Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes

Vaclav Janovec,Thomas F Baumert,Iva Pichova,Jaroslav Chlupac,Pavel Dostalik,Ivan Hirsch,Kamila Clarova,Laurence Chaperot,Tomas Hofman,Jan Hodek,Barbora Lubyova,Jiri Fronek,Jan Weber,Sarah Durand

doi:10.1038/s41598-020-69614-7

Abstract

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

Highlights

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro
We found that conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with a dual-acting agonist of TLR7/8 (R848) and TLR2/7 (CL413) were more potent drivers of inhibition of hepatitis e and s antigens (HBeAg and HBsAg) production from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with agonists specific only for TLR7 (GS-9620, CL264) or TLR9 (CpG-A, CpG-B)
We determined the levels of selected cytokines secreted into supernatants of PBMCs stimulated for 16 h by different agonists of TLR7 (CL264-CM, GS-9620[L]-CM (50 nM)), TLR7/8 (R848-CM, GS-9620[H]CM (10 μM)), TLR9 (CpG-A-CM, CpG-B-CM) and a TLR2/7 dual agonist (CL413-CM) (Fig. 1, linear plot, Supplementary Fig. S1, logarithmic plot)

Summary

Introduction

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. A side-by-side comparison of a large panel of cytokines in vitro revealed that proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6, are as efficient as IFNs at inhibiting HBV replication[16,17,18] Both IFNs and proinflammatory cytokines control HBV replication and contribute to HBV cure in different m odels[17,19]. It was shown that GS-9620 (vesatolimod), an agonist of endosomal TLR7, which is preferentially expressed in plasmacytoid dendritic cells (pDCs)[26,27,28,29,30] but not in primary hepatocytes (PHH)[23,31,32], significantly reduced viremia and cccDNA expression, and led to functional cure in animal m odels[20,21,22]. IFN-I secreted by TLR7-agonist-stimulated PBMCs was identified as the major substance inhibiting HBV production without reducing cccDNA levels[24]

Methods

Results

Conclusion