Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

Abstract

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice.

Figure 1

Biochemical and functional analysis of Flu-VLPs. A. Incorporation of HA, NA and M2 proteins from H7N1 (A/Chicken/FPV/Rostock/1934) or H5N1 (A/Thailand/KAN-1/04) influenza viruses on MLV retroviral core particles, as indicated. Purified VLPs were loaded on SDS-PAGE and incorporation levels of the different proteins were determined by Western Blot analysis using polyclonal sera against H7N1-HA, H5N3-HA, H7N1-NA and M2 proteins. HA₀: HA precursor protein, HA₁: HA surface subunit; HA₂: HA transmembrane subunit; NA: neuraminidase; CA: MLV capsid protein. The difference in the molecular weight of H5 vs. H7 HA₂, of ca. 3 kDa, was due to the presence of an additional glycan for H7 HA₂. B. Infectious titres obtained with Flu-VLPs incorporating surface proteins from H7N1 or H5N1 influenza viruses or with VSV-VLPs incorporating the VSV-G of vesicular stomatitis virus (VSV), as indicated. VLP-containing supernatants were used to infect 10⁵ TE671 target cells plated in 12-well for 6 hr at 37°C. The transduction efficiency of GFP, determined as the percentage of GFP-positive cells, was measured by fluorescence-activated cell sorter (FACS) analysis 72 hr after infection, as previously described [22]. The transduction titres, provided as GFP-transducing units (t.u.)/ml, were calculated by using the formula: Titre = %inf × (10⁵/100) × d, where "d" is the dilution factor of the viral supernatant and "%inf" is the percentage of GFP-positive cells as determined by FACS analysis using dilutions of the viral supernatant that transduced between 0.1% and 5% of GFP-positive cells. Due to the use of a FACS method to monitor transduced cells, the determination of GFP-positive cell below 0.1%, i.e., corresponding to transduction titres below 10³ t.u./ml in our experimental conditions, could not be accurately be determined. The background levels of transduction were therefore fixed at 10³ t.u./ml in all experiments.

Figure 2

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.