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 (Figure 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 (Figure S1). 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 (Figure 1e and Figure S2). DLS test results showed that the diameter of these Cap-3M2e VLPs were larger than that of Cap VLPs (Figure 1f). 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 (Figure 1g). 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 (Figure 1f and 1g).
3.2 Humoral immune effects
All Cap-3M2e VLPs groups induced high levels of total M2e-specific antibodies (Figure S3a). 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 (Figure 2a). 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 (Figure 2a). The Cap-shaM2e VLPs and Cap-sahM2e VLPs groups induced higher anti-swine IAV M2e antibodies than other groups (Figure 2b). Anti-swine IAV M2e antibody in the Cap-shaM2e VLPs group was 6.4 times that of the Cap-ashM2e VLPs group (Figure 2b). Meanwhile, anti-avian IAV M2e antibodies in the Cap-ahsM2e VLPs and Cap-ashM2e VLPs were the highest (Figure 2c). The anti-avian IAV M2e antibody in Cap-ahsM2e VLPs group was 8 times that of the Cap-sahM2e VLPs group (Figure 2c). However, the PCV2-specific antibodies in all groups were consistent, indicating that the insertion of three copies of M2e in different orders did not affect the immunogenicity of the Cap VLPs (Figure S3b).