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The Journal of Immunology, 2000, 164: 6435-6443.
Copyright © 2000 by The American Association of Immunologists

Plasmid DNA Encoding IFN-{alpha}1 Antagonizes Herpes Simplex Virus Type 1 Ocular Infection Through CD4+ and CD8+ T Lymphocytes1

Sansanee Noisakran* and Daniel J. J. Carr2,{dagger}

* Departments of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112; and {dagger} Departments of Ophthalmology, Microbiology, and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was undertaken to further characterize the anti-viral efficacy of a plasmid DNA encoding IFN-{alpha}1 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The initial exposure to herpes simplex virus type 1 (HSV-1)3 infection in the cornea elicits a strong innate and adaptive immune response to the pathogen. Chemokines generated following HSV-1 infection (1) play a role in promoting chemotaxis of leukocytes to the site of infection, but also facilitate tissue pathology by attracting neutrophils to the site, as recently shown with macrophage inflammatory protein-1{alpha} and macrophage inhibitory protein-2 (2, 3). Neutrophils are an immediate and important cellular component of the innate immune response that are necessary in controlling viral replication and spread into peripheral tissues (4, 5). The infiltration of neutrophils into the cornea has previously been shown to be associated with the presence of NK cells (6). NK cells are also involved in controlling acute HSV-1 infection (7, 8) either through the direct cytolysis of virally infected cells or through the secretion of anti-viral cytokines, including IFN-{gamma} (9). Likewise, T lymphocytes are found to infiltrate ocular tissue following HSV-1 infection, predominantly expressing the CD4+ phenotype (10). The infiltration of CD4+ T lymphocytes is thought to promote local tissue destruction as a result of IL-2 and IFN-{gamma} production, facilitating the continuous infiltration of neutrophils into the site of infection (11, 12). However, the absence of IFN-{gamma} or the receptor for IFN-{gamma} promotes HSV-1 replication and increases the susceptibility to virus-induced mortality (13, 14), suggesting a key role for this cytokine in HSV-1-mediated pathogenesis and neuronal apoptosis (15). Other cytokines produced locally or by infiltrating leukocytes in response to corneal infection with HSV-1 include the proinflammatory cytokines (IL-1ß, IL-6, IL-12, and TNF-{alpha}), IL-5, IL-10, and IFN-{alpha} (16, 17, 18, 19, 20).

Following the initial infection and replication within the cornea, HSV-1 travels to the trigeminal ganglion (TG) by retrograde transport and establishes a latent infection, again eliciting a potent immune response during this process involving chemokines and cytokines and the infiltration of primarily CD8+ T lymphocytes, macrophages, granulocytes, and {gamma}{delta} T cells (21, 22, 23, 24, 25). Although Ab enhances the likelihood of viral clearance and survival of the host (26, 27, 28), {gamma}{delta} T lymphocytes and CD8+ T cells may ultimately be responsible for viral clearance within the nervous system and resistance to developing encephalitis (29, 30, 31, 32).

In some patients the primary concern of ocular HSV-1 infection is the tissue destruction associated with the infection and reactivation of the latent virus, resulting in stromal keratitis and eventually blindness. A recent report suggesting that HSV-1-mediated stromal keratitis is a result of molecular mimicry between an epitope found within the unique long 6 protein of HSV-1 and self Ag within the cornea (33) emphasizes the need to control the acute infection, the establishment of latency, and reactivation of the virus. To this end a number of vaccines have been developed to protect the host from HSV-1 infection (34, 35, 36), one of which has been found to exacerbate the infection (37). Although there are currently no commercially available vaccines for HSV-1, some of the recently developed candidate vaccines have been found to reduce the incidence of viral reactivation (38, 39). Another approach in establishing resistance to HSV-1 infection or reducing inflammation as a result of ocular infection is through the use of plasmid constructs encoding cytokines. IL-10, a Th2 cytokine that has previously been shown to reduce the severity of corneal disease (40), has been used as a prototype cytokine to reduce ocular inflammation through the naked DNA approach (41). Likewise, type I IFNs (IFN-{alpha} and IFN-ß), which have been found to control HSV-1 infection (42, 43, 44), have also been employed in controlling ocular HSV-1 infection following the application of plasmid DNA encoding them (45, 46). The present study was undertaken to further characterize the protective effect mediated by the IFN-{alpha}1 transgene against HSV-1-induced encephalitis following topical application to the cornea.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virus and cells

CV-1 African green monkey kidney, hybridoma 53-6.72 (anti-Lyt 2, a lymphocyte differentiation Ag found on the surface of cytolytic T cells) and hybridoma GK1.5 (anti-L3T4, a T cell surface Ag expressed by Th/inducer cells) cell lines were obtained from American Type Culture Collection (Manassas, VA). CV-1 cells were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (Life Technologies), an antibiotic/antimycotic solution (Life Technologies), and gentamicin (final concentration, 20 µg/ml culture medium; Life Technologies). The hybridoma cells were cultured in AIM-V medium (Life Technologies) supplemented with 5% FBS (Life Technologies). The cell cultures were incubated at 37°C in an atmosphere of 5% CO2 and 95% humidity. HSV-1 (McKrae) stock was prepared as previously described (23).

