Topical application of the cornea post-infection with plasmid DNA encoding interferon-alpha1 but not recombinant interferon-alphaA reduces herpes simplex virus type 1-induced mortality in mice.

A study was undertaken to compare the efficacy of recombinant interferon (rIFN)-alphaA to plasmid DNA encoding IFN-alpha1 against ocular herpes simplex virus type 1 (HSV-1) infection. The topical application of rIFN-alphaA (100-300 units/eye) onto the cornea of mice subsequently infected 24 h later with HSV-1 antagonized viral-induced mortality. The enhancement in cumulative survival in the rIFN-alphaA-treated mice correlated with a reduction of viral titers recovered in the eye and trigeminal ganglion (TG) at 3 and 6 days post-infection. The protective effect was site-specific such that when rIFN-alphaA was administered orally or intranasally, no efficacy against HSV-1 was observed. However, the protective effect was time-dependent. Specifically, when the rIFN-alphaA (100-1000 units/eye) was administered at 24 h post-infection, no protective effect was observed against HSV-1 compared to the vehicle-treated group. In contrast, plasmid DNA (100 microg/eye) containing the IFN-alpha1 transgene showed significant protection when topically applied 24 h post-infection. Although the transgene was found to traffic distal from the site of application (eye), including the trigeminal ganglion and the spleen where CD11b(+) and CD11c(+) cells express the transgene, the migration of the transgene did not correlate with efficacy. Collectively, the results suggest that naked DNA encoding type I IFN applied post-infection provides a greater degree of protection against ocular HSV-1 infection in comparison with recombinant protein effectively antagonizing viral replication and spread.

Type I interferons and herpes simplex virus infection: a naked DNA approach as a therapeutic option?

Herpes simplex virus (HSV) types 1 and 2 are highly successful human pathogens that can elicit blinding herpetic keratoconjunctivitis, fatal sporadic encephalitis, aseptic meningitis, and increase the risk of acquiring additional sexually transmitted diseases. Type I interferons (IFN) play a significant role in controlling HSV pathogenesis by antagonizing viral replication and spread. Taking advantage of the susceptibility of HSV to IFNs, a novel approach of employing plasmid DNA cassettes expressing type 1 IFNs to antagonize viral pathogenesis has been undertaken. This review will describe recent work in our lab and those of others using naked DNA encoding cytokines to antagonize HSV replication and virus trafficking or immune-mediated pathogenesis as a result of viral assault to ocular tissue.

ICAM-1 is required for resistance to herpes simplex virus type 1 but not interferon-alpha1 transgene efficacy.

The purpose of this study was to determine the role of ICAM-1 in ocular herpes simplex virus type 1 (HSV-1) infection. Wild-type and ICAM-1 knockout mice were assessed for resistance to ocular HSV-1 infection in the presence of naked DNA plasmid vector or plasmid DNA encoding interferon-alpha1 topically applied to the cornea of the mice. Wild-type mice showed greater resistance to HSV-1 infection compared to ICAM-1 knockout mice as measured by cumulative survival. The absence of ICAM-1 did not affect the efficacy of the interferon-alpha1 transgene against ocular HSV-1. Both ICAM-1 and wild-type mice treated with the transgene showed a reduction in viral load and antigen expression in the trigeminal ganglion compared to the plasmid vector-treated counterparts. In contrast, the presence of the transgene reduced the number of infiltrating cells into the cornea in comparison to plasmid vector DNA controls in the wild-type mice but not in the ICAM-1 knockout mice. Collectively, these results suggest that the IFN-alpha1 transgene can restore resistance against HSV-1 infection in ICAM-1-deficient mice.

The application of a plasmid DNA encoding IFN-alpha 1 postinfection enhances cumulative survival of herpes simplex virus type 2 vaginally infected mice.

