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Abstract The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant) resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding. more...

Objective: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment., Design: We identified TLR8 -expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients., Results: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12 ) and downregulated genes associated with the KC identity (eg, SPIC ). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition., Conclusion: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection., Competing Interests: Competing interests: FZ and BT received grants from Assembly, Beam Therapeutics, Blue Jay and JnJ; FZ had consulting activities with Assembly, Blue Jay and GSK. YCH receives grants from Gilead Sciences and Sysmex. FZ is an associate editor of the journal. SD, LL, NK, JJW, AHL, and SPF are or were employees of Gilead Sc., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.) more...

Cutaneous leishmaniasis (CL), caused by protozoan parasites of the Leishmania genus, is prevalent in tropical and subtropical regions, with important morbidity, particularly in low- to middle-income countries. Current systemic treatments, including pentavalent antimonials and miltefosine, are associated with significant toxicity, reduced efficacy, and are frequently ineffective in cases of severe or chronic CL. Immunotherapies leverage the immune system to combat microbial infection and offer a promising adjunct or alternative approach to the current standard of care for CL. However, the heterogeneous clinical presentation of CL, which is dependent on parasite species and host immunity, may require informed clinical intervention with immunotherapies. This review explores the clinical and immunological characteristics of CL, emphasising the current landscape of immunotherapies in in vivo models and clinical studies. Such immune-based interventions aim to modulate immune responses against Leishmania , with additive therapeutic effects enabling the efficacy of lower drug doses and decreasing the associated toxicity. Understanding the mechanisms that underlie immunotherapy for CL provides critical insights into developing safer and more effective treatments for this neglected tropical disease. Identifying suitable therapeutic candidates and establishing their safety and efficacy are essential steps in this process. However, the feasibility and utility of these treatments in resource-limited settings must also be considered, taking into account factors such as cost of production, temperature stability, and overall patient access. more...

Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens., Competing Interests: Potential conflicts of interest . M. M., S. D., T. G., R. C., C. L., A. T., J. P., S. P. F., and S. B. are or were employees of Gilead Sciences. S. S., S. A. E., D. D., H. L., and K. O. are employees of Hookipa Pharma. M. D. R. has financial relationships with CaroGen Corporation, received royalties from a Yale University patent, and received research funding from Gilead Sciences. Al; other authors reported no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...

Objective: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function., Design: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients., Results: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2., Conclusions: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability., Competing Interests: Competing interests: CF, Grant: Gilead, Abbvie. Consultant: Gilead, Abbvie, Vir Biotechnology, Arrowhead, Transgene, BMS; PL: advisor and speaker bureau for Gilead, Roche, BMS, GSK, MSD, Arrowhead, Alnylam, Spring Bank, Janssen, EIGER, Myr Pharma; AL: Consultant: MyrPharma, Gilead; MM: advisory board for Abbvie. SD, SPF, AG, JM, MG and LL are employees of and stock-holders in Gilead Sciences. The remaining authors disclose no conflicts., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.) more...

Background and Aims: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBV-infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators., Approach and Results: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosal-associated invariant T cells, CD4 + follicular helper T cells, and, in about 50% of patients, HBV-specific CD8 + T cells expressing interferon-γ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferon-γ and TNF-α, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4 + regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNF-related apoptosis-inducing ligand + NK cells and degranulation of arginase-I + polymorphonuclear MDSCs., Conclusions: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBV-specific CD8 + T cells, CD4 + follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.) more...

Background and Aims: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus., Approach and Results: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688., Conclusions: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.) more...

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate ( R )-2-((2-amino-7-fluoropyrido[3,2- d ]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, ( R )- 7 ). Potent TLR8 agonism (IL-12p40 EC 50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC 50 > 50 μM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:( R )- 7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral ( R )- 7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with ( R )- 7 , supporting the clinical development of ( R )- 7 for the treatment of CHB. more...