Plasmid DNA construct

Plasmid pCMV-ß (vector) was purchased from Clontech (Palo Alto, CA). This eukaryotic expression vector (7.2 kb) contains Escherichia coli ß-galactosidase (a reporter gene) expressed under the control of a human CMV immediate-early promoter/enhancer, an RNA splice donor and acceptor sequence, and an SV40 late polyadenylation signal. Plasmid pCMV-IFN-{alpha}1 was generated as previously described (45). The production and purification of large scale DNA preparations were performed as described previously, with minor modifications (47).

Infection of mice

Female ICR mice (25–34 g; Harlan Sprague-Dawley, Indianapolis, IN) were anesthetized by injection of 0.1 ml of PBS containing xylazine (2 mg/ml; 6.6 mg/kg) and ketamine (30 mg/ml; 100 mg/kg) i.p. Corneas were scarified with a 25-gauge needle, and tear film was blotted with tissue before inoculating with 450 PFU/eye of HSV-1 (McKrae strain). To determine the viral dose response of the IFN-{alpha}1 transgene, varying doses of HSV-1 (150, 450, 900, and 4500 PFU/eye for the ICR mice receiving the plasmid DNA construct) were employed. The infection was verified by swabbing the eyes 2–3 days postinfection (PI) and placing the swabs in CV-1 monolayer cultures to observe any cytopathic effect. Animals were handled in accordance with the National Institutes of Health guidelines on the Care and Use of Laboratory Animals (Publication 85–23, revised 1996). All procedures were approved by the Louisiana State University Health Sciences Center and University of Oklahoma Health Sciences Center institutional animal care and use committees.

Administration of plasmid DNA construct

ICR mice were anaesthetized, and mouse corneas were subsequently scarified with a 25-gauge needle as well as blotted with tissues before placing 100 µg of either pCMV-ß (vector) or pCMV-IFN{alpha}1/eye in 3 µl of PBS (pH 7.4). In one experiment mice were administered 150 PFU/eye and subsequently treated 24 or 48 h PI with 100 µg/eye pCMV-ß or pCMV-IFN-{alpha}1. In other experiments, the plasmid DNA constructs were topically administered 24 h before ocular challenge with HSV-1.

Purification of anti-L3T4 and anti-Lyt2 mAb

Rat mAbs to L3T4 and Lyt 2 Ags used in the CD4+/CD8+ T cell depletion study were purified as follows. Supernatants secreted from the hybridoma cell cultures (either 53-6.72 or GK1.5 clones) were collected and clarified by centrifugation at 1,000 x g for 5 min. The Ig fraction was precipitated in a final concentration of 40% ammonium sulfate (Sigma, St. Louis, MO) at 4°C overnight. The precipitate was separated from the supernatant by centrifugation at 10,000 x g for 15 min and resuspended in a minimum amount of deionized water required to dissolve the precipitate. The resultant solution was dialyzed overnight at 4°C in 200 vol of PBS (pH 7.4) using the Spectra/Por membrane (m.w. cutoff, 6,000–8,000; The Spectrum Co., Gardena, CA) and concentrated (100-fold from the original volume) using polyethylene glycol (Mr cutoff, 15–20 kDa; Sigma). The purified Abs were stored as aliquots and kept at -20°C (for short term storage).

In vivo depletion of CD4+ and CD8+ T lymphocytes

ICR mice were i.p. injected with 100 µl of normal rat serum (control), or rat anti-mouse Lyt 2 or L3T4 Ab every other day for 6 days. On day 6 post-treatment, all mice were topically administered pCMV-IFN-{alpha}1 (100 µg/eye), with the exception of a nontreated group that received PBS and served as another control group. Twenty-four hours following the topical treatment with the plasmid DNA, the mice were ocularly infected with HSV-1 (450 PFU/eye) and observed for cumulative survival. To assess the success of the depletion of T lymphocyte populations, spleen cells were removed and analyzed for the percentages of CD3+CD4+ and CD3+CD8+ cells using FITC- and PE-labeled Abs to CD3, CD4, and CD8 markers and a FACSCalibur instrument (Becton Dickinson, Mountain View, CA).

