The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Härle, P.
Right arrow Articles by Carr, D. J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Härle, P.
Right arrow Articles by Carr, D. J. J.
The Journal of Immunology, 2001, 166: 1803-1812.
Copyright © 2001 by The American Association of Immunologists

The Application of a Plasmid DNA Encoding IFN-{alpha}1 Postinfection Enhances Cumulative Survival of Herpes Simplex Virus Type 2 Vaginally Infected Mice1

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{alpha}1 introduced into the vaginal lumen postinfection (PI). Mice infected with HSV-2 intravaginally and treated intravaginally 24 h later with 100 µg DNA encoding IFN-{alpha}1 showed enhanced survival (10/15) in comparison to mice treated with 100 µg plasmid DNA vector alone (3/10) or vehicle (4/27). In contrast, mice receiving recombinant IFN-{alpha}A (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-{alpha}1 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-{alpha}1 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Herpes simplex virus type 2 (HSV-2)3 is a common genital pathogen that has been conservatively projected to result in 500,000 new cases annually (1). Recent studies assessing seroprevalence rates in the U.S. suggest that as much as 33% of the adult population is infected with HSV-2 (2, 3). A recent study of over 2300 patients found the acquisition of new HSV-2 cases reaching a rate of 5.1 cases per 100 person-years, suggesting the continued prevalence of this viral pathogen in the U.S. population (4). Although both HSV-1 and HSV-2 can infect the genital tract, HSV-2 is more severe with an increase in the frequency of recurrence (5). Primary genital herpes is manifested by the appearance of macules, papules, and vesicles resulting in the development of pustules and ulcers. Complications following primary genital herpetic infection include sacral radiculomyelitis that can lead to urinary retention, neuralgias, and meningoencephalitis. Following the acute infection, HSV-2 will establish a latent infection within the neurons of the sensory ganglia (sacral ganglia) and periodically reactivate with clinical or subclinical presentation. On average the frequency of recurrences is four to five episodes per year (1). Several case control studies have shown an association of HIV-1 and HSV-2 in both the homosexual and heterosexual population with the relative risk of acquiring HIV-1 infection in HSV-2-infected individuals increasing 1.5- to 2-fold compared with noninfected controls (6, 7, 8, 9, 10).

The immune response to HSV-2 vaginal infection in the human population is not well understood due to the unique immune vaginal environment (11). To this end, a mouse animal model has been developed to more closely study the immune response to vaginal HSV-2 infection (12, 13). In this model, susceptibility to HSV-2 was induced following the administration of a progesterone derivative, DePo-Provera (Upjohn, Kalamazoo, MI) s.c. These mice become infected with HSV-2 with an up-regulation of MHC class II expression in the vaginal epithelium, the infiltration of CD8+ cells in the epithelium and stroma, and the occurrence of plasma cells in the stroma and lymphoid nodules (13). Most migrating cells appear to be lymphoid in nature with no evidence that macrophages or Langerhans cells are trafficking out of the tissue (14). Although the initial infection of the genital tract by HSV-2 in progesterone-treated mice appears to be similar to the initial infection in humans, susceptible epithelial cells in mice are present in both vagina and cervix whereas they are mainly in the cervix of humans (15). The infection is controlled by local innate and adaptive immunity including neutrophils (16) and CTLs (17, 18, 19, 20). In one study surveying HIV+ to HIV- patients, the frequency of recurrence of HSV-2 to the prevalence of precursor CTLs was highly correlated (21). Other effector cells (e.g., NK cells) and cytokines such as IFN-{gamma} have been suggested to control HSV-2 infection in the vagina (22).

Vaccine development is ongoing to generate a protective IgG response against HSV-2 as IgA is not required for protection against vaginal HSV-2 infection (15, 23, 24). Vaccines using recombinant HSV-2 glycoproteins including glycoprotein (g)B and gD have been found to provide adequate protection against subsequent vaginal infection with HSV-2 in a guinea pig model (25) but clinical outcomes have not been forthcoming. Using an attenuated virus, another group found promising results measuring survival and clinical scores whether the virus was inoculated s.c. or intranasally (26).

The use of gene therapy as a potential approach to treat vaginal HSV-2 infection has also been investigated. One of the first studies used a plasmid CMV (pCMV) expressing HSV-2 gD administered i.m. in guinea pigs 64, 43, and 22 days before vaginal infection with HSV-2 (strain MS). The results showed that animals treated with the pCMV expressing HSV-2 gD had a reduction in viral replication, decreased severity of the primary infection, and a reduction in recurrent disease compared with animals receiving the control plasmid (27). These results also correlated with high serum titers to HSV-2. In another study, coinjection of cDNA encoding IL-12 i.m. was found to augment the protective effect of the HSV-2 gD DNA vaccination against genital HSV-2 infection as measured by cumulative survival and severity of clinical presentation using inbred and outbred mice (28). Using this same strategy, these investigators also found that Th1 cytokine DNA cassettes (i.e., IL-2, IL-12, IL-15, and IL-18) codelivered with HSV-2 gD DNA augmented the efficacy of the vaccine alone against genital HSV-2 infection, whereas Th2 cytokine DNA cassettes (i.e., IL-4 or IL-10) tended to increase the rate of morbidity and mortality in recipients (29). Taken together, these results suggest that promoting a Th1 cytokine profile may facilitate the protective effect of conventional or DNA vaccines that are used against HSV-2 infection. However, none of these studies address the therapeutic application of gene therapy in vaginal HSV-2 infection. This study presents results that illustrate the potential application of naked DNA administration as a therapeutic adjuvant or option to currently prescribed treatment regimens against vaginal HSV-2 infection.


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

Vero cells and L929 cells were originally obtained from the American Type Culture Collection (Manassas, VA). These cells were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (Life Technologies), an antibiotic/antimycotic solution (Life Technologies), and gentamicin (a final concentration of 20 µg/ml culture media; Life Technologies). The cell cultures were incubated at an atmosphere of 37°C, 5% CO2, and 95% humidity. A clinical isolate of HSV-2 was obtained from University Hospital (New Orleans, LA). An additional HSV-2 isolate (strain MS) was obtained from American Type Culture Collection. Both stocks were prepared as described (30).

Plasmid DNA construct

pCMV-{beta} (vector) was purchased from Clontech Laboratories (Palo Alto, CA). This eukaryotic expression vector (7.2 kb) contains Escherichia coli {beta}-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. pCMV-IFN{alpha}1 was generated as previously described (31). The production and purification of large scale DNA preparations was as described previously, with minor modifications (32).