Using a hormonally induced susceptibility mouse model to investigate vaginal HSV type 2 (HSV-2) infection, a study was undertaken to determine the efficacy of a plasmid DNA encoding IFN-alpha1 introduced into the vaginal lumen postinfection (PI). Mice infected with HSV-2 intravaginally and treated intravaginally 24 h later with 100 microg DNA encoding IFN-alpha1 showed enhanced survival (10/15) in comparison to mice treated with 100 microg plasmid DNA vector alone (3/10) or vehicle (4/27). In contrast, mice receiving recombinant IFN-alphaA (5-500 U/vagina) 24 h PI showed no significant survival in comparison to the vehicle (saline)-treated group. The protective effect was time dependent in that mice receiving the IFN-alpha1 transgene 48 h PI succumbed at a rate similar to the plasmid DNA vector-treated group. The increase in cumulative survival elicited by the transgene corresponded with a reduction in viral replication and Ag expressed in the vaginal epithelium early (i.e., 3 days PI) during acute infection and replicating virus recovered in the spinal cord day 7 PI. By day 7 PI, HSV-2 glycoprotein B transcript expression was no longer detectable in vaginal tissue from the IFN-alpha1 transgene-treated group (0/8) compared with levels expressed in plasmid vector-treated controls (4/6 mice surveyed were positive). Collectively, these results suggest the application of DNA encoding type I IFN is an effective and alternative approach to currently prescribed therapies in controlling vaginal HSV-2 infection by antagonizing viral replication.

Plasmid DNA encoding IFN-alpha 1 antagonizes herpes simplex virus type 1 ocular infection through CD4+ and CD8+ T lymphocytes.

The present study was undertaken to further characterize the anti-viral efficacy of a plasmid DNA encoding IFN-alpha1 against ocular herpes simplex virus type 1 (HSV-1) infection. In mice ocularly treated with plasmid DNA encoding IFN-alpha 1, the efficacy of the transgene was inversely proportional to the amount of virus used to infect the mice. Ocular treatment of mice with the IFN-alpha 1 transgene was the only mucosal route tested that showed efficacy against ocular HSV-1 infection compared with vaginal or intranasal delivery. Mice treated with the plasmid DNA encoding IFN-alpha 1 showed a significant reduction in viral Ag expression in the eyes and trigeminal ganglion that correlated with a reduction in immune cell infiltration into the cornea and iris on days 3 and 6 postinfection, as evidenced by immunohistochemical staining. Depleting mice of either CD4+ or CD8+ T lymphocytes completely blocked the resistance to herpes simplex virus type 1-induced mortality in mice treated with the IFN-alpha 1 transgene. In the absence of infection, the application of naked DNA encoding IFN-alpha 1 significantly increased the levels of IL-6- and IFN-gamma-inducible protein 10 transcript expression in the corneas 24 h post-treatment. Expression of the plasmid construct following topical application in the eye included the rectus muscles proximal to the cornea as well as the spleen. Collectively, the protective efficacy of the IFN-alpha 1 transgene against ocular HSV-1 infection is dependent upon the local or distal participation of CD4+ and CD8+ T lymphocytes early in the course of the infection, suggesting an indirect effect of the transgene against HSV-1-induced mortality.

Therapeutic efficacy of DNA encoding IFN-alpha1 against corneal HSV-1 infection.

PURPOSE: Type I interferons (IFN-alpha and -beta) are an innate immune component that plays a critical role in controlling herpes simplex virus type 1 (HSV-1) infection. We have previously shown that topical administration of a plasmid DNA encoding IFN-alpha1 onto mouse corneas prior to ocular HSV-1 infection provided prophylactic efficacy against HSV-1-induced encephalitis. As a result, the present study was undertaken to investigate the kinetics of the efficacy mediated by the IFN-alpha1 transgene following ocular challenge with HSV-1. METHODS: Mice were ocularly infected with a lethal dose of HSV-1 (450 plaque forming units/eye, McKrae strain) following corneal scarification and topically administered the pCMV-IFN-alpha1 transgene or pCMV-beta (plasmid vector) starting at 12, 24, or 48 hr post infection. Cumulative survival of infected mice was recorded. In addition, the effect of the transgene on viral replication and viral gene expression was determined from tissues 3 and 6 days post infection by plaque assay and RT-PCR respectively. RESULTS: Mice treated with the pCMV-IFNalpha1 transgene survived to a greater degree compared to mice topically administered the plasmid vector alone in a time-dependent manner. The protective effect correlated with a decrease in the viral load and expression of HSV-1 immediate early and early gene transcripts, infected cell protein-27 and thymidine kinase respectively in the trigeminal ganglion 6 days post infection. CONCLUSION: These results suggest that the application of plasmid DNA encoding IFN-alpha1 transgene is beneficial as a therapeutic approach when applied early after HSV-1 infection of the corneas.

IFN-alpha1 plasmid construct affords protection against HSV-1 infection in transfected L929 fibroblasts.

