HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noisakran, S. Articles by Carr, D. J. J. Search for Related Content PubMed PubMed Citation Articles by Noisakran, S. Articles by Carr, D. J. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Herpes Simplex

Ectopic Expression of DNA Encoding IFN-1 in the Cornea Protects Mice from Herpes Simplex Virus Type 1-Induced Encephalitis¹

Sansanee Noisakran^*, Iain L. Campbell and Daniel J. J. Carr^2,*

^* Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, New Orleans, LA 70112; and Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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- and IFN-ß) 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-1 against ocular HSV-1 infection following in situ transfection of mouse cornea with a naked IFN-1-containing plasmid DNA. Topical administration of the IFN-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-1-transfected eyes expressed a fivefold increase in MHC class I mRNA over vector-treated controls. The protective efficacy of the IFN-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-ß antagonized the protective effect of the IFN-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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ocular infections caused by herpes simplex virus type 1 (HSV-1)³ are extremely common and can lead to encephalitis as well as to herpetic keratoconjunctivitis (1). Local immune responses to the corneal HSV-1 infection include the infiltration of mononuclear cells such as CD4⁺ and CD8⁺ T cells, macrophages, and neutrophils; the activation of NK cells; and the production of cytokines (2, 3, 4). Although there have been several studies assessing immunization strategies against HSV-1 infection, currently there is no successful vaccine commercially available. DNA immunization, a novel vaccination approach that induces both humoral and cellular immune responses, has been applied successfully to combat many infectious diseases (5, 6, 7, 8). Studies evaluating DNA vaccination against HSV-1 infection using different types of viral vectors that carry HSV-1 Ag-encoding genes have had varying degrees of success (9, 10, 11, 12). Although this approach is effective in generating an immune response, it is likely that viral vectors can reduce gene expression by killing transfected cells. In addition, the production of Ab can be induced, which modifies the immune response to the repeated administration of this type of vaccine (13).

Naked DNA vaccination against HSV-1 infection is another approach that has been developed using a plasmid construct consisting of a mammalian cell promoter, a polyadenylation site, a selective marker (e.g., drug-resistance gene), and an Ag-encoding gene as the immunogen. One in vitro study demonstrated that a plasmid construct encoding the immediate-early protein, infected cell polypeptide 27 (ICP27), of HSV-1 was capable of inducing HSV-1-specific CD8⁺ CTL generation (14). In a zosteriform model, i.m. immunization with glycoprotein B-encoded plasmid DNA construct has been shown to elicit CD4⁺ T cell activation and protect mice against acute HSV-1 infection (15). In another study, dendritic cells transfected with DNA as a delivery system were found to be more effective in comparison with the administration of naked DNA alone (16).

The concept of DNA immunization has been applied recently to gene therapy using vector constructs that encode cytokine genes. Presumably, following in vivo transfection, host cells take up plasmid DNA encoding the gene of interest; as a result, in situ expression of the transgene would either antagonize the microbial infection or alternatively, reduce the destructive inflammatory process associated with the infection. Recently, the possibility of using in vivo DNA transfection as a therapeutic option was supported by evidence showing that the topical administration of plasmid DNA encoding IL-10 reduced the incidence of herpetic stromal keratitis (17). To further explore this alternative approach, the present study was undertaken to characterize the efficacy of the topical administration of a plasmid encoding murine IFN-1 in mice. IFN- consists of multiple subtypes that are acid-stable, classically induced in cells by viruses or synthetic polyribonucleotides, and show potent antiviral and immunoregulatory functions (18). The IFN-1 subtype was investigated in the present study because it has been shown previously to have the greatest degree of protection against another herpesvirus, CMV, compared with other IFN- subtypes (19).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus and cells

Vero and CV-1 African green monkey kidney cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 5% FBS (Life Technologies, Gaithersburg, MD) and an antibiotic/antimycotic solution (Life Technologies) in an atmosphere of 37°C/5% CO₂/95% humidity. HSV-1 (McKrae strain) stock was prepared as described previously (20).

Infection of mice

Female ICR mice (weight 25–34 g, Harlan-Sprague-Dawley, Indianapolis, IN) were anesthetized by an i.p. injection of 0.1 ml of PBS containing xylazine (2 mg/ml; 6.6 mg/kg) and ketamine (30 mg/ml; 100 mg/kg). Corneas were scarified with a 25-gauge needle, and tear film was blotted with tissue. Mice were inoculated with 450 plaque-forming units (PFU)/eye of HSV-1 (McKrae strain). Infection was verified by swabbing the eyes at 2–3 days postinfection (PI) and placing the swabs in CV-1 monolayer cultures to observe for a cytopathic effect.

Animals were handled in accordance with the National Institutes of Health guidelines on the Care and Use of Laboratory Animals (publication no. 85-23, revised 1996). All procedures were approved by the Louisiana State University Medical Center Institutional Animal Care and Use Committee.

Plasmid DNA construct

Plasmid pCMV-ß (vector) was purchased from Clontech Laboratories (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-1 was generated as follows: A 690-bp HindIII-EcoRI fragment of mouse IFN-1 cDNA (a kind gift of Dr. E. Zwarthoff, Erasmus University, Rotterdam, The Netherlands) was excised from pGEM-4. After the addition of NotI linkers, this fragment was cloned in the NotI site of pCMV; thus, the generated construct was called pCMV-IFN-1. The plasmid DNA constructs were transformed into shortINVF'-competent E. coli cells according to the manufacturer’s instructions (Invitrogen, San Diego, CA). The production and purification of large-scale DNA preparations was as described previously, with minor modifications (21).

