Macrophages are important determinants of acute ocular HSV-1 infection in immunized mice.

PURPOSE: To determine the effect of macrophage depletion on herpes simplex virus type (HAV)-1 replication in the eye and on the establishment of latency in trigeminal ganglia (TG) of immunized and ocularly infected mice. METHODS: BALB/c mice were immunized with five HSV-1 glycoprotein DNA genes or were sham immunized. The virulent HSV-1 strain KOS was used as a positive vaccine control. Immunized mice were depleted of their macrophages by dichloromethylene diphosphonate (Cl(2)MDP) injection. After ocular infection with the HSV-1 strain McKrae, virus replication in the eye, blepharitis, corneal scarring, and dermatitis were determined. Finally, the copy numbers of latency-associated transcript (LAT) and CD4(+) and CD8(+) T-cell transcripts in the TGs of surviving mice 30 days after infection were determined by RT-PCR. RESULTS: Depletion of macrophages in immunized mice increased HSV-1 replication in the eye of infected mice between days 1 and 5 after ocular infection. Depletion of macrophages did not alter the HSV-1-induced death or corneal scarring in immunized mice. Macrophage depletion, however, resulted in increased blepharitis in immunized mice. Finally, macrophage depletion had no effect on the establishment of latency in immunized mice, as the TGs from both depleted and mock-depleted mice were negative for the presence of the LAT transcript. CONCLUSIONS: In immunized mice during primary HSV-1 ocular infection, macrophages play an important role in vaccine efficacy against HSV-1 replication in the eye and blepharitis in infected mice. During the latent stage of HSV-1 infection, however, macrophage depletion failed to have any observable effect on HSV-1 latency in the TGs of infected mice.

Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines.

PURPOSE: To compare the effectiveness of immunization with "naked" DNA corresponding to the genes encoding five HSV-1 glycoproteins, gB, gC, gD, gE, and gI (5gP DNA), with immunization with the five glycoproteins (5gP protein). Also, to compare immunization of 5gP protein in Montanide ISA 720 (SEPPIC, Paris, France), an adjuvant recently approved for use in humans, with immunization of 5gP protein in Freund's adjuvant. METHODS: BALB/c mice were vaccinated with 5gP DNA or 5gP protein emulsified in ISA 720 or Freund's adjuvant. Neutralizing antibody titers were determined by plaque-reduction assays. IL-2, -4, and -12 and IFN-gamma levels were determined by ELISA after in vitro stimulation of spleen cells. After ocular challenge with 2 x 10(5) plaque-forming units [pfu] per eye of HSV-1 strain McKrae, virus replication in the eye, survival, blepharitis, corneal scarring, and latency were determined. RESULTS: Neutralizing antibody titers (approximately 1:800-1:1200), corneal scarring (trace) and survival (100%) were similar for all vaccine groups, including 5gP DNA. Compared with the other vaccine groups, the 5gP DNA group had less ocular virus replication, as judged both by maximum virus titer and time of viral clearance. ISA 720 appeared more effective than Freund's against ocular virus replication and eye disease. The 5gP DNA-vaccinated mice had less blepharitis and latency than any other group and had the highest levels of IL-12 and IFN-gamma. All vaccine groups had similar levels of IL-2. CONCLUSIONS: The 5gP DNA vaccine appeared to be more effective than the corresponding protein subunit vaccine, regardless of adjuvant. Emulsification of the 5gP protein in ISA 720 appeared to be more effective than emulsification in Freund's adjuvant.

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

Prevention of herpes keratitis by monoclonal antibodies specific for discontinuous and continuous epitopes on glycoprotein D.

Seven monoclonal antibodies (mAb) specific for defined discontinuous and continuous epitopes on glycoprotein D of herpes simplex virus type 1 (HSV-1) were surveyed for their capacity to protect against virus-induced corneal disease in a murine ocular infection model. A known amount of purified mAb was transferred passively to BALB/c mice 24 hr after topical infection with HSV-1 on their scarified corneas. At high doses (50-136 micrograms), all seven mAbs protected against the development of persistent necrotizing stromal keratitis. Significant protection was also observed at low doses (20 micrograms) with two mAbs to discontinuous epitopes and two mAbs to continuous epitopes. Selected high-dose mAbs also were able to reduce the severity of blepharitis. These results indicated that at least seven different antigenic sites on glycoprotein D can serve as targets for effective antibody therapy in the murine model of HSV-1 ocular infection.