Background: Selgantolimod is a novel oral, selective Toll-like receptor 8 (TLR8) agonist in development for the treatment of chronic hepatitis B (CHB). TLR8 is an endosomal innate immune receptor and a target for treatment of viral infections. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selgantolimod in healthy volunteers., Methods: Of 71 subjects enrolled, 59 received a single dose of selgantolimod (0.5, 1.5, 3 or 5 mg) or placebo, and 12 were evaluated for food effect. Safety, PK and PD activity by induction of cytokines, chemokines and acute phase proteins were assessed. PK/PD analyses were conducted., Results: Single doses of 0.5-5 mg were generally safe. No serious adverse events (AEs) or AEs leading to discontinuation were reported, and most were Grade 1 in severity. Selgantolimod displayed rapid absorption and dose-proportional PK and PD activity. Food had minimal effect on PK but resulted in diminished PD activity. In PK/PD analyses, near-saturation of induction for most evaluated biomarkers occurred at the 5-mg dose., Conclusions: Single doses of up to 5 mg selgantolimod were safe and induced dose-dependent PD responses. These data support evaluation of selgantolimod in combination with other agents in future clinical studies of CHB. Australian New Zealand Clinical Trials Registration: ACTRN12616001646437. more...

Yellow fever virus (YFV), a deadly human pathogen, is the prototype of the genus Flavivirus. Recently, YFV re-emerged in Africa and Brazil, leading to hundreds of deaths, with some cases imported to China. Prophylactic or therapeutic countermeasures are urgently needed. Previously, several human monoclonal antibodies against YFV were screened out by phage display. Here, we find that one of them, 5A, exhibits high neutralizing potency and good protection. Crystallographic analysis of the YFV envelope (E) protein in its pre- and post-fusion states shows conformations similar to those observed in other E proteins of flaviviruses. Furthermore, the structures of 5A in complex with the E protein in both states are resolved, revealing an invariant recognition site. Structural analysis and functional data suggest that 5A has high neutralization potency because it interferes with virus entry by preventing both virus attachment and fusion. These findings will be instrumental for immunogen or inhibitor design., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.) more...

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection., Methods: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined., Results: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH., Conclusions: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB., Lay Summary: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.) more...

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects., Methods: Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH)., Results: GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8 + T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8 + T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8 + T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b + myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment., Conclusion: The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV., Lay Summary: New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.) more...

Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or "adjuvant" potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies. more...

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells., Competing Interests: All authors except Dr. Michel Strubin are employees of Gilead Sciences, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. more...

Background & Aims: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV)., Methods: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules., Results: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures., Conclusions: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients., (Copyright © 2015 European Association for the Study of the Liver. All rights reserved.) more...

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response. more...

A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event. more...

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1(-/-) mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1(-/-) cells and mice. IRF-1(-/-) mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1(-/-) macrophages supported enhanced WNV replication but infection was unaltered in IRF-1(-/-) fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8(+) T cell expansion. Although markedly fewer CD8(+) T cells were observed in naïve animals as described previously, remarkably, IRF-1(-/-) mice rapidly expanded their pool of WNV-specific cytolytic CD8(+) T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8(+) T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8(+) T cell response. more...

The host determinants that contribute to attenuation of the naturally occurring nonpathogenic strain of West Nile virus (WNV), the Kunjin strain (WNV(KUN)), remain unknown. Here, we show that compared to a highly pathogenic North American strain, WNV(KUN) exhibited an enhanced sensitivity to the antiviral effects of type I interferon. Our studies establish that the virulence of WNV(KUN) can be restored in cells and mice deficient in specific interferon regulatory factors (IRFs) or the common type I interferon receptor. Thus, WNV(KUN) is attenuated primarily through its enhanced restriction by type I interferon- and IRF-3-dependent mechanisms. more...

