Synthesis and purification of M1:HBcAg VLPs.

<p>M1 was coupled to HBcAg (residues 1–142) using the heterobifunctional cross-linker sMBS (Pierce). Western Blot indicating the coupled product is recognised by both the anti-M1 and anti-HBcAg antibodies. (B) Following conjugation, the M1:HBcAg was purified from unbound M1 protein by ultracentrifugation. The resulting coupled product was analysed by SDS-PAGE under reducing conditions followed by silver stain. Following purification no contaminating M1 was observed in the purified product. HBcAg (C) and M1:HBcAg (D) were stained with uranyl acetate and analysed by electron microscopy. The morphology of HBcAg was retained following coupling and purification.</p

Colocalisation of M1:HBcAg and Endocytic markers.

<p>Day 6 immature DCs were incubated with M1:HBcAg on ice to allow binding and the HBcAg was fluorescently labelled. The cells were subsequently incubated at 37°C to allow internalisation. At the indicated timepoints, cells were harvested, fixed, permeabilised and stained for endocytic markers (in green) for analysis by confocal microscopy. M1:HBcAg colocalised with the early endosome marker EEA1 within 10 minutes and the late endosomal marker LAMP1 from 30 minutes. Following internalisation, M1:HBcAg was shown to colocalise with MHC I. The frequency of pixel colocalisation (on the left) was determined by Manders coefficient from 4–8 cells per treatment. Statistical significance was determined by the unpaired student <i>t</i>-test; *p&lt;0.05, **p&lt;0.005.</p

M1-conjugation improves CD4⁺ responses to the universal T_H-epitope TTp2.

<p>TTp2 was coupled to M1 under oxidising conditions and coupling confirmed by SDS-PAGE (A). PBMC were treated with M1:TTp2 or molar equivalents of M1, TTp2 or mixed M1 and TTp2 in an IFNγ Elispot Assay (BD Biosciences). Responses from 10 individuals are shown (B). Statistically significant differences in the response observed for M1-coupled peptide and controls are denoted by * (determined by t Test). Activation of purified CD4⁺ cells (C) in the presence of the indicated antigen was assessed by Elispot assay and compared to PBMC depleted of CD4 cells in (D). Data representative of one individual's response is given showing the mean nd standard deviation of duplicate samples. Statistical significance is indicated by * where p&lt;0.05; statistical significance was determined by the student t-test.</p

M1:HBcAg is presented on MHCI and activates HBcAg-specific CD8+ T cells.

<p>Immature DCs were pulsed with M1:HBcAg or the control antigens for 60 minutes at room temperature. Following extensive washing, the cells were incubated overnight at 37°C. Antigen-loaded cells or untreated cells (UT) were incubated with HBc_18–27 cells for 6 hours in the presence of Brefeldin A and monensin. CD107a (A&amp;C) expression and IFNγ (B&amp;D) production were measured in parallel. Results in A and B indicate the T cell activation as a percentage of the positive control of DCs loaded with the molar equivalent of C_18–27 peptide. Data are representative of 4 individual experiments. M1:HBcAg treatment resulted in significantly more T cell clones expressing CD107a and IFNγ than the control antigens. *Denotes statistical significance determined by student <i>t</i>-test, &lt;0.05. A representative data set from one individual is presented in C &amp; D showing mean and standard deviation of duplicate samples.</p

Clinical data of HCV patients at time of biopsy.

<p>Clinical data of HCV patients at time of biopsy.</p

IFN-α reduces hepatocyte sensitivity to CTL cytotoxicity.

<p>Hepatocytes have been described as expressing low to no MHC Class I. As expected, IFN-α treatment increased the levels of MHC Class I on HepG2 (A) and HHL (B); filled curve represents an isotype control, the solid line represents the MCH Class I expression. C) HepG2 cells were stimulated for 16 hours with a serial dilution of IFN-α at 0 IU/ml (closed circles), 10 IU/ml (open diamonds), 100 IU/ml (open squares), and 1000 IU/ml (open inverted triangles), prior to cytotoxicity assay with the CTL line 2. Treatment with IFN-α reduced the HepG2 cells sensitivity to CTL cytotoxicity in a dose dependent manner. D) This phenomenon was also found with the novel human hepatocyte cell lines (HHL). HHL-17 cells were either left untreated (closed circles) or stimulated for 16 hours with 1000 IU/ml (open inverted triangles) prior to co-incubation with the CTL line 1.</p

Expression of the granzyme B inhibitor, serine proteinase inhibitor 9 (PI-9).

<p>A) RT-PCR revealed that IFN-α stimulated cells, HepG2 and HHL-9 cells, were able to upregulate PI-9. GAPDH expression was analysed as a positive control. Bi and ii) This was confirmed by FACS analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods and materials</a>. Upregulation of PI-9 was shown to be dose dependent.</p

Expression of PI-9 in liver tissue from patients with chronic hepatitis C.

<p>Liver specimens obtained from diagnostic biopsy were stained for PI-9 expression as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods</a>. A) Representative negative isotype control. B–D) PI-9 was detected in the majority of hepatocytes. Stronger PI-9 expression, as expected, was seen within the mononuclear infiltrate (highlighted by red arrows), while all the hepatocytes stained positively, albeit at a lower level (the strongest are highlighted by blue arrows).</p

IFN-α treated hepatocytes remain susceptible to FASL-induced apoptosis.

<p>Aii) Induction of apoptosis by FASL was assessed across a concentration range. HepG2 cells, either stimulated with IFN-α (1000 IU/ml) (closed inverted triangles) or left un-stimulated (open diamonds), were incubated with cross-linked rFASL at concentrations ranging from 0.1 pg/ml to 2 µg/ml. Apoptosis was assessed by Annexin V binding and propidium iodine (P.I) incorporation. Ai) Raw FACS data is shown. Bii) HepG2 cells, either un-stimulated (open and closed circles), stimulated with 100 IU/ml (open and closed squares) IFN-α or 1000 IU/ml (open and closed inverted triangles) IFN-α, were incubated with 1 µg/ml (left, open symbols) or 2 µg/ml (right, closed symbols) cross-linked rFASL over a time course of up to 6 hours. Apoptosis was assessed by the cytosolic presence of the activated form of caspase 3. Bi) Raw FACS data.</p

Analysis of alternative triggers for PI-9 expression.

<p>A) The up-regulation of PI-9 was also found to be triggered by other inflammatory cytokines. HepG2 cells were treated for 16 hours with either IFN-γ at 100 IU/ml, or IL-1β at 50 ng/ml. GAPDH expression was used as a positive control. B) Stimulation of the HepG2 cell line with either IFN-γ (open squares) or IL-1β (open triangles) also inhibited CTL killing compared to the un-stimulated HepG2 cells (closed circle). C) HepG2 cells were left un-infected (Nil), or infected with either a baculovirus expressing a sub-genomic replicon (NS-replicon), or with a control baculovirus expressing LacZ (+Control). PI-9 expression was analysed by RT-PCR. PI-9 was strongly up-regulated only in the cells expressing the sub-genomic replicon. GAPDH expression was used as a positive control.</p