This indicates that the HCV particles released from infected-HepaRG cells (HCV-RG) are indeed infectious. To determine the buoyant density distribution of HCV RNA, E1E2 and core antigens, the viral preparations from media collected at days 28 and 42 Panobinostat datasheet p.p. (Fig. 1A,b) were pooled and subjected to iodixanol gradient density centrifugation. Figure 1D shows that the HCV-RG particles had a relatively homogeneous distribution between 1.06 and 1.12 g/mL. They expressed E1E2 envelope proteins and contained RNA and core antigen. In addition, the positive fractions reacted with polyclonal antibodies against apoE (++, P/N ratio = 5-6) and
apoB (+, P/N ratio = 3-4), suggesting that host lipoproteins could be associated with these particles mimicking circulating HCV.14 Immunohistochemistry experiments were performed to investigate intracellular expression of HCV E1E2 and core antigens (Ag) in infected-HepaRG cells at 28 and 56 BMN 673 cell line days p.p. (infection 1). Figure 1E shows that the HCVsp-infected HepaRG cells at D28 p.p. exhibited a very strong staining of cytoplasm and perinuclear regions for E1E2 Ag (a). Fifty to sixty percent of cells were positive. Core Ag staining (b) appeared
also in the cytoplasm possibly around lipid droplets. Some cells were labeled both in the cytosol and the nucleus. Control HCV(−) uninfected HepaRG cells were clearly negative in the presence of D32.10 (a) or C7.50 (b), as well as HCV-infected cells in the presence of a control IgG1 antibody (not shown). Positively stained infected cells exhibited morphological features of hepatocytes.5 Altogether, these results indicate that the human DOK2 HepaRG cells can be infected with HCVsp when proliferated and do produce de novo infectious lipoprotein-associated enveloped complete HCV particles for up to 6 weeks when differentiated. To investigate whether the unique E1E2-specific D32.10 mAb inhibits HCV infection, the infection experiment (infection 3) was performed after preincubation of HCVsp with D32.10 at a 0.5 μg/mL concentration. Figure 2 shows that the D32.10 mAb completely inhibited HCV RNA production in HepaRG culture supernatants.
The total amount of HCV RNA remained at very low levels throughout the follow-up of the infection from day 1 to day 21 in the presence of D32.10 with a mean inhibition of 80.5 ± 11.6% (Fig. 2A). When HCV RNA was quantified by qPCR, 5log10 copies/mL were detected at day 21 after control infection. The preincubation of the inoculum with D32.10 reduced by ≈97% the extracellular HCV RNA (−2 log10, Fig. 2B). To further support that control-infected HepaRG cells produced viral particles, iodixanol density gradient analysis was performed from HCV RNA-associated particles present in the culture media collected at days 14 and 21 (Fig. 2C). As seen previously (infection 1), both HCV RNA, E1E2, and core antigens were recovered as a major peak between 1.