Efficacy of Topical Cidofovir on Multiple Adenoviral Serotypes in the New Zealand Rabbit Ocular Model

Abstract

purpose. The goal of the present study was to determine the efficacy of topical 0.5% cidofovir twice daily for 7 days on the replication of multiple adenovirus (Ad) serotypes of subgroup C (Ad1, Ad5, Ad6) in the New Zealand rabbit ocular model.

methods. In duplicate experiments for each serotype, a total of 20 rabbits (Ad5) or 16 rabbits each (Ad1 and Ad6) were inoculated topically in both eyes, with 1.5 × 10⁶ pfu/eye of the appropriate virus. Twenty-four hours later, the rabbits in each serotype group were randomly divided into two topical treatment groups: I, 0.5% cidofovir; II, control vehicle. Treatment was twice daily for 7 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14.

results. Compared to the control, treatment with 0.5% cidofovir reduced the following: mean Ad titer (days 1 to 7) for Ad1 (6.3 ± 20 × 10¹ versus 2.5 ± 3.9 × 10² pfu/ml; P < 0.0003), Ad5 (3.4 ± 5.8 × 10² versus 1.6 ± 2.0 × 10³ pfu/ml; P < 0.000001), and Ad6 (1.2 ± 5.1 × 10² versus 5.5 ± 14 × 10² pfu/ml; P = 0.015); reduced Ad-positive eyes/total for Ad1[ 45/128 (35%) versus 84/128 (66%); P = 0.000002], Ad5 [84/160 (53%) versus 131/152 (86%); P < 0.000001], and Ad6 [36/128 (28%) versus 82/128 (64%); P < 0.000001]: and reduced the duration of Ad shedding for Ad1 (4.9 ± 1.9 versus 9.3 ± 3.3 days; P < 0.00007), Ad5 (6.4 ± 2.8 versus 11.5 ± 2.3 days; P < 0.0001), and Ad6 (4.4 ± 2.1 versus 8.4 ± 2.5 days; P < 0.00004).

conclusions. Topical 0.5% cidofovir twice daily for 7 days demonstrated significant antiviral activity against multiple adenoviral serotypes (Ad1, Ad5, and Ad6) in the New Zealand rabbit ocular model. These in vivo data expand in vitro studies indicating the efficacy of cidofovir against different adenovirus serotypes and support its use in clinical trials.

Adenoviral (Ad) ocular infections remain the most common external ocular viral infection worldwide. ¹ Epidemic keratoconjunctivitis, pharyngeal conjunctival fever, and follicular conjunctivitis are three examples of highly contagious adenoviral ocular infections that are associated with community and medical facility epidemics. Although not permanently blinding, ocular adenoviral infections are associated with significant patient morbidity, including symptomatic distress, with visual disturbances that can last months to years. Of the 47 serotypes of human adenovirus, about one half of these are known to cause ocular disease in patients. ¹  

The studies of the pathogenesis and treatment of ocular adenoviral infections have been limited by the narrow host range exhibited by human adenoviruses. It has been previously determined that one serotype of human adenovirus, adenovirus type 5 (Ad5), has the ability to extend its host range to permit replication in the eyes of New Zealand rabbits. ^{2 3} Our Ad5 McEwen/New Zealand rabbit ocular model has been used to establish possible clinical guidelines for the use of corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs) in the treatment of acute adenoviral ocular infections. ^{4 5}  

Cidofovir is a highly promising broad spectrum antiviral with significant inhibitory activity against a number of DNA viruses [human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-)1, HSV-2, varicella zoster virus (VZV), and adenoviruses ]. ⁶ It has been previously demonstrated in prevention and treatment studies that topical administration of cidofovir, using various concentrations and treatment regimens, significantly reduced ocular viral titers and the duration of viral shedding in the Ad5/New Zealand rabbit ocular model. ^{7 8 9}  

To further evaluate the efficacy of cidofovir as a topical treatment for adenoviral ocular infections, the antiviral efficacy against a variety of adenoviral ocular serotypes in vitro and in vivo should be determined along with the optimum concentration and dosing regimen. Previous in vitro studies have demonstrated that cidofovir was an effective antiviral agent against a variety of ocular adenoviral serotypes. ^{6 9 10 11} However, no data have been published to date demonstrating the efficacy of cidofovir against multiple Ad serotypes in vivo. 

