Correlates of immunologic protection requisite for an efficacious herpes simplex virus 1 (HSV-1) vaccine remain unclear with respect to viral pathogenesis and clinical disease. In the present study, mice were vaccinated with a novel avirulent, live attenuated virus (0⌬NLS) or an adjuvanted glycoprotein D subunit (gD-2) similar to that used in several human clinical trials. Mice vaccinated with 0⌬NLS showed superior protection against early viral replication, neuroinvasion, latency, and mortality compared to that of gD-2-vaccinated or naive mice following ocular challenge with a neurovirulent clinical isolate of HSV-1. Moreover, 0⌬NLS-vaccinated mice exhibited protection against ocular immunopathology and maintained corneal mechanosensory function. Vaccinated mice also showed suppressed T cell activation in the draining lymph nodes following challenge. Vaccine efficacy correlated with serum neutralizing antibody titers. Humoral immunity was identified as the correlate of protection against corneal neovascularization, HSV-1 shedding, and latency through passive immunization. Overall, 0⌬NLS affords remarkable protection against HSV-1-associated ocular sequelae by impeding viral replication, dissemination, and establishment of latency. IMPORTANCEHSV-1 manifests in a variety of clinical presentations ranging from a rather benign "cold sore" to more severe forms of infection, including necrotizing stromal keratitis and herpes simplex encephalitis. The present study was undertaken to evaluate a novel vaccine to ocular HSV-1 infection not only for resistance to viral replication and spread but also for maintenance of the visual axis. The results underscore the necessity to reconsider strategies that utilize attenuated live virus as opposed to subunit vaccines against ocular HSV-1 infection. Herpes simplex virus 1 (HSV-1) is a highly successful human pathogen that results in approximately 40,000 new cases of severe visual impairment each year (1). In such cases, the immune response to the pathogen inadvertently mediates corneal pathology. Moreover, the morbidity associated with ocular infection results from episodic viral recrudescence (2, 3). This etiology is dependent upon reactivation of HSV-1 from latently infected neurons within the trigeminal ganglion (TG), which innervates the cornea and orofacial mucosae. Although gamma interferon (IFN-␥) and other cytokines secreted by T cells and other cornearesident cells facilitate viral clearance in the cornea, these soluble factors also recruit neutrophils and activate macrophages replete with proteases that instigate extracellular matrix remodeling and scar formation, thereby compromising visual acuity (4-10). Furthermore, protracted inflammatory responses sustained beyond clearance of the virus contribute to corneal neovascularization (1, 11). Consequently, developing HSV vaccines that elicit robust protection against infection without enhancing the risk for corneal immunopathology is an important clinical matter as no sanctioned HSV vaccine clinical trials to dat...

Herpes simplex virus type 1 (HSV-1) infection of the cornea induces VEGF-A-dependent lymphangiogenesis that continues to develop well beyond the resolution of infection. Inflammatory leukocytes infiltrate the cornea and have been implicated to be essential for corneal neovascularization, an important clinically relevant manifestation of stromal keratitis. Here, we report that cornea infiltrating leukocytes including neutrophils and T cells do not have a significant role in corneal neovascularization past virus clearance. Antibody mediated depletion of these cells did not impact lymphatic or blood vessel genesis. Multiple pro-angiogenic factors including IL-6, angiopoietin-2, HGF, FGF-2, VEGF-A, and MMP-9 were expressed within the cornea following virus clearance. A single bolus of dexamethasone (DEX) at day 10 pi resulted in suppression of blood vessel genesis and regression of lymphatic vessels at day 21 pi compared to control-treated mice. Whereas IL-6 neutralization had a modest impact on hemangiogenesis (day 14–21 pi) and lymphangiogenesis (day 21 pi) in a time-dependent fashion, neutralization of FGF-2 had a more pronounced effect on the suppression of neovascularization (blood and lymphatic vessels) in a time-dependent, leukocyte-independent manner. Furthermore, FGF-2 neutralization suppressed the expression of all pro-angiogenic factors measured and preserved visual acuity.

Herpes simplex virus 1 (HSV-1) is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously shown that HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of vascular endothelial growth factor A (VEGFA) dependent upon expression of VEGFR2 during acute infection. We hypothesized that other factors may be involved in lymphangiogenesis, with proinflammatory cytokines as the leading candidates. IMPORTANCEWe have identified at least two proinflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-␣ as additional proangiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

The capacity of licensed vaccines to protect the ocular surface against infection is limited. Common ocular pathogens such as herpes simplex virus type 1 (HSV-1) are increasingly recognized as major contributors to visual morbidity worldwide. Humoral immunity is an essential correlate of protection against HSV-1 pathogenesis and ocular pathology, yet the ability of antibody to protect against HSV-1 is deemed limited due to the slow IgG diffusion rate in the healthy cornea. We show that a live-attenuated HSV-1 vaccine elicits humoral immune responses that are unparalleled by a glycoprotein subunit vaccine vis-à-vis antibody persistence and host protection. The live-attenuated vaccine was utilized to assess the impact of immunization route on vaccine efficacy. The hierarchical rankings of primary immunization route with respect to efficacy were: subcutaneous ≥ mucosal > intramuscular. Prime-boost vaccination via sequential subcutaneous and intramuscular administration yielded greater efficacy than any other primary immunization route alone. Moreover, our data also support a role of complement in prophylactic protection as evidenced by intracellular deposition of C3d in the corneal epithelium of vaccinated animals following challenge and delayed viral clearance in C3-deficient mice. We also identify that the neonatal Fc receptor (FcRn) is upregulated in the cornea following infection or injury concomitant with increased antibody perfusion. Lastly, selective siRNA-mediated knockdown of FcRn in the cornea impeded protection against ocular HSV-1 challenge in vaccinated mice. Collectively, these findings establish a novel mechanism of humoral protection in the eye involving FcRn and may facilitate vaccine and therapeutic development for other ocular surface diseases.