Type I interferons (IFN-α/β) control viral infection by triggering the expression of genes that restrict transcription, translation, replication, and assembly. Many viruses induce IFN responses after recognition by cytoplasmic or endosomal RNA sensors (RIG-I-like RNA helicases [RLR] and Toll-like receptors [TLR]), which signal through the cognate adaptor signaling molecules IPS-1, TRIF, and MyD88. Recent studies have demonstrated that IPS-1-dependent induction of IFN-α/β downstream of RLR recognition restricts West Nile virus (WNV) infection in many cell types, whereas TRIF-dependent TLR3 signaling limits WNV replication in neurons. Here, we examined the contribution of MyD88 signaling to the control of WNV by evaluating IFN induction and virus replication in genetically deficient cells and mice. MyD88(-/-) mice showed increased lethality after WNV infection and elevated viral burden primarily in the brain, even though little effect on the systemic type I IFN response was observed. Intracranial inoculation studies corroborated these findings, as WNV spread more rapidly in the central nervous system of MyD88(-/-) mice, and this phenotype preceded the recruitment of inflammatory leukocytes. In vitro, increased WNV replication was observed in MyD88(-/-) macrophages and subsets of neurons but not in myeloid dendritic cells. MyD88 had an independent effect on recruitment of monocyte-derived macrophages and T cells into the brain that was associated with blunted induction of the chemokines that attract leukocytes. Our experiments suggest that MyD88 restricts WNV by inhibiting replication in subsets of cells and modulating expression of chemokines that regulate immune cell migration into the central nervous system. more...

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells. more...

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1(-/-) mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1(-/-) mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection. more...

Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors. more...

As a first line of defense after viral infection, host cells develop an intrinsic immune response to control virus dissemination and protect against serious infection. Recent experiments have shown a dominant role of the IFN-alpha/beta response in protection against lethal West Nile virus (WNV) by limiting the cellular and tissue tropism of infection. This review will focus on advances in identifying the host sensors that detect WNV and the adaptor molecules and signaling pathways that regulate the induction of IFN-alpha/beta defenses that limit WNV replication, spread and pathogenesis., (Copyright 2009 S. Karger AG, Basel.) more...

Protection against West Nile virus (WNV) infection requires rapid viral sensing and the generation of an interferon (IFN) response. Mice lacking IFN regulatory factor 3 (IRF-3) show increased vulnerability to WNV infection with enhanced viral replication and blunted IFN-stimulated gene (ISG) responses. IRF-3 functions downstream of several viral sensors, including Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Cell culture studies suggest that host recognizes WNV in part, through the cytoplasmic helicase RIG-I and to a lesser extent, MDA5, both of which activate ISG expression through IRF-3. However, the role of TLR3 in vivo in recognizing viral RNA and activating antiviral defense pathways has remained controversial. We show here that an absence of TLR3 enhances WNV mortality in mice and increases viral burden in the brain. Compared to congenic wild-type controls, TLR3(-/-) mice showed relatively modest changes in peripheral viral loads. Consistent with this, little difference in multistep viral growth kinetics or IFN-alpha/beta induction was observed between wild-type and TLR3(-/-) fibroblasts, macrophages, and dendritic cells. In contrast, a deficiency of TLR3 was associated with enhanced viral replication in primary cortical neuron cultures and greater WNV infection in central nervous system neurons after intracranial inoculation. Taken together, our data suggest that TLR3 serves a protective role against WNV in part, by restricting replication in neurons. more...

Type I interferon (IFN-alpha/beta) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7(-/-) mice. Compared to congenic wild-type mice, IRF-7(-/-) mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-alpha gene expression and protein production were reduced and viral titers were increased in IRF-7(-/-) primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-beta response remained largely intact. Our data suggest that the early protective IFN-alpha response against WNV occurs through an IRF-7-dependent transcriptional signal. more...

We recently reported increased prevalence of Lassa virus (LASV) infection in Mastomys natalensis during the rainy season in Guinea, West Africa. Here, the association of LASV prevalence with fecundity, fertility, and the age structure of the rodent population was analyzed using data from the previous study. The animals reproduced throughout the year, but highest fecundity was observed during the rainy season. Accordingly, the rodent population was aged at the beginning and young at the end of the rainy season. Lassa virus infection was observed in all age groups, including the very young, which is compatible with vertical transmission. However, since the prevalence of infection showed a trend with increasing age in the younger age groups, horizontal transmission by yet unknown mechanisms may also play a role in LASV transmission. Multivariate analysis did not show an association of LASV prevalence with any demographic variable studied. Rodent behavior influencing virus transmissibility and contaminated environment may therefore be responsible for spatial and seasonal variations in LASV prevalence in M. natalensis. more...

