PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Curr Eye Res. Author manuscript; available in PMC 2010 May 25.
Published in final edited form as:
PMCID: PMC2875692
NIHMSID: NIHMS201110

A Human Apolipoprotein E Mimetic Peptide Effectively Inhibits HSV-1 TK-Positive and TK-Negative Acute Epithelial Keratitis in Rabbits

Abstract

Purpose/Aim

To compare the antiviral effect of a peptide derived from human apolipoprotein E (1% apoEdp) with 1% trifluorothymidine against HSV-1 thymidine kinase (TK)-positive (HSV-1 McKrae) and with 3% foscarnet against HSV-1 TK-negative (KOS background) in the rabbit eye model of acute HSV-1 epithelial keratitis.

Materials and Methods

Topical treatment began 3 days postinfection and continued for five days. Rabbits were treated 5 times per day with1% apoEdp, 1% trifluorothymidine, 3% foscarnet, or PBS. Slit-lamp examination was performed on a masked basis from postinfection days 3–10 to determine the severity of epithelial keratitis.

Results

Topical treatment with 1% apoEdp was as effective as 1% trifluorothymidine against TK-positive and 3% foscarnet against TK-negative HSV-1 epithelial keratitis.

Conclusions

ApoEdp 1% has efficacy for the topical treatment of TK-positive and TK-negative ocular herpes in the rabbit eye model. Thus, apoEdp has the potential to be used for treatment of keratitis caused by TK mutants.

Keywords: Apolipoprotein E, Apolipoprotein E dimer peptide, epithelial keratitis, Foscarnet, herpes simplex virus, trifluorothymidine

INTRODUCTION

We previously described the in vivo therapeutic efficacy of a novel dimer peptide derived from human apolipoprotein E, apoEdp, that effectively blocks herpetic stromal keratitis (HSK) in a mouse eye model.1 This peptide is known to differ significantly from antiviral compounds in common use related to their mechanisms of action.2 For example, nucleoside analogs acyclovir and trifluorothymidine (TFT) require phosphorylation; therefore, their antiviral activity is not initiated until the infection has progressed to the point that viral thymidine kinase (TK) has been synthesized. By contrast, apoEdp acts by blocking viral attachment by mimicking the heparan sulfate proteoglycan necessary for initial attachment of virus to cells, and thus does not require processing by target cells to become active. 2

We reported that topical treatment of 1% apoEdp was potent as both an antiviral, measured through reduction of viral titers, and as anti-inflammatory, measured through the reduction of pro-inflammatory and angiogenic cytokines, without any local or systemic toxicity in mice starting at 1 day postinfection (PI).1 Given the promising therapeutic results obtained in the mouse model using this peptide, we extended our studies involving apoEdp to the rabbit ocular model of herpes keratitis, because this model is a good predictor for the treatment of human disease.3 In this study, we used the rabbit ocular model because it affords us the opportunity to follow the antiviral efficacy more accurately by masked slit-lamp examination. In addition to proving that the ApoE antiviral is effective against a TK-sensitive virus (wild type), this study also included a TK negative recombinant HSV-1; the importance of this TK negative virus is that it cannot be inhibited by the nucleoside analogs such as TFT (Viroptic) or acycloguanisine. This study compared the antiviral effect of 1% apoEdp with 1% TFT against HSV-1 TK positive (HSV-1 McKrae) and with 3% foscarnet against HSV-1 TK negative (KOS background) in the rabbit eye model.

MATERIAL AND METHODS

Antiviral Compounds and Viruses

The apoE dimer peptide (apoEdp) was synthesized (Genemed, San Antonio, TX) with a purity of greater than 97%. Commercial 1% TFT (Viroptic, Monarch Pharmaceuticals Inc., Bristol, TN) and 3% foscarnet (sodium phosphonoformate tribasic hexahydrate, Sigma, St. Louis, MO) were used as positive control treatments for HSV-1 McKrae and HSV-1 TK negative (mutant TKG7dG.1in KOS background4) [a kind gift from Professor D.M. Coen, Harvard Medical School, Boston, MA.], respectively. Both viruses were propagated on primary rabbit kidney cell monolayers and titered by plaque assay on African green monkey kidney cell (CV-1) monolayers.