In vitro virus inhibition assay

Mouse fibroblasts (9.6 x 105 cells) were transfected with 6–10 µg of pCMV (vector) or pCMV-IFN{alpha}1 in six-well cultures per well according to a calcium phosphate transfection protocol (33). Fresh medium was added onto each well 3 h before transfection. Four hours following transfection, the media were removed and the cultures were washed three times with PBS followed by an incubation at 37°C overnight in a 5% CO2, 95% humidity atmosphere. HSV-2 infection at a multiplicity of infection (MOI) of 0.5 or 1.0 was conducted 24 h following transfection. One hour after infection, the supernatant was removed and replaced with fresh culture medium. Two hundred microliters of supernatant were collected at 24 h postinfection (PI) for titration of infectious virus on Vero cells. Each experiment was conducted in triplicate and repeated four times.

Infection and treatment of mice

Female ICR mice (25–34 g, Harlan Sprague-Dawley, Indianapolis, IN) were injected s.c. with 2.0 mg of DepoProvera as previously described (20). Five days following the progesterone administration, the mice 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. and infected vaginally with 2000 PFU of HSV-2 (MS strain) or 8000 PFU of HSV-2 (clinical isolate) in a 20-µl volume of PBS. Twenty-four or 48 h PI, 100 µg of pCMV-IFN{alpha}1, pCMV vector, or vehicle (PBS) was administered into the vaginal lumen in a 20-µl volume. Alternatively, 5 or 500 U of mouse IFN-{alpha}A (PBL Biomedical Laboratories, New Brunswick, NJ) or vehicle was administered into the vaginal lumen 24 h PI in a 20-µl volume. Mice were then monitored for cumulative survival or evaluated at the indicated times PI. 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 Health Sciences Center and The University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committees.

Viral plaque assay

Supernatants from mouse fibroblasts transfected with the pCMV-IFN{alpha}1 or plasmid vector control were collected at 24 h following HSV-2 infection and were titered for infectious virus on Vero cell monolayers in a 36- to 48-h plaque assay. In addition, mice were sacrificed by CO2 asphyxiation at 3 or 7 days PI for evaluation of the effect of the pCMV-IFN{alpha}1 treatment on virus load in the vagina, uterus, spinal cord, and brain stem. Individual samples were excised and placed in RPMI 1640 (Life Technologies) medium containing 5% FBS (Life Technologies) and an antibiotic/antimycotic solution (Life Technologies). The tissue was homogenized in a Ten Broeck homogenizer (Bellco, Vineland, NJ) and following centrifugation (10,000 x g, 5 min), the clarified supernatants were assayed for infectious virus on Vero cell monolayers in a 36- to 48-h plaque assay.

IFN bioassay

Supernatants from L cells transfected with pCMV-{beta} (vector) or pCMV-IFN-{alpha}1 were collected 24 h posttransfection and assayed for IFN levels by bioassay as previously described (34). Supernatants from uninfected cells transfected with pCMV-{beta} (vector) or pCMV-IFN-{alpha}1 served as controls to establish basal levels of secreted biologically active IFN. As an additional control, recombinant IFN-{alpha}A (0.01–1000 U/ml) was applied to L cell cultures in half-log dilutions 24 h before infection with HSV-2 (MOI = 1.0) to establish the IC50 for the recombinant protein against the virus.

Immunohistochemical staining for HSV-2 Ags

Vaginas were collected from the vector- or IFN-{alpha}1 transgene-treated, HSV-2-infected mice on days 3 and 7 PI. The tissue was 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 tissue was 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 bath of water (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-2 Ag was detected using 3,3'-diaminobenzidine (peroxidase) Detection Kit (Ventana) and new fuchsin (Ventana). The stained sections were dehydrated in 100% ethanol, cleared using xylene, and coverslipped with permount.

Staining tissue for {beta}-galactosidase expression

Three days after the administration of plasmid DNA encoding {beta}-galactosidase into the vagina of mice, the animals were sacrificed and vaginal tissue was removed and fixed with 4% paraformaldehyde (Sigma, St. Louis, MO) 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-gal) containing 20 mM potassium ferricyanide, 2 mM MgCl2, 2 mg 5-bromo-4-chloro-3-indolyl-{beta}-D-galactoside/ml, and 120 µl of 10% Nonidet P-40 (Sigma) and 100 µl of 1% sodium deoxycholate per 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.

Quantification of {beta}-galactosidase activity in selected tissue

One or 2 days after the administration of plasmid DNA encoding {beta}-galactosidase intravaginally, the mice were sacrificed and the vagina, iliac-lumbar lymph nodes, and spinal cord were removed, weighed, and homogenized. {beta}-galactosidase levels were quantified in the lysates of homogenized tissue using a commercially available high sensitivity {beta}-galactosidase assay kit (Stratagene, La Jolla, CA). Infected, nontransfected mice served as controls in subtracting background from the experimental tissue.

RT-PCR

To determine the effect of the plasmid DNA construct on the local expression of cytokine, chemokine, and immune cell transcripts in infected tissue, RNA isolation and RT-PCR were conducted using vaginas and iliac-lumbar lymph nodes from uninfected or HSV-2-infected mice administered either the vector or the IFN-{alpha}1 transgene (100 µg/vagina). RNA was extracted from the vaginas and lymph nodes in Ultraspec RNA isolation reagent (Biotecx Laboratories, 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, IFN-{gamma}, and RANTES were as described previously (35). Primers for CD4, CD8, IL-4, IFN-{gamma}-induced NO synthase (iNOS), MHC class I, and TGF-{beta} were obtained from Clontech Laboratories (Palo Alto, CA). PCR primers for DNA polymerase were as follows: 5'-CAG TAC GGC CCC GAG TTC GTG A-3' (sense) and 5'-GTA GAT GGT GCG GGT GAT GTT-3' (antisense) as described (36). PCR primers for HSV-2gB2a were as follows: 5'-CTG GTC AGC TTT CGG TAC GA-3' (sense) and 5'-CAG GTC GTG CAG CTG GTT GC-3' (antisense) as described (37). 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).

ELISA for cytokine proteins in vaginal lavage fluid and tissue

Vaginal lavage fluid (50 µl/vagina) was collected from mice at 7 days PI and assayed for IFN-{gamma} using a commercially available ELISA kit (PBL Biomedical Laboratories, New Brunswick, NJ). In a similar fashion, clarified supernatants from homogenized vaginas or uteri taken from mice at 3 and 7 days PI treated with the plasmid vector or IFN-{alpha}1 transgene were weighed and assessed for IFN-{gamma} by ELISA. The sensitivity of the assay was 30 pg/ml with a corresponding coefficient ranging from .9500 to .9800, n = 6.

Splenic and lumbar lymph node NK activity

Cells were obtained from the spleen or lumbar lymph nodes of plasmid vector- or IFN-{alpha}1 transgene-treated mice on days 3 or 7 PI, enumerated using trypan blue, and evaluated for NK activity using a standard 4-h 51Cr-release microcytotoxicity assay with YAC-1 cells as targets as previously described (38).

