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.

CNRS UMR5309 Inserm U1209 CHU Grenoble Alpes IAB EFS Université Grenoble Alpes 38000 Grenoble France

Department of Anatomy 2nd Faculty of Medicine Charles University 15006 Prague Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University BIOCEV 25150 Vestec Czech Republic

Inserm Institut de Recherche Sur Les Maladies Virales Et Hepatiques UMRS 1110 Universite de Strasbourg 67000 Strasbourg France

Institute of Molecular Genetics of the Czech Academy of Sciences 14220 Prague Czech Republic

IOCB and Gilead Research Center Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science 16610 Prague Czech Republic

Pole Hepato Digestif Institut Hospitalo Universitaire Hopitaux Universitaires de Strasbourg 67000 Strasbourg France

Transplantation Surgery Department Institute for Clinical and Experimental Medicine 14021 Prague Czech Republic

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