The importance of IFNα2A (Roferon-A) in HSV-1 latency and T cell exhaustion in ocularly infected mice.

Published studies have generated compelling results indicating that type I IFN modulates function of HSV-1 latency-associated transcript (LAT). One member of type I IFN is IFNα2A also called Roferon-A). IFNα2A has been used in monotherapy or in combination therapy with other drugs to treat viral infections and different kinds of cancer in humans. The goal of this study was to determine whether the absence of IFNα2A affects primary and latent infections in ocularly infected mice. Therefore, we generated a mouse strain lacking IFNα2A expression (IFNα2A-/-). Ocular HSV-1 replication, IFN and immune cell expressions on days 3 and 5 post infection (PI), as well as eye disease, survival, latency-reactivation, and T cell exhaustion were evaluated in ocularly infected IFNα2A-/- and wild type (WT) control mice. Absence of IFNα2A did not affect other members of the IFNα family but it affected IFNß and IFNγ expressions as well as some immune cells on day 5 PI compared to WT mice. Viral replication in the eye, eye disease, and survival amongst ocularly infected IFNα2A-/- mice were similar to that of WT infected mice. The absence of IFNα2A significantly reduced the levels of latency and T cell exhaustion but not time of reactivation compared with control mice. Our results suggest that blocking IFNα2A expression may be a useful tool in reducing latency and the subsequent side effects associated with higher levels of latency.

Presence of CD80 and Absence of LAT in Modulating Cellular Infiltration and HSV-1 Latency.

CD80 is the best-known costimulatory molecule for effective T cell functions. Many different reports have summarized the role of CD80 in HSV-1 and its functions in maintaining adaptive immunity, which is the main player in causing herpes stromal keratitis (HSK). To determine the effects of absence or overexpression of CD80 in HSV-1 infection, we infected CD80-/- and WT mice with a recombinant HSV-1 expressing murine CD80 (HSV-CD80) in place of the latency associated transcript (LAT). Parental dLAT2903 virus lacking LAT was used as a control. After infection, critical components of infection like virus replication, eye disease, early cellular infiltrates into the corneas and trigeminal ganglia (TG), latency-reactivation in the infected mice were determined. Our findings reveal that the absence of CD80 in the CD80-/- mice infected with both viruses did not affect the viral titers in the mice eyes or eye disease, but it played a significant role in critical components of HSV-induced immunopathology. The WT mice infected with dLAT2903 virus had significantly higher levels of latency compared with the CD80-/- mice infected with dLAT2903 virus, while levels of latency as determined by gB DNA expression were similar between the WT and CD80-/- mice infected with HSV-CD80 virus. In contrast to the differences in the levels of latency between the infected groups, the absence of CD80 expression in the CD80-/- mice or its overexpression by HSV-CD80 virus did not have any effect on the time of reactivation. Furthermore, the absence of CD80 expression contributed to more inflammation in the CD80-/--infected mice. Overall, this study suggests that in the absence of CD80, inflammation increases, latency is reduced, but reactivation is not affected. Altogether, our study suggests that reduced latency correlated with reduced levels of inflammatory molecules and blocking or reducing expression of CD80 could be used to mitigate the immune responses, therefore controlling HSV-induced infection.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

The anti-apoptotic function of HSV-1 LAT in neuronal cell cultures but not its function during reactivation correlates with expression of two small non-coding RNAs, sncRNA1&2.

