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...

HSV-1 is the leading cause of sporadic viral encephalitis with mortality rates approaching 30% despite treatment with the antiviral drug of choice, acyclovir. Permanent neurological deficits are common in patients that survive but the mechanism leading to this pathology is poorly understood impeding clinical advancements in treatment to reduce central nervous system (CNS) morbidity. Using magnetic resonance imaging and type I IFN receptor deficient mouse chimeras, we demonstrate HSV-1 gains access to the murine brain stem and subsequently brain ependymal cells leading to enlargement of the cerebral lateral ventricle and infection of the brain parenchyma. A similar enlargement in the lateral ventricles is found in a subpopulation of herpes simplex encephalitic patients. Associated with encephalitis is an increase in CXCL1 and CXCL10 levels in the cerebral spinal fluid, TNF-α expression in the ependymal region and the influx of neutrophils of encephalitic mouse brains. Reduction in lateral ventricle enlargement using the anti-secretory factor peptide, AF-16, reduces mortality significantly in HSV-1 infected mice without any effect on expression of inflammatory mediators, infiltration of leukocytes, or changes in viral titer. Microglial cells but not infiltrating leukocytes or other resident glial cells or neurons are the principal source of resistance in the CNS during the first 5 days post infection through a TRIF-dependent, type I IFN pathway. Our results implicate lateral ventricle enlargement as a major cause of mortality in mice and speculate such an event transpires in a subpopulation of human herpes simplex virus encephalitic patients.

Collectively, these findings provide new insight into host defense in the cornea and the pathogenesis of HSV-1 infection by identifying previously unacknowledged MCs as protective innate sentinels for infection of the ocular surface and reinforcing that neutrophils are detrimental to corneal infection.

Type 1 interferons (IFNα/β) mediate immunologic host resistance to numerous viral infections including herpes simplex virus type 1 (HSV-1). The pathways responsible for IFNα/β signaling during the innate immune response to acute HSV-1 infection in the cornea are incompletely understood. Using a murine ocular infection model, we hypothesized that the stimulator of IFN genes (STING) mediates resistance to HSV-1 infection at the ocular surface and preserves the structural integrity of this mucosal site. Viral pathogenesis, tissue pathology, and host immune responses during ocular HSV-1 infection were characterized by plaque assay, esthesiometry, pachymetry, immunohistochemistry, flow cytometry, and siRNA transfection in wildtype C57BL/6 (WT), STING-deficient (STING−/−), and IFNα/β receptor-deficient (CD118−/−) mice at days 3–5 post infection. The presence of STING was critical for sustained control of HSV-1 replication in the corneal epithelium and neuroinvasion, but loss of STING had a negligible impact with respect to gross tissue pathology. Auxiliary STING-independent IFNα/β signaling pathways were responsible for maintenance of the corneal integrity. Lymphatic vessels, mast cells, and sensory innervation were compromised in CD118−/− mice concurrent with increased tissue edema. STING-dependent signaling led to the upregulation of tetherin, a viral restriction factor we identify is important in containing the spread of HSV-1 in vivo.

Whether complement dysregulation directly contributes to the pathogenesis of peripheral nervous system diseases, including sensory neuropathies, is unclear. We addressed this important question in a mouse model of ocular HSV-1 infection, where sensory nerve damage is a common clinical problem. Through genetic and pharmacologic targeting, we uncovered a central role for C3 in sensory nerve damage at the morphological and functional levels. Interestingly, CD4 T cells were central in facilitating this complement-mediated damage. This same C3/CD4 T cell axis triggered corneal sensory nerve damage in a mouse model of ocular graft-versus-host disease (GVHD). However, this was not the case in a T-dependent allergic eye disease (AED) model, suggesting that this inflammatory neuroimmune pathology is specific to certain disease etiologies. Collectively, these findings uncover a central role for complement in CD4 T cell-dependent corneal nerve damage in multiple disease settings and indicate the possibility for complement-targeted therapeutics to mitigate sensory neuropathies.

Type I interferon (IFNα/β)-driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to herpes simplex virus type 1 (HSV-1) are incompletely understood. Here we show that mice with deficiencies in IFNα/β signaling or STING exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFNα/β signaling and efficacy of early adaptive immune responses to HSV-1 were dissected using bone marrow chimeras and adoptive cell transfer approaches to profile clonal expansion, effector function, and recruitment of HSV-specific CD8+ T cells. Lymphotropic viral dissemination was commensurate with abrogated CD8+ T cell responses and pathological alterations of fibroblastic reticular cell (FRC) networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the TG during acute infection is conferred in part by STING and IFNα/β-signaling in both bone marrow-derived and resident cells, which coalesce to support a robust HSV-1-specific CD8+ T cell response.

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.

The cornea is essential for vision yet highly sensitive to immune-mediated damage following infection. Generating vaccines that provide sterile immunity against ocular surface pathogens without evoking vision loss is therefore clinically challenging. Here, we tested a prophylactic live-attenuated vaccine against herpes simplex virus type 1 (HSV-1), a widespread human pathogen that can cause corneal blindness. Parenteral vaccination of mice resulted in sterile immunity to subsequent HSV-1 challenge in the cornea and suppressed productive infection of the nervous system. This protection was unmatched by a relevant glycoprotein subunit vaccine. Efficacy of the live-attenuated vaccine involved a T-dependent humoral immune response and complement C3 but not Fcγ-receptor 3 or interferon-α/β signaling. Proteomic analysis of viral proteins recognized by antiserum revealed an unexpected repertoire dominated by sequestered antigens rather than surface-exposed envelope glycoproteins. Ocular HSV-1 challenge in naive and subunit-vaccinated mice triggered vision loss and severe ocular pathologies including corneal opacification, scar formation, neovascularization, and sensation loss. However, corneal pathology was absent in mice receiving the live-attenuated vaccine concomitant with complete preservation of visual acuity. Collectively, this is the first comprehensive report of a prophylactic vaccine candidate that elicits resistance to ocular HSV-1 infection while fully preserving the cornea and visual acuity.