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.

PurposeHerpes simplex virus type-1 (HSV-1) is a leading cause of neurotrophic keratitis, characterized by decreased or absent corneal sensation due to damage to the sensory corneal innervation. We previously reported the elicited immune response to infection contributes to the mechanism of corneal nerve regression/damage during acute HSV-1 infection. Our aim is to further establish the involvement of infiltrated macrophages in the mechanism of nerve loss upon infection.MethodsMacrophage Fas-Induced Apoptosis (MAFIA) transgenic C57BL/6 mice were systemically treated with AP20187 dimerizer or vehicle (VEH), and their corneas, lymph nodes, and blood were assessed for CD45+CD11b+GFP+ cell depletion by flow cytometry (FC). Mice were ocularly infected with HSV-1 or left uninfected. At 2, 4, and/or 6 days post infection (PI), corneas were assessed for sensitivity and harvested for FC, nerve structure by immunohistochemistry, viral content by plaque assay, soluble factor content by suspension array, and activation of signaling pathways by Western blot analysis. C57BL6 mice were used to compare to the MAFIA mouse model.ResultsMAFIA mice treated with AP20187 had efficient depletion of CD45+CD11b+GFP+ cells in the tissues analyzed. The reduction of CD45+CD11b+GFP+ cells recruited to the infected corneas of AP20187-treated mice correlated with preservation of corneal nerve structure and function, decreased protein concentration of inflammatory cytokines, and decreased STAT3 activation despite no changes in viral content in the cornea compared to VEH-treated animals.ConclusionsOur results suggest infiltrated macrophages are early effectors in the nerve regression following HSV-1 infection. We propose the neurodegeneration mechanism involves macrophages, local up-regulation of IL-6, and activation of STAT3.

Herpes simplex virus type 1 (HSV-1) infection of the cornea induces vascular endothelial growth factor (VEGF)-A-dependent lymphangiogenesis. However, the extent to which HSV-1-induced corneal lymphangiogenesis impacts the adaptive immune response has not been characterized. Here, we used floxed VEGF-A mice to study the importance of newly created corneal lymphatic vessels in the host adaptive immune response to infection. Whereas the mice infected with the parental virus (strain SC16) exhibited robust corneal lymphangiogenesis, mice that received the recombinant virus (SC16 ICP0-Cre) that expresses Cre recombinase under the control of infected cell protein 0 (ICP0), an HSV-1 immediate early gene, showed a significant reduction in lymphangiogenesis. There was no difference in virus recovered from the cornea of mice infected with SC16 vs SC16 ICP0-Cre. However, viral loads were significantly elevated in the trigeminal ganglia (TG) of mice with reduced corneal lymphangiogenesis. The increase in viral titer correlated with a significant loss of HSV-1-specific CD8+ T cells that traffic to the TG of mice infected with the recombinant virus. Intrastromal delivery of size exclusion dye (FITC-dextran) revealed a time-dependent defect in the ability of the lymphatic vessels in SC16 ICP0-Cre infected mice to transport soluble antigen from the cornea to the draining lymph nodes. We interpret these results to suggest that the newly created lymphatic vessels in the cornea driven by HSV-1 infection are critical in the delivery of soluble viral antigen to the draining lymph node and subsequent development of the CD8+ T cell response to HSV-1.

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHCligand discovery pipeline in FVB/NJ mice, identified and tracked H-2D q /PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

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