Immunogenicity of Flu-VLPs. A. Neutralising activity of S2 sera from mice immunised with Flu-VLPs incorporating the HA, NA and M2 proteins from H7N1 (lanes 1–8) or H5N1 (lanes 13–20) influenza viruses (H7-VLP and H5-VLP, respectively), or with VSV-VLPs harbouring the VSV-G glycoprotein (lanes 9–12). Some Flu-VLPs were treated at acidic pH5.3 (H7-VLP (A) and H5-VLP (A)) to induce irreversible conformational changes in the HA protein before injection (lanes 5–8 and lanes 13–16 for H7-VLPs and H5-VLPs, respectively). Sera from each mouse were incubated at 37°C for 40 min with H7-VLPs, H5-VLPs or VSV-VLPs harbouring a GFP marker gene, as indicated, and then used to infect TE671 target cells. The transduction titres determined in the presence of diluted mouse sera were calculated as described in Fig. 1. The results are expressed as the mean percentages (mean ± SD; n = 5) of neutralisation of the transduction titres determined with the immune sera relative to titres determine with S0 pre-immune sera. Sera or antibodies raised against FPV (fowl plague virus; H7N1 influenza virus) and VSV were used as controls in the neutralisation assays (anti-FPV and anti-VSV, respectively). B. Determination of the specificity of antibodies raised in S2 sera from mice immunised with Flu-VLPs by Western blot analysis. H7-VLPs (left) and H5-VLPs (right) were loaded onto SDS-PAGE and transferred to membrane after electrophoresis. Lanes of these membranes were individualised and separately incubated with S2 sera from immunised mice (see above) at a 1/500 serum dilution. S0: pre-immune serum; S. HA; control polyclonal rabbit serum raised H7N1 HA; S. M2; control serum raised against H7 M2; HA0: HA precursor protein; HA1: HA surface subunit; HA2: HA transmembrane subunit; NA: neuraminidase; M2: M2 matrix protein. C. The neutralising curves of sera from mice immunised with H7-VLPs and H5-VLPs, harvested two weeks after each injection (S1, S2, S3 and S4), were determined for different serum dilutions (1/20, 1/100, 1/500 and 1/2,500). The results are expressed as the mean percentages (mean ± SD; n = 5) of neutralisation of the transduction titres determined with the immune sera relative to titres determine with S0 pre-immune sera.