In previously published reports, our group demonstrated that the members of human adenovirus subgroup C (Ad1, Ad2, Ad5, and Ad6) were able to replicate in rabbit corneal organ culture. ¹² Recently, we demonstrated that Ad1, Ad2, and Ad6, along with Ad5, also have the ability to replicate and cause productive infections in the eyes of New Zealand rabbits. ¹³  

The goal of the present study was to determine the antiviral efficacy of topical 0.5% cidofovir administered twice daily for 7 days, on multiple Ad serotypes of subgroup C (Ad1, Ad5, and Ad6) in the New Zealand rabbit ocular model of adenoviral infection. 

Materials and Methods

Virus Serotypes and Cells

The reference adenovirus serotype (Ad6 ATCC) used in this study was purchased from the American Type Culture Collection (ATCC, Rockville, MD). The clinical adenoviral isolates were cultured from patients presenting with typical adenoviral ocular disease at the Eye and Ear Institute of Pittsburgh. The isolates were serotyped by serum neutralization and found to be types 1 and 5. The isolates, designated Ad1 Kmetz and Ad5 McEwen along with the Ad6 ATCC reference strain, were grown in A549 monolayers at 37°C, in a 5% CO₂–water vapor atmosphere, harvested, aliquoted, and frozen as a virus stock at −70°C. Before use, the stock viruses were titered using a standard plaque assay. 

A549 cells, an epithelial-like cell derived from human lung carcinoma, were grown and maintained in Eagle’s minimum essential medium with Earle’s salts (Sigma Cell Culture Reagents, St. Louis, MO), supplemented with 6% fetal bovine serum (Harlan Bioproducts for Science, Indianapolis, IN), 2.5 μg/ml amphotericin B, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin (Sigma). 

Experimental Drugs

Cidofovir [S-HPMPC; (S)-9-(3-hydroxy-2-phophonylmethoxypropyl)cytosine], was provided by Bausch and Lomb Pharmaceuticals, Inc., Tampa, Florida. A topical ocular formulation was prepared as a 0.5% solution. Control eye drops for cidofovir consisted of the vehicle alone. 

Animals

Six-week-old, 1-kg, female New Zealand albino rabbits were obtained from Myrtle’s Rabbitry (Thompson Station, TN). All animal studies conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. Institutional approval was obtained and institutional guidelines regarding animal experimentation were followed. 

Experimental Design

This study was performed in duplicate for each virus serotype. After appropriate systemic and topical anesthesia, ² a total of 20 rabbits, for both trials, for Ad5 McEwen and 16 rabbits each for Ad1 Kmetz and Ad6 ATCC were inoculated with 50 μl (1.5 × 10⁶ pfu/eye) of the appropriate virus in both eyes after 12 cross-hatched strokes of a no. 25 sterile needle. ⁷ Twenty-four hours after inoculation, each virus serotype group was divided into two topical treatment groups: I, 0.5% cidofovir (n = 10 rabbits for Ad5, n = 8 rabbits for Ad1 and Ad6); II, control drops (n = 10 rabbits for Ad5, n = 8 rabbits for Ad1 and Ad6). For each serotype tested, treatment of the cidofovir and control groups was administered in both eyes twice daily for 7 days. Ocular swabbing was performed on days 0, 1, 3, 4, 5, 7, 9, 11, and 14 after inoculation at least 1 hour after the second dose. Each eye was swabbed in the upper and lower fornicies with a cotton-tipped applicator, and the swab was placed in 1 ml of media and frozen at −70°C, pending plaque assay. The experimental design was masked with regard to the identity of the treatment drops, so that treatment, swabbing, and the determination of viral titers also was masked to those carrying out the experiment. 

Determination of Viral Titers (Plaque Assay)

The ocular samples to be titered were thawed and diluted serially (1:10) for two dilutions. Each dilution and the undiluted original sample were then inoculated onto A549 monolayers (0.1 ml per well) in duplicate wells of a 24-well plate. The virus was adsorbed for 3 hours at 37°C in a 5% CO₂–water vapor atmosphere. After adsorption, 1 ml of A549 media plus 0.5% methylcellulose was added to each well, and the plates were incubated at 37°C in a 5% CO₂–water vapor atmosphere. After 7 days incubation, the cells were stained with 0.5% gentian violet, and the number of plaques was counted under a dissecting microscope (25×). The viral titers were then calculated and expressed as plaque-forming units per milliliter (pfu/ml). 