Statistics

ANOVA and Tukey’s t test were used to determine significant (p < 0.05) differences between the IFN-{alpha}1 construct- and vector construct-treated groups. 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
 
Transfection of mouse L cells antagonizes HSV-2 replication

To determine the efficacy of the IFN-{alpha}1 transgene against HSV-2 infection under controlled conditions, we initially tested the plasmid construct in an in vitro transfection assay. L cells transfected with the plasmid DNA encoding IFN-{alpha}1 were found to antagonize HSV-2 replication compared with the plasmid vector control-transfected cells at MOI values of 0.5–1.0 (Fig. 1Go). The resistance to infection corresponded to detectable levels of bioactive IFN with supernatants from L cells transfected with the IFN-{alpha}1 transgene possessing high levels of bioactive IFN compared with cells transfected with the plasmid vector alone (Fig. 2Go). Moreover, the amount of bioactive IFN produced by the IFN-{alpha}1-transfected cells did not depend on HSV-2 infection. By comparison, the application of recombinant IFN-{alpha}A to L929 cells before HSV-2 infection was used to establish the IC50 in the bioassay. The curve generated shows an IC50 = 13.9 ± 4.9 U/ml for recombinant IFN-{alpha}A measured against HSV-2 (Fig. 3Go).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 1. IFN-{alpha}1-transfected L929 cells are resistant to HSV-2 infection. L929 cells were transfected with plasmid DNA control (vector) or plasmid DNA encoding IFN-{alpha}1 (14 pg DNA/cell). Following a 24-h incubation, the cells were infected with HSV-2 at an MOI of 0.5 or 1.0. At 24 h PI, supernatants were collected and assayed for infectious virus by plaque assay using monkey Vero cells as the indicator cell. **, p < .01 comparing the transgene- to vector-transfected cells.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. IFN production by L cells transfected with plasmid vector DNA or IFN-{alpha}1 plasmid DNA. L929 cells were transfected with 6 µg of plasmid DNA (vector) or plasmid DNA containing the IFN-{alpha}1 transgene (8.6 pg DNA/cell). Twenty-four hours posttransfection, the cells were infected with HSV-2 (MOI = 0.5). Twenty-four hours PI, the supernatant from each transfected cell culture was collected and assayed for IFN activity using a IFN bioassay. Universal IFN-{alpha} A/D was used to generate a standard curve to determine the content of each unknown. This experiment is a summary of four experiments. Uninfected, transfected cells served as controls for the viral infection.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 3. Murine IFN-{alpha}A antagonizes HSV-2 replication in a dose-dependent fashion. Mouse L929 fibroblasts were incubated 18 h with IFN-{alpha}A (0.01–1000 U/ml) and subsequently infected with HSV-2 at an MOI = 0.5. Twenty-four hours PI, the supernatants were collected and assayed for infectious virus by plaque assay. This figure is a summary of two experiments conducted in triplicate for each dose of IFN-{alpha}A. The IC50 was determined by sigmoidal fit analysis using Microcal Origin software (North Hampton, MA).

 
Plasmid gene expression

Because the in vitro study suggested the IFN-{alpha}1 transgene could induce a state of resistance in targeted cells to HSV-2 infection and generate biologically active IFN, the plasmid DNA encoding IFN-{alpha}1 was evaluated for efficacy against HSV-2 infection in vivo. Initially, the gene was assessed for location following vaginal administration using the vector construct expressing the LacZ reporter gene. The results show the reporter gene to be expressed in the epithelial lining of the vagina 3 days postadministration (Fig. 4Go). To further evaluate the reporter gene expression, tissue from the vagina, regional lymph nodes, and spinal cord were assayed for reporter gene content in HSV-2-infected mice. Due to high background levels, the reporter gene content could not be determined for transfected vaginal tissue. However, both the lymph node and spinal cord were found to express the reporter gene above nontransfected tissue within 24 h posttransfection in HSV-2-infected tissue, suggesting that the plasmid DNA trafficks to the tissue either by locally transfected cells or independent of cells (Fig. 5Go). Because type I IFNs up-regulate MHC class I expression, the expression of the MHC class I gene was used as a surrogate marker to determine the production of a biologically active transgene product in situ. Vaginal tissue transfected with the pCMV-IFN{alpha}1 construct showed a 41% increase in MHC class I gene expression in comparison to the plasmid vector control 24 h posttransfection (Fig. 6Go).



View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 4. Expression of {beta}-galactosidase protein in the vagina. Three days after topical application of the plasmid DNA encoding {beta}-galactosidase, the vagina was removed, fixed in 4% paraformaldehyde, and incubated overnight with the substrate X-gal at 37°C. After overnight incubation, the tissue was fixed in 3.7% formaldehyde in PBS and embedded in paraffin. Tissues were sectioned at 5 µm and stained with nuclear fast red. A, Normal vagina used as a negative control at a magnification of x200. B, Vagina transfected with the plasmid construct encoding {beta}-galactosidase at a magnification of x200. It should be noted that in retrieving the vaginas from the mice the tissue was inverted.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. {beta}-galactosidase reporter gene expression in the lymph node and spinal cord following intravaginal administration. HSV-2-infected mice (n = 2/group/experiment) were administered with pCMV containing the {beta}-galactosidase reporter gene intravaginally (100 µg DNA/20 µl PBS) 24 h PI. Twenty-four and 48 h posttransfection, mice were sacrificed and vagina, iliac-lumbar lymph nodes (LN), and spinal cord (SC) were removed, weighed, and assayed for {beta}-galactosidase activity. The results summarize two experiments. Bars represent the mean ± SEM.

 


View larger version (74K):
[in this window]
[in a new window]
  		FIGURE 6. Administration of a plasmid DNA encoding IFN-{alpha}1 augments MHC class I gene expression in vaginal tissue 24 h PI. Progesterone-treated mice (n = 2/group) were administered 100 µg DNA plasmid vector or plasmid containing the IFN-{alpha}1 transgene. Twenty-four hours PI, the mice were sacrificed and vaginal tissue was assessed for MHC class I mRNA levels by RT-PCR. This figure is a representative of two experiments with similar outcomes. The MHC class I gene expression is enhanced 41% over vector-treated controls based on image analysis using the MHC class I to GAPDH amplicon ratio as the point of reference.