Immunohistochemical staining for HSV-1 Ags

To investigate the effect of the topical administration of plasmid DNA construct on HSV-1 Ag expression, eyes and TG were collected from the vector- or IFN-{alpha}1 transgene-treated, HSV-1-infected mice on days 3 and 6 PI. Eyes and TG from uninfected mice were used as negative controls. The tissues were fixed in alcoholic Z-fix (Anatech, Battle Creek, MI) at room temperature for 48 h, transferred to 70% ethanol, and processed on a MUP Processor (Ventana Medical Systems, Tucson, AZ) through graduated alcohols, XS-3 xylene substitute (Statlab Medical Products, Lewisville, TX), and paraffin (Shandon Lipshaw, Pittsburgh, PA). The processed tissues were subsequently embedded in peel-away paraffin, allowed to cool at room temperature, and cut at a thickness of 5 µm using a Leitz 1512 microtome (Global Medical Instrumentation, St. Paul, MN). The sections were placed into a water bath (53°C), transferred onto slides, and air-dried. After deparaffinization, the sections were rehydrated and placed on a Ventana Nexes ES IHC Staining System (Ventana). Prediluted rabbit anti-mouse polyclonal Ab directed against HSV (types 1 and 2; Ventana) and biotinylated goat anti-rabbit IgG (Ventana) were used as a primary and secondary Ab, respectively. The presence of HSV-1 Ag was detected using the diaminobenzidene (peroxidase) detection kit (Ventana) and Ventana Red (alkaline phosphatase detection kit, Vetana). The stained sections were dehydrated in 100% ethanol, cleared using xylene, and coverslipped with Permount.

Staining tissue for ß-galactosidase expression

Three days after topical application of plasmid DNA encoding ß-galactosidase onto the cornea of mice, the animals were sacrificed, and various tissues were removed and fixed with 4% paraformaldehyde (Sigma) in PBS (pH 7.2) for 1 h at 4°C. The tissues were then washed four times in PBS and reacted overnight in substrate solution (X-galactostidase) containing 20 mM potassium ferrocyanide, 20 mM potassium ferricyanide, 2 mM MgCl2, 2 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactoside/ml, 120 µl of 10% Nonidet P-40 (Sigma), and 100 µl of 1% sodium deoxycholate/20 ml. After the reaction, the tissues were washed in PBS, fixed in 3.7% formaldehyde, and embedded in paraffin. Tissue was processed in 5-µm sections and stained with Nuclear Fast Red.

Immunohistochemical staining for immune cell infiltration

To determine the effect of the plasmid DNA construct treatment on the infiltration of immune cells during the acute HSV-1 infection, the eyes and TG from infected mice on days 3 and 6 PI were processed as described above. The paraffin-embedded tissue sections (5 µm) were stained with hematoxylin and eosin using the MUP Processor (Ventana). The stained sections were dehydrated in 100% ethanol, cleared in xylene, and coverslipped with Permount.

Plaque assay

To determine the kinetics of infectious HSV-1 shedding in the tear films of the infected mice during the acute phase of infection, mouse eyes were swabbed, and the cotton applicators were placed in 500 µl of culture media (RPMI 1640 containing 5% FBS and a antimycotic/antibiotic solution; Sigma) for 1 h. The samples were serially diluted and placed (100 µl) onto CV-1 cell monolayers in 96-well culture plates. After a 1-h incubation at 37°C in 5% CO2 and 95% humidity, the supernatants were discarded, and 75 µl of an overlay solution (0.5% methylcellulose in culture medium) was added on top of the monolayers. The cultures were incubated at 37°C in 5% CO2 and 95% humidity for 24–48 h to observe plaque formation, and the amount of infectious virus was reported as PFU per swab.