The purpose of the present study was to evaluate the resistance against herpes simplex virus type 1 (HSV-1) using an interferon-alpha1 (IFN-alpha1) transgene in specifically targeted cells in vitro. Transfection of mouse fibroblast L929 cells with an IFN-alpha1 plasmid construct reduced viral load and viral gene expression in a time-dependent fashion. Supernatants from IFN-alpha1-transfected cells augmented natural killer (NK) cell activity, and such an effect was antagonized with neutralizing antibody to IFN-alpha/beta. In addition, transfected cells displayed an increase in the IFN inducible genes (2',5'-oligoadenylate synthetase [2',5'-OAS], T cell-specific guanine nucleotide triphosphate-binding protein, IFN regulatory factor 1 [IRF-1], and major histocompatibility complex [MHC] class I) compared with plasmid vector-treated controls. Collectively, these results show that IFN-alpha1 transfection of cells in vitro induces or upregulates a spectrum of IFN-regulated genes involved in the direct or indirect antiviral action of this cytokine. In addition, the transgene significantly increases the resistance of transfected cells in vitro to HSV-1 infection.

The relationship between interleukin-6 and herpes simplex virus type 1: implications for behavior and immunopathology.

Cytokines are hormones once thought to be restricted to the immune system produced solely by hematopoietic-derived cells and acting on receptors expressed by cells of the immune system. However, it is now clear that many cytokines are produced not only by lymphocytes, monocytes, granulocytes, and dendritic cells but are also synthesized by cells outside the realm of the immune system in response to stimuli that may not be associated with immune homeostasis. In fact, there is evidence supporting a role of selected cytokines modifying behavior and neuroendocrine function. Recently, a potential relationship between the cytokine interleukin (IL)-6 and herpes simplex virus type 1 (HSV-1) reactivation has been found. This article discusses the relevance of these findings and considers the potential impact that HSV-1 infection has on behavior and chronic inflammatory processes that can occur in the nervous system during "latent" virus infection as a result of chronic IL-6 expression.

Lymphocytes delay kinetics of HSV-1 reactivation from in vitro explants of latent infected trigeminal ganglia.

A persistent immune response to herpes simplex virus type 1 (HSV-1) is evidenced by the expression of cytokine transcripts along with infiltrating mononuclear cells in the ganglia of latently infected mice. In trigeminal ganglion (TG) explant co-cultures, the presence of nonirradiated or irradiated primed splenocytes significantly reduced HSV-1 reactivation as defined by secreted infectious HSV-1 found in the supernatants of TG explant cultures. Primed splenocytes depleted of CD4+ or CD8+ cells did not antagonize HSV-1 reactivation. Cytokines including interleukin (IL)-2, IL-6, IL-10, and IL-12 were all detected in the TG explant cultures containing splenocytes. To further study the role of cytokines in HSV-1 reactivation, dissociated TG cell cultures were treated with exogenous recombinant cytokines including IFN-alpha or -gamma, IL-4, 6, 10, 12 or tumor necrosis factor (TNF)-alpha at concentrations ranging from 2.0 pg to 2.0 ng/culture (or 0.3-300 units/culture for the IFNs). While no cytokines tested at any concentration significantly modified HSV-1 reactivation, neutralizing antibody to IL-6, but not to IFN-alpha/beta, significantly antagonized HSV-1 reactivation. Collectively, the results suggest that IL-6 is directly or indirectly involved in HSV-1 reactivation in TG explant cultures.

Ectopic expression of DNA encoding IFN-alpha 1 in the cornea protects mice from herpes simplex virus type 1-induced encephalitis.

A novel approach to combat acute herpes simplex virus type 1 (HSV-1) infection has recently been developed by administration with a plasmid DNA construct encoding cytokine genes. Cytokines, especially type I IFNs (IFN-alpha and IFN-beta) play an important role in controlling acute HSV-1 infection. The purpose of the present study was to investigate the potential efficacy of ectopically expressed IFN-alpha 1 against ocular HSV-1 infection following in situ transfection of mouse cornea with a naked IFN-alpha 1-containing plasmid DNA. Topical administration of the IFN-alpha 1 plasmid DNA exerted protection against ocular HSV-1 challenge in a time- and dose-dependent manner and antagonized HSV-1 reactivation. In addition, IFN-alpha 1-transfected eyes expressed a fivefold increase in MHC class I mRNA over vector-treated controls. The protective efficacy of the IFN-alpha 1 transgene antagonized viral replication, as evidenced by the reduction of the viral gene transcripts (infected cell polypeptide 27, thymidine kinase, and viral protein 16) and viral load in eyes and trigeminal ganglia during acute infection. The administration of neutralizing Ab to IFN-alpha beta antagonized the protective effect of the IFN-alpha 1 transgene in mice. Collectively, these findings demonstrate the potential of using naked plasmid DNA transfection in the eye to achieve ectopic gene expression of therapeutically active agents.