Multiple functions are associated with HSV-1 latency associated transcript (LAT), including establishment of latency, virus reactivation, and antiapoptotic activity. LAT encodes two sncRNAs that are not miRNAs and previously it was shown that they have antiapoptotic activity in vitro. To determine if we can separate the antiapoptotic function of LAT from its latency-reactivation function, we deleted sncRNA1 and sncRNA2 sequences in HSV-1 strain McKrae, creating ΔsncRNA1&2 recombinant virus. Deletion of the sncRNA1&2 in ΔsncRNA1&2 virus was confirmed by complete sequencing of ΔsncRNA1&2 virus and its parental virus. Replication of ΔsncRNA1&2 virus in tissue culture or in the eyes of WT infected mice was similar to that of HSV-1 strain McKrae (LAT-plus) and dLAT2903 (LAT-minus) viruses. The levels of gB DNA in trigeminal ganglia (TG) of mice latently infected with ΔsncRNA1&2 virus was intermediate to that of dLAT2903 and McKrae infected mice, while levels of LAT in TG of latently infected ΔsncRNA1&2 mice was significantly higher than in McKrae infected mice. Similarly, the levels of LAT expression in Neuro-2A cells infected with ΔsncRNA1&2 virus was significantly higher than in McKrae infected cells. Reactivation in TG of ΔsncRNA1&2 infected mice was similar to that of McKrae and time of reactivation in both groups were significantly faster than dLAT2903 infected mice. However, levels of apoptosis in Neuro-2A cells infected with ΔsncRNA1&2 virus was similar to that of dLAT2903 and significantly higher than that of McKrae infected cells. Our results suggest that the antiapoptotic function of LAT resides within the two sncRNAs, which works independently of its latency-reactivation function and it has suppressive effect on LAT expression in vivo and in vitro.

Absence of CD80 reduces HSV-1 replication in the eye and delays reactivation but not latency levels.

Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80-/- mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80-/- mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.

Binding of herpesvirus entry mediator (HVEM) and HSV-1 gD affect reactivation but not latency levels.

Previously we reported that the HSV-1 latency associated transcript (LAT) specifically upregulates the cellular herpesvirus entry mediator (HVEM) but no other known HSV-1 receptors. HSV-1 glycoprotein D (gD) binds to HVEM but the effect of this interaction on latency-reactivation is not known. We found that the levels of latent viral genomes were not affected by the absence of gD binding to HVEM. However, reactivation of latent virus in trigeminal ganglia explant cultures was blocked in the absence of gD binding to HVEM. Neither differential HSV-1 replication and spread in the eye nor levels of latency influenced reactivation. Despite similar levels of latency, reactivation in the absence of gD binding to HVEM correlated with reduced T cell exhaustion. Our results indicate that HVEM-gD signaling plays a significant role in HSV-1 reactivation but not in ocular virus replication or levels of latency. The results presented here identify gD binding to HVEM as an important target that influences reactivation and survival of ganglion resident T cells but not levels of latency. This concept may also apply to other herpesviruses that engages HVEM.

IL-17A expression by both T cells and non-T cells contribute to HSV-IL-2-induced CNS demyelination.

Previously we reported that a recombinant HSV-1 expressing murine IL-2 (HSV-IL-2) causes CNS demyelination in different strains of mice and in a T cell-dependent manner. Since TH17 cells have been implicated in CNS pathology, in the present study, we looked into the effects of IL-17A-/- and three of its receptors on HSV-IL-2-induced CNS demyelination. IL-17A-/- mice did not develop CNS demyelination, while IL-17RA-/-, IL-17RC-/-, IL-17RD-/- and IL-17RA-/-RC-/- mice developed CNS demyelination. Adoptive transfer of T cells from wild-type (WT) mice to IL-17A-/- mice or T cells from IL-17A-/- mice to Rag-/- mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFß did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A-/- brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that "neuron projection extension involved in neuron projection guidance" and "ensheathment of neurons" pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination.

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.

Blocking Autophagy in M1 Macrophages Enhances Virus Replication and Eye Disease in Ocularly Infected Transgenic Mice.

Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 (IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The in vivo and in vitro responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice. IMPORTANCE Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.

Herpes Simplex Virus 1 Glycoproteins Differentially Regulate the Activity of Costimulatory Molecules and T Cells.