Mechanisms of protection against herpes simplex virus type 1-induced retinal necrosis by in vitro-activated T lymphocytes.

In BALB/c mice, acute retinal necrosis occurs in the uninoculated eye 8 to 10 days following uniocular anterior chamber inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1). Retinitis in the uninjected eye can be prevented if HSV-1-specific immune effector cells that have been restimulated with virus in vitro are administered intravenously within 1 day of anterior chamber inoculation of virus. We explored further the mechanism of protection afforded by these activated immune effector cells. The results of our studies revealed that optimal protection from retinitis required in vitro restimulation, since infusion of 50 x 10(6) HSV-1-primed but nonrestimulated cells could not protect as well as 10 x 10(6) activated cells. Analysis of both restimulated and nonrestimulated cells showed that only in vitro-restimulated cells were cytotoxic to HSV-1-infected syngeneic target cells. From these studies, we concluded that the ability to kill virus-infected target cells contributed to optimal protection achieved by intravenous administration of activated immune effector cells. Furthermore, T-cell subset depletion of activated immune effector cells demonstrated that both L3T4+ and Lyt-2+ T cells in the transfer inoculum contributed to protection. Additional studies revealed that although the transferred immune effector cells reached the injected eye within 24 h, virus replication in the injected eye was not affected. In the uninjected eye, virus titers were low, consistent with protection of this eye from retinitis. Taken together, the virus recovery results suggest that the interaction of virus with intravenously administered HSV-1-specific immune effector cells which limits virus spread and/or replication of virus probably occurred within the central nervous system and prevented the second wave of virus from entering the uninoculated eye.

Experimental keratitis in rabbits by human HSV-1 variants: prevention and treatment.

The efficacy of different therapies and vaccine preparations was assessed for treating or preventing herpetic ocular keratitis induced by experimental inoculation in rabbits with two HSV-1 variants that display different pathogenetic potential. Early administration of acyclovir (ACV) promoted fast healing and prevented neurologic involvements: alpha-interferon (alpha-IFN) was less efficient than ACV; combined therapy with both drugs increased the antiviral effects. In an attempt to prevent the disease, rabbits were vaccinated with a slightly pathogenic HSV-1 variant or with a secreted form of an engineered HSV-1 glycoprotein gB (gB-1s) and were subsequently challenged with a highly pathogenic HSV-1 variant. Immunization of rabbits with gB-1s was much more efficient than immunization with live virus in reducing the severity of herpetic keratitis and in preventing CNS disease.

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.

Herpes simplex virus glycoprotein D. Protective immunity against murine herpetic keratitis.

%$%%protective effect of glycoprotein D (gD) immunization against murine herpetic keratitis was investigated. gD was purified by affinity chromatography using anti-gD monoclonal antibodies. Prior immunization with gD was shown to be effective in protecting mice from both the development of stromal keratitis and the spread of the virus to the central nervous system. The level of serum antibodies for virus neutralization, as well as for complement-dependent cytolysis (CDC), was significantly elevated in gD-immunized animals. Cellular immunity, however, was not detected. These results indicate that two antibody-mediated defense mechanisms--virus neutralization and CDC--were responsible for the protective effect observed in our study.

Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.

Mice were treated with serum containing antibodies to herpes simplex virus type 1 (HSV-1) or normal serum, 1 day before inoculation on the cornea with HSV-1 strain McKrae. As expected, without passive immunization, mice developed high levels of serum neutralizing antibody. By contrast, in passively immunized animals, such antibody became undetectable by 29 days after inoculation of serum, in spite of the virus infection. There was no difference between passively immunized mice and those given normal serum in the duration of shedding of virus in tears and the duration and severity of corneal epithelial disease. However, non-immunized mice had a high incidence of mortality and developed disease of the iris, corneal stroma and lids, and their corneas became opaque and vascularized. In non-immunized animals, the timing of isolation of virus from nervous tissues and the sequence of appearance of virus antigens in ocular tissues indicate that the disease of deeper eye tissue was caused by virus spreading from the nervous system back to the eye. Restriction of such spread in passively immunized animals seems the likely explanation for their protection from death and severe ocular damage. Despite this restricted spread, passively immunized animals had a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion. However, in comparison with mice given normal serum, there was a far lower incidence of such infection in the other two parts of this ganglion and in the superior cervical ganglion. Since passively immunized animals have a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion and their eyes are normal, they will prove useful in studies involving induction of recurrent disease.