RT-PCR

To determine the effect of the plasmid DNA construct on the local expression of cytokine, chemokine, and immune cell transcripts in uninfected tissue, RNA isolation and RT-PCR were conducted using the eyes from mice topically administered either the vector or the IFN-{alpha}1 transgene (100 µg/eye) for 24 h before the collection of targeted tissue. As previously described (23), RNA was extracted from the eyes in Ultraspec RNA isolation reagent (Biotecx, Houston, TX). First-strand cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). PCR was performed in a thermal cycler (Ericomp Delta cycler, Ericomp, San Diego, CA) with 30–35 cycles of 94°C (45 s-1 min)->57–65°C (45 s-1 min)->72°C (40 s-2 min). PCR primers for GAPDH, IL-6, IL-10, IL-12 (p40), IFN-{gamma}, CD4, and CD8 were as described previously (23, 45). PCR primers for IL-2 were 5'-TCCACTTCAAGCTCTACAG-3' (sense) and 5'-GAGTCAAATCCAGAACATGCC-3' (antisense). Primers for IL-4 were 5'-CAGTGATGTGGACTTGGACTCATTCATGGTGC-3' (sense) and 5'-CCAGCTAGTTGTCATCCTGCTCTTCTTCTCG-3' (antisense). Primers for IFN-{gamma}-induced nitric oxide synthase (Nos-2) were 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3' (sense) and 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' (antisense). Primers for RANTES were 5'-GAAGATCTCTGCAGCTGCCCT-3' (sense) and 5'-GCTCATCTCCAAATAGTTGA-3' (antisense). Primers for JE/monocyte chemoattractant protein-1 and macrophage inflammatory protein-1ß were previously described (1). Primers for IFN-{gamma}-inducible protein-10 (IP-10) were 5'-CAGCACCATGAACCCAAGTGC-3' (sense) and 5'-GCTGGTCACCTTTCAGAAGACC-3' (antisense). Oligonucleotide primers were synthesized by the Louisiana State University Health Sciences Center Core Laboratories (New Orleans, LA). The PCR products were visualized by ethidium bromide-stained agarose gel (2%) electrophoresis, and the gel densitometry was analyzed using a Bio-Rad 1000 image documentation system (Bio-Rad, Hercules, CA).

Statistics

One-way ANOVA and Tukey’s t test were used to determine significant (p < 0.01 and 0.05) differences between the IFN-{alpha}1 construct- and vector construct-treated groups relative to the expression of cytokine and chemokine transcripts and immune cell infiltration. The significant difference (p < 0.01) in viral gene expression between the vector- and the IFN-{alpha}1 transgene-treated groups was determined by {chi}2 test. Mann-Whitney U test was used to determine the significant (p < 0.05) difference in the cumulative survival studies. All statistical analysis was performed using the GBSTAT program (Dynamic Microsystems, Silver Spring, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasmid gene expression

Previous studies have found the expression of a reporter gene (ß-galactosidase) following the application of plasmid DNA at mucosal surfaces (including ocular and intranasal routes) to occur both locally and at sites distant to the original site of administration (41, 48). Our results found gene expression in the rectus muscles surrounding the eye following topical application of the plasmid DNA vector to the scarified cornea of anesthetized animals with no evidence of expression at other sites within the eye (Fig. 1GoA). However, consistent with the expression of the reporter gene distal to the original site of application (49), ß-galactosidase expression was also detected in the spleen of mice (Fig. 1GoB).



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  		FIGURE 1. Expression of ß-galactosidase protein in tissue sections. Four days after topical application of the plasmid DNA encoding ß-galactosidase, the eyes and spleen were removed, fixed in 4% paraformaldehyde, and incubated overnight with the substrate X-galactosidase at 37°C. After overnight incubation, the tissues were fixed in 3.7% formaldehyde in PBS and embedded in paraffin. Tissues were sectioned at 5 µm and stained with Nuclear Fast Red. A, Eye transfected with the plasmid construct encoding ß-galactosidase. Magnification: left panel, x40; right panel, x400. Normal spleen and spleen from a mouse transfected with the plasmid construct encoding ß-galactosidase. Magnification, x400 for both normal spleen (left panel) and spleen from the transfected mouse.

 
Effects of IFN-{alpha}1 transgene on the local expression of cytokine, chemokine, and immune cell transcripts

Different methods of DNA delivery have previously been found to influence Th cells, Ab, and innate immune responses (48). Specifically, the inoculation of DNA by saline injection, but not by gene gun, tends to generate Th 1 lymphokines and complement-dependent Abs (48). Likewise, IFN-{alpha} has been shown to play an important role in differentiation toward the Th 1 response (50, 51). As a result, the present study was undertaken to determine the effects of topical administration of naked plasmid DNA encoding IFN-{alpha}1 on the local immune profile, which, in turn, might participate in the protective mechanism against HSV-1 infection. The results show that topical administration of the IFN-{alpha}1 transgene significantly (p < 0.05) enhanced the levels of IL-6 and IP-10 transcript expression in mouse eyes compared with plasmid vector treatment alone (Fig. 2Go). However, the expression levels of immune cell (CD4 and CD8) and other cytokine as well as chemokine transcripts evaluated were not modified or were not detected in the eyes following treatment with the plasmid DNA encoding IFN-{alpha}1 compared with that of the vector-treated group (data not shown).



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  		FIGURE 2. The IFN-{alpha}1 transgene augments the levels of IL-6 and IP-10 transcript expression in the eye. Mice were topically administered 100 µg/eye of pCMV-ß (V) or IFN-{alpha}1 ({alpha}1). Twenty-four hours after the plasmid DNA construct treatment the eyes were removed and processed for RNA. RT-PCR was then conducted using primers specific for GAPDH, IL-6, and IP-10. Results are a summary of two independent experiments (n = 3/group/experiment). The values are a pixel ratio of IL-6 or IP-10 mRNA to GAPDH mRNA ± SEM. *, p < 0.05, comparing the levels of IL-6 and IP-10 transcript expression in the eye from the IFN-{alpha}1 transgene-treated group to that in the vector-treated group by ANOVA and Tukey’ t test.

