Bepirovirsen induces innate immune activation in the liver potentially through TLR8 signaling

BACKGROUND & AIMS: Bepirovirsen is an unconjugated antisense oligonucleotide in Phase 3 trials for chronic hepatitis B virus (HBV) infection. Post hoc analyses from a Phase 2a study (chronic HBV participants) indicated immediate cytokine production upon dosing of bepirovirsen, leading to the hypothesis that bepirovirsen may have innate immune stimulatory activity via pattern recognition receptors (PRRs) in hepatic non-parenchymal cells, alongside its known direct antiviral mechanism in hepatocytes. METHODS: Cytokine production was evaluated in Phase 2a serum and plasma samples using Myriad RBM, Olink, or MSD panels. Preclinical studies used PBMCs from healthy donors (MSD, flow cytometry) and PRR-overexpressing HEK or THP-1 cells to assess innate immune activation by bepirovirsen. Cytokine protein levels were measured with Luminex or MSD in wild-type (WT) and hTLR8 transgenic mice, while gene expression (RT-qPCR, RNAscope) was analyzed in vivo exclusively in hTLR8 transgenic mice from Shanghai Model Organisms Center (SMOC). RESULTS: Many pro-inflammatory cytokines for immune cell recruitment and activation were upregulated in bepirovirsen-treated participants. Similar findings were seen preclinically in PBMCs from healthy donors following bepirovirsen treatment in vitro. Analysis of innate immune activation in PRR-overexpressing cells revealed weak, yet positive (p = 0.00085), induction only when human Toll-like receptor 8 (TLR8) was present. In hTLR8 transgenic mice, but not wild-type mice, bepirovirsen treatment induced pro-inflammatory cytokines, similar to the TLR8 agonist selgantolimod. Kupffer cells and macrophages were identified as the primary responders to bepirovirsen in hTLR8 mouse livers. CONCLUSIONS: Clinical and preclinical data show that bepirovirsen elicits an innate immune response, evidenced by induction of inflammatory cytokines. This effect is mediated at least in part by TLR8 agonism, complementing its established antiviral mechanism. IMPACT AND IMPLICATIONS: The aim of this study was to characterize additional mechanisms of action for bepirovirsen, including immune activation through PRRs, based on the hypothesis that bepirovirsen may elicit an innate immune response in addition to its antiviral mechanism. Using Phase 2a study samples we show that bepirovirsen induces IFN-γ, TNF, and other cytokines, while in vitro we demonstrate that that bepirovirsen harbors innate immune stimulatory activity, potentially through human TLR8. Furthermore, we show that the innate immune activation induced by bepirovirsen is distinct from that of other ASOs. This study demonstrates that bepirovirsen can induce an innate immune response, likely through TLR8 agonism, in addition to its known antiviral mechanism; however, the clinical impact of this immunomodulatory effect remains unclear and requires further investigation.