Protection of mice from herpes simplex virus-induced retinitis by in vitro-activated immune cells.

A form of acute retinal necrosis occurred in the contralateral eyes of susceptible mice 1 week after each received a uniocular injection of live herpes simplex virus type 1 (HSV-1) in the anterior chamber. Although these mice did not develop systemic delayed hypersensitivity to virus antigens, their sera contained virus-specific antibodies at the time contralateral retinitis occurred. These findings suggest that systemic immunity might not be able to protect against contralateral retinitis. To explore this possibility further, we examined lymph nodes and spleens of intraocularly infected mice to determine whether their lymphoid tissues contained primed HSV-1-specific cytotoxic T cells. Virus-specific cytotoxic T cells were readily identified in these mice. We wondered why successful immune priming did not confer protection against HSV-1 retinitis. We examined this issue by evaluating the capacity of in vitro-generated, HSV-1-specific effector T cells to prevent retinitis by infusing these cells by various routes and at various times into mice that received an intracameral injection of HSV-1. The results revealed that virus-specific effector cells could prevent contralateral retinitis if injected intravenously or into the anterior chamber of the contralateral eye at the same time that virus was injected into one eye. However, the effector cells failed to prevent retinitis if they were injected into the same eye that received HSV-1 or if their intravenous administration was delayed until 24 h after the HSV-1 injection into the eye. We concluded that immune T cells can protect against contralateral retinal necrosis caused by uniocular injection of HSV-1 into the anterior chamber but only if they are administered during the first 24 h after virus infection. We propose that a retinitis-inducing process is set in motion during this early time interval postinfection. Once the process has been initiated and established, it is no longer susceptible to immune intervention. It would appear that mice that are susceptible to contralateral retinitis fail to mobilize a protective response quickly enough to ward off the establishment of the retinitis-inducing process and its disastrous eventuality.

Oral acyclovir reduces the incidence of recurrent herpes simplex keratitis in rabbits after penetrating keratoplasty.

To determine if acyclovir sodium prevents postoperative herpes simplex virus type 1 (HSV-1) recurrences, 21 rabbits harboring latent HSV-1 underwent uniocular autograft penetrating keratoplasty. All operated-on eyes were treated with topical and subconjunctival dexamethasone sodium phosphate. Ten of the 21 rabbits also received oral acyclovir (intravenous acyclovir was given at the time of surgery). Postoperatively, 9 (82%) of 11 operated-on eyes in rabbits not treated with acyclovir had positive HSV-1 ocular cultures. In acyclovir-treated rabbits, however, none of the 10 operated-on eyes had positive ocular cultures. In addition, 9 (82%) of 11 of the operated-on eyes had geographic ulcers develop in the non-acyclovir-treated rabbits, compared with 1 (10%) of 10 in the acyclovir-treated rabbits. Finally, stromal keratitis appeared in 5 (56%) of 9 of the operated-on eyes in non-acyclovir-treated rabbits and 1 (12%) of 8 of the operated-on eyes in acyclovir-treated rabbits. The results of this study indicate that acyclovir significantly lowered the incidence of HSV-1 ocular shedding, geographic ulceration, and stromal keratitis in a rabbit autograft penetrating keratoplasty model.

Quantitation of purified monoclonal antibody needed to prevent HSV-1 induced stromal keratitis in mice.

A purified IgG2a monoclonal antibody with a neutralizing titer of 10(4) and specificity for gD was evaluated for its therapeutic potential in a murine ocular infection model. BALB/c mice, infected on the scarified cornea with 10 times the HSV-1 strain RE concentration needed to produce severe and persistent stromal opacity, were given a single inoculation of antibody intraperitoneally 24 hours later. The animals were then followed for corneal disease development. Antibody, at concentrations as low as 10 micrograms per mouse, was strikingly effective at preventing corneal opacity. Furthermore, the corneas, once clear, remained clear whereas the controls developed +3 to +5 stromal disease which was still present 60 days post-infection. Animals that had been treated and recovered from infection were resistant to subsequent HSV-1 challenge on the opposite cornea. These results demonstrate the therapeutic potential of systemically administered microgram quantities of anti-gD antibody.

Immunomodulation of experimental murine herpes simplex keratitis: I. UV-HSV protection.

