4 Discussion

The M2e is a highly conserved candidate epitope in different subtypes of IAV and offers potential to develop universal vaccines, if it can be appropriately presented and sensed by host immune system. Displaying multiple copies of M2e molecules on the surface of nanoparticles is an effective means to increase the level of anti-M2e antibodies. Although M2e is conserved among IAV, there are still some differences among strains. Particularly, M2e derived from different species varies considerably (Schepens, De Vlieger, & Saelens, 2018). Therefore, nanoparticles usually display M2e of human, swine and avian IAV in tandem to increase the broad spectrum.
Affinity maturation of antibodies requires stable and full display of epitopes rather than transient state (Dormitzer, Ulmer, & Rappuoli, 2008). However, M2e adopt at least two transformed conformations (Cho et al., 2016; Cho et al., 2015). In addition, due to the existence of flexible links between different M2e, the instability of M2e conformation is exacerbated. Therefore, the relatively stable one in the multiple copies of M2e is more easily recognized by the immune system and eventually induces higher levels of antibodies.
VLPs, as a kind of nanoparticles with precisely defined three-dimensional structure, provide a large number of sites for M2e that can be accurately inserted (Rodriguez-Limas, Sekar, & Tyo, 2013). The N-terminal, C-terminal and loops are usually the prominent sites of VLPs suitable for insertion into M2e. However, loops are generally flexible structures and can only tolerate peptides of limited length (D. Wang et al., 2018). In this study, the C-terminal of Cap protein protrudes from the surface of Cap VLPs, and participates in the formation of linear and conformational neutralization epitopes, indicating that the C-terminus can be efficiently recognized by immune system (Khayat et al., 2011; Lekcharoensuk et al., 2004). Therefore, it can be utilized as an insertion site for multiple copies of M2e. Results showed that the M2e which near the C-terminal induced higher levels of species-specific anti-M2e antibodies. It was because the immune system efficiently recognized the C-terminal of Cap protein, thereby efficiently recognizing adjacent M2e. The level of species-specific anti-M2e antibodies induced by two M2e that away from the C-terminal are low and there no significant difference. This is due to the indefinite spatial conformation of M2e and the wobble of the flexible link leading to the decline of the immune system’s recognition ability.
Therefore, it is necessary to selectively display M2e of IAV of species-specific in the most prominent and relatively fixed position of nanoparticles based the immune target of universal IAV vaccines, so as to induce a more efficient immune effect. For example, when chickens inject with universal IAV nanovaccines, M2e of the avian IAV need to be displayed in a prominent and stable position on nanoparticle.