 
The efficacy of the IFN-{alpha}1 transgene against ocular HSV-1 infection is dependent on the infectious dose of virus

To investigate the dose-dependent nature of this resistance, the resistance to ocular HSV-1 infection in mice topically treated with the IFN-{alpha}1 transgene could be overridden by increasing the viral inoculum. Specifically, mice topically treated with the IFN-{alpha}1 construct showed increasing sensitivity to the lethal effects of elevating infectious viral doses (Fig. 3Go). For example, whereas 100% of mice treated with the IFN-{alpha}1 transgene 24 h before infection with 150 PFU/eye of HSV-1 survived the infection, only 22% (two of nine) survived the infection at 4500 PFU/eye of HSV-1 (Fig. 3Go). Because the efficacy of the transgene was appreciably greater at a lower viral inoculum (i.e., 50% lethal dose), an experiment was performed to determine whether the protective effect of topically applying the plasmid DNA encoding IFN-{alpha}1 could be delayed. Mice infected with 150 PFU/eye and treated 24 h PI with the IFN-{alpha}1 transgene showed an enhanced cumulative survival rate compared with mice treated with the plasmid vector (Fig. 4Go). However, the protective effect was time dependent, in that when the transgene was applied 48 h PI there was no significant efficacy against ocular HSV-1 infection (Fig. 4Go).



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  		FIGURE 3. The efficacy of IFN-{alpha}1 transgene against ocular HSV-1 infection is dependent upon the infectious dose of virus. Mice were topically administered 100 µg/eye of either pCMVß (vector) or pCMVIFN-{alpha}1 and 24 h later infected with HSV-1 at a dose of 150 PFU/eye (A), 450 PFU/eye (B), 900 PFU/eye (C), or 4500 PFU/eye (D). The cumulative survival of the infected mice was determined within a 30-day period. Results are a summary of three experiments for doses of 150, 450, and 900 PFU/eye (n = 3/group/experiment) and two experiments for doses of 4500 PFU/eye (n = 3 group/experiment).

 


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  		FIGURE 4. Therapeutic efficacy of the IFN-{alpha}1 transgene in enhancing the cumulative survival of HSV-1-infected mice. Mice were infected with HSV-1 (150 PFU/eye) in 3 µl of PBS following scarification of the cornea. Twenty-four or 48 h PI, 100 µg/eye of pCMV-ß (vector) or pCMVIFN-{alpha}1 was topically applied to the eye, and the mice were monitored for cumulative survival. This figure is a summary of three experiments (n = 5/group/experiment).

 
Effects of IFN-{alpha}1 transgene on HSV-1 Ag expression in the infected tissues during acute HSV-1 infection

Topical administration of the IFN-{alpha}1 transgene has previously been shown to antagonize viral replication in the eye and TG, as evidenced by the recovery of virus from infected tissues (45). To further investigate the influence of the IFN-{alpha}1 construct on the expression of HSV-1 Ags in the infected tissues during the acute phase of infection, mice topically treated with either pCMV-ß (vector) or pCMV-IFN{alpha}1 and subsequently (24 h later) infected with HSV-1 were inspected for viral Ag expression in the eyes and TG 3 and 6 days PI. The results show that even though a similar percentage of eyes stained positively for HSV-1 Ag comparing the vector- to IFN-{alpha}1 transgene-treated mice on day 3 PI, the number of viral Ag positively staining foci per section from the IFN-{alpha}1 transgene-treated mice was less than that from the vector-treated mice (Table IGo and Fig. 5GoA). Within the TG on day 3 PI, there was a 50% reduction in the number of TG positive for HSV-1 Ag and a reduction in the size of the foci in the mice treated with the IFN-{alpha}1 transgene compared with the plasmid vector-treated control (Table IGo and Fig. 5GoB). Treatment with the IFN-{alpha}1 transgene resulted in a significant reduction of viral Ag expression and number of foci in the eyes and TG 6 days PI compared with the vector controls (Table IGo and Fig. 5Go, C and D).


