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.