In vivo antiviral efficacy of a dipeptide acyclovir prodrug,val-val-acyclovir,against HSV-1 epithelial and stromal keratitis in the rabbit eye model

PURPOSE: A dipeptide prodrug of the antiviral nucleoside acyclovir (ACV), val-val-ACV (VVACV), was evaluated in vivo as a potential drug candidate for improving antiviral efficacy against herpetic epithelial and stromal keratitis. METHODS: The effect of 1% VVACV on epithelial keratitis induced by inoculation of HSV-1 strain McKrae (25 microL of 10(5) plaque-forming units [PFU]) in the scarified rabbit cornea and stromal keratitis induced by intrastromal injection of HSV-1 strain RE (10 microL of 10(5) PFU) was compared with that of 1% trifluorothymidine (TFT) and balanced salt solution as the vehicle control. Both eyes of 10 rabbits were used in each treatment group. Lesions were evaluated by slit lamp examinations over a 2-week period after infection. Aqueous humor samples and corneas were analyzed for drug concentrations at the end of each experiment. Cytotoxicity of VVACV in comparison with val-acyclovir (VACV), ACV, and TFT was evaluated in cellular proliferation assays. RESULTS: The dipeptide prodrug VVACV demonstrated excellent activity against HSV-1 in the rabbit epithelial and stromal keratitis models: 1% VVACV was as effective as 1% TFT. The prodrug was also less cytotoxic than TFT, which is the only effective drug currently licensed and routinely used for topical treatment of ocular herpes infections in the United States. CONCLUSIONS: The less cytotoxic and highly water-soluble prodrug VVACV, which showed excellent in vivo activity against HSV-1 in rabbit epithelial and stromal keratitis, is a promising drug candidate for treatment of ocular HSV infections.     

Efficacy of glycoprotein inhibitors alone and in combination with trifluridine in the treatment of murine herpetic keratitis

The present study examined the anti-herpetic effect of the glycoprotein inhibitors, hydroxynorvaline and 2-deoxyglucose, alone and in combination with trifluridine on murine ocular herpes. Following ocular inoculation with a large dose of HSV-1 RE strain (10(6) pfu), ICR mice were treated during the acute infection with different therapeutic regimens, and their efficacy was evaluated by ocular virus titers, clinical grading of blepharo-conjunctivitis and histological evaluation of stromal keratitis and iridocyclitis. The results following a large dose HSV-1 inoculum demonstrated that trifluridine was the best single therapeutic agent. Hydroxynorvaline and 2-deoxyglucose had no effect at all. Combination therapy of the glycoprotein inhibitors with trifluridine was no better than trifluridine alone. The mouse HSV-1 keratitis model proved to be an effective, economical alternative to the rabbit model for the evaluation of new antiviral agents.     

Protective immunity against herpetic ocular disease in an outbred mouse model

Immunization of outbred mice by subdermal (footpad) inoculation with the F strain of herpes simplex virus type 1 (HSV-1) induces an immune response which protects the animals against herpetic ocular disease and encephalitis, and reduces the incidence of latent trigeminal ganglion infections following corneal challenge with the RE strain of HSV type 1. The protective effects are proportional to the dose of virus used for immunization. Heat-killed virus preparations also protected the mice against encephalitis and stromal keratitis, but failed to prevent epithelial keratitis and establishment of latency.     

Ocular safety and efficacy of an HSV-1 gD vaccine during primary and latent infection

