Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection

Abstract

West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. Previous studies demonstrated NITD008 as a potent antiviral for dengue virus, however this drug was associated with preclinical toxicity. The ability of NITD008 to block WNV replication is only shown in Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25mg/kg at days 1-6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3-6 and days 5-9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis.

Keywords: Adenosine analogue inhibitor; Antiviral; Co-treatment; Histone deacetylase inhibitor; Inflammation; West Nile virus.

Figure 1

NITD008 protects against lethal challenge with WNV. (A–C) C57BL/6 mice inoculated with 1000 PFU WNV (NY99) via footpad injection were administered with different doses of NITD008 via oral gavage twice per day from day 1 to 6 after inoculation and observed for clinical symptoms and mortality. Clinical score criteria: 1, ruffled fur; 2, ruffled fur with distinct hunched back; 3, paresis/difficulty walking; 4, paralysis; 5, moribund/euthanized; (D) WNV titers measured in the serum at day 3 after infection using plaque assay. Error bar represent ± SEM from at least 4 mice/group. **p< 0.005, *p<0.05

Figure 2

Protective effect is lost when NITD008 treatment is delayed. WNV-infected mice were administered with vehicle or 10 mg/kg NITD008 on either days 1 to 6, day 3 to 6 or days 5 to 9 and observed for (A) mortality and (B) clinical symptoms. Error bar represent ± SEM. **p< 0.005, *p<0.05

Figure 3

NITD008 blocks WNV dissemination to peripheral tissues and brain. WNV-infected mice treated with vehicle or 10 mg/kg NITD008 on days 1–6 after inoculation were sacrificed at day 5 (spleen and kidney) and day 8 (brain) after infection and WNV titers were analyzed in the tissue homogenates using plaque assay. Dotted line- detection limit of plaque assay. Error bar represent ± SEM from at least 4 mice/group. *p<0.05

Figure 4

Co-treatment of SAHA with NITD008 improves outcome of lethal challenge with WNV: (A) SAHA was administered on days 3–6 or 5–9 after WNV inoculation, once per day and survival was monitored (B) Survival of mice treated with 100 mg/kg of SAHA or 10 mg/kg NITD008 alone was compared with mice treated with 100 mg/kg of SAHA and 10 mg/kg NITD008 on days 5–9 after inoculation (C) WNV titers in the brain at day 9 after infection from mice treated with SAHA (days 3–6 and 5–9) and SAHA plus NITD008 (days 5–9) using qRT-PCR. Error bars represent ± SEM from at least 4 mice/group.

Figure 5

Treatment with NITD008 and SAHA attenuates inflammatory mediators in infected mouse brains. Brains harvested from mice treated with NITD008 and SAHA were used to analyze the levels of mRNA of TNF-α and MIP-1α. The data from at least 4mice/group was normalized to GAPDH and is represented as fold change as compared to un-infected controls.

Figure 6

Treatment with NITD008 and SAHA reduces markers of WNV-associated neuropathology in the brain: (A) H&E staining of PFA perfused brains depicts shrunken neuron body (white arrows) with light pink cytoplasmic staining representing degenerating neurons in the brain of WNV infected mice at day 9 after inoculation. NITD008 treatment demonstrated significant improvement in the neuron body (white arrow head). (B) TUNEL positive cells (red) were prominent in the brain of WNV-infected mice at day 9 after infection, which reduced significantly in the brains from mice treated with early NITD008 and combined treatment of NITD008 + SAHA. (C) Quantitative representation of TUNEL positive cells from at least 5 fields per coverslip from two independent experiments. *p<0.05 compared to WNV infected brains.