 
Efficacy of administration of plasmid DNA encoding IFN-{alpha}1 against vaginal HSV-2 infection

The therapeutic application of the IFN-{alpha}1 transgene was investigated in mice vaginally infected with HSV-2. The IFN-{alpha}1 transgene applied to the vaginal lumen 24 h PI significantly protected mice from HSV-2-mediated mortality in comparison to all other treated groups of animals (Fig. 7Go). By comparison, 5–500 U of IFN-{alpha}A applied in a similar fashion (i.e., as a single bolus administered 24 h PI) showed no protective effect against HSV-2 infection compared with the saline- or plasmid vector-treated group as measured by cumulative survival (Fig. 7Go). The protective effect of the transgene against HSV-2 infection was time dependent because HSV-2-infected mice administered the transgene 48 h PI showed no increase in survival compared with mice treated with saline or plasmid vector alone (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 7. Plasmid DNA encoding IFN-{alpha}1 enhances the cumulative survival of mice infected with HSV-2. Mice (n = 10–27/group) were infected with HSV-2 (two experiments were conducted using the clinical isolate (8000 PFU/vagina) and two experiments were conducted using strain MS (2000 PFU/vagina) with similar outcomes) and subsequently administered 5.0–500.0 U/vagina IFN-{alpha}A, 100 µg plasmid DNA alone (vector), 100 µg plasmid DNA encoding IFN-{alpha}1, or saline in a 20-µl volume 24 h PI. Mice were then recorded for survival. *, p < 0.05 comparing the IFN-{alpha}1 transgene-treated groups to other groups as determined by the Mann-Whitney U test.

 
Plasmid DNA encoding IFN-{alpha}1 antagonizes viral replication during acute infection

Because the IFN-{alpha}1 transgene enhanced survival of HSV-2-infected mice, the effect on local viral replication was determined. Although there was no difference in the clearance rate (occurring by day 12 PI) comparing the IFN-{alpha}1 transgene-treated to plasmid vector- or saline-treated mice measuring recoverable virus in the vaginal lavage at times PI (data not shown), IFN-{alpha}1 transgene-treated mice revealed a 3-fold reduction in infectious virus isolated from the tissue at 3 days PI compared with the plasmid vector-treated animals (Table IGo). Consistent with this observation, immunohistochemical staining of vaginal tissue for virus found a reduction in HSV-2 Ag expression in the IFN-{alpha}1-treated mice compared with the vector-treated group (Fig. 8Go). By day 7 PI, there was no significant difference in the vaginal viral load recovered from infected mice treated with the plasmid DNA encoding IFN-{alpha}1 compared with the plasmid vector-treated group (Table IGo). However, there was a significant difference in the viral load recovered in the spinal cord (Table IGo). Specifically, 6/6 plasmid vector-treated mice had recoverable HSV-2 in the spinal cord compared with 5/11 IFN-{alpha}1 transgene-treated animals. In addition, there was a 3-fold decrease in the viral load (Table IGo). In a similar fashion, 2/6 plasmid vector-treated mice had recoverable virus in the brain stem day 7 PI compared with 0/11 IFN-{alpha}1 transgene-treated mice. At the molecular level, 4/6 plasmid vector-treated mice had detectable HSV-2 gB mRNA compared with 1/6 mice treated with the IFN-{alpha}1 transgene day 3 PI as determined by RT-PCR (Table IIGo). However, tissue isolated from both groups day 3 PI had similar levels of DNA polymerase mRNA expression (Table IIGo). By day 7 PI, 0/8 mice treated with the IFN-{alpha}1 transgene had detectable levels of HSV-2 gB mRNA compared with 4/6 vector-treated animals (Table IIGo). However, there were again no significant differences in the DNA polymerase mRNA expression comparing the plasmid vector alone- to the IFN-{alpha}1-treated mice (Table IIGo). These results suggest that in the presence of the IFN-{alpha}1 transgene, the capacity of HSV-2 to replicate is diminished as a direct result of reduced expression of gB. HSV-2 gB has a central role in adsorption of virus as well as the penetration of the virus into the host cell (39). Collectively, the transgene reduces the spread as well as the replication of the virus from the vaginal tissue to peripheral and, ultimately, central nervous systems based on a reduction in viral yields in the vagina and spinal cord in line with the progression of the disease.


View this table:
[in this window]
[in a new window]
  		Table I. Administration of plasmid DNA encoding IFN-{alpha}1 24 h PI antagonizes vaginal HSV-2 replication early during the acute infection1

 


View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 8. Intravaginal application of the IFN-{alpha}1 transgene reduces HSV-2 Ag expression in the vagina day 3 PI. Mice were infected with 8000 PFU/vagina HSV-2 and subsequently administered with 100 µg/vagina of either pCMV-{beta} (vector) or pCMV-IFN{alpha}1 24 h PI. Vaginal tissue was obtained from the infected animals 72 h PI and assessed for the presence of HSV-2 Ag by indirect immunohistochemical staining. Uninfected vaginas served as negative controls. Results are a representative of two independent experiments (2 samples/group/experiment). Vector treatment (A) shows multiple sites of viral Ag (indicated by arrows) staining a dark purple compared with the IFN-{alpha}1 transgene treatment (B) with minimal viral Ag detected at a magnification of x200.

 

View this table:
[in this window]
[in a new window]
  		Table II. IFN-{alpha}1 transgene treatment reduces the expression of HSV-2 gB mRNA in the vagina1

 
Effects of IFN-{alpha}1 transgene on the immune response to vaginal HSV-2 infection

IFN-{alpha}{beta} reportedly influences a variety of immune events including the promotion of Th1 cell development and cytokine production (40, 41, 42), activated T cell survival (43), and virally induced/inducible NO synthetase activation (44). These results showing a reduction in HSV-2 replication and Ag expression following the application of the IFN-{alpha}1 transgene in infected mice may also modify the immune response of the host to the infection. To investigate the immune profile of the host during acute vaginal HSV-2 infection, vaginal and lumbar lymph nodes were assayed for expression of IL-4, IL-6, IL-10, IFN-{gamma}, iNOS, RANTES, TGF-{beta}, CD4, and CD8 transcript levels before and at times following HSV-2 infection. IL-4, IL-6, IL-10, and IFN-{gamma} were chosen to determine the predilection for Th1 to Th2 cytokine synthesis, whereas RANTES was chosen as one of the primary chemokines activating T cells. CD4 and CD8 transcript expression were measured to see whether modifications in the infiltration of one of these cell types occur in mice treated with the IFN-{alpha}1 transgene comparing the two plasmid DNA-treated groups. TGF-{beta} expression was assessed based on the amount present in uninfected vaginal tissue as well as the immunomodulatory capacity of TGF-{beta} on adaptive immune responses. With the exception of CD4, IL-6, RANTES, and TGF-{beta}, there was no detectable level of cytokine or CD8 immune cell transcripts in the vagina of uninfected mice (Table IIIGo). By day 3 PI, there was a significant rise in the expression of CD4 immune cell and RANTES transcripts in both plasmid vector- and IFN-{alpha}1 transgene-treated groups (Table IIIGo). With the exception of IL-4, all other cytokine and CD8 immune cell transcripts were detectable to varying degrees at this time point (Table IIIGo). By day 7 PI, IL-6, IFN-{gamma}, and iNOS mRNA levels increased in both groups of treated animals compared with day 3 PI levels. CD4 mRNA levels dropped considerably at the day 7 PI time point compared with day 3 PI levels in both plasmid vector- and IFN-{alpha}1 transgene-treated groups with no significant differences between the groups. However, CD8 expression was different comparing the plasmid vector- and IFN-{alpha}1 transgene-treated mice at day 7 PI in that 6/6 plasmid vector-treated mice had detectable levels of CD8 mRNA expressed in the vaginal tissue compared with 1/6 from the IFN-{alpha}1 transgene-treated group (Table IIIGo).


