Integrative Analysis of miRNA and circRNA Expression Profiles and Interaction Network in HSV-1-Infected Primary Corneal Epithelial Cells.

PURPOSE: Circular RNAs (circRNAs) are products of alternative splicing with roles as competitive endogenous RNAs or microRNA sponges, regulating gene expression and biological processes. However, the involvement of circRNAs in herpes simplex keratitis remains largely unexplored. METHODS: This study examines circRNA and miRNA expression profiles in primary human corneal epithelial cells infected with HSV-1, compared to uninfected controls, using microarray analysis. Bioinformatic analysis predicted the potential function of the dysregulated circRNAs and microRNA response elements (MREs) in these circRNAs, forming an interaction network between dysregulated circRNAs and miRNAs. RESULTS: A total of 332 circRNAs and 16 miRNAs were upregulated, while 80 circRNAs and six miRNAs were downregulated (fold change ≥2.0 and p < 0.05). Gene ontology (GO) and KEGG pathway analyses were performed on parental genes of dysregulated circRNAs to uncover potential functions in HSV-1 infection. Notably, miR-181b-5p, miR-338-3p, miR-635, and miR-222-3p emerged as pivotal miRNAs interacting with multiple dysregulated circRNAs. CONCLUSIONS: This comprehensive study offers insights into differentially expressed circRNAs and miRNAs during HSV-1 infection in corneal epithelial cells, shedding light on circRNA-miRNA interactions' potential role in herpes simplex keratitis pathogenesis.

Knockout of signal peptide peptidase in the eye reduces HSV-1 replication and eye disease in ocularly infected mice.

We previously reported that knocking out signal peptide peptidase (SPP), a glycoprotein K (gK) binding partner, in mouse peripheral sensory neurons reduced latency-reactivation in infected mice without affecting primary virus replication or eye disease. Since virus replication in the eye plays an essential role in eye disease, we generated a conditional knockout mouse lacking SPP expression in the eye by crossing Pax6 (paired box 6)-Cre mice that have intact Pax6 expression with SPPflox/flox mice. Significantly less SPP protein expression was detected in the eyes of Pax6-SPP-/- mice than in WT control mice. HSV-1 replication in the eyes of Pax6-SPP-/- mice was significantly lower than in WT control mice. Levels of gB, gK, and ICP0 transcripts in corneas, but not trigeminal ganglia (TG), of Pax6-SPP-/- infected mice were also significantly lower than in WT mice. Corneal scarring and angiogenesis were significantly lower in Pax6-SPP-/- mice than in WT control mice, while corneal sensitivity was significantly higher in Pax6-SPP-/- mice compared with WT control mice. During acute viral infection, absence of SPP in the eye did not affect CD4 expression but did affect CD8α and IFNγ expression in the eye. However, in the absence of SPP, latency-reactivation was similar in Pax6-SPP-/- and WT control groups. Overall, our results showed that deleting SPP expression in the eyes reduced primary virus replication in the eyes, reduced CD8α and IFNγ mRNA expression, reduced eye disease and reduced angiogenesis but did not alter corneal sensitivity or latency reactivation to HSV-1 infection. Thus, blocking gK binding to SPP in the eye may have therapeutic potential by reducing both virus replication in the eye and eye disease associated with virus replication.

Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro.

PURPOSE: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment. METHODS: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group. CONCLUSIONS: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs. TRANSLATIONAL RELEVANCE: This study offered a promising target for the cure of HSK.

Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice.

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

Characterization of Resident Corneal Plasmacytoid Dendritic Cells and Their Pivotal Role in Herpes Simplex Keratitis.

The presence and potential functions of resident plasmacytoid dendritic cells (pDCs) in peripheral tissues is unclear. We report that pDCs constitutively populate naïve corneas and are increased during sterile injuries or acute herpes simplex virus 1 (HSV-1) keratitis. Their local depletion leads to severe clinical disease, nerve loss, viral dissemination to the trigeminal ganglion and draining lymph nodes, and mortality, while their local adoptive transfer limits disease. pDCs are the main source of HSV-1-induced IFN-α in the corneal stroma through TLR9, and they prevent re-programming of regulatory T cells (Tregs) to effector ex-Tregs. Clinical signs of infection are observed in pDC-depleted corneas, but not in pDC-sufficient corneas, following low-dose HSV-1 inoculation, suggesting their critical role in corneal antiviral immunity. Our findings demonstrate a vital role for corneal pDCs in the control of local viral infections.