Statistical Analysis

After the completion of all experiments, the codes that masked the identity of the treatment were broken, and the data from each experiment were analyzed statistically. Because comparable results were obtained in each experiment, the data were then pooled to obtain a larger subject number and analyzed using analysis of variance (ANOVA), Fisher’s Exact Test, and χ² analyses. Significance was established at the P ≤ 0.05 confidence level. 

Results

Adenoviral Ocular Titers

The mean serial Ad ocular titers for both treatment groups for each serotype were determined by calculating the mean and SD of all ocular cultures per day. Compared to the control group, treatment with 0.5% cidofovir significantly reduced adenoviral titers for Ad1 Kmetz (Fig. 1) (days 3, 4, 5; P < 0.03), Ad5 McEwen (Fig. 2) (days 3, 4, 5, 7, 9, 11; P < 0.02), and Ad6 ATCC (Fig. 3) (days 4, 7; P < 0.04) (ANOVA). 

The mean Ad titers (days 1–7) were determined by calculating the mean and SD of all ocular cultures from both treatment groups for each serotype during the 7-day treatment period. Treatment with 0.5% cidofovir, compared to the control, significantly reduced the mean Ad titer (days 1–7) for Ad1 Kmetz [n = 80 for both groups; 6.3 ± 20 × 10¹ versus 2.5 ± 3.9 × 10² pfu/ml (P < 0.0003)], Ad5 McEwen [0.5% Cidofovir group, n = 100; control group, n = 98; 3.4 ± 5.8 × 10² versus 1.6 ± 2.0 × 10³ pfu/ml (P < 0.000001)], and Ad6 ATCC [n = 80 for both groups; 1.2 ± 5.1 × 10² versus 5.5 ± 14 × 10² pfu/ml (P = 0.015)] (ANOVA). 

Ad-Positive Eyes per Total

The number of Ad-positive eyes per total was determined for each serotype and treatment group by ascertaining the number of eye swabs that demonstrated a positive Ad culture per total number of cultures. Table 1 summarizes these results. Overall, from days 1 to 14, eyes treated with 0.5% cidofovir demonstrated fewer Ad-positive eyes than the control group for Ad1 Kmetz [45/128 (35%) versus 84/128 (66%); P = 0.000002], Ad5 McEwen [84/160 (53%) versus 131/152 (86%); P < 0.000001], and Ad6 ATCC[ 36/128 (28%) versus 82/128 (64%); P < 0.000001] (χ² analysis). 

Daily Ad-positive eyes are also displayed in Table 1 . Treatment with 0.5% cidofovir significantly reduced the number of Ad-positive eyes compared to the control group on days 5, 7, 9, and 11 (P < 0.02) for Ad1 Kmetz; days 7, 9, 11, and 14 (P < 0.02) for Ad5 McEwen; and days 3, 4, 5, 7, and 9 (P ≤ 0.0434) for Ad6 ATCC (Fisher’s Exact Test). 

Duration of Ad Shedding

The duration of Ad shedding was determined for each eye by discerning the final day on which a positive Ad culture was detected and calculating the mean and SD for both treatment groups for each serotype. Treatment with 0.5% cidofovir significantly reduced the mean duration of Ad shedding compared to the control group for the three serotypes tested: Ad1 Kmetz [n = 16 for both groups; 4.9 ± 1.9 versus 9.3 ± 3.3 days (P < 0.00007)]; Ad5 McEwen [n = 20 for 0.5% Cidofovir; n = 18 for control; 6.4 ± 2.8 versus 11.5 ± 2.3 days (P < 0.0001)]; Ad6 ATCC [n = 16 for both groups; 4.4 ± 2.1 versus 8.4 ± 2.5 days (P < 0.00004)] (ANOVA). 

Discussion

Cidofovir is a highly promising, broad-spectrum antiviral drug that is FDA approved for systemic treatment of CMV retinitis in patients with acquired immunodeficiency syndrome. Previous reports have demonstrated that cidofovir is an effective antiviral agent against a number of Ad serotypes in vitro. ^{6 9 10 11} Recent studies in the Ad5/New Zealand rabbit ocular model have demonstrated that topical administration of cidofovir, using various concentrations and treatment regimens, significantly reduced ocular adenoviral titers and the duration of adenoviral shedding in both prevention and treatment studies. ^{7 8 9} Topical antiviral therapy in the New Zealand rabbit model was initiated 24 hours after inoculation with high titers of Ad5. This time course differs from the clinical setting in which antiviral therapy would be initiated with the onset of clinical signs and symptoms usually after 1 week of subclinical infection. Based in part on these studies, evaluation of topical cidofovir for the treatment of adenoviral ocular infections is now being performed in clinical trials. 