Administration of plasmid DNA construct

After mice were anesthetized, corneas were scarified with a 25-gauge needle and blotted with tissue before 100 µg/eye of either pCMV-ß (vector) or pCMV-IFN-1 was administered at 24 h, 72 h, or 2 wk before HSV-1 injection. In addition, to test the prophylactic efficacy of the IFN-1 construct on acute HSV-1 infection, we anesthetized HSV-1-infected mice and topically treated their eyes with 100 µg/eye of either pCMV-ß (vector) or pCMV-IFN-1 at 24 h PI. For the dose-response study, mice were treated with either PBS or pCMV-IFN-1 (5, 25, 50, or 100 µg/eye) and infected with 450 PFU/eye of HSV-1 24 h later.

RT-PCR

RT-PCR was performed as described previously (20). Briefly, RNA was extracted from excised tissue 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 cycler, Ericomp, San Diego, CA) with 30–35 cycles of 94°C for 75 s, 57–65°C for 75 s, and 72°C for 30–45 s. PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), latency-associated transcript (LAT) RNAs, ICP27, IFN-, IL-6, and IL-10 were as described previously (20, 22). IFN- (consensus sequence for IFN-1, 2, and 7), CD4, and CD8 primer sequences were obtained from Clontech Laboratories. Primers for viral protein 16 (VP16) were 5'-GGACTCGTATTCCAGCTTCAC-3' (sense) and 5'-CGTCCTCGCCGTCTAAGTG-3' (antisense). Primers for thymidine kinase (TK) were 5'-ATGGCTTCGTACCCCTGCCAT-3' (sense) and 5'-GGTATCGCGCGGCCGGGTA-3' (antisense). Primers specific for the IFN-1 transgene were 5'-ATTCCCGCAGGAGAAGGTGGATGCCCCA-3' (sense) and 5'-GAGTAGTTACATAGAATAGTACA-3' (antisense) based on the published sequence for the murine IFN-1 cDNA sequence upstream primer starting at nucleotide 550 (GenBank accession no. X01974) and the downstream primer sequence starting at nucleotide 20 of the SV40 late region polyadenylation sequence, which was used as a 3' untranslated region in the pCMV-IFN-1 fusion gene construct (sequence according to Clontech Laboratories). Primers for JE/monocyte chemoattractant protein-1 (MCP-1) and the setting for the amplification of the specific products were as described previously (23). The primers for cytokine response gene-2 (CRG-2) were 5'-CAGCACCATGAACCCAAGTGC-3' (sense) and 5'-GCTGGTCACCTTTCAGAAGACC-3' (antisense).

Northern blot analysis

For Northern blot hybridization, total RNA (5 µg) was denatured, electrophoresed in 1% agarose/2.2 M formaldehyde gels, transferred to nylon membranes, and hybridized overnight at 45°C with ³²P-labeled cDNA probes. The probes used were a 0.6-kb KpnI-SacI fragment of a murine MHC class (H2D^b) cDNA (provided by Dr. P. Petersen, The Scripps Research Institute) and a 0.26-kb fragment of the murine ß-actin gene (24). For quantification, autoradiographs were scanned (Scanjet 4C/T, Hewlett Packard, San Jose, CA); band density was assessed with National Institutes of Health Image 1.57 software.

In vivo neutralization assay

In indicated experiments, mice received rabbit anti-mouse anti-IFN-ß (Access Biomedical, San Diego, CA; 1000 neutralizing units) or normal rabbit Ig at the time of infection and at 3 and 6 days PI as described previously (22).

Statistics

The Mann-Whitney U test was used to determine significant (p < 0.05) differences between the IFN-1 plasmid construct- and vector construct-treated groups relative to cumulative survival using the GBSTAT program (Dynamic Microsystems, Silver Springs, MD). All other statistical analyses comparing vehicle- or vector-treated mice with IFN-1 construct-treated animals involved ANOVA and Tukey’s post hoc t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transient expression of plasmid DNA encoding IFN-1 in eyes

To verify the exogenous expression of IFN-1 in eyes, mice were topically administered with 100 µg/eye of either pCMV-ß (vector control) or pCMV-IFN-1 at 24 h, 72 h, or 2 wk before sacrifice. The transgene was expressed in uninfected eyes in a time-dependent manner by RT-PCR (Fig. 1) and ribonuclease protection assay (data not shown). Likewise, IFN-1 transgene expression was detected in the eyes of IFN-1 construct-treated mice at 3 days PI but was absent in the vector-treated mice (Fig. 1). However, expression during infection was transient, disappearing at 6 days PI (Fig. 1). A possible explanation for this result is that the host-transfected corneal epithelial cells are compromised or destroyed as a result of the infection, leading to a loss in transgene expression during the acute phase of the infection.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Transient expression of IFN-1 construct in eyes. A, Mice (n = 2/experiment, repeated three times) were topically treated with 100 µg/eye of pCMV-IFN-1 and then sacrificed at 24 h, 72 h, or 2 wk posttreatment. RNA isolation as well as RT-PCR for GAPDH and the IFN-1 transgene were performed from tissue extracted from the eyes from each individual animal. B, Mice (n = 3/group, repeated three times) were treated with 100 µg/eye of either pCMV-ß (V) or pCMV-IFN-1 (1), infected with HSV-1 24 h later, and then sacrificed on days 3 and 6 PI to determine the expression of the IFN-1 transgene using RT-PCR. This figure shows a representative RT-PCR analysis. C, A summary of the RT-PCR results is shown as a pixel ratio of IFN-1 to GAPDH.