One potential complication of systemic herpes simplex virus (HSV) vaccination is that subsequent ocular infection may lead to increased immunogenic corneal scarring. Therefore, V52, a genetically engineered vaccinia virus that expresses the HSV-1 glycoprotein gD, was tested for ocular safety and for protection against ocular challenge with a stromal-disease-producing strain (McKrae) of HSV-1. To maximize immune response, rabbits were vaccinated by a series of inoculations. V52-vaccinated rabbits developed significant HSV-1 neutralizing antibody titers; however, they were not as high as those induced by vaccination with live HSV-1 McKrae. One month after the final vaccination, all rabbits were challenged ocularly. Eyes were monitored for 35 days for epithelial keratitis, stromal keratitis, and iritis. In no case was epithelial keratitis, stromal keratitis, or iritis significantly exacerbated by vaccination. The gD V52 recombinant vaccine provided protection against HSV-1 induced epithelial keratitis (P = 0.02) and long-term stromal scarring (P = 0.04). There was no significant reduction in the incidence of trigeminal ganglionic latency in the vaccinated rabbits (P greater than 0.05). Thus, our results indicate that V52, a gD recombinant vaccine probably is safe with regard to corneal scarring, and may provide a small amount of protection against ocular HSV-1 infection. The amount of protection provided was less than that reported in mice and guinea pigs. This suggests that to provide high levels of ocular protection in rabbits (and probably in humans), HSV-1 vaccines may have to elicit a more vigorous immune response than that produced by normal HSV-1 infection.     

Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits

Spontaneous ocular shedding and adrenergic induction of ocular shedding were examined in rabbits infected with ten strains of herpes simplex virus type 1 (HSV-1): McKrae, KOS, F, Rodanus, 17 Syn+, RE, E-43, SC-16, MacIntyre, and CGA-3. All ocular inoculations were with 50 microliter of HSV-1 with titers between 1-10 X 10(6) PFU/ml. All corneas, except those that received the McKrae strain, were scarified. Acute ocular infection was determined by slit-lamp biomicroscopy. Dendritic keratitis or geographic ulcers developed in all eyes of all rabbits within 10 days after ocular inoculation. All eyes of all surviving rabbits were swabbed for 20 consecutive days during days 20-39 postinoculation (PI). On PI day 19, no active lesions were present as judged by slit-lamp biomicroscopy. Ocular tear film was collected on a Dacron-tipped swab and placed on primary rabbit kidney cell monolayers. The cell monolayers were monitored for cytopathic effects consistent with HSV-1 infection. Spontaneous HSV-1 shedding was detected in some eyes from all groups of latently infected rabbits, except those infected with CGA-3. Spontaneous shedding (positive swabs/total swabs) of the other nine strains ranged from 0.7% to 15.7%. After PI day 42, the rabbit eyes received 6-hydroxydopamine by iontophoresis, followed for 5 days by topical application of 2% epinephrine. This procedure results in induced HSV-1 ocular shedding for a duration of 3-5 days in rabbits infected with the McKrae strain. In rabbits latently infected with KOS, F, RE, MacIntyre, and CGA-3, no induced HSV-1 shedding was detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus stromal keratitis is not titer-dependent and does not correlate with neurovirulence

We developed a murine model of ocular herpes simplex virus (HSV) disease which is particularly suited for testing stromal keratitis because most animals show some evidence of infection. Using this model, we characterized the ocular disease patterns caused by ten recent low-passage clinical isolates of HSV-1, as well as those caused by the established laboratory strains HSV-1 KOS and HSV-2 333. Viral strains were evaluated for their ability to cause stromal keratitis, blepharitis, vascularization of the cornea, and mortality. The model was not useful for scoring epithelial keratitis. The ocular disease caused by the recent isolates ranged from very mild disease to severe stromal keratitis. Some of the recent isolates caused disease as severe as the two laboratory strains. A comparison of the virulence characteristics expressed by various HSV strains indicated that the ability to cause stromal disease was correlated with vascularization of the cornea (correlation coefficient = 0.797, P less than 0.001) and was not correlated with the neurovirulence of the strains (correlation coefficient 0.045, P greater than 0.05). The severity of stromal keratitis was not dependent on the amount of inoculum over the range tested and a strain causing severe stromal keratitis caused severe ocular disease even when mixed with a nonstromal strain at ratios of 10:1, 100:1, and 1000:1.     