View this table:
[in this window]
[in a new window]
  		Table III. Transcript levels in the vagina of HSV-2-infected mice1

 
Similar to the infected vaginal tissue, uninfected lumbar lymph nodes expressed minimal levels of cytokines with the exception of IL-6, iNOS, RANTES, and TGF-{beta} (Table IVGo). As expected, CD4 and CD8 transcripts were prevalent in lymph node samples. The degree of expression of these transcripts is relative owing to the fidelity of the primer pair for the targeted amplicon because under basal conditions, there are more (percentage and absolute number) CD4+ T lymphocytes compared with CD8+ T cells in the lumbar lymph nodes (data not shown). At day 3 PI in both groups of treated mice, all transcript levels increased. By day 7 PI, CD4, CD8, IFN-{gamma}, and RANTES transcript levels were lower in comparison to day 3 PI levels, whereas IL-6, IL-10, iNOS, and TGF-{beta} levels were either similar to or elevated in comparison to those levels measured day 3 PI in both groups of treated mice (Table IVGo). However, there were no significant differences in the expression of cytokine or immune cell transcripts comparing the IFN-{alpha}1 transgene- to plasmid vector-treated group of animals at day 3 or 7 PI.


View this table:
[in this window]
[in a new window]
  		Table IV. Transcript levels in the lumbar lymph nodes of HSV-2-infected mice1

 
Mice receiving plasmid DNA encoding IFN-{alpha}1 show reduced IFN-{gamma} protein levels in vaginal tissue early during acute HSV-2 infection

IFN-{gamma} has been identified as a central mediator in enhancing T cell-mediated clearance mechanisms of HSV-2 from infected vaginal tissue (22). Likewise, IFN-{gamma}-stimulated macrophages produce NO that has been associated with anti-viral effects (45) including HSV infection (46). Therefore, IFN-{gamma} protein levels were measured in the vaginal tissue at times following HSV-2 infection. At 3 days PI, there was twice as much IFN-{gamma} in the vaginal tissue obtained from mice treated with the plasmid vector in comparison to IFN-{alpha}1 transgene-treated animals (Fig. 9Go). However, by day 7 PI, there was no significant difference in the level of IFN-{gamma} comparing the two groups of DNA-treated mice. Likewise, there was no difference in the level of IFN-{gamma} in vaginal lavage fluid comparing the plasmid vector (384 ± 94 pg/ml)- to the IFN-{alpha}1 transgene (378 ± 151 pg/ml)-treated groups at day 7 PI. IFN-{gamma} levels were also detected in the uteri of HSV-2-infected mice at day 7 but not day 3 PI. However, there were no differences in the levels of IFN-{gamma} comparing the IFN-{alpha}1 transgene (989 ± 134 pg/g tissue)- to plasmid vector (755 ± 161 pg/g tissue)-treated groups.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 9. HSV-2 infection elicits IFN-{gamma} within the vaginal tissue. Mice (n = 6–11/group/timepoint) were infected with 8000 PFU HSV-2 intravaginally and treated 24 h later with 100 µg of plasmid DNA alone (vector) or plasmid DNA encoding IFN-{alpha}1. At 3 or 7 days PI, mice were sacrificed and vaginal tissue was excised and homogenized in RPMI 1640. The supernatants from clarified homogenates were assayed for IFN-{gamma} content by ELISA in duplicate. Day 7 PI vaginal lavages were collected and assayed for IFN-{gamma} content by ELISA as well. *, p < 0.05 comparing the vector- to IFN-{alpha}1 transgene-treated group day 3 PI as determined by ANOVA and Tukey’s t test. This figure is a summary of two experiments.

 
Vaginal HSV-2 infection augments splenic but not lymph node NK activity

NK cells are responsive to IFN and have been shown to participate in controlling viral infections including HSV (47, 48). Therefore, NK cell activity was surveyed during vaginal HSV-2 infection in both the spleen and lumbar lymph nodes. There were no differences in the level of NK activity in either the lymph node or spleen comparing mice treated with the plasmid vector control to mice receiving the IFN-{alpha}1 transgene (Fig. 10Go). Likewise, there was no difference in the level of cytolytic activity comparing the day 3 to day 7 PI in the lymph node population. However, there was a significant rise in the splenic NK activity comparing day 3 to day 7 PI in either vector- or transgene-treated groups (Fig. 10Go).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 10. Splenic NK levels increase in response to vaginal HSV-2 infection day 7 PI. Mice (n = 6/group/time point) were infected with HSV-2 (8000 PFU/vagina) and treated with plasmid DNA alone (vector) or plasmid DNA encoding IFN-{alpha}1 24 h PI. Cells obtained from the iliac-lumbar lymph nodes or spleen at 3 or 7 days PI were assayed for NK activity. The results show percent cytolytic activity at a 100:1 E:T ratio. Similar results were found at all E:T ratios (i.e., 50:1, 25:1, and 12:1) tested. *, p < 0.05 comparing the day 7 to day 3 splenic NK activity as determined by ANOVA and Tukey’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies have shown the successful implementation of DNA vaccines expressing viral components in animal models as a prophylactic means to control lethal and recurrent HSV infection (49, 50, 51). However, this study illustrates the utility of cytokine gene therapy as a therapeutic approach to control a localized mucosal infection. The efficacy of the transgene is observed both in vitro and in vivo as measured by, among other parameters, viral replication. Because the in vitro study uses only transfected cells, the effect was direct, antagonizing viral replication at the transcriptional, translational, or posttranslational level. A previous study measuring the efficacy of pCMV-IFN{alpha}1-transfected L cells against HSV-1 found the protective effect to be modest with a 50% reduction in virus yield at an MOI of 0.5 and no significant reduction in virus replication at a higher (i.e., MOI = 1.0) virus inoculum (52). In that study, the transgene was found to antagonize viral transcription with a reduction in HSV-1 immediate-early, early, and late gene expression. In this study, the resistance of IFN-{alpha}1-transfected L929 cells to HSV-2 infection is more potent in comparison to the HSV-1 infection with >50% reduction in recoverable virus at an MOI = 1.0. The resistance to infection correlated with the production of bioactive IFN, suggesting that the transfected cells secrete a biologically active transgene product. Because only ~0.4% of the transfected cells express the transgene (52), the active secretion of IFN is required to elicit a state of resistance to HSV-2 in the recipient cells in culture.