In vivo transfection with plasmid DNA encoding IFN-1 protects mice against ocular HSV-1 challenge

To determine the in vivo efficacy of a plasmid IFN-1 construct against ocular HSV-1 infection, either pCMV-ß (vector) or pCMV-IFN-1 (100 µg/eye) were applied topically to the corneas of mice at 24 h, 72 h, or 14 days before ocular HSV-1 infection, or at 24 h PI. Topical administration of pCMV-IFN-1 at 24 h before infection significantly (p < 0.05) enhanced the survival of mice compared with vector-treated mice (Fig. 2). Treating mice with the IFN-1 construct at 72 before infection or at 24 h following HSV-1 infection had no significant effect against acute HSV-1 infection (Fig. 2). In addition, none of the mice receiving the IFN-1 construct at 2 wk before the infection survived in the period surveyed (Fig. 2). The data illustrate the transient nature of the protective effect, which is consistent with the expression of the transgene within the cornea. To determine the dose-dependent efficacy of the IFN-1 construct against HSV-1, mice were topically given either vehicle (PBS) or pCMV-IFN-1 (5, 25, 50, or 100 µg/eye) and subsequently challenged 24 h later with HSV-1. The results show that compared with mice receiving vehicle (PBS) or pCMV-IFN-1 (5 µg/eye), mice receiving higher concentrations of pCMV-IFN-1 (25–100 µg/eye) were protected against HSV-1 infection (Table I).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Topical administration with IFN-1 construct enhances cumulative survival of HSV-1-infected mice. Either pCMV-ß (vector) or pCMV-IFN-1 (100 µg/eye) was topically applied to the cornea of mice at the indicated time before or after HSV-1 infection. The infected mice were observed for survival for 30 days. Results either represent two to three experiments (n = 4–5/experiment) for pCMV-IFN-1 given at 24 h, 72 h, or 2 wk before HSV-1 infection or at 24 h following HSV-1 infection or represent four experiments for pCMV-ß (n = 8–10/experiment). *, p < 0.05, comparing IFN-1 construct treatment at 24 h before infection to vector treatment.

View this table: [in this window] [in a new window]   Table I. IFN-1 transgene protects mice from ocular HSV-1 infection in a dose-dependent fashion1

Effect of IFN-1 construct on the expression of MHC, viral transcripts, immune cell transcripts, and cytokine as well as chemokine transcripts

Type I IFNs possess many antiviral characteristics, including the induction or up-regulation of MHC class I molecules, which facilitates CTL recognition of virally infected cells. To determine whether application of the IFN-1 transgene onto the cornea elicited such an effect, mouse eyes were transfected with the pCMV-ß vector or with pCMV-IFN-1 and assessed for MHC class I mRNA expression. The results show a fivefold elevation in the expression of MHC class I RNA in eyes that were transfected with the IFN-1 transgene compared with the vector-treated group (Fig. 3, A and B). Equivalent amounts of RNA for each sample were analyzed based on the hybridization intensities for ß-actin (Fig. 3A). To further characterize the protective mechanism elicited by the IFN-1 construct, viral loads were assessed in the eyes and trigeminal ganglia (TG) during acute infection. There was a significant reduction in the amount of infectious virus recovered from the eyes of mice topically treated with pCMV-IFN-1 compared with vector-treated controls at 3 and 6 days PI (Fig. 4). Likewise, 7 of 12 mice treated with the vector had detectable virus in the cerebellum (100 PFU/cerebellum) compared with 3 of 12 mice treated with the pCMV-IFN-1 construct (10 PFU/cerebellum). Consistent with these results, viral transcript expression was also modified. Specifically, treatment with the IFN-1 construct reduced the expression of HSV-1 ICP27 and HSV-1 VP16 in the TG at 3 days PI, whereas the expression of TK was not detected (Fig. 5). In the eye, HSV-1 TK was not detected in the pCMV-IFN-1-treated cornea (Fig. 5). At a later timepoint during acute infection (i.e., day 6 PI), topical administration with the IFN-1 construct reduced the expression of all viral transcripts tested in both the eyes and TG (ICP27, TK, and VP16) (Fig. 5). Because cytokines and chemokines are detected in the eyes and TG during an acute, ocular HSV-1 infection (2, 3, 4, 20, 23) and the antigenic stimulus (in the form of infectious virus) is reduced in the pCMV-IFN-1-treated mice, cytokine and chemokine transcript levels were measured. The results show that application of the pCMV-IFN-1 at 24 h before infection reduced the expression of CRG-2 in the TG, of CD8 and MCP-1 in the eyes, and of IL-6 in both the eyes and TG at 3 days PI (Fig. 5). The expression of MCP-1 in the TG was unchanged following treatment with the IFN-1 construct. The expression of CD8 was not detected in the TG from vector-treated or IFN-1-treated mice at 3 days PI. At the later timepoint during acute infection (i.e., 6 days PI), topical administration of the IFN-1 construct to mice resulted in a reduced expression of IL-10 and CD8 in the TG (Fig. 5). In addition, the expression of IL-6 in the TG was also reduced in the IFN-1 construct-treated mice. No other transcripts measured were found to be different when vector-treated mice were compared with IFN-1 construct-treated mice day 6 PI (Fig. 5).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. IFN-1 transgene augments MHC class I RNA expression in the eyes of mice. Mice (n = 2/group) were treated with either pCMV-ß or pCMV-IFN-1 (100 µg/eye). Next, mice were sacrificed at 24 h posttreatment, and RNA was isolated from the eyes of the mice and assayed for expression of MHC class I and ß-actin by Northern blot gel analysis. A, Representative gel showing both MHC class I and ß-actin; B, densitometric analysis of the gel.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. In vivo expression of IFN-1 construct reduces viral load in the eyes and TG at 6 days PI. Mice were topically treated with 100 µg/eye of either pCMV-ß or pCMV-IFN-1 and infected with HSV-1 after 24 h. The viral load in the eyes and TG was determined at 3 and 6 days PI as described previously (35). Results are reported as the mean ± SEM from five experiments; n = 3 mice/experiment (pCMV-ß) and n = 3 mice/experiment (pCMV-IFN-1). *, p < 0.05, comparing the pCMV-IFN-1-treated group with the vector-treated control group.

