Fig 1: Recall antibody response induced by VLP vaccination to influenza.(A) Western blot analysis of VLP-induced humoral immunity against PR8 HA. The PR8 HA0 (11684-V08H, Sino Biological Inc.; expressed by baculovirus system) was treated with TPCK-trypsin (5 µg/mL) at 37°C for 15 min to induce cleavage into HA1 and HA2. The cleaved PR8 HA was admixed with purified H3N2-VLP (providing annexin A2 as loading control), serially diluted in two folds and quantified by western blot analysis. The antibody against the annexin A2 (ab41803, Abcam) in H3N2-VLP was used to normalize the loading amount of protein samples and transfer efficiency of western blotting. Mice vaccinated with various VLPs were bled before (Pre-), 4 days (Post-D4) or 14 days (Post-D14) after challenge as indicated. The antisera (500-fold dilution used in this assay) were analyzed by western blotting. HA0, HA1, and HA2 are labeled on the left. (B) Comparative analysis of cross-reactive anti-HA2 antibody elicited by VLP vaccination before challenge or recalled after homologous and heterologous viral challenges. VLP vaccines and strains of challenge virus are as indicated. (C), (D) The relative folds of antibody response specific to PR8 HA1 or HA2 in panel A and B were quantified and summarized correspondingly. Comparing the relative level of HA2 antibody before and after viral challenge, asterisk indicates statistic significance as used in Figure 1. (E) The HAI titers in vaccinated mice 4 days after PR8 infection were determined by standard methods. Scatter plot with mean values of the same group; bars, SEM. Type of VLP vaccine or PBS control is indicated.