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  		Table I. Immunohistochemical staining for HSV-1 Ags in the infected tissues following plasmid DNA administration1

 


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  		FIGURE 5. Topical application of the IFN-{alpha}1 transgene reduces HSV-1 Ag expression in the eyes and TG. Mice were topically administered 100 µg/eye of either pCMV-ß (vector) or pCMV-IFN{alpha}1 and 24 h later challenged with HSV-1 (450 PFU/eye). The eyes and TG were collected from the infected mice 3 and 6 days PI and assessed for the presence of HSV-1 Ag by indirect immunohistochemical staining. Uninfected eyes and TG served as negative controls. Results are a representative of two independent experiments (four to six samples per group per experiment). Left panel, vector treatment; right panel, IFN-{alpha}1 transgene treatment. A, Eye, day 3 PI. B, TG, day 3 PI. C, Eye, day 6 PI. D, TG, day 6 PI. Arrows indicate the presence of viral Ags. Magnification: eye, x40; TG, x400.

 
Effects of IFN-{alpha}1 transgene on immune cell infiltration in the infected tissues during acute HSV-1 infection.

Inflammation and tissue destruction following ocular HSV-1 infection correlate with the infiltration of leukocytes, including neutrophils, lymphocytes, macrophages, and NK cells (3, 4, 5, 12). To determine whether the infiltration of these cells into the eye is modified by the topical application of the IFN-{alpha}1 transgene during the acute HSV-1 infection, eye sections were enumerated for infiltrating cells 3 and 6 days PI. Compared with the basal levels of cells in the corneal epithelium, iris, and ciliary body from uninfected eyes (44 ± 4), the number of infiltrating cells was significantly (p < 0.01) increased following HSV-1 infection in both plasmid vector- and IFN-{alpha}1 transgene-treated groups (Fig. 6Go). However, the topical administration of the IFN-{alpha}1 transgene was associated with a significant (p < 0.01) reduction in the number of infiltrating cells in the corneal epithelium, iris, and ciliary body 3 days PI (137 ± 13 cells; n = 35 sections) compared with that in the vector controls (251 ± 24 cells; n = 44 sections; Fig. 6GoB). Likewise, there was a significant (p < 0.01) decrease in the number of the infiltrating cells detected in the cornea of IFN-{alpha}1 transgene-treated mice (197 ± 20 cells; n = 42 sections) compared with that in vector-treated mice (341 ± 22 cells; n = 34 sections) 6 days PI (Fig. 5CGo). Similar results were found when inspecting infiltrating cells in the iris and ciliary body from plasmid vector- and IFN-{alpha}1 transgene-treated mice, in that fewer infiltrating cells were detected in these tissues of the IFN-{alpha}1 transgene-treated group compared with the plasmid vector-treated group (data not shown).



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  		FIGURE 6. IFN-{alpha}1 transgene reduces the infiltration of leukocytes into ocular tissue during acute HSV-1 infection. Mice were topically treated with either pCMV-ß (vector) or pCMV-IFN{alpha}1 24 h before corneal HSV-1 infection. The eyes were removed from the infected mice 3 and 6 days PI, and the tissues were cross-sectioned and processed for hematoxylin-eosin staining. The number of cells infiltrating cells the corneas of the infected mice that had received the plasmid DNA construct were assessed and compared with those in uninfected mice (control). Results are a representative of two independent experiments (n = 4–6/group/experiment). A, Uninfected eye. B and C, Infected eye on day 3 PI (B) and day 6 PI (C) that had been treated with the vector construct (left panel) or the IFN-{alpha}1 construct (right panel). Magnification, x200.

 
Resistance to HSV-1 by the IFN-{alpha}1 transgene requires the presence of CD4+ and CD8+ T lymphocytes

The nature of HSV-1 antagonism induced by the IFN-{alpha}1 transgene could be at the level of the targeted tissue (52) or, alternatively, could be an indirect effect involving other immune mediators. In characterizing the involvement of T lymphocytes, CD4- and CD8-depleted mice were evaluated for resistance to ocular HSV-1 infection in the presence or the absence of the IFN-{alpha}1 transgene. Mice depleted of CD4 or CD8 T lymphocytes remained so until day 7 PI, when 15–20% of the normal level of depleted cells was achieved (Table IIGo). NK cells (defined as CD3-NK1.1+) were not modified during this treatment regimen, maintaining a level of 4.1 ± 0.7%. Compared with the control serum-treated mice administered the plasmid DNA encoding IFN-{alpha}1, both CD4- and CD8-depleted animals administered the transgene were sensitive to HSV-1 infection, similar to the saline-treated group, as measured by cumulative survival (Fig. 7Go). In addition, CD8-depleted mice treated with the plasmid DNA encoding IFN-{alpha}1 showed a level of recoverable virus in the eye film similar to that in nondepleted mice treated with the IFN-{alpha}1 transgene. However, CD4-depleted mice treated with the IFN-{alpha}1 transgene exhibited higher levels of virus similar to levels in nontransgene treated animals (Fig. 8Go).


