Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don't harbor immunostimulatory properties in primary isolated liver cells

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.

Figure 1. Schematic experimental procedure and NAP description.

Primary human hepatocytes (PHH), Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC) and peripheral blood mononuclear cells (PBMC) were stimulated with NAPs for 6 h to analyze cytokine gene expression by qRT-PCR and for 24 h to analyze cytokine secretion by ELISA (A). Overview of nucleic acid polymers (NAPs) used in this study (B).

Figure 2. Nucleic acid polymers (NAPs) were efficiently taken up by different liver cell types.

The identity of primary human hepatocytes (PHH) (A), Kupffer cells (KC) (B) and liver sinusoidal endothelial cells (LSEC) (C) was assessed by immunofluorescent staining of cell type-specific markers albumin (A), CD68 (B) and LYVE-1 (C) (green), respectively. Nuclei were counterstained with DAPI (blue). Uptake of NAPs was visualized using Cy3-labeld (red) REP 2055 [0.01 μM], REP 2139 [0.05 μM] and REP 2165 [0.05 μM]. Immunofluorescence staining was detected with a laser scanning microscope (LSM; Axiovert 100 M; Zeiss, Jena, Germany) at 20 × magnification. Image analysis was performed with LSM Image Browser (Zeiss). Scale bar 50 μm.

Figure 3. Cell type-specific expression of innate immune genes in response to NAP treatment in vitro.

Primary human hepatocytes (PHH, n = 3–5), Kupffer cells (KC, n = 3–5), liver sinusoidal endothelial cells (LSEC, n = 3–6) and peripheral blood mononuclear cells (PBMC, n = 3–6) were stimulated with DNA-based (REP 2006 and REP 2055) and RNA-based (REP 2139 and REP 2165) NAPs or immunostimulatory controls (TLR3 agonist Poly(I:C); TLR7/8 agonist ssRNA40 [ssRNA] and TLR9 agonist CpG ODN2216 [CpG ODN]) for 6 h. RNA was extracted, and gene expression of interleukin 6 (IL6), tumor necrosis factor (TNF), interferon alpha 4 (IFNA4) and interferon lambda 2 (IFNL2) was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Values represented mean ± SEM (normalized to 100,000 copies of beta actin (ACTB) mRNA). Group size n = 3–6 cell preparations. Statistically significant changes compared to untreated controls are reported for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); w/o, without treatment.

Figure 4. Cell type-specific cytokine secretion in response to NAP treatment in vitro.

Primary human hepatocytes (PHH, n = 3–5), Kupffer cells (KC, n = 3–6) and peripheral blood mononuclear cells (PBMC, n = 3) were stimulated with DNA-based (REP 2006 and REP 2055) and RNA-based (REP 2139 and REP 2165) NAPs or immunostimulatory controls (TLR3 agonist Poly(I:C); TLR7/8 agonist ssRNA40 [ssRNA] and TLR9 agonist CpG ODN2216 [CpG ODN]) for 24 h. Supernatants were collected and secretion of interleukin 6 (IL6), tumor necrosis factor (TNF), interferon alpha 4 (IFNA4) and interferon lambda 2 (IFNL2) was quantified by enzyme-linked immunosorbent assay (ELISA). Values represented mean ± SEM. Group size n = 3–6 cell preparations. Statistically significant changes compared to untreated controls are reported for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); w/o, without treatment.