Rabbit Keratitis Studies

The mildly scarified corneas of New Zealand White rabbits (1.5–2.5 kg) were inoculated with 25 µL of a suspension of HSV-1 strain McKrae or HSV-1 TK negative (1 × 105 PFU). Animal care and treatment in this investigation were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the protocol was approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center in New Orleans. Treatment began 3 days PI when the disease was well established and continued for five consecutive days. Three groups of 5 rabbits were treated using either 1% apoEdp, 1% TFT, or 3% foscarnet; another group received a vehicle control (sterile PBS). Before the start of this experiment, we evaluated the toxicity of 1% apoEdp and 3% foscarnet in a separate group of normal rabbit eyes (n=6) to ensure that the drugs were safe and well tolerated. We also did safety and tolerance studies on reepithelialization of rabbit corneas following cross hatch scarification as well as a full 3-mm diameter corneal epithelial button removed. We compared the 1% dipeptide given five times a day to the vehicle give five times per day. There was no difference in the reepithelialization (cross hatch method design) as well as the migration in the corneal epithelial cells (corneal button) between the 1% ApoE dipeptide and the vehicle.

Both eyes of all rabbits were used. Each compound (50 µL) was applied topically five times per day every 2 hours starting at 8 AM and ending at 4 PM. Before treatment, the rabbit groups were balanced based on slit-lamp examination (SLE) scores to assure the same average score. SLE was performed in a masked fashion once a day from PI days 3 though 10. The ocular lesions were characterized as deep punctuate lesions, dendritic lesions, or geographical epithelial defects, according to the tenets described previously.5

Statistics

For slit-lamp scores, the Student’s t-test was used for comparison among groups and the results were expressed as mean ± standard error of mean (SEM). A value of p < 0.05 was considered to be statistically significant.

RESULTS

This is the first rabbit ocular study using topical treatment with apoEdp; 1% apoEdp given topically five times a day demonstrated therapeutic efficacy against epithelial keratitis induced by TK-positive and TK-negative HSV-1. Against HSV-1 McKrae, topical treatment with 1% apoEdp significantly reduced severity of epithelial erosion when compared with the placebo (Figure 1). The severity of keratitis in apoEdp-treated eyes and in the TFT-treated eyes was significantly reduced on PI days 6–10. On PI day 10, there was no significant difference between the apoEdp- and TFT-treated eyes.

Figure 1
Slit-lamp scores for 1% apoEdp and trifluorothymidine (TFT) in a rabbit model of epithelial keratitis induced by HSV-1 McKrae. Significant difference (p<0.05) for *apoEdp and †TFT compared with placebo (PBS).

Against the HSV-1 TK-negative mutant, 1% apoEdp was as effective as 3% foscarnet in reducing the severity of keratitis in rabbit eyes. On PI days 7 and 10 there was no significant difference between the 1% apoEdp-treated and 3% foscarnet-treated eyes.

DISCUSSION

Epithelial keratitis is the most common clinical presentation of ocular infection by HSV, accounting for 70% to 80% of all cases.6,7 The recurrence rate of HSV epithelial keratitis may be greater in diabetics, atopes, corneal transplant recipients, and people with immunosuppression. 811 Topical application of TFT is the gold standard in the treatment of HSV keratitis, and foscarnet eye drops (3%) are equivalent to TFT eye drops in the treatment of acyclovir-resistant cases,12 although cytotoxicity limits their use.13,14 Herpes infections continue to be prevalent; in the HEDS15 trial, acyclovir prevented only 40% of recurrences and resistance to nucleoside analogs has been reported,16 especially in immunocompromised transplant recipients and AIDS patients. Therefore, it is important to explore new treatment options with alternative mechanisms of antiviral action.

In this study, the severity of keratitis caused by a TK-negative mutant of HSV-1 was less severe than wild type HSV-1 McKrae; the TK-negative mutant virus had a HSV-1 KOS background and KOS is known as a less virulent strain compared with McKrae. Despite this difference in pathogenicity between the two viruses, regardless of virus strains used, from PI days 6–10, topical apoEdp effectively reduced the severity of epithelial keratitis to a significantly decreased level compared with placebo-treated eyes. In our mouse study, we also observed a delay in antiviral activity, i.e., clearance of infectious virus (see Fig. 4, IOVS 49:4263–4268, 2008).

The treatment currently available for moderate or severe cases of HSK includes topical corticosteroids to prevent irreversible corneal damage. However, this treatment has the potential for complications such as corneal melting, glaucoma, and cataract.17 We previously reported that the topical application of 1% apoEdp in mice had a potent anti-inflammatory effect without any toxicity. 1

Our results demonstrated that a novel topical ocular antiviral peptide (apoEdp) exhibited similar therapeutic effects compared with foscarnet and TFT with no observed toxicity. We have tested this against both HSV-1 and HSV-2 and have found concentrations in which there is high synergism (data not shown). We suggest that a combination treatment of herpetic epithelial keratitis with topical apoEdp along with an oral antiviral nucleoside could emerge as an improvement over single-drug treatment or as an alternative treatment for clinically resistant cases.

