Identification of Hemagglutinin Mutations Caused by Neuraminidase Antibody Pressure

Abstract

The balance in the functions of hemagglutinin (HA) and neuraminidase (NA) plays an important role in influenza virus genesis. However, whether and how N2 neuraminidase-specific antibodies may affect the attributes of HA remains to be investigated. In this study, we examined the presence of amino acid mutations in the HA of mutants selected by incubation with N2-specific monoclonal antibodies (MAbs) and compared the HA properties to those of the wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus. The higher NA inhibition (NI) ability of N2-specific MAbs was found to result in greater proportions of mutations in the HA head. The HA mutations affected the thermal stability, switched the binding preferences from α2,6-linked sialic acid receptor to α2,3-linked sialic acid receptor, and promoted viral growth in mouse lungs. These mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition (HI) titers. The evolutionary analysis also proved that some HA mutations were highly correlated with NA antibody pressure. Our data demonstrate that HA mutations caused by NA-specific antibodies affect HA properties and may contribute to HA evolution. IMPORTANCE HA binds with the sialic acid receptor on the host cell and initiates the infection mode of influenza virus. NA cleaves the connection between receptor and HA of newborn virus at the end of viral production. The HA-NA functional balance is crucial for viral production and interspecies transmission. Here, we identified mutations in the HA head of H9N2 virus caused by NA antibody pressure. These HA mutations changed the thermal stability and switched the receptor-binding preference of the mutant virus. The HI results indicated that these mutations resulted in significant antigenic drift in mutant HA. The evolutionary analysis also shows that some mutations in HA of H9N2 virus may be caused by NA antibody pressure and may correlate with the increase in H9N2 infections in humans. Our results provide new evidence for HA-NA balance and an effect of NA antibody pressure on HA evolution.

Keywords: antigenic drift; hemagglutinin mutations; influenza virus; neuraminidase antibody pressure; receptor binding preferences.

FIG 1

NA antibody pressure resulted in mutations in the HA head. (A) NI abilities of N2-specific MAbs against WT XXM virus were tested in the Mu-NANA assay. Each concentration was tested in duplicates, and the data are represented as the mean values ± standard errors of the means (SEM). (B) HA mutations caused by incubation with N2-specific MAbs in chicken embryos were examined by HA sequencing and analyzed with Chromatogram software. Substitutions in nucleotide codons encoding the amino acids at positions 166, 198, and 234 are highlighted with rectangles. (C) A model of the locations of mutant residues on HA was generated by Swiss PdbViewer/DeepView with the HA monomer (PDB code 1JSH). The structure is shown in side view (left) and top view (right). The mutant residues and sialic acid receptor are marked with different colors on the HA head.

FIG 2

HA mutations changed the thermal stability of HA and switched the receptor tropism. (A) Nonreducing allantoic fluid of each mutant virus was tested by Western blotting. HA trimer, HA dimer, HA0 (HA monomer), and HA1 are indicated on the left. (B) HA titers of the mutant viruses incubated at 56°C for different times were measured with 0.5% chicken red blood cells (cRBCs). (C) Two synthetic glycan receptors, α2,3SLN-PAA-biotin and α2,6SLN-PAA-biotin, were used to test the binding preference of the WT virus and mutant virus. Wells of 96-well plates were coated with serially diluted glycan receptors (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, and 0.07 μg/ml) and the plates further incubated with each virus. OD650nm, optical density at 650 nm.

FIG 3

NA antibody pressure breaks the HA-NA balance and affects receptor-binding preference. The sialic acid receptors are shown in the form of blue diamonds. The blue see-saw is used to show HA-NA function balance. The inhibition of NA by NA-specific antibodies results in dysfunction of NA, which further leads to the imbalance of HA-NA function. When the pressure transmits to the flexible HA, HA mutations take place in the HA head and change the receptor-binding preferences for survival under NA antibody pressure.

FIG 4

HA mutations caused by NA antibody improve viral growth. (A) Viral growth kinetics of the WT XXM virus and the mutants in MDCK cells. All data are presented as the mean values ± SEM of three duplicates from two independent experiments. The data for the mutant viruses and the WT virus were compared by two-way ANOVA in GraphPad Prism 5 (*, P < 0.05; **, P < 0.001; ***, P < 0.0001). (B) Body weight changes of mice (n = 5 per group) infected with the WT XXM virus and the mutants. All data are represented as mean values ± SEM. (C) Viral loads in lungs from the mice on days 3 and 6 postinfection. Lungs of 3 mice were tested each time. The ratio of positive samples to total samples is shown above each column. (D) The HI titers of serum samples (n = 5 per group) cross-reacting with the WT XXM virus and mutants were measured in an HI assay. (E) Histological lesions in the lungs from the infected mice on day 6 postinfection. Representative images of hematoxylin and eosin (H&E)-stained lung tissues are shown in ×200 magnification.

FIG 5

The effect of NA antibody pressure on HA evolution. The major branch of the phylogenetic tree of full-length HA and NA genes of H9N2 IAVs isolated in China from 1994 to 2020 was generated with Nextstrain (https://nextstrain.org/flu/avian/h9n2). Viruses in the phylogenetic trees are colored according to the hosts. The NA mutations selected by antibody pressure are indicated as markers for NA (left). The HA mutations at positions 166, 168, 198, 201, 220, and 234 are indicated on the HA branch (right). HA and NA genes belonging to the same virus are connected by lines.