View larger version (77K): [in this window] [in a new window]   FIGURE 5. Expression of viral, immune cell, cytokine, and chemokine transcripts in the eyes and TG after treatment with IFN-1 construct. Mice were topically treated with either pCMV-ß or pCMV-IFN-1 (100 µg/eye) and infected with HSV-1 (450 PFU/eye) after 24 h. The expression of viral (ICP27, TK, and VP16), immune cell (CD4 and CD8), cytokine (IL-6, IL-10, IFN-, and IFN-), and chemokine (CRG-2 and MCP-1) transcripts was determined by RT-PCR from the eyes and TG of infected mice at 3 or 6 days PI. Data are representative of three experiments; n = 2–3 samples/transcript/experiment (pCMV-ß) and n = 3 samples/transcript/experiment (pCMV-IFN-1).

Anti-IFN-ß Ab blocks the protective effect elicited by the IFN-1 construct

To further ensure that the protective effect elicited by the transgene was due to IFN- rather than to the induction of an immune response to the plasmid DNA (25, 26, 27, 28), neutralizing Ab to IFN-ß or control rabbit Ig was administered to mice undergoing transgene administration. Whereas the pCMV-IFN-1-treated mice administered control Ab showed an increased cumulative survival compared with the vehicle-treated group, pCMV-IFN-1-treated mice administered anti-IFN-ß succumbed to infection at the same rate as the untreated group of mice (Fig. 6).

View larger version (14K): [in this window] [in a new window]   FIGURE 6. The efficacy of pCMV-IFN-1 against HSV-1 infection is antagonized in the presence of anti-IFN-ß Ab. Mice were administered PBS or pCMV-IFN-1 (100 µg/eye) at 24 h before infection with HSV-1 (450 PFU/eye). At the time of infection and again at 3 and 6 days PI, mice (n = 5/group/experiment) were administered anti-IFN-ß or rabbit control Ab i.p. Mice were monitored and recorded for survival. This figure is a summary of two experiments. *p < 0.05 comparing control Ig-treated with other groups on days 10–30 PI and comparing control Ig-treated with the saline-treated group on day 9 PI.

Topical administration of the IFN-1 construct suppresses HSV-1 reactivation

To determine the effect of a topical administration of naked plasmid DNA on the establishment of HSV-1 latency and reactivation, TG explant cocultures were established using HSV-1-infected mice that had survived into viral latency (i.e., day 30 PI). The results show that the TG from mice that had been administered the IFN-1 construct either at 72 h before infection or at 24 h PI reactivated to a level similar to the vector control (Fig. 7). However, none of the TG from mice treated with the IFN-1 construct at 24 h before infection reactivated (Fig. 7). LAT expression was weakly detected in the TG from nonreactivated explant cultures obtained from the mice pretreated with pCMV-IFN-1 at 24 h before infection as determined by RT-PCR compared with samples from mice treated with the transgene PI or vector-treated mice (Fig. 7). These results suggest that the presence of the transgene may reduce the establishment of latent HSV-1 in the sensory ganglia.

