Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro.

Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.