Neutralisation of VLP cell entry by sera from vaccinated animals.

<p>(A) Huh7 cells (5×10⁵ cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E₈Pam₂Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×10⁵) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.</p

Cell-mediated responses elicited by vaccination.

<p>HLA-A2k^b transgenic mice (n = 3 per group) were inoculated (100 µl) subcutaneously at the base of the tail on days 0 and 14 with 30 µg of VLPs alone, emulsified with an equal amount of complete freund’s adjuvant (CFA) or pre-mixed with 30 µg of E₈Pam₂Cys in saline. Splenocytes were obtained 28 days later and restimulated for 7 days in the presence of 10 µg VLPs or an irrelevant HCV-derived HLA-A2-restricted epitope not part of the VLP construct. The frequency of peptide-specific T cells producing IFN-γ was determined in an ELISPOT assay. Each bar represents the average number of IFN-γ producing T cells and standard deviation in each group after subtracting non-specific responses from corresponding samples stimulated with the irrelevant peptide.</p

Antibody responses elicited by vaccination.

<p>Groups of BALB/c mice (n = 5 per group) were inoculated (100 µl) sub-cutaneously at the base of the tail with 20 µg of VLPs alone, pre-incubated with an equal volume of aluminium hydroxide (13 mg/ml) or 60 µg of E₈Pam₂Cys in saline on days 0 and 21. (A) VLP-specific antibody levels in sera prepared from blood taken on days 21 (white circles) and 35 (black circles) were then determined by ELISA. Individual animal antibody titres are presented with the mean value represented by the horizontal bar. An additional dose of each formulation was also administered to 3 mice from each group on day 56 and antibody titres determined on day 63 (grey circles). (B) E2-specific antibody titres in these animals were also determined at this same time point.</p

Dendritic cell uptake of fluorescenated VLPs.

<p>CD11c⁺ MHC Class II⁺ splenocyte-derived cultured D1 dendritic cells (2×10⁵ cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E₈Pam₂Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×10⁴ cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.</p

Dendritic cell maturation.

<p>(A) D1 dendritic cells (2×10⁵ cells) were incubated with VLPs (5 µg) alone or formulated with E₈Pam₂Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class II^high expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E₈Pam₂Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E₈Pam₂Cys, or (F) Alhydrogel.</p