View larger version (37K): [in this window] [in a new window]   FIGURE 7. Treatment with pCMV-IFN-1 during acute HSV-1 infection antagonizes HSV-1 reactivation in TG explants. TG were obtained from mice at 30 days PI and cocultured with CV-1 indicator cells. A, Supernatants from the cultures were collected daily for 11 days and assessed for infectious virus by plaque assay using CV-1 cells; n = 4 (pCMV-ß or pCMV-IFN-1 treatment at 72 h before infection), n = 6 (pCMV-IFN-1 treatment at 24 PI), and n = 16 (pCMV-IFN-1 treatment at 24 before infection). B, After the 11-day survey, cultures were collected and assayed for LAT and GAPDH expression by RT-PCR. Lane 1, TG culture from a mouse pretreated (72 h) with pCMV-IFN-1 before infection; lanes 2–10, TG cultures from a mouse pretreated (24 h) with pCMV-IFN-1 before infection; lanes 11–13, TG cultures from a mouse treated at 24 h PI with pCMV-IFN-1. The lane marked with a "-" represents the primer control; the lane marked with a "+" represents the positive control. GAPDH represents the level of cDNA input for all samples analyzed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmid DNA delivery is an important subclass of gene therapy that shows promise, as it is thought to be qualitatively safer than virus vectors due to the potential for less adverse effects (39). Because previous studies have identified murine IFN- as a key component controlling the HSV-1 replication that is generated in vivo following active immunization (40, 41), and because transgenic mice expressing IFN-1 under the control of a glial fibrillary acidic protein (GFAP) (expressed primarily in astrocytes) promoter (GFAP-IFN-1) are resistant to HSV-1 infection (42), a study was undertaken to investigate the potential efficacy of the IFN-1 transgene in ocular HSV-1 infection using a pCMV delivery system. In the present study, the expression of the IFN-1 transgene in the cornea is transient and short-lived in infected animals. However, the pCMV-IFN-1 plasmid construct but not the vector plasmid protected mice from HSV-1-induced encephalitis in a dose- and time-dependent manner when applied to the cornea before infection. The protective effect is mediated through the expression of the transgene rather than through exposure to plasmid DNA, as indicated in experiments using neutralizing Ab to IFN-ß that showed that IFN-1-treated mice that were administered the neutralizing Ab but not control Ab succumbed to the infection similar to controls.

Ectopic expression of IFN-1 in the eye was found to reduce viral replication, as evidenced by a reduction in the viral load and expression of viral genes during the course of acute infection. Consistent with these findings, in vitro studies have found that type I IFNs block HSV-1 immediate-early gene expression (43, 44) or elicit the production of defective, noninfectious HSV-1 particles from infected cells (45). Consequently, the data suggest that placement of the IFN-1 transgene in the eye before infection prevented the replication and spread of the virus from the origin of infection (cornea) to the sensory ganglia and possibly the central nervous system. The measurement of viral loads in the cerebellum showed a reduction in HSV-1 (6 days PI), which supports this point. Because the immune response to acute infection results in significant pathology (46), a reduction in viral replication and spread would reduce the inflammatory response to the infection. In fact, the findings in this study showed a reduction in some chemokines (MCP-1 and CRG-2), cytokines (IL-6 and IL-10), and immune cell (CD8) transcripts in the eye and TG of mice treated with pCMV-IFN-1 as evidenced on day 6 PI. Although there was no apparent difference in the expression of IFN- in the TG when vector-treated mice and pCMV-IFN-1-treated mice were compared at 6 days PI, IL-10 levels were reduced in the pCMV-IFN-1-treated mice. Recently, IL-10 has been reported to reduce HSV-1-elicited IFN-1 (47), suggesting that expression of the transgene may protect mice not only by reducing HSV-1 gene expression and viral load but also by reducing IL-10 levels that could suppress the local production of virally induced IFN-.

HSV-1 eludes immune surveillance by blocking Ag processing and presentation by infected cells (48, 49, 50). However, in GFAP-IFN-1 transgenic mice that express elevated levels of MHC class I within the central nervous system (51), susceptibility to HSV-1 infection as measured by viral loads and viral gene expression is greatly reduced (42). In a similar manner, the IFN-1 transgene-induced elevation in the expression of MHC class I within the eye coincided with an increased resistance to HSV-1 infection. Collectively, the data suggest that another means by which mice administered the pCMV-IFN-1 construct become resistant to HSV-1 infection may reside with the enhanced expression of MHC class I in the eye and presumably with increased Ag presentation.

In addition to protection against acute HSV-1 infection, topical administration of the IFN-1 construct at 24 h before infection reduced the establishment of latency as evidenced by LAT expression and the lack of viral reactivation in TG explant cultures. The weak detection of LAT in TG from the mice treated with the pCMV-IFN-1 at 24 h before infection compared with TG from mice treated with the pCMV vector or treated with pCMV at 72 h before infection suggests that either fewer neurons/ganglia were infected in the TG of mice pretreated (24 h) with the transgene or, alternatively, fewer HSV-1 genome copy numbers/neuron were present. These results are consistent with recent observations correlating the expression of LAT within sensory ganglia to HSV-1 reactivation (52, 53).

The application of cytokine gene therapy in controlling viral replication is illustrated in the present report. The present study showed that preexposing mice to the IFN-1 transgene at 24 h before infection significantly enhanced the survival of mice. A single bolus of naked DNA did not protect the animals when applied 3–14 days before infection or at 24 h PI. We cannot exclude the possibility that the lack of protection seen in the mice treated with the transgene PI may have been due to the reduced level of plasmid uptake and subsequent expression of transgene, because it was not formally proven that the IFN-1 plasmid was incorporated and expressed at a level similar to that seen for the mice treated with the transgene before infection. Administration of the transgene before infection followed scarification of the cornea to facilitate the growth of new tissue and uptake of plasmid DNA. Although this approach has little practical value in treating patients presenting with herpetic stromal keratitis, it is possible that a greater degree of protection in HSV-1-infected mice may be realized by increasing the frequency of administration of the IFN-1 transgene after infection. The present study only treated mice with a single application before or after infection. Furthermore, facilitating the uptake of plasmid DNA by alternative means may also increase the efficacy of the transgene through a greater degree of incorporation into the target tissue.

