intranasally infected with 10e+5 PFU of the A/Anhui/1/2013 H7N9) virus. Mice were monitored daily for weight loss and survival (C and D, respectively) and viral lung titers in selected mice at day 4 post infection (B). Weight loss and lung viral titer was presented as mean± standard deviation (A&C). *p<0.05, ****p<0.0001.