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Topical Administration of HSV gD-IL-2 DNA Is Highly Protective Against Murine Herpetic Stromal Keratitis

Inoue, Tomoyuki M.D.; Inoue, Yoshitsugu M.D.; Hayashi, Kozaburo M.D.; Yoshida, Atsushi M.D.; Nishida, Kohji M.D.; Shimomura, Yoshikazu M.D.; Fujisawa, Yukio Ph.D.; Aono, Aki Ph.D.; Tano, Yasuo M.D.

Basic Investigations

Purpose. To evaluate the immunopreventive effect of eyedrops that contain gD-IL-2 DNA (a chimeric gene of the glycoprotein D gene of herpes simplex virus type 1 (HSV-1) and human interleukin-2 (IL-2) on murine herpetic keratitis.

Methods. A plasmid containing gD-IL-2 (pHDLneo1) was constructed. The eyedrops containing 90 μg/10 μL of the DNA was instilled bilaterally into the conjunctival sacs of BALB/c mice on days 0 and 7. Three weeks after the last administration, neutralizing antibody, delayed-type hypersensitivity (DTH), and 51 Cr-release from infected targeted cells by lymphocytes from the cervical lymph nodes and spleen were determined. Immunized mice were challenged with HSV-1, after which the clinical signs of the corneal epithelia and stroma were scored.

Results. Specific neutralizing antibody was raised and prominent DTH reaction was elicited from immunized mice. Lymphocytes obtained from the local lymph nodes and spleen vigorously potentiated the cytotoxic activity against the virus-infected cells. Clinically, the development of stromal keratitis was completely inhibited, but prevention or reduction of HSV-1 epithelial lesions was not demonstrated statistically.

Conclusion. Topical immunization with a DNA vaccine encoding gD-IL-2 totally prevented the development of herpetic stromal keratitis. This procedure is a simple and convenient method for possible clinical application in the future.

From the Department of Ophthalmology, Osaka University Medical School, Osaka (T.I., Y.I., A.Y., K.N., Y.T.); Kobe Institute of Health, Hyogo (K.H.); Department of Ophthalmology, Kinki University School of Medicine, Osaka (Y.S.); Research and Development Division, Biotechnology Research Laboratories, Takeda Chemical Industries, Osaka (Y.F., A.A.), Japan.

Submitted April 24, 2001.

Revision received September 5, 2001.

Accepted September 11, 2001.

Supported in part by a Grant-in-Aid for Scientific Research by the Ministry of Education, Science and Culture, Japan, no. 12470365, a grant from Osaka Eye Bank, Osaka, and a grant from Kobe City Health and Welfare Administration.

None of the authors has any proprietary interest in this article.

Address correspondence and reprint requests to Dr. T. Inoue, Department of Ophthalmology, Osaka University Medical School, 2-2 Yamadaoka, Suita 565-0871, Japan. E-mail:

Repeated attacks of reactivated herpes simplex virus type 1 (HSV-1) on the cornea eventually induce sight-threatening stromal keratitis. 1,2 Therefore, prevention and elimination of recurrent viral assaults before the induction of blinding immune responses are critical for the preservation of vision. HSV-1 glycoprotein D (gD) has been shown to be a most effective vaccine candidate because it can confer powerful protection in immunized animals. 3 Thus, Inoue et al. 4 immunized mice with gD protein and inhibited stromal keratitis, and Hinuma et al. 5,6 linked interleukin-2 (IL-2) to gD to form a chimeric protein, gD-IL-2, expecting an adjuvant effect. They obtained more efficient protection in experimental animals than with gD alone. Recently, we studied the efficacy of DNA vaccines encoding gD and/or gD-IL-2 by subconjunctival injections. 7 Plasmid gD-IL-2 induced a more potent cellular immunity than gD plasmid and prevented the development of stromal keratitis. 7

A recent study demonstrated that topical administration of HSV gB DNA developed effective immunity against lethal HSV encephalitis. 8 Accordingly, we evaluated the preventive effect of the conjunctival instillation of eyedrops that contained DNA encoding gD-IL-2 on stromal keratitis.

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Eight-week-old female BALB/c mice (H-2 d ) were used. They were bred in our laboratory and treated humanely in accordance with the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research.

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The CHR3 strain of HSV-1 was propagated in green monkey kidney (GMK) cells. At maximum cytopathic effect (CPE), the virus was harvested by freezing and thawing three times. After centrifugation at 3,000 rpm for 10 minutes, supernatant was aliquoted and stocked at −80° before use.

The virus 7 was titrated using GMK monolayers on 96-well microplates with antibody overlay method (virus titer = 3 × 10 6 plaque-forming unit/mL).

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DNA Preparation

The expression plasmids (pHSGneo, pHDLneo1) were the same as those used previously. 7 Briefly, a truncated gD DNA fragment (277 amino acids) obtained from HSV-1, Miyama strain, which had been ligated to a mature human IL-2 gene fragment dissociated from an IL-2 expression plasmid, was inserted into the expression vector (pHSGneo). The isolated plasmid (pHDLneo1) contained the truncated gD-IL-2 fusion gene (410 amino acids) under the control of MuLV LTR and SV40 early promoter. The vector plasmid (pHSGneo) was prepared for use as a control.

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Immunization Protocol

pHDLneo1, containing 90 μg/10 μL DNA, was administered topically to the conjunctival sac bilaterally on days 0 and 7. Mice treated with the vector alone (pHSGneo) were used as negative controls.