Collectively, the present study serves to illustrate that naked DNA plasmid construct technology may provide an alternative therapeutic option in treating localized infections in an attempt to influence the immune response and promote a favorable outcome in the host. Moreover, the transient nature of transgene expression suggests that the introduction of foreign DNA into selective target tissue may be short-lived depending upon the infectious disease and the time of application. One advantage to decreased expression of the transgene is that an immune response to the product is potentially reduced. Recent findings suggest that multiple exposures to plasmid DNA containing transgenes encoding chemokines generate a humoral immune response to the transgene product (54). Finally, the application of cytokine gene therapy may prove to be equally or better suited for other microbial pathogens, sites of infection, or the control of an unwarranted inflammatory response (55).

	Footnotes

 
¹ This work was supported by U.S. Public Health Service Grants NS35470 (to D.J.J.C.) and MH47680 (to I.L.C.).

² Address correspondence and reprint requests to Dr. Daniel J. J. Carr, Department of Microbiology and Immunology, Louisiana State University Medical Center, Box P6-1, 1901 Perdido Street, New Orleans, LA 70112-1393.

³ Abbreviations used in this paper: HSV-1, herpes simplex virus type 1; TG, trigeminal ganglia; PFU, plaque-forming unit; PI, postinfection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICP27, infected cell polypeptide 27; TK, thymidine kinase; VP16, viral protein 16; LAT, latency-associated (RNA) transcripts; MCP-1, monocyte chemotactic protein-1; CRG-2, cytokine response gene-2; GFAP, glial fibrillary acidic protein.

