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.