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.

1,25-Dihydroxyvitamin D3 prolongs graft survival without compromising host resistance to infection or bone mineral density.

BACKGROUND: Recently, we have shown that 1,25-dihydroxyvitamin D3 prolongs graft survival in mice and rats when the donor and recipient differ at two or more major histocompatability loci. Among the most serious side effects encountered with the currently available transplantation antirejection drugs are an increased susceptibility to infection and decreased bone mineralization. Our results suggest that 1,25-dihydroxyvitamin D3 prolongs graft survival without these side effects of bone loss and susceptibility to infection. METHODS: We compared the ability of 1,25-dihydroxyvitamin D3-treated, nontreated, or cyclosporine (CsA)-treated mice to resist infection with Candida albicans and herpes simplex virus-1. To determine bone density, femurs were collected from nontreated, 1,25-dihydroxyvitamin D3-treated (50 ng/mouse/day), or CsA-treated (25 mg/kg/day) mice, and bone ash was determined. RESULTS: Here we show that 1,25-dihydroxyvitamin D3 treatment does not increase the susceptibility of the host to fungal or viral infection. Furthermore, CsA causes bone loss, whereas 1,25-dihydroxyvitamin D3 actually increases bone mass. CONCLUSIONS: The use of 1,25-dihydroxyvitamin D3 and its analogs to increase transplant survival will avoid bone loss and opportunistic infection, two important disadvantages of the most widely used transplant antirejection drugs--CsA and the glucocorticoids.

Mixed infection with herpes simplex virus type 1 generates recombinants with increased ocular and neurovirulence.

The authors used the method of mixed ocular infection and subsequent in vivo selection to isolate Herpes simplex virus type 1 intratypic recombinants with increased ocular virulence and neurovirulence. Four recombinants were studied in some detail (DRG1A3, DRG2A2, DRG3A3, and DRG4A1). The recombinants had lethal doses in 50% of animals tested (LD50) at least 2-3 log units lower than either parent virus (OD4 and CJ394) and caused significantly more severe stromal keratitis, vascularization of the cornea, and blepharitis than either parent. Studies on the ability of DRG1A3 and DRG4A1 to replicate in the eye, trigeminal ganglia, and brain showed that these recombinants replicated to higher titers (1-3.5 log units) than the parents in all three tissues. One of the parents, OD4, spread to the central nervous system with the same kinetics as CJ394, DRG1A3, and DRG4A1 but had a restricted ability to replicate in all tissues, which may account for its lack of virulence. The other parent, CJ394, was nonneurovirulent but replicated to titers which were only 1-1.5 log units lower than the neurovirulent recombinants. These recombinants should be useful in studying virulence determinants in herpetic ocular infections.

A herpes simplex type 1 ICPO deletion mutant demonstrates diminished pathogenicity during acute ocular infection in different host animals.

We compared a previously characterized herpes simplex type 1 alpha 0 deletion mutant, dlx3.1, which produced no functional ICP0, with its wild-type parental strain, KOS, during acute ocular infection of different host animals. Acute pathogenicity of the viral strains in NZ rabbits, Balb/c and A/J mice was evaluated by keratitis scores, ocular and trigeminal ganglionic viral titers, and host survival. We found that dlx3.1 was significantly less pathogenic than KOS. Host differences proved very important in the evaluation of acute pathogenicity. A species-dependent enhancement of ocular pathogenicity was demonstrated for dlx3.1 following a larger viral inoculum and host immunosuppression. We conclude that alpha 0 gene function appears to play an important role during acute ocular pathogenicity of HSV-1 in animal models. Furthermore, in vivo pathogenicity studies contribute important information in the evaluation of essential viral gene function.

A microscopic study of herpes simplex virus retinopathy in mice.

ICR white mice were inoculated with herpes simplex virus (HSV) type I in the anterior chamber of one eye. Animals were killed at intervals of 2, 4, 6, 8, and 10 days and both eyes were obtained for light and electron microscopic study of retinal changes. HSV retinopathy developed in 42 (91%) of 46 inoculated eyes. Fourteen (88%) of sixteen noninoculated eyes examined after the sixth postinoculation day developed HSV retinopathy. The earliest signs of retinopathy in the inoculated eye were peripheral retinal vasculitis and inflammatory cells throughout the nerve fiber layer on day 2. No virus was found in retinal tissue until day 4, at which time disruption of outer retinal layers (outer nuclear layer and layer of rods and cones) was observed in the peripheral retina. The earliest signs of retinopathy in the noninoculated eye were isolated foci of outer retinal disruption in the posterior retina on day 6. The inflammation accompanying early retinal changes of HSV retinopathy were more severe in the inoculated eye. Electron microscopy of both eyes revealed viral particles in the inner nuclear and ganglion cell layers at the time of outer retinal disruption, but viral particles were seen only rarely in the outer retinal layers at this stage. Early disruption of normal retinal architecture may be due to infection and destruction of Muller cells. The retinopathy progressed in both eyes to total destruction of the retina by day 10. Viral infection of the retinal pigment epithelium occurred, but viral particles were seen only rarely in the underlying choroid. This model may be useful for the study of HSV retinopathy in humans.

Effects of cyclosporine A on clinical and immunological parameters in herpes simplex keratitis.

The immunosuppressive effects of cyclosporine A (CyA) on the clinical and antiviral immune responses were examined in experimental herpes simplex virus (HSV) keratitis in the rabbit in order to clarify the role that immune lymphocytes play in herpetic stromal disease. Cyclosporine A was administered intramuscularly to rabbits daily starting from the time of corneal infection with HSV until day 14 postinfection. Control HSV-infected rabbits received daily injections of the solvent vehicle alone. HSV-infected rabbits receiving CyA treatment showed more severe and persistent stromal keratitis, and a greater incidence and duration of virus recovery from the cornea. Suppression of cellular immune responses to T cell mitogens, B cell mitogens (anti-rabbit immunoglobulins), and HSV antigens were observed in the CyA treatment group. These results show that in CyA-treated HSV-infected rabbits the antiviral immune responses are inhibited. Acute viral infections with cytopathic viruses such as HSV may therefore be more dramatic, suggesting that CyA may facilitate the potentiation of HSV infections ordinarily suppressed by immune cells.

Herpetic keratitis in athymic (nude) mice.

The inflammatory response to herpes simplex virus infection of the cornea was studied in athymic nude (nu/nu) and heterozygote (nu/+) BALB/c mice. Although athymic mice were highly susceptible to HSV infection and died 13 to 17 days after corneal inoculation, they failed to develop necrotizing keratitis of the cornea. Heterozygote mice survived the initial virual infection, but many of these mice developed necrotizing keratitis and permanent corneal scarring. Light and electron microscopy showed numerous inflammatory cells (polymorphonuclear leukocytes and lymphocytes) in the corneas of heterozygote mice, but not in the athymic mice. These studies suggest that the immune system plays a dual role in herpes simplex virus infection of the cornea: protection against dissemination of the virus and immunopathogenesis of necrotizing keratitis in the cornea.