An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus

Leung, Ho-Chuen; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Zhao, Han-Jun; Cheung, Chung-Yan; Ng, Fai; Huang, Jian-Dong; Zheng, Bo-Jian

Amino acid mutations at positions 10 and 11 of M2e reduce the monoclonal antibody binding affinity, 32 whereas virus mutants at M2e position 10 can escape the protection of M2e antibodies. 33 Moreover, M2e peptide variants at positions 10, 14, and 16 significantly reduce reactive antibody-binding titer. 19 These findings indicate that although M2e is relatively conserved among different subtypes of influenza virus, amino acid mutations at certain positions could indeed affect the efficacy of M2e vaccination. Notably, there are five amino acid differences at positions 13, 14, 18, 21, and 24 between H5N1-M2e and H7N9-M2e, which accounted for approximately 21% (5/24) of the total amino acids of M2e ( Figure 1 ). However, these amino acid variations did not abolish the efficacy of the vaccination. The protection against lethal challenge by H7N9 virus (Figures 3 and 4) was not affected, despite the fact that the amino acid variations between H5N1-M2e and H7N9-M2e included a mutation at position 14, which has been reported to affect the antibody cross-reaction. 19 To examine how the amino acid variations may affect the structure of the M2e peptide, the 3D structures of M2e-A/Vietnam/1194/04 (H5N1), M2e-A/Hong Kong/156/97 (H5N1), M2e-A/Anhui/01/13 (H7N9), and M2e-A/Beijing/501/09 (H1N1) were analyzed using the online software PEP-FOLD ( Figure 5 ). The 3D structures of M2e-A/ Vietnam/1194/04 (H5N1) and M2e-A/Anhui/01/13 (H7N9) showed that the middle regions were similarly folded as an ''open'' structure, although there were five amino acid differences between these two M2e sequences (Figure 1) . Although there was only one amino acid difference between M2e-A/Vietnam/1194/04 (H5N1) and M2e-A/ Beijing/501/09 (H1N1), the 3D structure of M2e-A/Beijing/501/09 (H1N1) showed a relatively more ''open'' structure ( Figure 5 ). However, M2e-A/Hong Kong/156/97 (H5N1), which contained amino acid variations at positions 10, 14, and 16 ( Figure 1 ) and had low cross-reactivity with the H5N1-M2e-induced antibody ( Figure 2C ), had the same region folded as a hairpin structure. According to the 3D prediction model, the 12th amino acid, arginine, and the 15th amino acid, tryptophan, in the structure of M2e-A/Hong Kong/156/97 (H5N1) are bulky and are located at the top of the hairpin, which may provide steric hindrance that blocks the access to the lower region of the structure. This structure may limit the antibody-binding ability and the antigen processing by the immune system. By contrast, the 12th-position arginine and the 15th-position tryptophan of the other three M2e structures orientate in such a way that they would not block the lower access. These observations may explain why the H5N1-M2e vaccine-induced antibody could still react with M2e that contained variations at positions 13, 14, 18, 21, and 24, but it could not react with M2e containing amino acid variations at positions 10, 14, and 16, because their 3D structure were not compatible.