Comparison of the different vaccination strategies in terms of their capacity to induce neutralizing antibodies against various HCVcc genotypes.

<p>Comparison of the different vaccination strategies in terms of their capacity to induce neutralizing antibodies against various HCVcc genotypes.</p

Cross-neutralizing properties against HCVcc of antibodies induced by immunization with chimeric particles harboring the E1E2 heterodimer, compared with those of antibodies induced by a mixture or different sequential immunization combinations of chimeric particles harboring the E1 and E2 proteins separately.

<p>Five-fold dilutions of rabbit sera collected on days 0 and 56 were first incubated with HCVcc harboring HCV envelope glycoproteins derived from strains of various genotypes for 1 hour at 37°C, which were then used to infect Huh7.5 cells for 6 hours. Infection levels were determined after 48 hours of incubation at 37°C, in a FFU staining assay. The percentage neutralization in the presence of the post-immune serum (day 56) was compared with that in the presence of pre-immune serum (day 0) from the same rabbit. The assay was performed in duplicate and the results are expressed as mean values.</p

Humoral immune responses induced in rabbits immunized with chimeric particles harboring the E1E2 heterodimer, compared with a mixture or different sequential immunization combinations of chimeric particles harboring the E1 and E2 proteins separately.

<p>Specific anti-E1 (A) and anti-E2 (B) responses were evaluated with “in-house” ELISAs on rabbit sera collected at various time points. Anti-HBsAg responses (C) on these serum samples were evaluated with a routine immunoassay (Abbott Laboratories). Sera from rabbits immunized with the adjuvant alone or with Engerix were used as negative and positive controls, respectively. Black arrows indicate the time at which immunization with 4 x 15 μg of immunogens occurred. Results are expressed as the difference in optical density (E1- or E2-β-gal) and anti-HBsAg titer (mIU/ml), respectively.</p

Comparison of the cross-neutralizing properties against HCVcc of two pairs of equal-amount mixtures of an anti-E1 rabbit serum and an anti-E2 rabbit serum with those of these sera used individually.

<p>HCVcc harboring HCV envelope glycoproteins derived from strains of various genotypes were first incubated for 1 hour at 37°C with five-fold serial serum dilutions of rabbit sera collected on day 56, or mixtures. To prepare these mixtures, we selected from our previous immunization experiments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151626#pone.0151626.ref025" target="_blank">25</a>] two animals from each group with sera presenting optimal cross-neutralizing properties against heterologous HCV strains of various genotypes. Sera from rabbits immunized with the adjuvant alone or with Engerix were used as negative controls. They were then incubated with Huh7.5 cells for 6 hours. Infection levels were determined after 48 hours of incubation at 37°C, in a FFU staining assay. The percentage neutralization was determined by subtracting the infectious titer obtained with the pre-immune serum (day 0) from that obtained with the post-immune serum (day 56) from the same rabbit. The assay was performed in duplicate, and the results are expressed as mean values.</p

Principle underlying correlative scanning electron microscopy-transmission electron microscopy (CSEMTEM).

<p>(A): SEM acquisition, at low magnification, made it possible to locate a microcarrier of particular interest (white arrow), i.e. bearing a cell with chimeric viral particles at its surface. (B): Mapping of the whole carbon disc with the multiscan module was performed, to identify the domain of interest on the disk (white circle). (C and D): After resizing (white circle in C), the sample containing the microcarrier of interest was examined under a stereo microscope (D). (E): After inclusion in Epon resin, thin sections (500 nm) of the resized sample were cut with an ultramicrotome and stained with toluidine blue, making it possible to identify the microcarrier of interest by light microscopy. (F): Ultrathin sections (70 nm) of this block were then cut for analysis of the resized sample by TEM, making it possible to visualize cells at the surface of the selected microcarrier (inset in F). The microcarrier of interest (arrows in A, D, E and F) can be followed during all these steps, through the recording of SEM, optical and TEM photographs. Scale bars : 200 µm in A, D and E; 2 mm in B and C; 100 µm in F.</p

TEM analysis of Vero cells selected by the CSEMTEM method.

<p>(A): Electron-dense chimeric viral particles were observed at the cell surface (black arrows). (B, C and D): Several released particles were observed at the site of small-vesicle exocytosis at the plasma membrane (white arrows). Scale bars: 0.2 µm in A, B and C; 0.1 µm in D.</p

Effect of LB-piVe infection on apoptosis-related genes expression.

<p>50μg of total protein samples were resolved by SDS-PAGE in Novex 4–20% Tris-glycine polyacrylamide gels and then transferred to Hybond ECL membranes. Blots were probed with anti-caspase-3, anti-cleaved PARP1, anti-BIK, and anti- Bcl-xL. Horse radish peroxidase-conjugated secondary antibodies were used for detection and developed with the Femto Chemiluminescence Substrate kit. GAPDH was used as internal loading control. Negative controls: naïve Huh7.5 cells (C), and Huh7.5 cells treated with UV-LB-piVe (UV).</p

LB-piVe infection triggers exposure of phosphatidylserine on the surface of infected cells.

<p>(A) UV-LB-piVe (left panel) or LB-piVe infected (right panel) cells and Staurosporine-treated (center panel) were incubated with PE Annexin V in a buffer containing 7-Amino-Actinomycin D (7-AAD), and analyzed by flow cytometry. Numbers in the quadrants indicate the percentage of cells in the corresponding areas. (B) Representation of the frequency of live, early apoptotic, late apoptotic and necrotic cells from (A).</p

Analysis by negative staining electron microscopy and immunogold labeling of the chimeric yellow fever virus/dengue virus (YFV/DENV) particles secreted in the cell supernatant.

<p>(A): Numerous 50 to 60 nm spherical particles that had morphological characteristics of a typical flavivirus were observed by regular negative staining electron microscopy. (B): The specificity of these particles was confirmed by immunogold labeling with an anti-DENV E glycoprotein. Scale bars : 0.5 µm in A ; 100 nm in B.</p

Apoptotic signaling pathways triggered by LB-piVe infection.

<p>Pathway analysis was performed with the Pathway Studio 9 software. Using the PCR Array data, a network was generated in which the genes and gene products are represented by nodes. The shapes of the nodes represent the protein’s primary function and the connection lines indicate the type of interaction (colored lines: direct regulation; dotted gray lines: indirect regulation). Color intensity reflects the level of gene expression in LB-piVe infected cells versus control if the fold-change ≥1.2 (red: up-regulated; blue: down-regulated; grey: no change in expression).</p