Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus

Abstract

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016‒2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.

Keywords: Antigenic change; H7N9; HA; Influenza A virus; Neutralization; mouse monoclonal antibody.

Figure 1

In vivo protective efficacy in mice. Three mice per group were intraperitoneally injected with the indicated antibodies at 15 mg/kg. One day later, the mice were intranasally challenged with 10 mouse lethal dose 50 (MLD₅₀) of Anhui/1. Body weight and survival were monitored daily for 14 days. A mouse anti-NP mAb at 15 mg/kg served as a negative control.

Figure 2

Phylogenetic tree of HA sequences derived from human H7N9 viruses. The evolutionary history was inferred using the Neighbor-Joining method with Kimura distances. Five major clusters are shown as a collapsed branch. A/Netherlands/219/2003 is defined as an outgroup. The Yangtze River Delta and Pearl River Delta lineages are circulating in China. Highly pathogenic H7N9 viruses, which harbor multiple basic amino acids in the HA cleave site, are included in the Yangtze River Delta lineage.

Figure 3

Amino acid substitutions potentially involved in evasion from neutralizing mAbs. (A) Alignment of H7-HA sequences. Amino acid sequences of HA derived from the human H7N9 viruses tested in Table 5 were aligned. Since all tested mAbs targeted the HA head, the HA head sequences are shown. Each colored circle indicates each position on the HA structure. (B) Amino acid substitution sites mapped onto the H7-HA molecule. Amino acid mutations that were identified from escape mutant viruses (A135V, G144E, and L226Q) and substitutions that appear to be important for evasion from mAb recognition (I130T, T132A, A135T, and R140K) were mapped onto the three-dimensional (3D) structure of the H7-HA trimer (PDB; 4LCX) by using the molecular graphics system PyMOL. Cyan indicates the receptor binding site.