A/J mice were immunized subcutaneously with ultraviolet light (UV) inactivated herpes simplex virus type-1, MP strain (HSV-MP). Control A/J mice were immunized subcutaneously either with media (unimmunized controls) or with live HSV-MP (immunized controls). Immunized and control mice were challenged ocularly with either MP or mP strain HSV-1 after corneal scarification and were followed for 3 weeks post corneal challenge. The mice were observed during this time period for signs of herpes simplex keratitis (HSK), lid lesions and encephalitis. At the time of sacrifice, the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these studies demonstrated that immunization with UV inactivated HSV (UV-HSV) gave the same protection against keratitis and encephalitis as immunization with live virus. Furthermore, the cocultivation assays indicated that immunization with either live HSV-1 or UV inactivated HSV-1 protected against the establishment of latency.

Immunomodulation of experimental murine herpes simplex keratitis: II. Glycoprotein D protection.

Subcutaneous immunization with purified HSV-1 glycoprotein D (gD) protects susceptible A/J mice against keratitis and encephalitis following corneal HSV-1 challenge. Mice were immunized with gD, in complete Freund's adjuvant, 3.0 micrograms/mouse followed by two booster doses of 1.5 micrograms/mouse at weeks 2 and 4. Control groups of A/J mice were injected with either complete Freund's adjuvant (unimmunized controls) or live HSV-1 MP strain (immunized controls). All mice were challenged ocularly at week 5 with HSV-1, F strain (6.5 x 10(3) PFU) after corneal scarification. None of the 16 animals immunized with gD developed stromal keratitis; only 3 out of 12 animals immunized with live HSV-1 developed a stromal keratitis; 13 out of 16 CFA primed unimmunized mice developed severe stromal keratitis within 14 days post corneal challenge, and 3 out of 16 control CFA primed animals died within 16 days post corneal challenge. At the time of sacrifice (3 weeks post corneal challenge), the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these experiments indicate that immunization with gD produces protection against latent ganglionic infection in 56% of the immunized animals, and provides protection against keratitis and death following HSV corneal challenge.

Hypothetical vaccination of the Dutch population with a herpes simplex virus vaccine: estimation of the profitability using a demographic projection model.

The apparently increasing evidence of herpes simplex virus infections of the genital tract has focused attention on preventing the infection by vaccination. Herpes genitalis is not, however, the most quantitatively important clinical manifestation of herpes simplex virus infections. Because 41% of the hospitalized patients are younger than 20 years, vaccination of birth cohorts would be more favourable. In this paper the financial benefits of a hypothetical herpes simplex virus vaccination were calculated with the use of a population projection model. For the Netherlands, if the price of the hypothetical herpes simplex virus vaccine equals the cost price of the mumps component of the combined mumps-measles-rubella vaccine, the herpes vaccine would be profitable within 8 years.

Contact lens disinfection to prevent transmission of viral disease.

Current FDA standards for contact lens disinfecting systems require that viricidal activity be demonstrated against just one strain of herpes simplex virus, type 1. Small and nonenveloped viruses (e.g., adenoviruses) may be more resistant to disinfection than herpes simplex virus (HSV); however, the efficacy of disinfection systems against these other agents is not routinely tested. Currently approved methods of chemical and thermal lens disinfection do appear to be efficient means to inactivate HSV and the human immunodeficiency virus, both of which have lipid envelopes.

Effect of immunization and immunosuppression on induced ocular shedding and recovery of herpes simplex virus in infected rabbits.

Immunization and immunosuppression were evaluated during latent ocular herpes simplex virus, type 1 (HSV-1) infection in the rabbit, using the following parameters: (1) ability to recover virus from preocular tearfilm cultures; (2) reactivation of latent infection by direct electrical stimulation; and (3) recovery of virus from latently infected ganglia by whole-cell co-cultivation. Immunization prior to ocular inoculation of virus significantly reduced both the titer of virus shed into the tearfilm and the duration of virus shedding during primary ocular infection. Half of the non-immunized control rabbits died secondary to virus encephalitis, whereas none of the immunized rabbits died. The immunized rabbits could not be induced to shed virus by electrically stimulating the trigeminal ganglion directly. Immunosuppression of latently infected rabbits with high-dose cyclophosphamide (300 mg kg-1) enhanced virus shedding in the tearfilm and increased mortality due to viral encephalitis. Low-dose cyclophosphamide immunosuppression (40 mg kg-1) did not increase mortality because of viral encephalitis. Tearfilm virus shedding secondary to electrical induction in high-dose and low-dose cyclophosphamide animals was higher than that of control, non-immunosuppressed animals.