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  		Table II. Phenotypic profile of mice depleted of CD4 or CD8 T lymphocytes1

 


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  		FIGURE 7. CD4+ and CD8+ T lymphocytes are required in the protective mechanism mediated by IFN-{alpha}1 transgene. Mice were i.p. injected with normal rat serum or rat IgG specific for either mouse CD4+ or CD8+ T lymphocytes three times (days 1, 4, and 6) in a 6-day period. On day 6 post-treatment mice were administered either pCMV-IFN-{alpha}1 (100 µg/eye) or PBS before a subsequent challenge with HSV-1 (450 PFU/eye) 24 h later. Mice that had not received the rat Ig, but were topically treated with PBS and infected with HSV-1, served as negative controls. The cumulative survival of the infected mice was assessed up to 30 days PI. Results are a summary of two or three experiments (n = 4–5/group/experiment). *, p < 0.05, comparing the cumulative survival of the IFN-{alpha}1 transgene-treated mice receiving the control Ab to that of the IFN-{alpha}1 transgene-treated mice depleted of the CD4+ or CD8+ T cells or to that of the vehicle-treated mice, as assessed by Mann-Whitney U test.

 


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  		FIGURE 8. CD4+ T cells reduce viral shedding in the tear film as a result of topical application of the IFN-{alpha}1 transgene. CD4+- and CD8+-depleted mice treated with the plasmid DNA encoding IFN-{alpha}1 (100 µg/eye) were surveyed for viral shedding at times PI. Normal rat serum-treated mice topically administered the IFN-{alpha}1 transgene (control) served as the positive control, whereas nondepleted mice topically administered PBS served as the negative control. These results are a summary of two experiments (n = 4–5 mice/experiment). Viral clearance from the eyes did not differ between groups, occurring by day 7 PI.

 
Ocular route of IFN-{alpha}1 transgene application induces the highest degree of protection against HSV-1 infection compared with other mucosal sites of delivery

To determine the site-directed nature of the anti-viral efficacy of IFN-{alpha}1, plasmid DNA encoding IFN-{alpha}1 was administered by various mucosal routes to mice subsequently infected with HSV-1. All routes showed expression of the lacZ gene based on histochemical staining, indicating the success of the transfection (data not shown). The ocular route of administration proved superior to other mucosal sites, as evidenced by cumulative survival of the recipient animals receiving the naked DNA encoding IFN-{alpha}1 compared with those receiving the vector DNA alone (Fig. 9Go). Neither the intravaginal nor the intranasal route of administration had any protective effect against ocular HSV-1 infection.



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  		FIGURE 9. A comparison of the efficacy of plasmid DNA encoding IFN-{alpha}1 against ocular HSV-1 administered by different routes. Mice (n = 6/site) were administered plasmid vector (vector) or plasmid DNA encoding IFN-{alpha}1 by the ocular, i.n., or intravaginal route. Twenty-four hours postadministration, the mice were infected with HSV-1 (450 PFU/eye) and surveyed for cumulative survival. The results are representative of three experiments with similar outcomes. *, p < 0.05, comparing the group given transgene by the ocular route with vector-treated groups, as determined by Mann-Whitney U test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study further characterizing the resistance to HSV-1 infection elicited by targeted tissue transfected with the plasmid DNA encoding IFN-{alpha}1 found that both CD4+ and CD8+ T lymphocytes are necessary for the transgene to induce a significant anti-viral state in the host. These results are not surprising, in that cells transfected in vitro with the IFN-{alpha}1 transgene showed only a modest antagonism to HSV-1 infection, as measured by the quantity of virus measured following infection (52). However, transfected cells produced and secreted biologically active IFN-{alpha} that was found to augment NK cell activity (52). Taken together, these results suggest that the primary anti-viral effect mediated by the transgene is indirect, either promoting activated T cell longevity (53, 54) or enhancing the effector function of stimulated T cells. HSV-1 infection has been shown to induce a fratricide state among CTLs through the up-regulation of CD95 (55), which may facilitate immune evasion similar to HSV-1-mediated down-regulation of MHC class I molecules (56). Administration of the IFN-{alpha}1 transgene, which has previously been shown to augment MHC class I transcript expression (45), may also enhance T cell survival in the face of HSV-1 infection, subverting some of the immune-evading mechanisms associated with this virus. Whether the transgene modifies the ratio of Th1 to Th2 effector cells within the infected tissue has not been addressed and is the subject of a future study.