The effect of immunization with herpes simplex virus glycoprotein D fused with interluekin-2 against murine herpetic keratitis

PURPOSE: To evaluate the effect of vaccination with fusion protein(gD-IL-2) consisting of herpes simplex type1(HSV-1) glycoprotein D(gD) and human interleukin-2(IL-2), and the effect of plasmid DNA vaccine encoding gD-IL-2 against murine herpetic keratitis. METHODS: Plasmid containing gD-IL-2(pHDL-neol) was constructed, and gD-IL-2 peptide was purified. BALB/c mice were injected twice hypodermally or subconjunctivally with 1 microgram/0.1 ml of gD-IL-2 peptide, or twice subconjunctivally with 90 micrograms/0.05 ml of gD-IL-2 plasmid DNA. Neutralizing antibody titer and delayed-type hypersensitivity (DTH) against HSV-1 were measured. Immunized mice were challenged with CHR3 strain of HSV-1 via the cornea. The clinical picture of epithelial and stromal keratitis was scored. RESULTS: Stromal keratitis was inhibited in gD-IL-2 peptide- or gD-IL-2 DNA-immunized mice, but epithelial keratitis was not. It was confirmed that plasmid gD-IL-2 elicited significant serum virus neutralizing titer and DTH response. CONCLUSION: Vaccination with gD-IL-2 was effective against herpetic keratitis.     

Preventive effect of local plasmid DNA vaccine encoding gD or gD-IL-2 on herpetic keratitis

PURPOSE: The goal of this study was to evaluate the effectiveness of a local plasmid DNA vaccine encoding herpes simplex virus (HSV) type 1 glycoprotein D (gD) or gD-interleukin (IL)-2 (chimeric gene of gD and human IL-2) in preventing murine herpetic keratitis. METHODS: Plasmids containing gD (pHSDneo1), gD-IL-2 (pHDLneo1), or vaccine vector (pHSGneo) were injected subconjunctivally with BALB/c mice on days 0 and 7 (90 microgram x 2). Immunization was indicated by positive virus-neutralizing antibody titer, swollen pinna (due to delayed-type hypersensitivity [DTH] reaction), and release of (51)Cr from splenic and/or local cytotoxic effector cells on day 28. In another group of the immunized mice, corneas were challenged with HSV-1 (CHR3 strain, 10 microliter of 3 x 10(6) plaque-forming units [PFU]/ml). Mice were evaluated for clinical signs of epithelial or stromal keratitis on days 1 through 8 and days 10 and 14 or measured on days 2, 4, or 6 for viral titers in the eyes, trigeminal ganglia, and brain. RESULTS: All gD-DNA-injected mice obtained specific immunity. Furthermore, gD-IL-2-DNA elicited a higher DTH reaction and more vigorous cytotoxic effector cell activity. Stromal keratitis scores were lower for all immunized mice compared with control mice, although the difference in epithelial keratitis scores was not statistically significant. Viral titers in eyes, trigeminal ganglia, and brains were suppressed in all immunized mice. CONCLUSIONS: Local immunization with plasmid DNA encoding gD or gD-IL-2 induces humoral and cellular immunity against HSV-1 and inhibits development of stromal keratitis. gD-IL-2 DNA induces greater cell-mediated immunity than gD DNA alone. A plasmid encoding gD-IL-2 is therefore a promising candidate for a vaccine against HSV-1.     

Effect of aminoguanidine, a nitric oxide synthase inhibitor, on ocular infection with herpes simplex virus in Balb/c mice

PURPOSE. To study the effect of aminoguanidine (AMG), an inhibitor of nitric oxide production, on the ocular infection of Balb/c mice with herpes simplex virus (HSV) type 1 strain F and HSV-2 strain G. METHODS. Animals were treated with different amounts of AMG (0.5, 0.1, and 0.05 mg/mouse) by topical application in the eye from postinfection (PI) days -2 through +5, considering 0 the day of infection. At different PI days, development of herpetic keratitis was evaluated in treated and control mice. RESULTS. Treated animals showed a dose-dependent increase in ocular disease after viral infection, compared with control animals. Viral titers in ocular washings were higher in AMG-treated mice (PI day 2, HSV-1: AMG 0.5 mg, 1.3 x 10(3) plaque-forming units (PFU)/ml; control, 0. 22 x 10(2) PFU/ml, P < 0.025). At PI day 3, control corneas had only scattered inflammatory cells, whereas those from treated animals showed a conspicuous infiltrate consisting primarily of neutrophils. Viral titers were also higher in brains of treated mice. These animals died earlier and in a greater proportion than control animals (percentage of mortality, PI day 12, HSV-1: AMG 0.5 mg, 40% +/- 4%; control, 18% +/- 3%, P < 0.05). CONCLUSIONS. These data indicate an inhibitory effect of nitric oxide on HSV ocular infection.     