The in vivo application of the transgene was found to enhance the cumulative survival of mice to vaginal HSV-2 infection in a time-dependent fashion by antagonizing viral replication. Surprisingly, recombinant IFN-{alpha}A (5–500 U/vagina) applied at the identical time point as the transgene was not found to antagonize HSV-2 infection as measured by mortality. Because the recombinant IFN-{alpha} does antagonize HSV-2 replication in vitro, it was anticipated to successfully block HSV-2-induced mortality in vivo. The acidity of the vaginal secretions would not modify the activity of the molecule due to its acid-stable nature.

The transgene itself or in association with the plasmid backbone may elicit a potent host response to the viral pathogen. Unlike recombinant IFN applied and presumably acting locally, the transgene applied intravaginally is expressed not only in the local stratified epithelial layer of the vagina but is expressed in the regional lymph nodes and spinal cord within 24 h posttransfection. The trafficking of the transgene to sites distant from the original site of application may involve tissue macrophages or Langerhans cells (53). Consistent with this observation, the application of plasmid DNA intranasally has been identified in the bone marrow, spleen, draining lymph nodes, blood, liver, and lung, suggesting that the dissemination of the DNA is via the bloodstream (54). The expression of the transgene at a location distant from the original site of application may result in a "priming" effect as previously reported for IFN-{alpha} (55). Combined with the anti-viral activity of IFN-{alpha}, the DNA backbone of the plasmid may augment the effects of the transgene acting as an adjuvant either in the uptake of the transgene by immune cells or in driving the immune response (56, 57). However, because the plasmid vector DNA does not protect the mice against HSV-2-induced mortality, the plasmid DNA alone does not contribute to the efficacy in the absence of the transgene.

Mice treated with the IFN-{alpha}1 transgene showed a significant reduction in viral load and HSV-2 Ag expression during the early period of the acute infection (i.e., day 3–4 PI) in comparison to recipients receiving the plasmid vector. By reducing the viral load during the initial phase of infection, the transgene may allow the host additional time to respond to the pathogen in mounting a vigorous immune response. The reduction in virus during the initial stages of infection correlated with lower levels of IFN-{gamma} in the vaginal tissue of the transgene-treated animals. We interpret these results to suggest that IFN-{gamma} reflects the inflammation manifested by HSV-2 replication and tissue pathology and should not be misconstrued as solely a protective cytokine. These data are consistent with those found in ocular HSV-1 disease in which IFN-{gamma} is found to exacerbate the inflammation associated with the acute infection (58). However, similar to HSV-1 infection, IFN-{gamma} plays an important role in vaginal HSV-2 clearance (22). This suggestion is evident in this study in that by day 7 PI, levels of IFN-{gamma} in the vaginal tissue are elevated compared with day 3 PI levels in both groups of treated mice, and viral titers and viral Ag in the tissue have significantly dropped. The source of IFN-{gamma} could include any one of a number of resident or infiltrating leukocytes following HSV-2 infection including neutrophils (59). However, neutrophil depletion in HSV-2-infected mice does not alter the level of IFN-{gamma} (16), suggesting a more likely source to be T lymphocytes and NK cells.

Although the viral load in the vaginal tissue was not significantly different at day 7 PI comparing the plasmid vector- to IFN-{alpha}1 transgene-treated mice, there was a significant reduction in recoverable virus in the spinal cord and the complete absence of virus in the brain stem from IFN-{alpha}1 transgene-treated animals compared with the vector DNA-treated group. The finding of transgene trafficking to the spinal cord within 24 h PI most likely contributed in establishing an anti-viral state within this tissue. Only 1/11 control-treated mice had detectable HSV-2 in the spinal cord 3 days PI suggesting that HSV-2 retrograde transport to the dorsal root ganglion and subsequently spinal cord does not occur before 48 h after vaginal administration. Therefore, the expression of the transgene in the spinal cord 24 h posttransfection effectively antagonizes HSV-2 replication in this tissue. Likewise, the reduction in viral replication in the vagina early in the primary infection also impacts on the amount of virus that reaches the central nervous system (i.e., spinal cord). Accordingly, less virus replicates at the initial site of infection resulting in a reduction in virus that reaches the central nervous system that has been pre-exposed to the transgene. Ultimately, this cascade results in the effective blockade of virus reaching the spinal cord and brain and eliciting central nervous system inflammation and death.

Measuring cytokine, chemokine, and immune cell transcripts in vaginal tissue and lymph nodes did not result in discernable differences between the IFN-{alpha}1 transgene- and plasmid vector-treated groups with the exception of CD8 mRNA. The drop in the frequency of detecting CD8 transcripts in the IFN-{alpha}1 transgene-treated mice at day 7 PI may be the result of viral clearance as indicated in the absence of gB transcript expression. Based on the near absence of Th2 cytokines in the vagina (e.g., IL-4 and IL-10), the predominant cytokine expression is a Th1 profile following HSV-2 infection. In contrast, the regional (iliac-lumbar) lymph nodes expressed both Th1 and Th2 cytokine mRNA as determined by RT-PCR. However, the role regional lymph nodes play in vaginal HSV-2 infection is somewhat ambiguous as indicated by NK activity. Specifically, NK cell activity did not appreciably rise in cells isolated from the lymph node comparing day 7 to day 3 PI. Yet, splenic NK activity significantly rose in both transgene- and plasmid vector-treated groups comparing day 7 to day 3 PI. The increase in splenic NK activity may be the result of trafficking of effector cells from the vagina via the blood to the spleen or, alternatively, may reflect the response of the host to HSV-2 spread from the vaginal epithelium to surrounding tissue.

Collectively, this study illustrates the therapeutic potential of applying plasmid DNA encoding a type I IFN (IFN-{alpha}1) against vaginal HSV-2 infection upon the administration of the transgene intravaginally. A similar protective effect has also been reported when vaccine or DNA immunization against vaginal HSV infection has been applied locally (60, 61, 62). Clearly, additional studies are necessary to further define the role of local and regional immune responses to vaginal HSV-2 infection to optimize vaccine and DNA immunization protocols. This study focused on the application of naked DNA applied in one bolus PI as opposed to multiple applications or delivery systems such as cationic lipids or gene gun (63). Nevertheless, the initial success of this application warrants further studies to optimize conditions that facilitate maximum expression of the transgene in the targeted tissue.


