Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways

Abstract

HSV-1 is a significant human pathogen that can result in the loss of sight as a result of episodic reactivation of latent virus from sensory ganglion neurons. In this study the potential efficacy of anti-viral cytokine expression in preventing latent virus reactivation was investigated. Both type I (IFN-beta) and type II (IFN-gamma) IFN transgene expression following transduction of trigeminal ganglion explant cultures significantly reduced the incident of HSV-1 reactivation that in the case of IFN-beta was dependent on the presence of double stranded RNA-dependent protein kinase and RNase L. In vivo, expression of the IFN-gamma but not IFN-beta transgene significantly delayed and reduced the frequency of reactivation of latent mice exposed to UV light without discernable inflammation. This result is the first report that demonstrates the ability to block reactivation using an ectopic cytokine expression system and warrants further exploration as a means to prevent HSV-1 reactivation.

FIGURE 1

Ad:IFN-β and Ad:IFN-γ antagonize HSV-1 reactivation from TG explant cultures. TG from C57BL6 mice infected 30 days previously was removed, processed to single cell suspensions, and plated into 24 well plates at a concentration of 1-3 × 10⁵ cells/well in 1.0 ml of media (9). At the initiation of culture, cells were transduced with 5.0 TU of Ad:Null (empty vector), Ad:IFN-β, or Ad:IFN-γ. Non-transduced cultures received only vehicle (PBS). Starting 24 hr post culture, 200 μl of the supernatant was collected every 48 hr up to 11 days post culture condition and assayed for the presence and quantity of HSV-1 by plaque assay. (A) Summarizes the frequency of reactivation of latent infected wild type mouse TG explant cultures from 12-26 wells/condition from 3-4 experiments. **p<.01 comparing Ad:IFN-γ to Ad:Null day 7-11 post infection. *p<.05 comparing Ad:IFN-β to Ad:Null day 7-11 post infection. (B) Summarizes the frequency of reactivation of latent infected RNase L deficient mouse TG explant cultures from 10-19 wells/condition from 3 experiments with the exception of Ad:IFN-g which is taken from 6 wells. *p<.05 comparing Ad:IFN-β to Ad:Null day 9-11 post infection. (C) Summarizes the frequency of reactivation of latent infected PKR deficient mouse TG explant cultures from 12-19 wells/condition from 3 experiments with the exception of Ad:IFN-γ which is taken from 4 wells. **p<.01 comparing Ad:IFN-β to Ad:Null day 7-11 post infection. (D) Summarizes the frequency of reactivation of latent infected PKR/RNase L deficient mouse TG explant cultures from 13-22 wells/condition from 3 experiments. *p<.05 comparing Ad:IFN-γ to Ad:Null day 9-11 post infection.

FIGURE 2

Ad:IFN-γ antagonizes HSV-1 reactivation in vivo without inducing a localized immune response. Latently infected mice were exposed to UV light to induce reactivation as described (29). Immediately following UV light exposure, the cornea of mice were transduced with Ad:Null, Ad:IFN-β, or Ad:IFN-γ as described (26, 27). Mice were euthanized at the indicated time post UV light exposure and the eyes were harvested, homogenized, and the clarified supernatant was assessed for virus by plaque assay or the indicated cytokine/chemokine by ELISA. Each group consisted of 5-7 mice/group/time point for cumulative percent reactivation (A) and n=6-12 per group at each time point for CXCL10 (B), IFN-γ (C), and CCL2 (D) levels. *p≤ .05 comparing the Ad:IFN-γ to Ad:Null or vehicle-treated mice for percent cumulative reactivation at day 5-7 post reactivation. **p<.01 comparing the reactivated to non-reactivated group for CXCL10 and CCL2 chemokines at 4-7 days post reactivation for CXCL10 and day 4 post reactivation for CCL2.