A Single-Cycle Glycoprotein D Deletion Viral Vaccine Candidate, ΔgD-2, Elicits Polyfunctional Antibodies That Protect against Ocular Herpes Simplex Virus.

Herpes simplex virus 1 (HSV-1) is a leading cause of infectious blindness, highlighting the need for effective vaccines. A single-cycle HSV-2 strain with the deletion of glycoprotein D, ΔgD-2, completely protected mice from HSV-1 and HSV-2 skin or vaginal disease and prevented latency following active or passive immunization in preclinical studies. The antibodies functioned primarily by activating Fc receptors to mediate antibody-dependent cellular cytotoxicity (ADCC). The ability of ADCC to protect the immune-privileged eye, however, may differ from skin or vaginal infections. Thus, the current studies were designed to compare active and passive immunization with ΔgD-2 versus an adjuvanted gD subunit vaccine (rgD-2) in a primary lethal ocular murine model. ΔgD-2 provided significantly greater protection than rgD-2 following a two-dose vaccine regimen, although both vaccines were protective compared to an uninfected cell lysate. However, only immune serum from ΔgD-2-vaccinated, but not rgD-2-vaccinated, mice provided significant protection against lethality in passive transfer studies. The significantly greater passive protection afforded by ΔgD-2 persisted after controlling for the total amount of HSV-specific IgG in the transferred serum. The antibodies elicited by rgD-2 had significantly higher neutralizing titers, whereas those elicited by ΔgD-2 had significantly more C1q binding and Fc gamma receptor activation, a surrogate for ADCC function. Together, the findings suggest ADCC is protective in the eye and that nonneutralizing antibodies elicited by ΔgD-2 provide greater protection than neutralizing antibodies elicited by rgD-2 against primary ocular HSV disease. The findings support advancement of vaccines, including ΔgD-2, that elicit polyfunctional antibody responses.IMPORTANCE Herpes simplex virus 1 is the leading cause of infectious corneal blindness in the United States and Europe. Developing vaccines to prevent ocular disease is challenging because the eye is a relatively immune-privileged site. In this study, we compared a single-cycle viral vaccine candidate, which is unique in that it elicits predominantly nonneutralizing antibodies that activate Fc receptors and bind complement, and a glycoprotein D subunit vaccine that elicits neutralizing but not Fc receptor-activating or complement-binding responses. Only the single-cycle vaccine provided both active and passive protection against a lethal ocular challenge. These findings greatly expand our understanding of the types of immune responses needed to protect the eye and will inform future prophylactic and therapeutic strategies.

CD80 Plays a Critical Role in Increased Inflammatory Responses in Herpes Simplex Virus 1-Infected Mouse Corneas.

We recently reported that herpes simplex virus 1 (HSV-1) infection suppresses CD80 but not CD86 expression in vitro and in vivo This suppression required the HSV-1 ICP22 gene. We also reported that overexpression of CD80 by HSV-1 exacerbated corneal scarring in BALB/c mice. We now show that this recombinant virus (HSV-CD80) expressed high levels of CD80 both in vitro in cultured rabbit skin cells and in vivo in infected mouse corneas. CD80 protein was detected on the surface of infected cells. The virulence of the recombinant HSV-CD80 virus was similar to that of the parental strain, and the replication of HSV-CD80 was similar to that of control virus in vitro and in vivo Transcriptome analysis detected 75 known HSV-1 genes in the corneas of mice infected with HSV-CD80 or parental virus on day 4 postinfection. Except for significantly higher CD80 expression in HSV-CD80-infected mice, levels of HSV-1 gene expression were similar in corneas from HSV-CD80-infected and parental virus-infected mice. The number of CD8+ T cells was higher, and the number of CD4+ T cells was lower, in the corneas of HSV-CD80-infected mice than in mice infected with parental virus. HSV-CD80-infected mice displayed a transient increase in dendritic cells. Transcriptome analysis revealed mild differences in dendritic cell maturation and interleukin-1 signaling pathways and increased expression of interferon-induced protein with tetratricopeptide repeats 2 (Ifit2). Together, these results suggest that increased CD80 levels promote increased CD8+ T cells, leading to exacerbated eye disease in HSV-1-infected mice.IMPORTANCE HSV-1 ocular infections are the leading cause of corneal blindness. Eye disease is the result of a prolonged immune response to the replicating virus. HSV-1, on the other hand, has evolved several mechanisms to evade clearance by the host immune system. We describe a novel mechanism of HSV-1 immune evasion via ICP22-dependent downregulation of the host T cell costimulatory molecule CD80. However, the exact role of CD80 in HSV-1 immune pathology is not clear. In this study, we show that eye disease is independent of the level of HSV-1 replication and that viral expression of CD80 has a detrimental role in corneal scarring, likely by increasing CD8+ T cell recruitment and activation.