Macrophage-depletion influences the course of murine HSV-1 keratitis

PURPOSE. Herpes simplex virus type 1 infection of the cornea induces an immune-mediated disease termed "herpes stromal keratitis" (HSK) that is a major cause of blindness. In this study we investigated the influence of macrophage depletion by Cl(2) MDP encapsulated in liposomes (Cl(2) MDP-LIP) on the course of HSV-1 keratitis. METHODS. The corneas of BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). Mice groups received sub-conjunctival PBS or Cl( 2) MDP-LIP injections 7 and 2 days prior to infection. The eyes were studied clinically, histologically and immunohistochemically with F4/80 antibody at various time points after treatment. Clearance of the virus from the HSV-infected eyes was measured with a standard plaque assay. RESULTS. After subconjunctival Cl(2) MDP-LIP treatment, the HSV-1-induced epithelial keratitis was more severe (P < 0.05). The virus titers were significantly higher after macrophage depletion (day 7, P < 0.005). Stromal keratitis developed in 78.6% of HSV-1 infected PBS treated control mice ( n = 14) by day 14 after infection. By subconjunctival Cl(2) MDP-LIP treatment (n = 14) the incidence of stromal keratitis was reduced to 42.9%, and the keratitis was less severe (P < 0.05). CONCLUSIONS. The data demonstrate an influence of macrophages on the course of HSV-1 keratitis in mice. Macrophage depletion influence the viral replication in the cornea and the immune-mediated process of HSK.     

Interferon production and sensitivity of rabbit corneal epithelial and stromal cells

The induction of interferon and the ability of interferon to induce the antiviral state were studied using rabbit corneal epithelial and stromal cells which were cultured for fewer than five passages. Interferon titers in the range of 7000 units/ml were induced in epithelial cell cultures and 76,000 units/ml in stromal cell cultures treated with UV-inactivated bluetongue virus. The interferon induced was stable to pH 2.0 treatment and heating to 56 degrees C for 16 hr. Infection of epithelial and stromal cell cultures with various strains of herpes simplex virus type 1 showed that all strains tested replicated to equivalent titers in the respective cell types, and that no detectable interferon was induced in stromal cells and only trace amounts in epithelial cells. Exogenously supplied rabbit interferon induced the antiviral state in cultures of both cell types restricting the replication of not only encephalomyocarditis virus but also herpes simplex virus. Sixty to ninety units of rabbit interferon reduced HSV-1 virus replication by 50%. Human interferons had less than 27% of the antiviral activity in rabbit cells than they had in a human cell line. The data indicate that exogenously supplied interferon may act to reduce the severity of herpetic keratitis by directly inducing the antiviral state in corneal epithelial and stromal cells. However, interferon endogenously produced by rabbit corneal cells in response to HSV-1 infection probably plays a minor role in the pathogenesis of ocular HSV-1 infections.     