Astrocyte-targeted expression of IFN-alpha1 protects mice from acute ocular herpes simplex virus type 1 infection.

Type I IFNs (i.e., IFN-alpha and IFN-beta) play a key role in the host's innate defense against viral pathogens. To examine the biologic relevance of IFN-alpha to a viral pathogen within the confines of the nervous system, IFN-alpha1 transgenic mice whose transgene is under the control of the glial fibrillary acidic protein promoter (GFAP-IFN-alpha, astrocyte specific) were examined for resistance to an ocular herpes simplex virus type 1 (HSV-1) infection. GFAP-IFN-alpha mice expressed significantly higher levels of IFN-alphabeta (533 U) in the trigeminal ganglion compared with nontransgenic mice (70 U) 72 h postinfection that corresponded with a significant reduction in the mRNA expression of the HSV-1 immediate early gene infected cell polypeptide 27 and late gene VP16, as well as the chemokines monocyte-chemoattractant protein-1 and cytokine response gene-2 in the eye and trigeminal ganglion. Six days postinfection, the viral load and the expression of infected cell polypeptide 27, CD8, RANTES, IFN-gamma, and IFN-alpha mRNA levels were reduced in the trigeminal ganglion of GFAP-IFN-alpha mice compared with the wild-type mice. Following the establishment of HSV-1 latency (i.e., 30 days postinfection), only one of nine (11%) GFAP-IFN-alpha mice was found to be latent compared with seven of eight (88%) of the wild-type mice, as determined by the expression of the latency-associated transcript RNAs. Likewise, only three of nine GFAP-IFN-alpha mice screened showed seroconversion by day 30 postinfection compared with nine of ten wild-type mice screened. Collectively, the results show that the IFN-alpha1 transgenic mice are less susceptible to acute HSV-1 infection and the establishment of viral latency.

The persistent elevated cytokine mRNA levels in trigeminal ganglia of mice latently infected with HSV-1 are not due to the presence of latency associated transcript (LAT) RNAs.

Trigeminal ganglia (TG) from mice latently infected with wild type HSV-1 contain detectable levels of cytokine transcripts that are not present in TG from uninfected mice. This suggests that during HSV-1 neuronal latency, the immune system is stimulated by the production of one or more viral proteins. Since the LAT (latency associated transcript) gene is essential for wild type levels of spontaneous reactivation and is the only highly active viral gene during latency, the stimulation of cytokines may indicate the presence of a LAT encoded latency protein. We therefore compared the cytokine transcript profiles in the TG of mice latently infected with wild type and LAT negative viruses. Mice were latently infected with either: (1) the LAT null mutant dLAT2903; (2) its marker rescued virus dLAT2903R; or (3) the parental wild type HSV-1 strain McKrae. As expected, reactivation following explant cultivation of TG from latently infected mice was significantly decreased with dLAT2903 (P < 0.05)(40 +/- 8%, n = 24) compared with dLAT2903R (85 +/- 7.6%, n = 36) or the parental virus (70 +/- 10.0%, n = 36). The relative levels of various cytokines was determined by RT-PCR analysis of TG extracts. None of the cytokine transcripts detected in mice latently infected with the wild type or marker rescued viruses were missing or decreased in mice latently infected with the LAT null mutant 30 or 60 days post infection. There were also no differences in the HSV-1 antibody titers induced by the LAT negative virus compared to the LAT positive viruses. Thus, although LAT facilitated reactivation of HSV-1 from explanted mouse TG, expression of LAT during latency did not appear to be involved in persistent cytokine expression in TG. This suggests that during latency, HSV-1 does not produce a highly antigenic abundant LAT encoded protein.

Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress.

The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus. However, the immune profile of this culture system prior to and after stress has never been established. In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice. Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA. The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml). Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2. While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml). Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures. However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation. Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells. By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells. Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells. Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR. Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period. Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type 1.

Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.