Figure 2
Slit-lamp scores for 1% apoEdp and 3% foscarnet in a rabbit model of epithelial keratitis induced by HSV-1 TK-negative mutant (KOS) strain. Significant difference (p<0.05) for *apoEdp and †3% foscarnet compared with placebo (PBS).

ACKNOWLEDGMENTS

This work was supported in part by National Eye Institute Grants NEI R01 EY06311 (JMH), F32EY016316 (DMN), NIH NEI P30 EY002377 (LSU Eye Center Core Grant), Research to Prevent Blindness (RPB) Senior Scientific Award (JMH), LSUHSC Translational Research Initiative Grant (PSB), and an unrestricted departmental grant from Research to Prevent Blindness, New York, NY. This work was done at the LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, LA.

REFERENCES

1. Bhattacharjee PS, Neumann DM, Foster TP, et al. Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model. Invest Ophthalmol Vis Sci. 2008;49:4263–4268. [PubMed]
2. Dobson CB, Sales SD, Hoggard P, Wozniak MA, Crutcher KA. The receptor-binding region of human apolipoprotein E has direct anti-infective activity. J Infect Dis. 2006;193:442–450. [PubMed]
3. Kaufman HE, Heidelberger C. Therapeutic antiviral action of 5-trifluoromethyl-2'-deoxyuridine in herpes simplex keratitis. Science. 1964;145:585–586. [PubMed]
4. Griffiths A, Link MA, Furness CL, Coen DM. Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase. J Virol. 2006;80:6568–6574. [PMC free article] [PubMed]
5. Anand BS, Hill JM, Dey S, et al. In vivo antiviral efficacy of a dipeptide acyclovir prodrug, val-val-acyclovir, against HSV-1 epithelial and stromal keratitis in the rabbit eye model. Invest Ophthalmol Vis Sci. 2003;44:2529–2534. [PubMed]
6. Labetoulle M, Auquier P, Conrad H, et al. Incidence of herpes simplex virus keratitis in France. Ophthalmology. 2005;112:888–895. [PubMed]
7. Liesegang TJ. Herpes simplex virus epidemiology and ocular importance. Cornea. 2001;20:1–13. [PubMed]
8. Rezende RA, Hammersmith K, Bisol T, et al. Comparative study of ocular herpes simplex virus in patients with and without self-reported atopy. Am J Ophthalmol. 2006;141:1120–1125. [PubMed]
9. Kaiserman I, Kaiserman N, Nakar S, Vinker S. Herpetic eye disease in diabetic patients. Ophthalmology. 2005;112:2184–2188. [PubMed]
10. Remeijer L, Doornenbal P, Geerards AJ, Rijneveld WA, Beekhuis WH. Newly acquired herpes simplex virus keratitis after penetrating keratoplasty. Ophthalmology. 1997;104:648–652. [PubMed]
11. Hodge WG, Margolis TP. Herpes simplex virus keratitis among patients who are positive or negative for human immunodeficiency virus: an epidemiologic study. Ophthalmology. 1997;104:120–124. [PubMed]
12. Behrens-Baumann W. Phosphonoformate (foscarnet, PFA) versus trifluorothymidine in the treatment of keratitis dendritica in the human. A double blind, randomized, preliminary trial. Acta Ophthalmol (Copenh.) 1992;70:690–692. [PubMed]
13. Pavan-Langston D. Clinical disease herpetic infections. In: Smolin G, Throft RA, editors. The Cornea. Boston, MA: Little, Brown and Company; 1994. pp. 83–215.
14. Imperia PS, Lazarus HM, Dunkel EC, Pavan-Langston D, Geary PA, Lass JH. An in vitro study of ophthalmic antiviral agent toxicity on rabbit corneal epithelium. Antiviral Res. 1988;9:263–272. [PubMed]
15. Wilhelmus KR. The epidemiology of ocular infections. In: Jaeger EA, Tasman W, editors. Duane’s Foundations of Clinical Ophthalmology. Philadelphia: Lipincott-Raven; 1998.
16. Coen DM. Acyclovir-resistant, pathogenic herpesviruses. Trends Microbiol. 1994;2:481–485. [PubMed]
17. Wilhelmus KR, Gee L, Hauck WW, et al. Herpetic Eye Disease Study. A controlled trial of topical corticosteroids for herpes simplex stromal keratitis. Ophthalmology. 1994;101:1883–1895. [PubMed]