Supplementary Figure 1: NVR 3-778 can induce core protein assembly into capsid-like structures in vitro and inhibits HBV replication in HepG2.2.15 cells with a mean EC50 of 0.24 µM. (A) Electron microscopy of recombinant HBV core protein N-terminal domain, amino acids 1-149, incubated in the absence or presence of NVR 3-778; (B), in vitro assembly reaction of fluorescently labeled HBV core protein N-terminal domain; (C) antiviral activity of NVR 3-778 in HepG2.2.15 cells, measured as reduction of HBV DNA concentration in the culture supernatant.   Supplementary Figure 1 NVR 3-778 can induce core protein assembly into capsid-like structures in vitro and inhibits HBV replication in HepG2.2.15 cells with a mean EC50 of 0.24 µM. (A) Electron microscopy of recombinant HBV core protein N-terminal domain, amino acids 1-149, incubated in the absence or presence of NVR 3-778; (B) antiviral activity of NVR 3-778 in HepG2.2.15 cells, measured as reduction of HBV DNA concentration in the culture supernatant.

Supplementary Figure 2 On treatment change of body weight, h-Alb and ALT levels. Data from Study2, PBR-HI14-007. Mean values and standard deviation error bars are shown.

BID small molecule treatment (84 administrations by oral gavage) HBV infection (Genotype C) 7 14 21 28 35 42 DAY -7 -1 -63 peg-IFN injections BID small molecule treatment (84 administrations by oral gavage) Group assignment 41 Blood collection Liver collection A Animals found dead or moribund (n=11): Vehicle treated (n=2) NVR-3778 treated (n=3) 12 27 peg-IFN treated (n=1) 14 18 20 DAY NVR-3778 + ETV treated (n=5) 28 20 21 25 30 40 Supplementary Figure 3 Overview of times when individual mice were found dead or moribund, relative to the treatment schedule.

Supplementary Figure 4 A B C D E On treatment change of serum HBV DNA. Data from Study2, PBR-HI14-007. Serum HBV DNA levels at different time points and mean values are shown for individual animals treated with (A) vehicle, (B) ETV, (C) peg-IFN, (D) NVR 3-778 and (E) NVR 3-778 + peg-IFN. Area below the limit of quantification is shown as a shaded area

Supplementary Figure 5 A B C D E On treatment change of serum HBsAg and HBeAg. Data from Study2, PBR-HI14-007. Serum antigen levels at different time points and mean values are shown for individual animals treated with (A) vehicle, (B) ETV, (C) peg-IFN, (D) NVR 3-778 and (E) NVR 3-778 + peg-IFN.

Mouse Mouse Human Human Mouse Human Supplementary Figure 6 Immunohistochemical staining of HBV infected humanized mouse liver. Liver samples from Study2, PBR-HI14-007 were stained with DAPI (blue, nuclei), anti-hCK18 (red, human hepatocytes) and anti-HBcAg (green). (A), liver section of the chimeric liver, showing specific staining of the human hepatocyte areas with anti-hCK18 and anti-HBcAg antibodies; (B) HBcAg staining shown in the absence of CK18 staining; (C) close-up of co-staining with both antibodies.

Supplementary Figure 7 Induction of interferon sensitive genes (ISGs) by treatment with peg-IFN, but not by treatment with vehicle control, ETV or NVR 3-778. Data from Study2, PBR-HI14-007. Graphs show relative expression levels of the indicated genes as compared to GAPDH mRNA. HLA-E (MHC class I, E), TAP1 (Transporter 1, ATP-Binding Cassette, Sub-Family B (MDR/TAP), ABCB2, RING4), ISG15 (ubiquitin-like modifier, UCRP, IFI15, IP17), MXA (MX Dynamin like GTPase 1, IFI78), STAT1 (Signal Transducer And Activator Of Transcription 1 ), USP18 (Ubiquitin specific peptidase 18, ISG43, UBP43), OAS (2'-5'-oligoadenylate synthetase 1), CXCL10 (IP10, C-X-C motif chemokine 10), ISG20 (Interferon Stimulated Exonuclease Gene 20), RIG-I (retinoic acid-inducible gene I), IFI16 (IFN-gamma inducible protein 16, IFNGIP1), PKR (protein kinase RNA-activated, EIF2AK1, p68 kinase).