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Neutralizaton Assay

Serial fourfold dilutions of heat-inactivated (56°C, 30 minutes) murine sera, obtained from the orbital venous plexus 4 weeks after the first immunization, were incubated with an equal volume of the virus (2 × 10 3 PFU/mL) for 1 hour at 37°C. Residual PFUs of the infective virus was assayed on Vero cell monolayers. The virus-neutralizing antibody titer was determined as the reciprocal of the dilution causing a 50% plaque reduction.

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Delayed-Type Hypersensitivity Assay

Three weeks after the second immunization, gD-IL-2 DNA immunized or control mice were injected with 10 μL of the UV-inactivated HSV antigen (10 7 PFU/mL before inoculation) intradermally into the right ear pinna. The same amount of the control antigen (supernatant of GMK cell lysate) was inoculated into the left ear pinna. Forty-eight hours later, the degree of delayed-type hypersensitivity (DTH) response was measured and expressed as the difference in thickness between right and left ear pinnae. Mice that had been infected intraperitoneally with the live virus (1 × 10 4 PFU/mL) 2 weeks earlier were used as positive controls.

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Cytotoxic Effector Cell Assay

The spleen and local lymph nodes were excised from immunized and control mice 3 weeks after the second immunization. Individual cell suspensions (4 × 10 6 cells/mL) were prepared from them, then mixed with partially purified virus (CHR3 strain at a MOI of 1.0 PFU/cell) and incubated for 5 days. A total of 100 μL mixture of cultured cells (1 × 10 6 cells/well) and 51 Cr-labeled, HSV-infected 3T3 clone A31 cells (H-2 d , 1 × 10 4 cells/well) were incubated for 4 hours at 37°C. The amount of radioactivity released into the supernatant was counted by an auto-γ-spectrophotometer. 51 Cr-labeled, HSV-infected L929 cells (H-2 k ) were used as H-2 mismatched target cells. The specific 51 Cr release was calculated as follows: percentage specific lysis = [(sample release − control release]/[maximum release − control release)] × 100. The spontaneous release was below 5% of the maximum release. The positive control mice were prepared in the same way as in the DTH assay.

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Challenge Virus Infection of the Cornea and Clinical Observation

At 3 weeks after the second immunization, the cornea was scarified by 10 crisscross scratches with a 27-gauge needle. Ten microliters of the virus was instilled into the conjunctival sac. Eyes were observed with a hand-held slit-lamp biomicroscope by the same observer daily from days 1 through 8 postinfection (PI), and on days 10 and 14 PI. The severity of epithelial and stromal lesions was graded from 0 to 5 according to the criteria described in our previous paper. 4,7 The scoring of epithelial and stromal lesions was done in a masked fashion.

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Statistical Analysis

The Kruskal-Wallis one-way analysis 9 of variance (ANOVA) with Tukey's method was used for the data of the DTH and cytotoxic effector cell assays. One-way ANOVA on ranks was performed for serum-neutralizing antibody titer and clinical scoring with Tukey's method.

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Serum-neutralizing antibody titers were elevated significantly in gD-IL-2–immunized mice relative to control plasmid-immunized mice at 4 weeks after the first immunization (one-way ANOVA on ranks pass and Tukey's method, p < 0.05) (Fig. 1). Plasmid gD-IL-2 elicited a positive DTH response in immunized mice when challenged with ultraviolet light–inactivated HSV (one-way ANOVA pass and Tukey's method, p < 0.05) (Fig. 2). A significant rise in the systemic and local cytotoxic effector cell activity was detected in gD-IL-2–immunized mice (one-way ANOVA pass and Tukey's method, p < 0.05) (Fig. 3A and B). The spontaneous percentage of release of 51 Cr from the HSV-1 infected C-mismatched L929 cells or uninfected 3T3 clone A31 cells was less than 1% in any groups.

Clinically, stromal keratitis was completely inhibited in the immunized mice (one-way ANOVA on ranks pass and Tukey's method, p < 0.05) (Fig. 4B), although, unexpectedly, epithelial keratitis was not substantially improved in the gD-IL-2–immunized mice (Fig. 4A). In control plasmid-immunized mice on day 10 PI, the cornea was edematous with dense stromal opacities, whereas in gD-IL-2–immunized mice, the cornea maintained its transparency and stromal keratitis was completely inhibited (Fig. 5).

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Plasmid DNA encoding gD-IL-2 that was delivered topically to the conjunctival sac elicited humoral and cellular immunity against the coding HSV antigen. It totally inhibited corneal stromal keratitis and was as effective as that administered subconjunctivally. This is the first report of the topical DNA immunization coding for a viral component and its effect on the viral infection on the cornea. Previously, Daheshia et al. 8 demonstrated that local administration of eyedrops containing gB DNA developed HSV-specific humoral and cellular immunity and protected immunized animals from a lethal HSV infection. However, they did not discuss the preventive effect against herpetic stromal keratitis.

After the topical application of eyedrops, the hydrodynamics of tears and blinking instantly removed the ingredients from the corneal surface. Therefore, the effect of the plasmid DNA may well be attributable to its uptake through the conjunctival epithelium rather than corneal surface. Indeed, normal corneal epithelium lacks antigen-presenting cells, whereas the mucous membrane of the conjunctiva is rich in antigen-presenting cells.

Our results demonstrated that the mice immunized by eyedrops containing gD-IL-2 DNA were completely free of stromal opacification during the entire period of the challenge experiment. We surmise that this immunization protocol induced immunity strong enough to halt the spread of the virus before the occurrence of the cytokine storm in the affected corneal stroma.

Taking all these results together, the convenient topical administration of DNA vaccine encoding gD-IL-2 had an equivalent effect as subconjunctival injection. 7 Therefore, the topical application method of vaccination may have a promising future in clinical medicine.

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Gene therapy; Herpes simplex keratitis; Immunopathology; Immunotherapy; Virus infection

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