The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

Einleitung: Die chronische Hepatitis-B-Virus (HBV)-Infektion ist eine der Hauptursachen von leberassoziierter Morbidität und Mortalität weltweit. Das HBV-Oberflächenantigen (HBsAg) beeinträchtigt das angeborene als auch das adaptive Immunsystem, was die Chronifizierung der Infektion begünstigt. Ziel dieser Studie war es, Immunantworten in HBsAg-defizienten und HBsAg-exprimierenden transgenen HBV-Mausmodellen zu analysieren.

cytokine assay were used to characterize CD8+ T-cell function. RESULTS: Our analysis revealed several interesting insights: (1) Tetramer-based analysis of HBV-specific CD8+ and CD4+ T-cells co-expressing T-bet and Eomes demonstrate distinct phenotypical signatures in acute-resolving versus chronic HBV infection. (2) In contrast to T-bet single positive CD8+ T-cells, co-expression of T-bet and Eomes is strongly associated with vigorous antiviral effector function characterized by increased IFN-c release. (3) On contrary, CD8+ T-cells lacking both molecules virtually lose their ability to produce antiviral cytokines. CONCLUSION: Taken together our results suggest that Tbet and Eomes expressing T-cells represent a unique and highly functional T-cell subset, which induction will reflect a promising approach for further therapeutic interventions in chronic persisting viral infections.

Background and aims: It is likely that HBV surface antigen (HBsAg) impairs toll-like receptor (TLR) signaling leading to an attenuation of innate and adaptive immune responses. Here, we aimed to analyze TLR3 signaling in HBV transgenic mice lacking the HBs antigen (1.4 tgHBV-s-mut).

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Background: Chronic infection with Hepatitis B virus (HBV) is a global health problem, with over 350 million carriers worldwide and up to 1,000,000 deaths annually from HBV-associated cirrhosis or hepatocellular carcinoma. There is significant unmet medical need for new treatments yielding a clinical cure or disease suppression with limited dosing and limited adverse effects. RNA interference (RNAi) is a promising modality for the treatment of viral disease. RNAi trigger molecules, such as small interfering RNAs (siRNAs), can be readily designed to possess the specificity, potency, and flexibility to target any viral gene with high efficacy and without the side effects observed with existing anti-viral drugs. The challenge in using anti-HBV siRNAs as drugs is to safely and effectively deliver them to hepatocytes. To this end, we developed an siRNA delivery vehicle named Dynamic PolyConjugates (DPCs). Methods: 145 anti-HBV siRNAs designed to target conserved sequences in HBV serotypes A-D were synthesized and then screened in cultured cells to identify those with highest potency. To determine efficacy in vivo, lead siRNAs were formulated in DPCs and intravenously injected in mouse models of HBV infection. Studies to assess toxicity were conducted in rats and non-human primates (NHPs). Results: Four biweekly injections of anti-HBV siRNA DPCs in mice carrying a hepatocyte-specific reporter gene fused to HBV sequences resulted in a 3-4 log reduction in gene expression over 2 months without changes in cytokines or clinical chemistries. Single dose injections in a replication-competent, transient transgenic HBV mouse model resulted in greatly reduced serum HBsAg levels (> 2 logs) and serum levels of HBV DNA. Single dose studies using transgenic HBV mice revealed dramatic reductions in HBeAg levels in serum, and in viral RNAs and viral replication intermediates in the liver. Safety and tolerability studies using rats and NHPs revealed that DPCs have a wide safety margin. Conclusion: Formulation of potent anti-HBV siRNAs in DPCs allowed highly effective reduction of viral protein production and viral load in mouse models of HBV infection. Combined with the favorable results observed in toxicological studies, we anticipate that anti-HBV siRNA DPCs will become a viable therapeutic for patients with chronic HBV infection.