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Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

Interleukin 10 (IL-10), a moderator of Delayed Type Hypersensitivity (DTH) responses, has been demonstrated to be present late in acute HSV corneal infection and may help limit blinding inflammatory lesions there. In contrast, IL-10 is present early in the development of recurrent herpetic stromal keratitis (HSK) lesions in mice. To determine the role of IL-10 and DTH responses in recurrent HSK, we examined DTH responses and disease parameters in latently infected IL-10 knock out (KO) mice, and latently infected normal mice that were untreated or received anti-IL-10 antibodies or recombinant IL-10 following ultraviolet-B stimulated ocular HSV recurrence. Low DTH responses were associated with less severe corneal disease while high DTH responses were associated with greater corneal disease. In IL-10 KO mice, and in normal mice given anti-IL-10 antibodies, corneal opacification was increased and DTH responses were significantly prolonged. Normal mice receiving rIL-10 by ocular and intra-peritoneal routes had less severe corneal lesions. Our results indicate that IL-10 and DTH responses play an important role in the pathogenesis of recurrent HSK in mice.

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs− vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs−-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0·007 to 0·035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0·05) and delayed type hypersensitive responses were unaffected by vhs− vaccination. Combined with studies using different vaccination timing and vhs− genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.

Recurrent herpetic stromal keratitis (HSK) is a potentially blinding, immune-mediated disease. To better understand the immunopathology of recurrent HSK, we examined the cytokine profile of mouse corneas with the condition.The eyes of latently infected mice were examined for corneal pathology and cytokine content following UV-B-stimulated herpes simplex virus (HSV) reactivation.Peak HSV-induced corneal disease, manifested by stromal opacification, occurred 7-14 days after viral reactivation in latently infected mice. In qualitative RT-PCR analyses, IFNgamma, IL-10, IL-4, and IL-12 p40 mRNA were simultaneously expressed before and during recurrent HSK. Competitive, semi-quantitative RT-PCR evaluation of cytokine mRNA revealed highest IFNgamma expression before and during clinical disease with a decline thereafter. IL-4 levels peaked and declined before day 14, while IL-10 peaked on days 7 or 14 and paralleled IFNgamma at lower levels. Small amounts of IL-12 p40 mRNA were detected late in the disease course. ELISA evaluation of corneal extracts demonstrated similar results, featuring early expression of Th2 cytokines relative to disease.The presence of Th2 cytokines during early stages of recurrent herpetic corneal lesions indicate the presence of a mixed Th1 and Th2 cell infiltrate, which is likely associated with a memory response to viral antigens. These data suggest that disease resolution in corneas with recurrent HSK may depend upon the balance between destructive and protective cytokines at individual sites of viral recurrence.

The protective efficacy of a glycoprotein D subunit vaccine (gD2 SB AS4) was evaluated in a mouse model of human recurrent herpetic stromal keratitis (HSK). When administered before primary infection, gD2 SB AS4 protected mice against corneal pathology, mortality, and latency resulting from ocular viral challenge with herpes simplex virus type 1 (HSV-1) McKrae strain. In addition, gD2 SB AS4 significantly decreased postreactivation corneal disease. A control vaccine, gD2 alum, protected against acute ocular infection only. When administered after primary infection, gD2 SB AS4 vaccination decreased postreactivation ocular shedding but had no other significant effects. Vaccination with gD2 SB AS4 was associated with high anti-gD antibody responses and low delayed-type hypersensitivity responses. These results have identified a prophylactic vaccine, gD2 SB AS4, with activity against acute and recurrent HSK in mice and emphasize the need for vaccine evaluation in both primary and recurrent ocular herpetic disease models.

Herpes simplex virus 1 (HSV-1) infection of the cornea leads to a potentially blinding disease, termed herpetic stromal keratitis (HSK) that is characterized by lesions of an immunoinflammatory nature. In spite of the fact that HSK typically presents as a recurrent disease due to reactivation of virus which latently infects the trigeminal ganglia, most murine studies of HSK have employed a primary and not recurrent model of the disease. This report documents the several recurrent models of HSK that have been developed and how data generated from these models differs in some important aspects from data generated following primary infection of the cornea. Chief among these differences is the fact that recurrent HSK takes place in the context of an animal that has a preexisting anti-HSV immune response, while primary HSK occurs in an animal that is developing such a response. We will document both differences and similarities that derive from this fundamental difference in these models with an eye towards possible vaccines and therapies that demonstrate promise in treating HSK.

Summary Since exercise stress is associated with multiple changes in immune parameters, we evaluated the effect of acute exercise on 3 indices of immune function in the horse. Six unconditioned thoroughbred horses were subjected to a tread- mill-based exercise challenge. Exerci se intensity was determined by monitoring each horse's heart rate, plasma lactate, and cortisol levels. Concurrently, peripheral blood mono- nuclear cells were used to assess pokeweed mitogen (PWM) -induced blastogenesis, influenza-specific proliferation, and lymphokine activated killer (LAK) cell activity. Heart rate peaked during the gallop on an incline while lactate was highest at the end of the stress test (To) with values of 200 bpm and 9 mM/L respectively. Average plasma cortisol was great- est at TO and T+2o with values 76% higher than unstressed horses. Significantly (P

