Abstract
Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2′-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.
Highlights
Innate immunity in chronic viral hepatitis, primarily focusing on parenchymal and non-parenchymal murine liver cells have been described previously, indicating a diversification of TLR signaling pathways in Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) compared to ‘classical’ antigen-presenting cells, such as myeloid dendritic cells[8]
Since many of the antiviral effects of Nucleic acid polymers (NAPs) therapy in HBV infection are similar to those observed with immunotherapy, a more rigorous examination of immunostimulatory effects of NAPs optimized for therapeutic use was conducted in primary cultures of human parenchymal and non-parenchymal liver cells and peripheral blood mononuclear cells
Identity and NAP uptake by different liver cell types was confirmed by treatment of Primary human hepatocytes (PHH), KCs and LSECs with cyanine dye 3 (Cy3)-labelled NAPs (REP 2055 [0.01 μM], REP 2139 [0.05 μM] and REP 2165 [0.05 μM])
Summary
Innate immunity in chronic viral hepatitis, primarily focusing on parenchymal and non-parenchymal murine liver cells have been described previously, indicating a diversification of TLR signaling pathways in Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) compared to ‘classical’ antigen-presenting cells, such as myeloid dendritic cells[8]. That TLR agonist-induced expression of pro-inflammatory (TNF, IL6, IL1b), antiviral (IFNB1) and anti-inflammatory cytokines (IL10) in murine KCs, LSECs, and hepatocytes is cell-type specific[8,9]. Since many of the antiviral effects of NAP therapy in HBV infection are similar to those observed with immunotherapy, a more rigorous examination of immunostimulatory effects of NAPs optimized for therapeutic use was conducted in primary cultures of human parenchymal and non-parenchymal liver cells and peripheral blood mononuclear cells. Experimental setup and description of NAPs are depicted in (Fig. 1)
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