   Acknowledgments
 
We thank Cathy Sprencel for her technical assistance and Dr. Iain L. Campbell (Dept. Neuropharmacology, The Scripps Research Institute, La Jolla, CA) for initially providing the pCMV-IFN{alpha}1 construct.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants EY12409 (to D.J.J.C.). P.H. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (HA2993/1-1). Back

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

3 Abbreviations used in this paper: HSV-2, HSV type 2; g, glycoprotein; pCMV, plasmid CMV; MOI, multiplicity of infection; PI, postinfection; iNOS, IFN-{gamma}-induced NO synthase. Back

Received for publication May 9, 2000. Accepted for publication November 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Whitley, R. J., B. Roizman. 1997. Herpes simplex viruses. D. D. Richman, and R. J. Whitley, and B. Roizman, eds. Clinical Virology 375. Churchill Livingstone, New York.
  2. Siegel, D., E. Golden, A. E. Washington, S. A. Morse, M. T. Fullilove, J. A. Catania, B. Marin, S. B. Hulley. 1992. Prevalence and correlates of herpes simplex infections: the population-bases AIDS in multiethnic neighborhoods study. J. Am. Med. Assoc. 268:1702.[Abstract]
  3. Fleming, D. T., G. M. McQuillan, R. E. Johnson, A. J. Nahmias, S. O. Aral, F. K. Lee, M. E. St. Louis.. 1997. Herpes simplex virus type 2 in the United States, 1974 to 1994. N. Engl. J. Med. 337:1105.[Abstract/Free Full Text]
  4. Langenberg, A. G. M., L. Corey, R. L. Ashley, W. P. Leong, S. E. Straus. 1999. A prospective study of new infections with herpes simplex virus type 1 and 2. N. Engl. J. Med. 341:1432.[Abstract/Free Full Text]
  5. Lafferty, W. E., R. W. Coombs, J. Benedetti, C. Critchlow, L. Corey. 1987. Recurrences after oral and genital herpes simplex virus infections: influence of site of infection and viral type. N. Engl. J. Med. 316:1444.[Abstract]
  6. Holmberg, S. D., J. A. Stewart, A. R. Gerber. 1988. Prior herpes simplex virus type 2 infection as a risk factor for HIV infection. Am. J. Med. 259:1048.
  7. Staam, W. E., H. H. Handsfield, A. M. Rompalo, R. L. Ashley, P. L. Roberts, L. Corey. 1988. The association between genital ulcer disease and acquisition of HIV infection in homosexual men. J. Am. Med. Assoc. 260:1429.[Abstract]
  8. Keet, I. P., F. K. Lee, G. J. van Griensven, J. M. Lange, A. Nahmias, R. A. Coutinho. 1990. Herpes simplex virus type 2 and other genital ulcerative infections as a risk factor for HIV-1 acquisition. Genitourin. Med. 66:330.[Medline]
  9. Hook, E. W. 3rd, R. O. Cannon, A. J. Nahmias, F. F. Lee, Jr C. H. Campbell, D. Glasser, T. C. Quinn. 1992. Herpes simplex virus infection as a risk factor for human immunodeficiency virus infection in heterosexuals. J. Infect. Dis. 165:251.[Medline]
  10. McFarland, W., L. Gwanzura, M. T. Bassett, R. Machekano, A. S. Latif, C. Ley, J. Parsonnet, R. L. Burke, D. Katzenstein. 1999. Prevalence and incidence of herpes simplex virus type 2 infection among male Zimbabwean factory workers. J. Infect. Dis. 180:1459.[Medline]
  11. Mestecky, J., P. N. Fultz. 1999. Mucosal immune system of the genital tract. J. Infect. Dis. 179:S470.
  12. McDermott, M. R., B. J. Smiley, P. L. J. Brais, H. Rudzroga, J. Bienestock. 1984. Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2. J. Virol. 51:747.[Abstract/Free Full Text]
  13. Parr, M. B., L. Kepple, M. R. McDermott, M. D. Drew, J. J. Bozzola, E. L. Parr. 1994. A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2. Lab. Invest. 70:369.[Medline]
  14. King, N. J. C., E. L. Parr, M. B. Parr. 1998. Migration of lymphoid cells from vaginal epithelium to iliac nodes in relation to vaginal infection by herpes simplex virus type 2. J. Immunol. 160:1173.[Abstract/Free Full Text]
  15. Parr, M. B., E. L. Parr. 1997. Protective immunity against HSV-2 in the mouse vagina. J. Reprod. Immunol. 36:77.[Medline]
  16. Milligan, G. N.. 1999. Neutrophils aid in protection of the vaginal mucosae of immune mice against challenge with herpes simplex virus type 2. J. Virol. 73:6380.[Abstract/Free Full Text]
  17. McDermott, M. R., C. H. Goldsmith, K. L. Rosenthal, L. J. Brais. 1989. T lymphocytes in genital lymph nodes protect mice from intravaginal infection with herpes simplex virus type 2. J. Infect. Dis. 159:460.[Medline]
  18. Milligan, G. N., D. I. Bernstein. 1995. Analysis of herpes simplex virus-specific T cells in the murine female genital tract following genital infection with herpes simplex virus type 2. Virology 212:481.[Medline]
  19. Posavad, C. M., D. M. Koelle, L. Corey. 1996. High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex virus in persons with genital herpes. J. Virol. 70:8165.[Abstract]
  20. Parr, M. B., E. L. Parr. 1998. Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes. J. Virol. 72:2677.[Abstract/Free Full Text]
  21. Posavad, C. M. D. M., M. F. Shaughnessy Koelle, L. Corey. 1997. Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses. Proc. Natl. Acad. Sci. USA 94:10289.[Abstract/Free Full Text]
  22. Milligan, G. N., D. I. Bernstein. 1997. Interferon-{gamma} enhances resolution of herpes simplex virus type 2 infection of the murine genital tract. Virology 229:259.[Medline]
  23. Milligan, G. N., D. I. Bernstein. 1995. Generation of humoral immune responses against herpes simplex virus type 2 in the murine female genital tract. Virology 206:234.[Medline]
  24. Parr, M. B., G. R. Harriman, E. L. Parr. 1998. Immunity to vaginal HSV-2 infection in immunoglobulin A knockout mice. Immunol. 95:208.[Medline]
  25. Stanberry, L. R., D. I. Bernstein, R. L. Burke, C. Pachl, M. G. Myers. 1987. Vaccination with recombinant herpes simplex virus glycoproteins: protection against initial and recurrent genital herpes. J. Infect. Dis. 177:1069.
  26. Morrison, L. A., X. J. Da Costa, D. M. Knipe. 1998. Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune responses and protection from genital challenge. Virology 243:178.[Medline]