Over the past 70 years, multiple approaches to develop a prophylactic or therapeutic vaccine to control herpes simplex virus (HSV) infection have failed to protect against primary infection, reactivation, or reinfection. In contrast to many RNA viruses, neither primary HSV infection nor repeated clinical recurrence elicits immune responses capable of completely preventing virus reactivation; yet the 12 known HSV-1 glycoproteins are the major inducers and targets of humoral and cell-mediated immune responses following infection. While costimulatory molecules and CD4/CD8 T cells both contribute significantly to HSV-1-induced immune responses, the specific effects of individual HSV-1 glycoproteins on CD4, CD8, CD80, and CD86 activities are not known. To determine how nine major HSV-1 glycoproteins affect T cells and costimulatory molecule function, we tested the independent effects of gB, gC, gD, gE, gG, gH, gI, gK, and gL on CD4, CD8, CD80, and CD86 promoter activities in vitro. gD, gK, and gL had a suppressive effect on CD4, CD8, CD80, and CD86 promoter activities, while gG and gH specifically suppressed CD4 promoter activity. In contrast, gB, gC, gE, and gI stimulated CD4, CD8, CD80, and CD86 promoter activities. Luminex analysis of splenocytes and bone-marrow-derived dendritic cells (BMDCs) transfected with each glycoprotein showed differing cytokine/chemokine milieus with higher responses in splenocytes than in BMDCs. Our results with the tested major HSV-1 glycoproteins suggest that costimulatory molecules and T cell responses to the nine glycoproteins can be divided into (i) stimulators (i.e., gB, gC, gE, and gI), and (ii) nonstimulators (i.e., gD, gK, and gL). Thus, consistent with our previous studies, a cocktail of select HSV-1 viral genes may induce a wider spectrum of immune responses, and thus protection, than individual genes. IMPORTANCE Currently no effective vaccine is available against herpes simplex virus (HSV) infection. Thus, there is a critical need to develop a safe and effective vaccine to prevent and control HSV infection. The development of such approaches will require an advanced understanding of viral genes. This study provides new evidence supporting an approach to maximize vaccine efficacy by using a combination of HSV genes to control HSV infection.

Small Noncoding RNA (sncRNA1) within the Latency-Associated Transcript Modulates Herpes Simplex Virus 1 Virulence and the Host Immune Response during Acute but Not Latent Infection.

The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNß) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.

Absence of signal peptide peptidase in peripheral sensory neurons affects latency-reactivation in HSV-1 ocularly infected mice.

We previously reported that HSV-1 infectivity in vitro and in vivo requires HSV glycoprotein K (gK) binding to the ER signal peptide peptidase (SPP). Anterograde-retrograde transport via peripheral nerves between the site of infection (i.e., eye) and the site of latency (neurons) is a critical process to establish latency and subsequent viral reactivation. Given the essential role of neurons in HSV-1 latency-reactivation, we generated mice lacking SPP specifically in peripheral sensory neurons by crossing Advillin-Cre mice with SPPfl/fl mice. Expression of SPP mRNA and protein were significantly lower in neurons of Avil-SPP-/- mice than in control mice despite similar levels of HSV-1 replication in the eyes of Avil-SPP-/- mice and control mice. Viral transcript levels in isolated neurons of infected mice on days 2 and 5 post infection were lower than in control mice. Significantly less LAT, gB, and PD-1 expression was seen during latency in isolated neurons and total trigeminal ganglia (TG) of Avil-SPP-/- mice than in control mice. Finally, reduced latency and reduced T cell exhaustion in infected Avil-SPP-/- mice correlated with slower and no reactivation. Overall, our results suggest that blocking SPP expression in peripheral sensory neurons does not affect primary virus replication or eye disease but does reduce latency-reactivation. Thus, blocking of gK binding to SPP may be a useful tool to reduce latency-reactivation.

Herpes Simplex Virus 1 Small Noncoding RNAs 1 and 2 Activate the Herpesvirus Entry Mediator Promoter.

Herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) plays a significant role in efficient establishment of latency and reactivation. LAT has antiapoptotic activity and downregulates expression of components of the type I interferon pathway. LAT also specifically activates expression of the herpesvirus entry mediator (HVEM), one of seven known receptors used by HSV-1 for cell entry that is crucial for latency and reactivation. However, the mechanism by which LAT regulates HVEM expression is not known. LAT has two small noncoding RNAs (sncRNAs) that are not microRNAs (miRNAs), within its 1.5-kb stable transcript, which also have antiapoptotic activity. These sncRNAs may encode short peptides, but experimental evidence is lacking. Here, we demonstrate that these two sncRNAs control HVEM expression by activating its promoter. Both sncRNAs are required for wild-type (WT) levels of activation of HVEM, and sncRNA1 is more important in HVEM activation than sncRNA2. Disruption of a putative start codon in sncRNA1 and sncRNA2 sequences reduced HVEM promoter activity, suggesting that sncRNAs encode a protein. However, we did not detect peptide binding using two chromatin immunoprecipitation (ChIP) approaches, and a web-based algorithm predicts low probability that the putative peptides bind to DNA. In addition, computational modeling predicts that sncRNA molecules bind with high affinity to the HVEM promoter, and deletion of these binding sites to sncRNA1, sncRNA2, or both reduced HVEM promoter activity. Together, our data suggest that sncRNAs exert their function as RNA molecules, not as proteins, and we provide a model for the predicted binding affinities and binding sites of sncRNA1 and sncRNA2 in the HVEM promoter. IMPORTANCE HSV-1 causes recurrent ocular infections, which is the leading cause of corneal scarring and blindness. Corneal scarring is caused by the host immune response to repeated reactivation events. LAT functions by regulating latency and reactivation, in part by inhibiting apoptosis and activating HVEM expression. However, the mechanism used by LAT to control HVEM expression is unclear. Here, we demonstrate that two sncRNAs within the 1.5-kb LAT transcript activate HVEM expression by binding to two regions of its promoter. Interfering with these interactions may reduce latency and thereby eye disease associated with reactivation.

Essential role of M1 macrophages in blocking cytokine storm and pathology associated with murine HSV-1 infection.

Ocular HSV-1 infection is a major cause of eye disease and innate and adaptive immunity both play a role in protection and pathology associated with ocular infection. Previously we have shown that M1-type macrophages are the major and earliest infiltrates into the cornea of infected mice. We also showed that HSV-1 infectivity in the presence and absence of M2-macrophages was similar to wild-type (WT) control mice. However, it is not clear whether the absence of M1 macrophages plays a role in protection and disease in HSV-1 infected mice. To explore the role of M1 macrophages in HSV-1 infection, we used mice lacking M1 activation (M1-/- mice). Our results showed that macrophages from M1-/- mice were more susceptible to HSV-1 infection in vitro than were macrophages from WT mice. M1-/- mice were highly susceptible to ocular infection with virulent HSV-1 strain McKrae, while WT mice were refractory to infection. In addition, M1-/- mice had higher virus titers in the eyes than did WT mice. Adoptive transfer of M1 macrophages from WT mice to M1-/- mice reduced death and rescued virus replication in the eyes of infected mice. Infection of M1-/- mice with avirulent HSV-1 strain KOS also increased ocular virus replication and eye disease but did not affect latency-reactivation seen in WT control mice. Severity of virus replication and eye disease correlated with significantly higher inflammatory responses leading to a cytokine storm in the eyes of M1-/- infected mice that was not seen in WT mice. Thus, for the first time, our study illustrates the importance of M1 macrophages specifically in primary HSV-1 infection, eye disease, and survival but not in latency-reactivation.

Blocking HSV-1 glycoprotein K binding to signal peptide peptidase reduces virus infectivity in vitro and in vivo.

HSV glycoprotein K (gK) is an essential herpes protein that contributes to enhancement of eye disease. We previously reported that gK binds to signal peptide peptidase (SPP) and that depletion of SPP reduces HSV-1 infectivity in vivo. To determine the therapeutic potential of blocking gK binding to SPP on virus infectivity and pathogenicity, we mapped the gK binding site for SPP to a 15mer peptide within the amino-terminus of gK. This 15mer peptide reduced infectivity of three different virus strains in vitro as determined by plaque assay, FACS, and RT-PCR. Similarly, the 15mer peptide reduced ocular virus replication in both BALB/c and C57BL/6 mice and also reduced levels of latency and exhaustion markers in infected mice when compared with control treated mice. Addition of the gK-15mer peptide also increased the survival of infected mice when compared with control mice. These results suggest that blocking gK binding to SPP using gK peptide may have therapeutic potential in treating HSV-1-associated infection.

Suppression of CD80 Expression by ICP22 Affects Herpes Simplex Virus Type 1 Replication and CD8+IFN-γ+ Infiltrates in the Eyes of Infected Mice but Not Latency Reactivation.

Previously, we reported that herpes simplex virus type 1 (HSV-1) ICP22 binds to the CD80 promoter and suppresses its expression in vitro and in vivo. To better understand the impact of ICP22 binding to CD80 on HSV-1 infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40-amino-acid (aa) region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, which does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. The replication of this recombinant virus in vitro and in vivo was higher than that of the ICP22-null virus, but virus replication kinetics were lower than those of the wild-type (WT) control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in dendritic cells (DCs) and interferon gamma (IFN-γ) expression in CD8+ T cells but not CD4+ T cells in infected mouse corneas. In contrast to the significantly reduced virus replication in the eyes of ocularly infected mice, the levels of latency reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding appears to reduce virus replication and enhance CD8+IFN-γ+ infiltrates in corneas of infected mice, with no effect on latency reactivation. IMPORTANCE Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates the expression of the costimulatory molecule CD80 but not CD86. In this study, we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to the CD80 promoter. This recombinant virus replicated less efficiently in vitro and in vivo than did the WT control virus, although CD80-expressing CD11c+ cells and IFN-γ-expressing CD8+ T cells were increased. Interestingly, the levels of latency and reactivation in the two viruses were similar despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 with the CD80 promoter could be used to temper the immune response.

Absence of CD28-CTLA4-PD-L1 Costimulatory Molecules Reduces Herpes Simplex Virus 1 Reactivation.

We previously reported that herpes simplex virus 1 (HSV-1) ICP22 binds to CD80 and suppresses CD80 expression in vitro and in vivo. Similar to ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also bind to CD80. In this study, we asked whether, similar to ICP22-null virus, the absence of these costimulatory molecules will reduce HSV-1 infectivity. To test our hypothesis, CD28-/-, CD28-/- CTLA4-/-, PD-L1-/-, and wild-type control BALB/c mice were ocularly infected with HSV-1 strain KOS. Levels of virus replication in the eye, corneal scarring (CS), latency, and reactivation in infected mice were determined. Expression of different genes in the trigeminal ganglia (TG) of latently infected mice was also determined by NanoString and quantitative reverse transcription-PCR (qRT-PCR). In the absence of costimulatory molecules, latency levels were higher than those in wild-type control mice, but despite higher latency, a significant number of TG from infected knockout mice did not reactivate. Reduced reactivation correlated with downregulation of 26 similar cellular genes that are associated with inflammatory signaling and innate immune responses. These results suggest that lower reactivation directly correlates with lower expression of interferon signaling. Thus, despite having different modes of actions, we identified a similar function for CD28, CTLA4, and PD-L1 in HSV-1 reactivation that is dependent on their interactions with CD80. Therefore, blocking these interactions could be a therapeutic target for HSV-1-induced reactivation. IMPORTANCE Costimulatory molecules play an important role in activation of T cell responses, and T cells contribute to HSV-1-induced eye disease in the host. Similar to HSV-1 ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also bind to CD80. In this study, we have shown that the absence of these costimulatory molecules significantly reduced HSV-1 ex vivo reactivation. Therefore, inhibiting the binding of costimulatory molecules to CD80 could be used to reduce reactivation and, consequently, HSV-1-induced eye disease.

Type 2 Innate Lymphoid Cells Induce CNS Demyelination in an HSV-IL-2 Mouse Model of Multiple Sclerosis.

We previously reported that infection of different mouse strains with a recombinant HSV-1 expressing IL-2 (HSV-IL-2) caused CNS demyelination. Histologic examination of infected IL-2rα-/-, IL-2rß-/-, and IL-2rγ-/- mice showed demyelination in the CNS of IL-2rα-/- and IL-2rß-/- mice but not in the CNS of IL-2rγ-/--infected mice. No demyelination was detected in mice infected with control virus. IL-2rγ-/- mice that lack type 2 innate lymphoid cells (ILC2s) and ILCs, play important roles in host defense and inflammation. We next infected ILC1-/-, ILC2-/-, and ILC3-/- mice with HSV-IL-2 or wild-type (WT) HSV-1. In contrast to ILC1-/- and ILC3-/- mice, no demyelination was detected in the CNS of ILC2-/--sinfected mice. However, transfer of ILC2s from WT mice to ILC2-/- mice restored demyelination in infected recipient mice. CNS demyelination correlated with downregulation of CCL5 and CXCL10. This study demonstrates that ILC2s contribute to HSV-IL-2-induced CNS demyelination in a mouse model of multiple sclerosis.

Increased phagocytosis in the presence of enhanced M2-like macrophage responses correlates with increased primary and latent HSV-1 infection.

After HSV-1 infection, macrophages infiltrate early into the cornea, where they play an important role in HSV-1 infection. Macrophages are divided into M1 or M2 groups based on their activation. M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Macrophage phenotypes can shift between M1 or M2 in vitro and in vivo following treatment with specific cytokines. In this study we looked at the effect of M2 macrophages on HSV-1 infectivity using mice either lacking M2 (M2-/-) or overexpressing M2 (M2-OE) macrophages. While presence or absence of M2 macrophages had no effect on eye disease, we found that over expression of M2 macrophages was associated with increased phagocytosis, increased primary virus replication, increased latency, and increased expression of pro- and anti-inflammatory cytokines. In contrast, in mice lacking M2 macrophages following infection phagocytosis, replication, latency, and cytokine expression were similar to wild type mice. Our results suggest that enhanced M2 responses lead to higher phagocytosis, which affected both primary and latent infection but not reactivation.

Role of TH17 Responses in Increasing Herpetic Keratitis in the Eyes of Mice Infected with HSV-1.

Purpose: TH17 cells play an important role in host defense and autoimmunity yet very little is known about the role of IL17 in herpes simplex virus (HSV)-1 infectivity. To better understand the relationship between IL17 and HSV-1 infection, we assessed the relative impact of IL17A-deficiency and deficiency of its receptors on HSV-1 responses in vivo. Methods: We generated IL17RA-/- and IL17RA-/-RC-/- mice in-house and infected them along with IL17A-/- and IL17RC-/- mice in the eyes with 2 × 105 PFU/eye of wild type (WT) HSV-1 strain McKrae. WT C57BL/6 mice were used as control. Virus replication in the eye, survival, corneal scarring (CS), angiogenesis, levels of latency-reactivation, and levels of CD8 and exhaustion markers (PD1, TIM3, LAG3, CTLA4, CD244, and CD39) in the trigeminal ganglia (TG) of infected mice were determined on day 28 postinfection. Results: No significant differences in virus replication in the eye, survival, latency, reactivation, and exhaustion markers were detected among IL17A-/-, IL17RA-/-, IL17RC-/-, IL17RA-/-RC-/-, and WT mice. However, mice lacking IL17 had significantly less CS and angiogenesis than WT mice. In addition, angiogenesis levels in the absence of IL17RC and irrespective of the absence of IL17RA were significantly less than in IL17A- or IL17RA-deficient mice. Conclusions: Our results suggest that the absence of IL17 protects against HSV-1-induced eye disease, but has no role in protecting against virus replication, latency, or reactivation. In addition, our data provide rationale for blocking IL17RC function rather than IL17A or IL17RA function as a key driver of HSV-1-induced eye disease.