3.1 Characteristics of Cap-3M2e VLPs
Six different recombinant Cap-3M2e proteins were expressed in E. coli and purified using Ni2+-NTA column. SDS-PAGE and Western blot showed that these recombinant Cap-3M2e proteins were successfully expressed and purified (Fig. 1d). These recombinant Cap-3M2e proteins can be recognized well with the anti-PCV2 polyclonal antibody and 14C2 monoclonal antibody (Anti-IAV M2 protein), indicating that these recombinant proteins retained the reactogenicity of M2e and the Cap protein (Fig. 1e). Endotoxin levels in these recombinant proteins were less than 0.18 EU/mg.
TEM images showed that these recombinant Cap-3M2e proteins could self-assemble into VLPs (Fig. 2a). DLS test results showed that the diameter of these Cap-3M2e VLPs were larger than that of Cap VLPs (Fig. 2b). The zeta potential of these Cap-3M2e VLPs were lower than that of Cap VLPs, which were caused by the strong negative charge of M2e molecules (Fig. 2c). The particle size and surface charge of these Cap-3M2e VLPs are consistent, indicating that the permutation of M2e of IAV from different species does not affect the morphology and surface charge of these VLPs (Fig. 2).
3.2 Humoral immune effects
All groups induced consistent PCV2-specific antibodies and neutralizing antibodies, indicated that the insertion of three copies of M2e in different orders did not affect the immunogenicity of the Cap VLPs (Fig. 3a and 3b). Total M2e-specific antibodies in Cap-3M2e VLPs groups were consistent, indicated the permutation of M2e did not affect the level of total M2e-specific antibodies (Fig. 3c). However, there were significant differences in levels of antibodies against M2e derived from human, swine and avian IAV in sera of each group. The Cap-hsaM2e VLPs and Cap-hasM2e VLPs groups induced higher anti-human IAV M2e antibodies than others (Fig. 3d). Anti-human IAV M2e antibody in the Cap-hasM2e VLPs group was 7.3 times that of the Cap-sahM2e VLPs and Cap-ashM2e VLPs groups (Fig. 3d). The Cap-shaM2e VLPs and Cap-sahM2e VLPs groups induced higher anti-swine IAV M2e antibodies than other groups (Fig. 3e). Anti-swine IAV M2e antibody in the Cap-shaM2e VLPs group was 6.4 times that of the Cap-ashM2e VLPs group (Fig. 3e). Meanwhile, anti-avian IAV M2e antibodies in the Cap-ahsM2e VLPs and Cap-ashM2e VLPs were the highest (Fig. 3f). The anti-avian IAV M2e antibody in Cap-ahsM2e VLPs group was 8 times that of the Cap-sahM2e VLPs group (Fig. 3f).