Increased severity of herpes simplex virus type 1-induced keratitis in Hox A5 transgenic mice

PURPOSE: Herpes simplex virus type 1 is a major cause of stromal keratitis and blindness in humans. Understanding of the role of host genes in the pathogenesis of herpes stromal keratitis is limited. We used a transgenic mouse model to examine the effect of a host gene, Hox A5 (which binds to the TAATGARAT sequence in the promoter regions of HSV-1 immediate early genes and increases HSV-1 replication), on the pathogenesis of HSV-1 induced stromal keratitis. METHODS: Corneas of wildtype and Hox A5 transgenic mice were infected with HSV-1 strain F following corneal scarification. Clinical severity of keratitis was evaluated using slit-lamp biomicroscopy. Histologic severity of keratitis was determined by light microscopic evaluation and by computerized morphometry. Ocular viral replication was measured via plaque assay. RESULTS: Clinical lesions of stromal keratitis were more severe at 17 and 23 days post infection in Hox A5 transgenic mice than in wildtype mice. Histological evaluation and morphometric analysis confirmed that keratitis lesions were more severe in the transgenic mice. HSV-1 replication was approximately100-fold greater in the corneas of transgenic mice than in wildtype mice. CONCLUSIONS: Our results demonstrate that a host gene (Hox A5) can increase ocular replication of HSV-1 and alter the pathogenesis of herpetic stromal keratitis.     

Stromal keratitis and anterior uveitis due to herpes simplex virus-2 in a young child.

BACKGROUND: An uncommon case of stromal keratitis and anterior uveitis due to herpes simplex virus type 2 (HSV-2) is reported. CASE: The patient was a 3-year-old boy admitted for conjunctival injection of the right eye of unknown cause, accompanied by corneal opacity and anterior uveitis. OBSERVATIONS: High titers of antibodies against HSV and Epstein-Barr virus (EBV) were found in blood samples. Polymerase chain reaction (PCR) for the detection of HSV-1, -2, and EBV genome fragments was carried out using an anterior chamber sample as a template. An HSV-2 genome fragment was amplified by PCR. Administration of acyclovir and betamethasone was started, with the consequent elimination of corneal opacity, inflammatory cells, and keratic precipitates. CONCLUSION: PCR clearly showed that HSV-2 was the causative pathogen of the stromal keratitis and anterior uveitis in this young patient. Systemic EVB infection may induce systemic immunocompromised conditions that can lead to reactivation of HSV-2 followed by ocular disorders.     

The lack of effect of tunicamycin and 2-deoxy-D-glucose on corneal stromal herpes in rabbits

A rabbit model for herpes simplex virus (HSV) stromal keratitis, produced by intrastromal injection of live virus, was used to evaluate the effects of tunicamycin and 2-deoxy-D-glucose therapy. In vivo and in vitro evidence suggests that HSV strains that produce stromal disease secrete relatively large amounts of highly antigenic glycoproteins. Also, various studies have shown that tunicamycin and 2-deoxy-D-glucose inhibit the production of complete HSV-specific glycoproteins. Thus, these drugs might be capable of mitigating the clinical manifestations of HSV stromal keratitis by reducing the antigenic load. However, when topical therapy with tunicamycin and/or 2-deoxy-D-glucose was begun in rabbit eyes, the day after intrastromal inoculation of live RE strain HSV and several days before the appearance of stromal disease, no difference in the clinical course of herpetic ocular disease was seen between the experimental (treated) and control (untreated) groups.     

The effect of immunization with herpes simplex virus glycoprotein D fused with interleukin-2 against murine herpetic keratitis

PURPOSE: To evaluate the preventive effect of vaccination using fusion protein (gD-IL-2) consisting of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD), and human interleukin-2 (IL-2), and plasmid DNA encoding gD-IL-2 against murine herpetic keratitis. METHODS: Plasmid containing gD-IL-2 (pHDLneo1) was constructed, and gD-IL-2 peptide was purified. BALB/c mice were injected hypodermally or subconjunctivally twice with 1 microg/0.1 mL of gD-IL-2 peptide, or subconjunctivally twice with 90 microg/0.05 mL of gD-IL-2 plasmid DNA. Neutralizing antibody titer and delayed-type hypersensitivity (DTH) against HSV-1 were measured. Immunized mice were challenged with CHR3 strain of HSV-1 into the cornea. Clinical manifestations of the epithelial and stromal keratitis were scored. RESULTS: Stromal keratitis was inhibited in gD-IL-2 peptide- or DNA-immunized mice; however, epithelial keratitis was not. It was confirmed that plasmid gD-IL-2 elicited significant virus neutralizing titer in sera and DTH response. CONCLUSION: Vaccination with gD-IL-2 was effective against murine herpetic keratitis.     

