Derek J. Royer

Microglia and a Functional Type I IFN Pathway Are Required To Counter HSV-1–Driven Brain Lateral Ventricle Enlargement and Encephalitis

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 neurologic deficits are common in patients that survive, but the mechanism leading to this pathology is poorly understood, impeding clinical advancements in treatment to reduce 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 anti-secretory factor peptide 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 d postinfection through a Toll/IL-1R domain-containing adapter inducing IFN-β–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 HSV encephalitic patients.

Thrombocytopenia as an adverse effect of complementary and alternative medicines, herbal remedies, nutritional supplements, foods, and beverages*

Background:  Thrombocytopenia is a well‐recognized adverse effect of many drugs. However, the association of thrombocytopenia with complementary/alternative medicines, herbal remedies, nutritional supplements, foods, and beverages has been rarely described, except for reports of thrombocytopenia caused by quinine‐containing beverages.

Granulocytes in Ocular HSV-1 Infection: Opposing Roles of Mast Cells and Neutrophils

PURPOSE The contributions of mast cells (MCs) to immunologic defense against pathogens in the eye are unknown. We have characterized pericorneal MCs as tissue-resident innate sentinels and determined their impact on the immune response to herpes simplex virus type-1 (HSV-1), a common ocular pathogen. METHODS The impact of mast cells on the immune response to HSV-1 infection was investigated using MC-deficient Kit(W-sh) mice. Virus titers, inflammatory cytokine production, eicosanoid profiles, cellular immune responses, and ocular pathology were evaluated and compared with C57BL/6J mice during an acute corneal HSV-1 infection. RESULTS Corneas of Kit(W-sh) mice have higher viral titers, increased edema, and greater leukocyte infiltration following HSV-1 infection. Following infection, cytokine profiles were slightly elevated overall in Kit(W-sh) mice. Eicosanoid profiles were remarkably different only when comparing uninfected corneas from both groups. Neutrophils within infected corneas expressed HSV-1 antigen, lytic genes, and served as a disease-causing vector when adoptively transferred into immunocompromised animals. Myeloid-derived suppressor cells did not infiltrate into the cornea or suppress the expansion, recruitment, or cytokine production by CD8+ T cells following acute HSV-1 infection. CONCLUSIONS 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.

A Highly Efficacious Herpes Simplex Virus 1 Vaccine Blocks Viral Pathogenesis and Prevents Corneal Immunopathology via Humoral Immunity

ABSTRACT 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 (0ΔNLS) or an adjuvanted glycoprotein D subunit (gD-2) similar to that used in several human clinical trials. Mice vaccinated with 0ΔNLS 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, 0ΔNLS-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, 0ΔNLS affords remarkable protection against HSV-1-associated ocular sequelae by impeding viral replication, dissemination, and establishment of latency. IMPORTANCE HSV-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.

The Current State of Vaccine Development for Ocular HSV-1 Infection

Herpes simplex virus type 1 continues to be the leading cause of infectious corneal blindness. Clinical trials for vaccines against genital HSV infection have been ongoing for more than three decades. Despite this, no approved vaccine exists, and no formal clinical trials have evaluated the impact of HSV vaccines on eye health. We review here the current state of development for an efficacious herpes simplex virus type 1 vaccine and call for involvement of ophthalmologists and vision researchers.

The Neonatal Fc Receptor and Complement Fixation Facilitate Prophylactic Vaccine-Mediated Humoral Protection against Viral Infection in the Ocular Mucosa

The capacity of licensed vaccines to protect the ocular surface against infection is limited. Common ocular pathogens, such as 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 Ab 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 Ab persistence and host protection. The live-attenuated vaccine was used to assess the impact of the immunization route on vaccine efficacy. The hierarchical rankings of primary immunization route with respect to efficacy were s.c. ≥ mucosal > i.m. Prime-boost vaccination via sequential s.c. and i.m. administration yielded greater efficacy than any other primary immunization route alone. Moreover, our data support a role for 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 Ab perfusion. Lastly, selective small interfering RNA–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.

Impact of type 1 interferon on the safety and immunogenicity of an experimental live-attenuated herpes simplex virus type 1 vaccine in mice.

ABSTRACT Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus 1 (HSV-1) mutant lacking the nuclear localization signal (NLS) on the ICP0 gene (0ΔNLS) is sensitive to inhibition by interferon beta (IFN-β) in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0ΔNLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFN-α/β) signaling on virulence and immunogenicity of HSV-1 0ΔNLS and uncover a probable sex bias in the induction of IFN-α/β in the cornea during HSV-1 infection. Our data show that HSV-1 0ΔNLS lacks neurovirulence even in highly immunocompromised mice lacking the IFN-α/β receptor. These studies support the translational viability of the HSV-1 0ΔNLS vaccine strain by demonstrating that, while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0ΔNLS does not induce significant tissue pathology. IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic “cold sores” to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Here, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

Herpesvirus-Associated Lymphadenitis Distorts Fibroblastic Reticular Cell Microarchitecture and Attenuates CD8 T Cell Responses to Neurotropic Infection in Mice Lacking the STING-IFNα/β Defense Pathways

Type I IFN (IFN-α/β)–driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to HSV type 1 (HSV-1) are incompletely understood. In this study, we show that mice with deficiencies in IFN-α/β signaling or stimulator of IFN genes (STING) exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFN-α/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 networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the trigeminal ganglia during acute infection is conferred in part by STING and IFN-α/β signaling in both bone marrow–derived and –resident cells, which coalesce to support a robust HSV-1–specific CD8+ T cell response.

Corneal Epithelial Cells Exhibit Myeloid Characteristics and Present Antigen via MHC Class II

Purpose To explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial–mesenchymal transition (EMT). Methods Corneas were harvested from mice 6 days following scratch injury, ragweed pollen–induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells. Results Ectopic expression of classic “myeloid” antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell–cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro. Conclusions This study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple “myeloid” antigens warrants careful evaluation of flow cytometry data involving corneal digests.