The Role of C21orf91 in Herpes Simplex Virus Keratitis.

Background and Objectives: This paper aims to describe the single nucleotide polymorphisms (SNPs) of C21orf91 rs1062202 and rs10446073 in patients with herpetic keratitis by evaluating corneal sub-basal nerves, as well as the density of Langerhans cells (LC) and endothelium cells (EC) during the acute phase of the disease. Materials and Methods: A prospective clinical study included 260 subjects: 70 with herpetic eye disease, 101 with previous history of herpes labialis-but no history of herpetic eye disease-and 89 with no history of any herpes simplex virus (HSV) diseases. All subjects underwent a complete ophthalmological examination including in vivo laser scanning confocal microscopy (LSCM) of the central cornea. C21orf91 rs1062202 and rs10446073 were genotyped using the real-time polymerase chain reaction (PCR) method with the Rotor-Gene Q real-time PCR quantification system. SNPs were determined using TaqMan genotyping assay, according to the manufacturer's manual. Results: The C21orf91 rs10446073 genotype GT was more frequent in the HSV keratitis group, compared with healthy controls (20.0% vs. 7.9%), OR 2.929[1.11-7.716] (p <0.05). The rs10446073 genotype TT was more frequent in healthy controls (12.4% vs. 1.4%), OR 22.0[2.344-260.48] (p <0.05). The rs10446073 genotype GT increased the risk of EC density being less than 2551.5 cell/mm2, OR 2.852[1.248-6.515] (p <0.05). None of the SNPs and their genotypes influenced the LC density and corneal sub-basal nerve parameters (p >0.05). Conclusions: Our study reports a new association between herpetic keratitis and human gene C21orf91, with the rs10446073 genotype GT being more common in herpetic keratitis patients and increasing the risk for the disease by a factor of 2.9.

IL28B Genetic Variations in Patients with Recurrent Herpes Simplex Keratitis.

Background and objectives: Recurrent herpes simplex keratitis (HSK) is the most common cause of corneal blindness in the developed world. A relationship between host gene polymorphisms and the recurrence of herpes simplex virus (HSV) infection has previously been proposed. Thus, the aim of this study was to investigate a potential association between the IL28B host genotype and recurrent HSK. Materials and Methods: Eighty patients older than 18 years of age of both genders with a history of recurrent herpes simplex labialis (HSL) were considered for inclusion. Seventy-five of these patients were found to be seropositive for HSV-1 and were subsequently enrolled in the study. Twenty-four of the enrolled patients also had a history of recurrent HSK associated with severe corneal scarring and visual acuity deterioration. Total DNA was isolated from whole blood samples. A single-nucleotide polymorphism (SNP) rs12979860 near the IL28B gene on chromosome 19 was genotyped. Results: A significant association was observed between recurrent HSK and two SNPs of the IL28B genotype (CCrs12979860 and CTrs12979860, p < 0.01). The variation CCrs12979860 showed a significantly greater association with HSK (16 out of 26 patients) compared with CTrs12979860 (8 out of 34 patients). Conclusion: Seropositive individuals with a history of recurrent HSK are likely to have the CC IL28B genotype. This genotype may be related to incomplete control of the infection and more frequent periodical viral shedding along the first nerve branch of the trigeminal ganglion, which clinically manifests as recurrent herpes keratitis. The clinical manifestation of recurrent HSV-1 infection seems to be influenced by polymorphism of the IL28B genotype.

The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection.

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

Transcriptional Profile and Integrative Analyses of Long Noncoding RNAs in Primary Human Corneal Epithelial Cells in Response to HSV-1 Infection.