Recently, our group demonstrated that Ad1, Ad2, and Ad6, along with Ad5, can replicate and cause productive infections in the eyes of New Zealand rabbits. ¹³ Although all these serotypes are members of a single subgroup of adenoviruses (subgroup C), they are the only adenoviruses known that have the genetic capability to extend their host range to replicate in the eyes of New Zealand rabbits. The serotypes most commonly associated with human adenoviral ocular disease (Ad3, 4, 7) did not replicate in rabbit corneal organ culture ¹² and (Ad8, 19) did not replicate in New Zealand rabbit eyes. ¹³ Nevertheless, we believe that information obtained from the use of the subgroup C adenoviruses will be useful to demonstrate antiviral efficacy of topical cidofovir against multiple Ad serotypes in vivo. 

The results of this study clearly confirms and extends our previous work by demonstrating that topical cidofovir reduces Ad5, Ad1, and Ad6 ocular titers and the number of Ad-positive eyes and shortens the duration of ocular shedding in vivo. The treatment regimen of 0.5% cidofovir twice daily for 7 days clearly delivered a therapeutic dose of cidofovir. The present study further strengthens the preclinical data that support the continued development of cidofovir as the first broad spectrum antiviral agent for the treatment of adenoviral and HSV ocular infections. ¹⁴  

 Supported by National Institutes of Health Grant EY05232, NIH Core Grant for Vision Research EY08098, The Eye and Ear Foundation of Pittsburgh, and Bausch and Lomb Pharmaceuticals, Inc., Tampa, Florida.

 Submitted for publication May 28, 1999; revised August 27, 1999; accepted September 15, 1999.

 Commercial relationships policy: C2, C3.

 

Figure 1.

 

Serial ocular titers (mean ± SD) for Ad1 Kmetz, (n = 16/d) in both the 0.5% Cidofovir (○) and control (▿) groups. *Denotes the days on which the 0.5% Cidofovir group demonstrated significantly lower (P < 0.03) mean ocular titers than the control group.

Figure 2.

 

Serial ocular titers (mean ± SD) for Ad5 McEwen (n = 20/d) in both the 0.5% Cidofovir (○) and control (▿) groups. However, for days 7 to 14, n = 18 in the control group, due to the loss of one rabbit. *Denotes the days on which the 0.5% Cidofovir group demonstrated significantly lower (P < 0.002) mean ocular titers than the control group.

Figure 3.

 

Serial ocular titers (mean ± SD) for Ad6 ATCC (n = 16/d) in both the 0.5% Cidofovir (○) and control (▿) groups. *Denotes the days on which the 0.5% Cidofovir group demonstrated significantly lower (P < 0.04) mean ocular titers than the control group.

Table 1.

 

Adenovirus-Positive Eyes per Total, Days 1–14

Serotype and Treatment	Day															Total
		1	3	4	5	7	9	11	14
Ad1 cidofovir	5/16	14/16	13/16	8/16*	4/16*	1/16*	0/16*	0/16	45/128*
	(31)	(88)	(81)	(50)	(25)	(6)	(0)	(0)	(35)
Ad1 control	7/16	16/16	16/16	15/16	13/16	7/16	6/16	4/16	84/128
	(44)	(100)	(100)	(94)	(81)	(44)	(38)	(25)	(66)
Ad5 cidofovir	20/20	20/20	20/20	16/20	3/20*	1/20*	3/20*	1/20*	84/160*
	(100)	(100)	(100)	(80)	(15)	(5)	(15)	(5)	(53)
Ad5 control	19/20	20/20	20/20	20/20	18/18, †	16/18	11/18	7/18	131/152
	(95)	(100)	(100)	(100)	(100)	(89)	(61)	(39)	(86)
Ad6 cidofovir	7/16	11/16*	9/16*	6/16*	1/16*	1/16*	1/16	0/16	36/128*
	(44)	(69)	(56)	(38)	(6)	(6)	(6)	(0)	(28)
Ad6 control	7/16	16/16	16/16	15/16	14/16	8/16	5/16	1/16	82/128
	(44)	(100)	(100)	(94)	(88)	(50)	(31)	(6)	(64)

 Percentages are in parentheses.

*   P < 0.05 when compared to appropriate control group (Fisher’s Exact Test or χ² analyses).

†  One rabbit died prior to day 7 culture.