Interferon regulatory factor (IRF)-3 is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-3 promotes antiviral control by inducing interferon (IFN)-beta, near normal levels of IFN-alpha and IFN-beta were observed in IRF-3(-/-) mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-3 modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-3-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-3 coordinates an antiviral response, we infected IRF-3(-/-) mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-3(-/-) mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-3(-/-) macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-alpha and IFN-beta production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-3(-/-) neurons lacked induction of host defense genes and had blunted IFN-alpha and IFN-beta production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-3 responses protect against WNV infection through both IFN-dependent and -independent programs. more...

Based on empiric surveillance data, the incidence of human Lassa fever (LF) cases in Guinea and other West African countries has been reported to increase during the dry season compared to the rainy season. To investigate possible links with the ecology of the rodent reservoir of the virus, we conducted a 2-year longitudinal survey of Mastomys natalensis in a region of high human Lassa virus (LASV) seropositivity in Guinea. Standardized rodent trapping with similar trapping efforts between seasons was performed in three villages and 53.5% (601/1123) of the animals were identified as M. natalensis using morphometric and molecular criteria. Mean trapping success (TS) of M. natalensis was always higher inside houses than in proximal cultivations. In the dry season, mean TS increased 2-fold inside houses and decreased up to 10-fold outside (p < 0.0001), suggesting aggregation of rodents inside houses due to restricted food supply. 14.5% (80/553) of M. natalensis were tested positive for Lassa virus by reverse transcription-polymerase chain reaction (RT-PCR; range, 5%-30%) and prevalence of the virus was two to three times higher in rodents captured in the rainy season than in the dry season (p < 0.05). Inside houses, however, the LASV prevalence fluctuated nonsignificantly with season. These data suggest that in Guinea the risk of LASV transmission from rodents to humans is present both in the rainy and the dry season, reflected by the occurrence of LF cases throughout the year. In the dry season, however, the increased risk of humans encountering Mastomys and their excreta inside of houses may result in an increase of human Lassa fever cases. more...

PCR screening of 1,482 murid rodents from 13 genera caught in 18 different localities of Guinea, West Africa, showed Lassa virus infection only in molecularly typed Mastomys natalensis. Distribution of this rodent and relative abundance compared with M. erythroleucus correlates geographically with Lassa virus seroprevalence in humans. more...

Human monoclonal antibody fragments neutralizing wild-type and vaccine strains of yellow fever (YF) virus (genotypes West Africa I + II, East/Central Africa, 17D-204-WHO) were generated by repertoire cloning from YF patients. Analysis of virus escape variants identified amino acid (aa) 71 in domain II of the envelope glycoprotein (E) as the most critical residue for neutralization, with aa 153-155 in domain I contributing to the epitope. These data confirm the previous mapping of YFV neutralizing epitopes using mouse monoclonal antibodies but suggest that a conformational epitope could be formed by amino acids from domains I and II opposing each other in the dimeric form of the E protein. While the sera of the YF patients showed up to 10-fold reduced neutralizing activity against the 17D escape variants, sera from 17D vaccinees retained their neutralizing titers. Mutations in this major neutralizing epitope of YFV thus do not seem to carry the risk of immune escape in persons immunized with the YFV-17D vaccine. more...

To study the contribution of inflammatory mediators to the pathogenesis of yellow fever (YF), the serum levels of several cytokines and chemokines were measured in 7 patients with fatal YF (f-YF), 11 patients with nonfatal hemorrhagic YF (nf/h-YF), and 18 patients with nonfatal nonhemorrhagic YF (nf/nh-YF). The levels of interleukin (IL)-6, monocyte chemoattractant protein-1, interferon-inducible protein (IP)-10, tumor necrosis factor- alpha , and IL-1 receptor antagonist (IL-1RA) were all statistically significantly higher in the patients with f-YF than in those with nf/nh-YF. In patients with nf/h-YF, only levels of IP-10 and IL-1RA were significantly elevated. The high levels of pro- and anti-inflammatory cytokines and chemokines in serum from patients with f-YF are reminiscent of those seen in patients with bacterial sepsis. This finding has implications for the understanding of the pathophysiology of YF and the development of therapeutic strategies. more...