  27. Bourne, N., L. R. Stanberry, D. I. Bernstein, D. Lew. 1996. DNA immunization against experimental genital herpes simplex virus infection. J. Infect. Dis. 173:800.[Medline]
  28. Sin, J.-I., J. J. Kim, R. L. Arnold, K. E. Shroff, D. McCallus, C. Pachuk, S. P. McElhiney, M. W. Wolf, S. J. Pompa-de Bruin, T. J. Higgins, et al 1999. IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge. J. Immunol. 162:2912.[Abstract/Free Full Text]
  29. Sin, J.-I., J. J. Kim, J. D. Boyer, R. B. Ciccarelli, T. J. Higgins, D. B. Weiner. 1999. In vivo modulation of vaccine-induced immune responses toward a Th1 phenotype increases the potency and vaccine effectiveness in a herpes simplex virus type 2 mouse model. J. Virol. 73:501.[Abstract/Free Full Text]
  30. Harland, J., S. M. Brown. 1998. HSV growth, preparation, and assay. S. M. Brown, and A. R. MacLean, eds. Herpes Simplex Virus Protocols 1. Humana Press, Totowa, NJ.
  31. Noisakran, S., I. L. Campbell, D. J. J. Carr. 1999. Ectopic expression of DNA encoding IFN-{alpha}1 in the cornea protects mice from herpes simplex virus type 1-induced encephalitis. J. Immunol. 162:4184.[Abstract/Free Full Text]
  32. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. C. Nolan, ed. In Molecular Cloning: A Laboratory Manual Vol 1:133. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  33. Chen, C., H. Okayama. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745.[Abstract/Free Full Text]
  34. Ishakawa, R., C. A. Biron. 1993. IFN induction and associated changes in splenic leukocyte distribution. J. Immunol. 150:3713.[Abstract]
  35. 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]
  36. Rogers, B. B., S. L. Josephson, S. K. Mak. 1991. Detection of herpes simplex virus using the polymerase chain reaction followed by endonuclease cleavage. Am. J. Pathol. 139:1.[Abstract]
  37. Cone, R. W., A. C. Hobson, Z. Brown, R. Ashley, S. Berry, C. Winter, L. Corey. 1994. Frequent detection of genital herpes simplex virus DNA by polymerase chain reaction among pregnant women. J. Am. Med. Assoc. 272:792.[Abstract]
  38. Carr, D. J. J., S. Mayo, B. M. Gebhardt, P. Porter. 1994. Central {alpha}-adrenergic involvement in morphine-mediated suppression of splenic natural killer activity. J. Neuroimmunol. 53:53.[Medline]
  39. Spear, P. G.. 1985. Glycoproteins specified by herpes simplex viruses. B. Roizman, ed. In The Herpesviruses vol. 3:315. Plenum Publishing, New York.
  40. Parronchi, P., M. De Carli, R. Manetti, C. Simonelli, S. Sampognaro, M.-P. Piccinni, D. Macchia, E. Maggi, G. Del Prete, S. Romagnani. 1992. IL-4 and IFN ({alpha} and {gamma}) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones. J. Immunol. 149:2977.[Abstract]
  41. Schandene, L., G. F. Cogan, P. Stordeur, A. Crusiaux, B. Kennes, S. Romagnani, M. Goldman. 1996. Recombinant interferon-{alpha} selectively inhibits the production of interleukin-5 by human CD4+ T cells. J. Clin. Invest. 97:309.[Medline]
  42. McRae, B. L., R. T. Semnani, M. P. Hayes, G. A. van Seventer. 1998. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J. Immunol. 160:4298.[Abstract/Free Full Text]
  43. Marrack, P., J. Kappler, T. Mitchell. 1999. Type I interferons keep activated T cells alive. J. Exp. Med. 189:521.[Abstract/Free Full Text]
  44. Schoneboom, B. A., J. S. Lee, F. B. Brieder. 2000. Early expression of IFN-{alpha}{beta} and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice. J. Interferon Cytokine Res. 20:205.[Medline]
  45. Karupiah, G., Z. Xie, R. M. L. Buller, C. Nathan, C. Duate, J. MacMicking. 1993. Inhibition of viral replication by interferon-{gamma}-induced nitric oxide synthase. Science 261:1445.[Abstract/Free Full Text]
  46. MacLean, A., X.-Q. Wei, F.-P. Huang, U. A. H. Al-Alem, W. L. Chan, F. Y. Liew. 1998. Mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced Th1 cell responses. J. Gen. Virol. 79:825.[Abstract]
  47. Gidlund, M., A. Orn, H. Wigzell, A. Senik, I. Gresser. 1978. Enhanced NK activity in mice injected with interferon and interferon inducers. Nature 273:759.[Medline]
  48. Habu, S., K. Akamatsu, N. Tamaoki, K. Okumura. 1984. In vivo significance of NK cell on resistance to virus (HSV-1) infections in mice. J. Immunol. 133:2743.[Abstract]
  49. Bourne, N., L. R. Stanberry, D. I. Bernstein, D. Lew. 1996. DNA immunization against experimental genital herpes simplex virus infection. J. Infect. Dis. 173:800.
  50. Kriesel, J. D., S. L. Spruance, R. A. Daynes, B. A. Araneo. 1996. Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease. J. Infect. Dis. 173:536.[Medline]
  51. Ghiasi, H. R., R. Kaiwar, A. Nesburn, S. Slanina, S. Wechsler. 1992. Baculovirus-expressed glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) protects mice against lethal intraperitoneal and lethal ocular HSV-1 challenge. Virology 188:469.[Medline]
  52. Noisakran, S., I. L. Campbell, D. J. J. Carr. 2000. IFN-{alpha}1 plasmid construct affords protection against HSV-1 infection in transfected L929 fibroblasts. J. Interferon Cytokine Res. 20:107.[Medline]
  53. La Cava, A., R. Billetta, G. Gaietta, D. B. Bonnin, S. M. Baird, S. Albani. 2000. Cell-mediated DNA transport between distant inflammatory sites following intradermal DNA immunization in the presence of adjuvant. J. Immunol. 164:1340.[Abstract/Free Full Text]
  54. Chun, S., M. Daheshia, S. Lee, S. K. Eo, B. T. Rouse. 1999. Distribution fate and mechanism of immune modulation following mucosal delivery of plasmid DNA encoding IL-10. J. Immunol. 163:2393.[Abstract/Free Full Text]
  55. Friedman, R. M.. 1966. Effect of interferon treatment on interferon production. J. Immunol. 98:872.
  56. Jakob, T., P. S. Walker, A. M. Krieg, M. C. Udey, J. C. Vogel. 1999. 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]
  57. Chattergoon, M. A., T. M. Robinson, J. D. Boyer, D. B. Weiner. 1998. 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]