Received for publication August 5, 1998. Accepted for publication January 6, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Whitley, R. J.. 1996. Herpes simplex viruses. B. N. Fields, and D. M. Knipe, and P. M. Howley, eds. In Fields Virology Vol. 2:2297. Lippincott-Raven Publishers, Philadelphia, PA.
2. Shimeld, C., J. L. Whiteland, N. A. Williams, D. L. Easty, T. Hill. 1997. Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1. J. Gen. Virol. 78:3317.[Abstract]
3. Liu, T., Q. Tang, R. L. Hendricks. 1996. Inflammation infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection. J. Virol. 70:264.[Abstract]
4. Tumpey, T. M., S. Chen, J. E. Oakes, R. N. Lausch. 1996. Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea. J. Virol. 70:898.[Abstract]
5. Wang, B., K. E. Ugen, V. Srikantan, M. G. Agadjanyan, K. Dang, Y. Refaeli, A. I. Sato, J. Boyer, W. V. Williams, D. B. Weiner. 1993. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90:4156.[Abstract/Free Full Text]
6. Donnelly, J. J., J. B. Ulmer, M. A. Liu. 1994. Immunization with DNA. J. Immunol. Methods 176:145.[Medline]
7. Yokoyama, M., J. Zhang, J. L. Whitton. 1995. DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection. J. Virol. 69:2684.[Abstract]
8. Donnelly, J. J., J. B. Ulmer, J. W. Shiver, M. A. Liu. 1997. DNA vaccines. Annu. Rev. Immunol. 15:617.[Medline]
9. Martin, S., B. T. Rouse. 1987. The mechanism of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: clearance of local infection. J. Immunol. 138:3431.[Abstract]
10. Ghiasi, H., S. Bahri, A. B. Nesburn, S. L. Wechsler. 1995. Protection against herpes simplex virus-induced eye disease after vaccination with seven individually expressed herpes simplex virus 1 glycoproteins. Invest. Ophthalmol. Vis. Sci. 36:1352.[Abstract/Free Full Text]
11. Gallichan, W. S., D. C. Johnson, F. L. Graham, K. L. Rosenthal. 1993. Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B. J. Infect. Dis. 168:622.[Medline]
12. Hariharan, M. J., D. A. Driver, K. Townsend, D. Brumm, J. M. Polo, B. A. Belli, D. J. Catton, D. Hsu, D. Mittelstaedt, J. E. McCormack, et al 1998. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector. J. Virol. 72:950.[Abstract/Free Full Text]
13. Yang, Y., Q. Li, H. C. J. Ertl, J. M. Wilson. 1995. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69:2004.[Abstract]
14. Rouse, R. J. D., S. K. Nair, S. L. Lydy, J. C. Bowen, B. T. Rouse. 1994. Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simple virus proteins. J. Virol. 68:5685.[Abstract/Free Full Text]
15. Manickan, E., R. J. D. Rouse, Z. Yu, W. S. Wire, B. T. Rouse. 1995. Genetic immunization against herpes simplex virus: protection is mediated by CD4⁺ T lymphocytes. J. Immunol. 155:259.[Abstract]
16. Manickan, E., S. Kanangat, R. D. J. Rouse, Z. Yu, B. T. Rouse. 1997. Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells. J. Leukoc. Biol. 61:125.[Abstract]
17. Daheshia, M., N. Kuklin, S. Kanangat, E. Manickan, B. T. Rouse. 1997. Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10. J. Immunol. 159:1945.[Abstract]
18. Baron, S. B., S. K. Tyring, W. R. Fleischmann, D. H. Coppenhaver, D. W. Niesel, G. R. Klimpel, J. S. Stanton, T. K. Hughes. 1991. The interferons: mechanisms of action and clinical applications. J. Am. Med. Assoc. 266:1375.[Abstract/Free Full Text]
19. Yeow, W., C. M. Lawson, M. W. Beilharz. 1998. Antiviral activities of individual murine IFN- subtypes in vivo: intramuscular injection of IFN expression constructs reduces cytomegalovirus replication. J. Immunol. 160:2932.[Abstract/Free Full Text]
20. Halford, W. P., B. M. Gebhardt, D. J. J. Carr. 1996. Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1. J. Immunol. 157:3542.[Abstract]
21. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. In Molecular Cloning: A Laboratory Manual, Vol. 1. C. Nolan, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.33.
22. Daigle, J., D. J. J. Carr. 1998. Androstenediol antagonizes herpes simplex virus type 1-induced encephalitis through the augmentation of type I IFN production. J. Immunol. 160:3060.[Abstract/Free Full Text]
23. Su, Y. H., X. T. Yan, J. E. Oakes, R. N. Lausch. 1996. Protective antibody therapy is associated with reduced chemokine transcripts in herpes simplex virus type 1 corneal infection. J. Virol. 70:1277.[Abstract]
24. Tokunaga, K., H. Taniguchi, K. Yoda, M. Shimizu, S. Sakiyama. 1986. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal ß-actin mRNA. Nucleic Acids Res. 14:829.
25. Chattergoon, M. A., T. M. Robinson, J. D. Boyer, D. B. Weiner. 1997. Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells. J. Immunol. 160:5707.[Abstract/Free Full Text]
26. Krieg, A. M., L. Love-Homan, A.-K. Yi, J. T. Harty. 1997. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428.[Abstract/Free Full Text]
27. Jakob, T., P. S. Walker, A. M. Krieg, M. C. Udey, J. C. Vogel. 1998. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J. Immunol. 161:3042.[Abstract/Free Full Text]
28. Yamamoto, S., T. Yamamoto, S. Shimada, E. Kuramoto, O. Yano, T. Kataoka, T. Tokunaga. 1992. DNA from bacteria, but not vertebrates, induces interferons, activates natural killer cells, and inhibits tumor growth. Microbiol. Immunol. 36:983.[Medline]
29. Samuel, C. E.. 1991. Antiviral actions of interferon: interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183:1.[Medline]
30. Muller, U., U. Steinhoff, L. F. L. Reis, S. Hemmi, J. Pavlovic, R. M. Zindernagel, M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264:1918.[Abstract/Free Full Text]
31. Gutterman, J. U.. 1994. Cytokine therapeutics: lessons from interferon . Proc. Natl. Acad. Sci. USA 91:1198.[Abstract/Free Full Text]
32. Krown, S. E.. 1998. Interferon-: evolving therapy for AIDS-associated Kaposi’s sarcoma. J. Interferon Cytokine Res. 18:209.[Medline]
33. Jr Fleischmann, W. R., J. Masoor, T. Y. Wu, C. M. Fleischmann. 1998. Orally administered IFN- acts alone and in synergistic combination with intraperitoneally administered IFN- to exert an antitumor effect against B16 melanoma in mice. J. Interferon Cytokine Res. 18:17.[Medline]
34. Beilharz, M. W., W. McDonald, M. W. Watson, J. Heng, J. McGeachie, C. M. Lawson. 1997. Low-dose oral type I interferons reduce early virus replication of murine cytomegalovirus in vivo. J. Interferon Cytokine Res. 17:625.[Medline]
35. Lecciones, J. A., N. H. Abejar, E. E. Dimaano, R. Bartolome, S. Cinco, N. Mariano, M. E. Yerro, S. Cobar, B. Fuggan. 1998. A pilot double-blind, randomized, and placebo-controlled study of orally administered IFN--n1 (Ins) in pediatric patients with measles. J. Interferon Cytokine Res. 18:647.[Medline]
36. Nagao, Y., K. Yamashiro, N. Hara, Y. Horisawa, K. Kato, A. Uemura. 1998. Oral-mucosal administration of IFN- potentiates immune response in mice. J. Interferon Cytokine Res. 18:661.[Medline]
37. Arao, Y., Y. Ando, M. Narita, T. Kurata. 1997. Unexpected correlation in the sensitivity of 19 herpes simplex virus strains to types I and II interferons. J. Interferon Cytokine Res. 17:537.[Medline]
38. Taylor, J. L., S. D. Little, W. J. O’Brien. 1998. The comparative anti-herpes simplex virus effects of human interferons. J. Interferon Cytokine Res. 18:159.[Medline]
39. Felgner, P. L.. 1997. Nonviral strategies for gene therapy. Sci. Am. 276:102.[Medline]
40. Hendricks, R. L., P. C. Weber, J. L. Taylor, A. Koumbis, T. M. Tumpey, J. C. Glorioso. 1991. Endogenously produced interferon protects mice from herpes simplex virus type 1 corneal disease. J. Gen. Virol. 72:1601.[Abstract/Free Full Text]
41. Halford, W. P., L. A. Veress, B. M. Gebhardt, D. J. J. Carr. 1997. Innate and acquired immunity to herpes simplex virus type 1. Virology 236:328.[Medline]
42. Carr, D. J. J., L. A. Veress, S. Noisakran, I. L. Campbell. 1998. Astrocyte targeted expression of interferon-1 protects mice from acute ocular herpes simplex virus type 1 infection. J. Immunol. 161:4859.[Abstract/Free Full Text]
43. Straub, P., I. Domke, H. Kirchner, H. Jacobsen, A. Panet. 1986. Synthesis of herpes simplex virus proteins and nucleic acids in interferon-treated macrophages. Virology 150:411.[Medline]
44. Mittnacht, S., P. Straub, H. Kirchner, H. Jacobsen. 1988. Interferon treatment inhibits onset of herpes simplex virus immediate-early transcription. Virology 164:201.[Medline]
45. Munoz, A., L. Carrasco. 1984. Formation of non-infective herpesvirus particles in cultured cells treated with human interferons. J. Gen. Virol. 65:1069.[Abstract/Free Full Text]
46. Tang, Q., W. Chen, R. L. Hendricks. 1997. Proinflammatory functions of IL-2 in herpes simplex virus corneal infection. J. Immunol. 158:1275.[Abstract]
47. Payvandi, F., S. Amrute, P. Fitzgerald-Bocarsly. 1998. Exogenous and endogenous IL-10 regulate IFN- production by peripheral blood mononuclear cells in response to viral stimulation. J. Immunol. 160:5861.[Abstract/Free Full Text]
48. Lewandowski, G. A., D. Lo, F. E. Bloom. 1993. Interference with major histocompatibility complex class II-restricted antigen presentation in the brain by herpes simplex virus type 1: a possible mechanism of evasion of the immune response. Proc. Natl. Acad. Sci. USA 90:2005.[Abstract/Free Full Text]
49. York, I. A., C. Roop, D. W. Andrews, S. R. Riddell, F. L. Graham, D. C. Johnson. 1994. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8⁺ T lymphocytes. Cell 77:525.[Medline]
50. Hill, A., P. Jugovic, I. York, G. Russ, J. Bennink, J. Yewdell, H. Ploegh, D. Johnson. 1995. Herpes simplex virus turns off the TAP to evade host immunity. Nature 375:411.[Medline]
51. Akwa, Y., D. E. Hassett, M.-L. Eloranta, K. Sandberg, E. Masliah, H. Powell, J. L. Whitton, F. E. Bloom, I. L. Campbell. 1998. Transgenic expression of IFN- in the central nervous system of mice protects against lethal neurotropic viral infection but induces inflammation and neurodegeneration. J. Immunol. 161:5016.[Abstract/Free Full Text]
52. Sawtell, N. M., D. K. Poon, C. S. Tansky, R. L. Thompson. 1998. The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain-specific and correlates with reactivation. J. Virol. 72:5343.[Abstract/Free Full Text]
53. Sawtell, N. M.. 1998. The probability of in vivo reactivation of herpes simplex virus type 1 increases with the number of latently infected neurons in the ganglia. J. Virol. 72:6888.[Abstract/Free Full Text]
54. Youssef, S., G. Wildbaum, G. Maor, N. Lanir, A. Gour-Lavie, N. Grabie, N. Karin. 1998. Long-lasting protective immunity to experimental autoimmune encephalomyelitis following vaccination with naked DNA encoding C-C chemokines. J. Immunol. 161:3870.[Abstract/Free Full Text]
55. Chun, S., M. Daheshia, N. A. Kuklin, B. T. Rouse. 1998. Modulation of viral immunoinflammatory responses with cytokine DNA administered by different routes. J. Virol. 72:5545.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. J. J. Carr, L. Tomanek, R. H. Silverman, I. L. Campbell, and B. R. G. Williams RNA-Dependent Protein Kinase Is Required for Alpha-1 Interferon Transgene-Induced Resistance to Genital Herpes Simplex Virus Type 2 J. Virol., July 15, 2005; 79(14): 9341 - 9345. [Abstract] [Full Text] [PDF]

