4 Discussion

High variability is the most prominent feature of IAV, which brings huge difficulties to vaccine development. Thus, the elicitation of antibody responses against sequence-conserved epitopes that could protect diverse virus strains is a high priority. 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.
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 (Wang et al., 2018). In this study, the N-terminal of the Cap protein is located inside the VLPs, which is difficult to be recognized by BCR and is not suitable for displaying B cell epitopes. On the contrary, 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.
Although a high affinity antibody that recognizes a pathogen’s protective epitope may also bind a structurally unstable peptide, immunization with the peptide is unlikely to elicit high titer protection antibodies against the pathogen. This is because upon immunization with the peptide, any B cell that bears an immunoglobulin that recognizes one of the many conformations of the peptide can be stimulated to expand. There is no possible to selectively activate and amplify those B cells bearing immunoglobulins that bind multiple conformations of the peptide. Furthermore, the flexible peptide could guide the affinity maturation of antibodies down many alternative paths. Affinity maturation of antibodies requires stable and full display of epitopes rather than transient state (Dormitzer, Ulmer, & Rappuoli, 2008). M2e adopt at least two transformed conformations (Cho et al., 2016; Cho et al., 2015). In the former, M2e bound to a protective mAb with residues Ser2-Leu3-Leu4-Thr5-Glu6 forming a N-terminal β turn. In the latter, M2e adopts a horseshoe-like conformation that is stabilized in its core by the tryptophan residue at position 15. In addition, due to the existence of flexible links between different M2e, the instability of multi-copy M2e conformation is exacerbated. Therefore, the relatively stable one in the these M2e is more easily recognized by the B cells and eventually induces higher levels of antibodies.
In order to clarify the difference between the M2e specific antibody levels of IAV from various species, we first tested the PCV2 specific antibody levels. The neutralizing antibody level of these Cap-3M2e VLPs immunization groups were consistent with the Cap VLPs group, indicating the difference of M2e antibody level in these Cap-3M2e VLPs immunization groups was related to the arrangement of M2e but not to the Cap VLPs vector. Results showed that the M2e which near the C-terminal of Cap protein induced higher levels of species-specific anti-M2e antibodies. This is because the C-terminal conformation of the Cap protein is stable and can be efficiently recognized by the immune system, thus the conformation of M2e which near the C-terminal is more stable and can be more effectively recognized by the immune system than that of other 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. The precise mechanism by which M2e-specific antibodies provide protection is controversial. But it is recognized that the protective effect is positively correlated with the level of M2e-specific antibodies. As shown in Fig. 4, groups with the highest level of species-specific M2e antibody showed the best protective effect during the challenge experimental. In general, 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 pigs inject with universal IAV nanovaccines, M2e of the swine IAV (SIV) need to be displayed in a prominent and stable position on nanoparticle.
Previous studies showed that the neutralization antibody level induced by Cap VLPs at 20 µg was consistent with that of the commercial PCV2 vaccine (Ingelvac CircoFLEX®, Boehringer Ingelheim)(Ding, Jin, Chen, et al., 2019; Ding, Jin, Zhou, et al., 2019). Therefore, in this study, in order to emphasize the effect of different M2e permutations on influenza virus vaccine efficacy, we used Cap VLPs instead of the commercial PCV2 vaccine and IAV VLPs as the control group. Previous studies have clearly shown that Cap-M2e VLPs nanovaccine induce high levels of PCV2-specific neutralizing antibodies and M2e-specific antibodies in mice and pigs, and significantly reduce SIV titers in pigs’ respiratory tract. Cap-3M2e VLPs nanovaccine can induce higher levels of M2e-specific antibodies than Cap-M2e VLPs nanovaccine in mice, and induce high levels of PCV2-specific neutralizing antibodies consistent with commercial vaccine in mice and pigs (Ding, Jin, Chen, et al., 2019; Ding, Jin, Zhou, et al., 2019). Based on this research, the Cap-3M2e VLPs nanovaccine can be further upgraded to Cap-shaM2e VLPs nanovaccine to increase the immune effectiveness in pigs. Based on previous research, we speculate that the Cap-shaM2e VLPs nanovaccine have the potential ability to defend against the challenge of SIV and PCV2 in pigs. Therefore, recommendations for future studies include evaluation of cellular and humoral immunity in pig model and evaluation of protection against SIV and PCV2.