The role that interferon-gamma (IFNgamma) plays during herpetic stromal keratitis (HSK) has not been definitively determined. In primary HSK most reports suggest that IFNgamma may help control viral replication and contribute to corneal pathology. However, its role in recurrent HSK has not been directly addressed. The present study addresses its role in recurrent HSK by comparing HSK in latently infected normal and IFNgamma gene knockout (GKO) on the C57BL/6 background. We initially evaluated HSK following primary infection and observed that GKO mice had higher tear film virus titers, but virtually identical ocular disease as normal mice. In contrast, following reactivation of latent virus, GKO mice had a greater incidence and severity of opacity, neovascularization, and blepharitis. Interestingly, the incidence of reactivation after UV-B exposure was equivalent in GKO and normal mice, but virus shedding was increased in the GKO groups. We also observed diminished delayed-type hypersensitivity responses in GKO mice, as expected. These data indicate that IFNgamma is important for the control of virus replication in both primary and recurrent ocular HSV infection in C57BL/6 mice. The enhanced recurrent disease seen in GKO mice may be the result of increased viral titers and persistence in these mice which act to prolong the stimulation of an inflammatory response.

In primary ocular herpes simplex virus (HSV) infection, nitric oxide may function to control viral replication and herpetic stromal keratitis (HSK) lesions. Recurrent HSK, manifested as corneal opacity and neovascularization, is the potentially blinding sequel to primary infection. Here, we assess the effects of nitric oxide synthase inhibition on a mouse model of recurrent HSK. In preliminary primary infection experiments, NIH inbred mice treated with aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), experienced no changes in post-infection tear, brain, or ganglia virus titers, but encephalitis-related mortality was elevated. After UV-B stimulated viral reactivation, iNOS inhibition did not affect virus shedding or clinical disease. In contrast to primary HSK, there was no exacerbation of mortality in recurrent disease. Our findings indicate that nitric oxide can be neuroprotective without antiviral effects in primary HSK, and does not play a significant role in the pathogenesis of recurrent HSK. Compared with data from other mouse strains, this work suggests that there may be a genetic component to the importance of NO in controlling ocular HSV infection.

Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Δ41Δ29, has combined vhs and replication (ICP8−) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs−strain (BGS41). Here, we tested the hypothesis that Δ41Δ29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Δ41Δ29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Δ41Δ29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Δ41Δ29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Δ41Δ29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.

The proliferative response of mononuclear cells from MS patients and normal control subjects to intact and delipidated myelin membranes was examined. The mean frequency of recognition in both groups of human subjects was greater for delipidated myelin than for intact myelin. Human T cell lines established using intact or delipidated myelin as the antigen were highly heterogeneous in response, and were each able to recognize myelin basic protein and myelin proteolipid protein peptides. However, there was no difference in the frequency of recognition of either form of myelin membrane when MS patients were compared to control subjects. Our results suggest that the presentation of delipidated forms of membrane proteins might enhance the response to myelin antigens in vivo, and be relevant to demyelinating diseases.

CCL2 and CCL3 are proinflammatory chemokines that are produced during the early stages of inflammation and are known to stimulate the migration of mononuclear cells to the site of inflammation,. Previous studies addressing the role of these chemokines during primary herpetic stromal keratitis (HSK), have suggested that CCL2 is involved in reducing corneal disease and that CCL3 is involved in promoting this disease. We addressed the role that these chemokines play in a recurrent model of HSK. Results from these studies did not demonstrate a significant role for CCL2 except for very early time points following reactivation of virus. Surprisingly, mice deficient in CCL3 did not have significantly reduced recurrent disease, , but in fact showed significantly enhanced disease. This argues that CCL3 might play an ameliorative role during recurrent HSK. In addition, we observed that these same CCL3 deficient mice showed increased resistance to viral-induced mortality following infection with HSV-1. Taken together, these results suggest that CCL3 plays a significant protective role during recurrent HSK and is involved in enhancing lethality.

Purpose To review the changing epidemiology of herpes simplex virus infection, emerging patterns of herpetic ocular disease, and the challenges and promise of herpes simplex virus vaccine therapy. Design Perspective. Methods Literature review. Results An epidemic increase in genital herpes simplex type 2 infection is reflected in a 30% increase in HSV-2 antibodies in the United States since 1976. Approximately one in four people in the United States over age 30 is infected with HSV-2. Primary acquisition of herpes simplex type 1 is becoming progressively delayed in many industrialized countries, in contrast to developing nations where the virus is acquired early in life and is ubiquitous. Changes in sexual behavior among young adults have been associated with a recent increase in genital HSV-1 infection, resulting from oral-genital rather than genital-genital contact. Clinical trials of HSV vaccines using selected herpes simplex virus type 2 proteins mixed in adjuvant have shown limited efficacy in seronegative women, but not in men. Conclusions The recent epidemic of genital herpes simplex type 2 infection is likely to result in an increase in neonatal ocular herpes and in delayed cases of acute retinal necrosis syndrome. The increase in genital HSV-1 may lead to industry production of vaccines that contain components of both HSV-1 and HSV-2 targeted toward limiting genital disease and transmission. As newer herpes simplex vaccines become available, ophthalmologists must be vigilant that a boost in immunity against HSV does not have a paradoxical effect in exacerbating break-through cases that develop immune-mediated herpes simplex stromal keratitis.