				B. Sainz Jr and W. P. Halford Alpha/Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1 J. Virol., October 11, 2002; 76(22): 11541 - 11550. [Abstract] [Full Text] [PDF]

				D. J. J. Carr and S. Noisakran The Antiviral Efficacy of the Murine Alpha-1 Interferon Transgene against Ocular Herpes Simplex Virus Type 1 Requires the Presence of CD4+, {alpha}/{beta} T-Cell Receptor-Positive T Lymphocytes with the Capacity To Produce Gamma Interferon J. Virol., August 12, 2002; 76(18): 9398 - 9406. [Abstract] [Full Text] [PDF]

				D. J.J. Carr, P. Härle, and B. M. Gebhardt The Immune Response to Ocular Herpes Simplex Virus Type 1 Infection Experimental Biology and Medicine, May 1, 2001; 226(5): 353 - 366. [Abstract] [Full Text]

				P. Harle, S. Noisakran, and D. J. J. Carr The Application of a Plasmid DNA Encoding IFN-{{alpha}}1 Postinfection Enhances Cumulative Survival of Herpes Simplex Virus Type 2 Vaginally Infected Mice J. Immunol., February 1, 2001; 166(3): 1803 - 1812. [Abstract] [Full Text] [PDF]

				S. Noisakran and D. J. J. Carr Plasmid DNA Encoding IFN-{alpha}1 Antagonizes Herpes Simplex Virus Type 1 Ocular Infection Through CD4+ and CD8+ T Lymphocytes J. Immunol., June 15, 2000; 164(12): 6435 - 6443. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noisakran, S. Articles by Carr, D. J. J. Search for Related Content PubMed PubMed Citation Articles by Noisakran, S. Articles by Carr, D. J. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Herpes Simplex