PURPOSE: Long noncoding RNAs (lncRNAs) have been demonstrated to have important regulatory functions in diverse cellular processes; however, the role of lncRNAs in the pathogenesis of herpes simplex keratitis (HSK) remains poorly understood. METHODS: Primary human corneal epithelial cells (HCECs) were infected with herpes simplex virus-1 (HSV-1) and the total RNAs extracted from both the infected group and the mock-infected group subjected to microarray analysis to identify the differential expression of lncRNAs and mRNAs. We also performed bioinformatic analysis including gene ontology (GO) analysis, pathway analysis and co-expression network analysis. RESULTS: Compared with mock-infected group, the expression of thousands of lncRNAs and mRNAs were significantly changed, and the microarray results were validated by qRT-PCR. The most enriched GOs targeted by up-regulated transcripts were defense response, intrinsic component of plasma membrane and cytokine activity，and the most enriched GOs targeted by the down-regulated transcripts were cellular metabolic process, intracellular part and poly (A) RNA binding. Pathway analysis indicated that the most correlated pathways for up- and down-regulated transcripts were cytokine-cytokine receptor interaction and RNA transport, respectively. CONCLUSIONS: Our study identified the genome-wide profile of lncRNAs and mRNAs expression in primary corneal epithelial cells with HSV-1 infection. These transcriptomic data together with subsequent bioinformatic analysis will provide us with novel clue to the insight into molecular mechanism and potential therapeutic targets of HSK. Further studies are expected to verify the potentially functional genes and pathways and explore the critical lncRNAs. ABBREVIATIONS: Long noncoding RNAs: lncRNAs; herpes simplex virus-1: HSV-1; herpes simplex virus keratitis: HSK; human corneal epithelial cells: HCECs.

Lack of TNF-α signaling through p55 makes the mice more susceptible to acute infection but does not alter state of latency and reactivation of HSV-1.

TNF-α has been shown to play an important role in pathogenesis and latency of HSV-1 infections. TNF-α signals through TNFR1 (p55) and TNFR2 (p75), and signaling through p55 generally results in gene activation leading to induction of inflammatory responses. Here, we studied the role of TNF-α signaling in latent virus reactivation in p55-knock out (KO) mouse model of ocular HSV-1 infection. We found that KO mice are more susceptible to HSV-1 infection compared to wild type C57Bl/6 mice. While the absence of TNFRI signaling enhanced the ganglion latent DNA content by two folds, there was no difference in the maintenance and reactivation of latent HSV-1. Strikingly, interfering with inflammatory responses through PGE2 synthesis by treating latently infected wild type mice with indomethacin (COX inhibitor) prior to UV-exposure prevented HSV-1 reactivation. These results suggest that reactivation of latent HSV-1 might result from the cumulative effects of a cascade of inflammatory cytokines including TNF-α.

Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus.

BACKGROUND: Epithelial cells play important roles as a critical barrier in protecting the cornea from microbial pathogens infection. METHODS: In this study, we were aiming to investigate the role of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) in corneal epithelial cells in response to Herpes Simplex Virus type 1 (HSV-1) infection and to elucidate the underlying mechanisms. RESULTS: We found the expression of TRIM32 was increased after infected with HSV-1 both in murine corneas and cultured human epithelial (HCE) cells. Furthermore, knockdown of the expression of TRIM32 significantly aggravated HSV-1 induced herpetic stromal keratitis (HSK) in mice and promoted the replication of HSV-1 in cultured HCE cells. We also observed that silencing of TRIM32 resulted in the decreased expression of IFN-ß and suppressed activation of interferon regulatory factor 3 (IRF3) both in vivo and in vitro. Finally, we found TRIM32 positively regulate IFN-ß production in corneal epithelial cells through promoting K63-linked polyubiquitination of stimulator of interferon genes (STING). CONCLUSION: In conclusion, our data suggested that TRIM32 as a crucial positive regulator of HSV-1 induced IFN-ß production in corneal epithelial cells, and it played a predominant role in clearing HSV-1 from the cornea.

Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology.

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV-1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4+ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT-RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV-1, could control the severity of SK lesions. Treatment with AT-RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT-RvD1-treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT-RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL-1ß, IL-6, IL-12, CXCL1, MCP-1, MIP-2, vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase 9 (MMP-9), and proinflammatory miRNA, such as miR-155, miR-132, and miR-223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT-RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings demonstrate that AT-RvD1 treatment could represent a useful strategy for the management of virus-induced immunopathological lesions.