Penetrating keratoplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used

The increased incidence of corneal graft failure in patients with herpes simplex virus (HSV) keratitis may be due in part to reactivation of latent HSV following surgical corneal trauma and postoperative corticosteroid therapy. To determine the onset, frequency, and nature of HSV recurrences following penetrating keratoplasty (PKP), 21 HSV type 1 (HSV-1) latently infected rabbits underwent unilateral autograft PKP. Opposite unoperated eyes served as HSV-1 latently infected controls. Corneal autografts were performed so that immunologic graft rejection would not be confused with recurrent HSV-1 stromal disease. After PKP, 11 of the 21 eyes were treated with dexamethasone. Ocular cultures and slit-lamp examinations were performed daily for the first postoperative 8 days and every other day thereafter for 82 days. Nine (82%) of the 11 dexamethasone-treated PKP eyes, 2 (20%) of the PKP eyes not treated with dexamethasone, and 3 (17%) of the 18 unoperated eyes had positive HSV-1 ocular cultures. Geographic ulcers appeared only in the PKP eyes treated with dexamethasone; 9 (82%) of the 11 PKP eyes treated with dexamethasone developed geographic ulcers. Between the 24th and 90th postoperative days, stromal keratitis appeared in 5 (56%) of the 9 PKP eyes treated with dexamethasone and in 2 (25%) of the 8 PKP eyes not treated with dexamethasone. Autograft PKP with postoperative corticosteroids significantly increased HSV-1 ocular shedding, epithelial ulceration, and stromal keratitis. This experimental model provides a useful tool to further investigate the development and treatment of HSV-1 epithelial and stromal recurrences after PKP.     

Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease

This study evaluated the continued presence of herpes simplex virus (HSV) nucleic acid sequences after resolution of acute herpetic stromal keratitis in the rabbit ocular model. Forty-four rabbits were inoculated bilaterally with 10(5) plaque-forming units of RE strain HSV-1 by intrastromal injection. All eyes were cultured for the presence of HSV during acute disease and immediately before the animals were killed. Full-thickness corneal buttons were then removed and processed for in situ hybridization with a 3H-labelled HSV DNA probe representing the full-length HSV genome. HSV nucleic acid sequences were detected autoradiographically at all time intervals examined. HSV nucleic acid sequences were localized in the epithelium and the anterior stromal keratocytes during acute disease and in all corneal layers during latent infection. Retention of HSV nucleic acid sequences, either HSV DNA or HSV RNA, or both, in corneal tissues (epithelium, stroma, and endothelium) may be a contributing factor in the development of HSV-induced stromal keratitis.     

Virus proteins in herpetic keratitis

We examined the differences in protein expression among several strains of herpes simplex virus that cause epithelial and stromal keratitis in the rabbit eye. The isolates were grown in HEp-2 cells in the presence of various radioactive isotopes and were analyzed by SDS-PAGE. The in vitro expression of viral specified proteins was studied with antiserum to the purified virion (heterologous) and with the strain's homologous antiserum made during herpetic ocular disease.We found: (1) similarity among the strains in their in vitro expression of viral proteins; (2) polypeptide differences in the areas of ICP 9/10-ICP 14 and ICP28/29 and 30, when these proteins were immune precipitated with their homologous antisera; (3) more abundant glycosylated polypeptides in the RE (stromal disease-producing) strain than in the F (epithelial disease-producing) strain; (4) secretion of the major glycoproteins (gC, gD and gA, gB) into the extracellular fluid by all tested strains. However, the amounts of the glycoproteins secreted seemed to be characteristic of the individual strain. Those strains that secreted the larger amounts of glycoproteins were the strains that are known to produce stromal disease in the rabbit eye, which suggests that one determinant in the induction of stromal disease may be the number of glycosylated polypeptides as well as the secretion of glycoproteins in a soluble antigenic form.