1 Introduction
Influenza A virus (IAV) causes huge economic loss to the global
husbandry industry and poses a significant threat to public health. The
IAV genome is composed of eight single-stranded negative-sense RNA
fragments, resulting in abnormally high frequency of gene mutations and
recombination, which bring great difficulties to the development of
universal vaccines (Lowen, 2017). IAV
occasionally cross species boundaries and pose lethal threats to other
species (Long, Mistry, Haslam, & Barclay,
2019). In particular, cross-species transmissions between humans, swine
and birds are more frequent, such as 2009 pandemic H1N1 swine influenza,
highly pathogenic H5N1 and H7N9 avian influenza jumped into humans,
causing great panic (Gao, 2018).
Therefore, it is urgent to establish IAV universal vaccines.
Hemagglutinin (HA) and neuraminidase (NA) are the main vaccine candidate
antigens. However, due to the abnormally high mutation rate, current
influenza vaccines can only provide effective protection against
epidemic strains consistent with their HA and NA. Ectodomain of matrix
protein 2 (M2e) is the most conservative and protective viral antigen
and can generate hetero-subtype immunity against multiple virus strains
and subtypes. However, M2e is difficult to be recognized by the immune
system due to the low molecular weight, the low abundance and the steric
blocking by HA and NA on the surface of IAV
(Kolpe, Schepens, Fiers, & Saelens,
2017). Various effective strategies for improving M2e antibody levels
have been proposed, in particular, some nanoparticle-based nanovaccines
show exciting immune effects, such as ferritin, virus-like particles
(VLPs), and gold nanoparticles (Kolpe et
al., 2017). To further improve the level of M2e-specific antibodies,
nanoparticles usually display multi-copy of M2e. Tandem expression of
M2e from human, swine and avian IAV are the most common pattern
(Deng, Chang, et al., 2018;
Deng, Mohan, et al., 2018;
Ding, Jin, Chen, et al., 2019;
K. H. Kim et al., 2018;
M. C. Kim, Lee, et al., 2013;
M. C. Kim et al., 2015;
M. C. Kim, Song, et al., 2013;
Petukhova et al., 2013;
Qi et al., 2018;
Q. Wang et al., 2020;
Y. Wang et al., 2020;
Yong, Yeap, Ho, Omar, & Tan, 2015).
Porcine circovirus type 2 (PCV2), one of the smallest mammalian viruses,
causes pigs severe immunosuppression and huge economic losses. The
unique capsid (Cap) protein of PCV2 can self-assemble to form VLPs,
which is an ideal candidate for PCV2 vaccines. What is more, the Cap
VLPs can carry foreign epitopes and have the potential to become
nanovaccines scaffold. Our previous research shown that Cap was capable
of carrying three copies of M2e at its C-terminal without impairing VLPs
formation and Cap-3M2e VLPs induced high levels of M2e-specific
antibodies and PCV2-specific neutralizing antibodies in mice and pigs
(Ding, Jin, Chen, et al., 2019).
However, due to the uncertainty of the crystal structure of M2e and
immune systems tend to efficiently recognize stable protruding domains
on the surface of nanoparticles, it is worth investigating whether the
permutation of M2e of IAV from different species has an impact on the
immune effect of nanovaccines.
In this study, we displayed the M2e derived from IAV of humans, swine
and avian in different order (six orders) at the C-terminal of porcine
circovirus type 2 (PCV2) Cap VLPs to explore the effect of different M2e
arrangement on the immune effect of the universal vaccine. Results
showed that the M2e closest to surface of Cap VLPs induced the highest
M2e-specific antibodies and conferred the best protection against IAV of
corresponding species. This result
will help to develop more efficient universal influenza vaccines.