In the absence of infection, transfection of mouse cornea with the IFN-{alpha}1 transgene augments the expression of both IP-10 and IL-6 mRNA. IP-10 is induced in macrophages by type I and II IFNs (57) and displays anti-viral activity (58) as well as induces NK cell migration and augments NK cell activity (59). Consequently, IP-10 may serve as another participant in the protection against lethal HSV-1-induced encephalitis. Similar to IP-10, IL-6 may antagonize HSV-1 replication in the eye. Specifically, transgenic mice expressing IL-6 in the peripheral and central nervous systems are highly resistant to ocular HSV-1 infection (60), while the susceptibility to infection is enhanced in mice lacking IL-6 (61). Furthermore, astrocyte cultures that produce IL-6 in response to HSV-1 generate more virus in the presence of neutralizing Ab to IL-6 than cultures treated with control serum (D. J. J. Carr, unpublished observation). Consequently, in the present model the induction of an anti-viral state in the cornea may have been established before the introduction of HSV-1. The timing of this anti-viral state is crucial to ocular HSV-1 infection, in that when the naked DNA encoding IFN-{alpha}1 is applied to the eyes at time points surpassing 12 h PI, there is no demonstrable protection against the infection (62) unless a reduction in viral inoculation is employed. Therefore, it is important that delivery of the naked DNA to the eye is immediate within the first round of replication of the virus (18–24 h PI) to establish resistance to HSV-1 replication and spread.

The topical application of the cornea with the IFN-{alpha}1 transgene not only antagonized the expression of HSV-1 Ags in the infected tissues, but it also reduced the infiltration of leukocytes into the cornea during the acute stage of infection. The correlation between viral Ag expression and the infiltration of cells suggests that viral Ag stimulates localized cells to secrete chemokines and other inflammatory mediators that promote extravasation of leukocytes into the cornea. However, ocular immunopathology by HSV-1 depends on viral replication (63). Consistent with this observation, the IFN-{alpha}1 transgene reduces HSV-1 replication in the eye (45). Therefore, by reducing viral replication, less Ag is present to stimulate the inflammatory process and thereby reduce chemotaxis of cells to the infected area. If this hypothesis is correct, it is doubtful that T cells confront HSV-1 at the site of infection, but, instead, provide protection peripheral to the origin of replication in the lymphoid tissue within the conjunctiva, lacrimal glands, or TG. Evidence suggests that the virus reaches the TG of transgene-treated animals, but replication is dramatically reduced, with little indication that latency is established (45). Because the transgene is expressed in immune organs distant from the original site of administration (i.e., the spleen), it is possible that T lymphocytes activated by the transgene product may also participate in controlling the spread of the virus to the peripheral nervous system.

It seems likely that there are two phases in the protective response essential for controlling ocular HSV-1 infection following the topical application of the IFN-{alpha}1 transgene. The initial phase is mediated by the direct action of the transgene product against the virus as well as the augmentation of the activity of other nonspecific immune mediators, including NK cells (64) and macrophages (65). In turn, macrophages act at the level of the late phase in concert with IFN-{alpha}1, presenting Ag to T lymphocytes and enhancing T lymphocyte function (66). Within the ganglion, the CD8+ T cells along with macrophages and {gamma}{delta} T cells may ultimately control the replication and spread of the virus in virally infected cells (24, 30, 31, 32).

Previous findings show that immunization with HSV-1 glycoproteins intranasally (i.n.) protected mice from the pathological manifestations associated with ocular HSV-1 infection (67). Therefore, an experiment was conducted comparing the efficacy of protection by the IFN-{alpha}1 transgene administered at different mucosal sites. It was anticipated that the introduction of the plasmid DNA encoding IFN-{alpha}1 i.n. would provide significant protection against ocular HSV-1 infection. This assumption was based on previous reports showing a reduction of HSV-1 ocular pathogenesis following the introduction of naked DNA encoding IL-10 i.n. (68). However, in the present study only when the transgene was applied to the ocular mucosa was protection against HSV-1 infection realized. The efficacy of the transgene against HSV-1 could be overridden by increasing the viral dose by >1 log over the 50% lethal dose used to infect the mice, indicating a threshold of protection. Nevertheless, these findings illustrate the potential clinical application of cytokine gene therapy as has previously been shown for HSV-1, but also includes other applications, including cancer treatment (69, 70).


   Acknowledgments
 
We thank Dr. Iain L. Campbell (Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA) for his critical evaluation of this manuscript.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant EY12409 (to D.J.J.C.) and a unrestricted grant from Research to Prevent Blindness, Inc. Back

2 Address correspondence and reprint requests to Dr. Daniel J. J. Carr, Department of Ophthalmology, DMEI #415, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Boulevard, Oklahoma City, OK 73104. Back

3 Abbreviations used in this paper: HSV-1, herpes simplex virus type 1; TG, trigeminal ganglion; PI, postinfection; IP-10, IFN-{gamma}-inducible protein-10; i.n., intranasally. Back

Received for publication January 18, 2000. Accepted for publication April 7, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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