CXCL9 compensates for the absence of CXCL10 during recurrent Herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is a disease that is typically associated with reactivation of a latent HSV-1 infection. This disease is driven, in part, by chemokines that recruit leukocytes to the cornea. Surprisingly, neutralization of CXCL10 significantly reduced disease, while B6-CXCL10-/- mice exhibited worse disease compared with similarly infected wild-type controls. We hypothesized that compensatory up-regulation of CXCL9 occurs in the absence of CXCL10. Analysis of CXCL9 expression in HSV-1-infected B6 mice and B6-CXCL10-/- mice revealed significantly more CXCL9 in B6-XCL10-/- mice. Treatment of B6 and B6-CXCL10-/- mice with neutralizing antibodies to CXCL9 reduced HSK scores in B6-CXCL10-/-, but not B6 mice. We conclude that CXCL10 production worsens HSK and that CXCL9 may compensate in CXCL10-deficient animals. These studies identify the critical role that CXCL10 plays in the pathogenesis of recurrent HSK, and that CXCL9 displays its importance when CXCL10 is absent.

Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine. IMPORTANCE: Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8+ T cells (CD8+ TEM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14286-294,VP13/14504-512, and VP13/14544-552) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8+ T-cell epitopes from ASYMP individuals.

Impaired Fas-Fas Ligand Interactions Result in Greater Recurrent Herpetic Stromal Keratitis in Mice.

Herpes simplex virus-1 (HSV-1) infection of the cornea leads to a potentially blinding condition termed herpetic stromal keratitis (HSK). Clinical studies have indicated that disease is primarily associated with recurrent HSK following reactivation of a latent viral infection of the trigeminal ganglia. One of the key factors that limit inflammation of the cornea is the expression of Fas ligand (FasL). We demonstrate that infection of the cornea with HSV-1 results in increased functional expression of FasL and that mice expressing mutations in Fas (lpr) and FasL (gld) display increased recurrent HSK following reactivation compared to wild-type mice. Furthermore, both gld and lpr mice took longer to clear their corneas of infectious virus and the reactivation rate for these strains was significantly greater than that seen with wild-type mice. Collectively, these findings indicate that the interaction of Fas with FasL in the cornea restricts the development of recurrent HSK.

Indoleamine 2,3-dioxygenase 1 in corneal endothelial cells limits herpes simplex virus type 1-induced acquired immune response.

BACKGROUND: Corneal endothelial cells are known to be targets of herpes simplex virus type 1 (HSV-1) infection; however, the pathogenesis of HSV infections of the endothelial cells has not been definitively determined. The purpose of this study was to examine an unrecognised strategy of corneal endothelial cells to protect themselves from HSV-1 infection. METHODS: Immortalised human corneal endothelial cells (HCEn) were infected with HSV-1. Based on the global transcriptional profile, the expression of indoleamine 2,3-dioxygenase 1 (IDO1) was determined using real-time PCR and western blots. To examine whether IDO1 has any antiviral role, we tested whether viral replication was affected by blocking the activity of IDO1. The immune modulatory role of IDO1 was analysed to determine whether IDO1 might contribute to modulating the recall responses of HSV-1-sensitised CD4(+) T cells. RESULTS: IDO1 was strongly expressed in HCEn cells after HSV-1 infection. IDO1 blockade did not significantly restrict viral transcription or replication, arguing against a previously recognised antiviral role for IDO1. When HCEn cells were examined for antigen-presenting function, HSV-1-primed HCEn cells stimulated the proliferation of allogeneic CD4(+) T cells and interleukin 10 (IL-10) secretion. When the recall response to HSV-1 was measured by the mixed lymphocyte reaction, the HCEn-stimulated CD4(+) T cells modulated and limited the recall response. When IDO1 was silenced in HCEn cells, the HCEn-mediated immune modulatory activity and regulatory T-cell activation were reduced. Overexpression of IDO1 promoted immune modulatory activity, which was partly conveyed by IL-10. CONCLUSIONS: IDO1 induced by HSV-1 infection limits and dampens excessive acquired immune responses in corneal endothelial cells.

Role of miR-155 in the pathogenesis of herpetic stromal keratitis.

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4(+) T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 and interferon-γ receptor α-chain levels in activated CD4(+) T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1-infected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity.