Controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin E1

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by HSV-1 infection and a common cause of blindness in humans. The inflammatory lesions are primarily perpetuated by neutrophils with the active participation of CD4(+) T cells. Therefore, targeting these immune cell types represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin E1 (RvE1), an endogenous lipid mediator, was shown to promote resolution in several inflammatory disease models. In the current report, we determined whether RvE1 administration begun at different times after ocular infection of mice with HSV could influence the severity of SK lesions. Treatment with RvE1 significantly reduced the extent of angiogenesis and SK lesions that occurred. RvE1-treated mice had fewer numbers of inflammatory cells that included Th1 and Th17 cells as well as neutrophils in the cornea. The mechanisms by which RvE1 acts appear to be multiple. These included reducing the influx of neutrophils and pathogenic CD4(+) T cells, increasing production of the anti-inflammatory cytokine IL-10, and inhibitory effects on the production of proinflammatory mediators and molecules, such as IL-6, IFN-γ, IL-17, KC, VEGF-A, MMP-2, and MMP-9, that are involved in corneal neovascularization and SK pathogenesis. These findings are, to our knowledge, the first to show that RvE1 treatment could represent a novel approach to control lesion severity in a virally induced immunopathological disease.     

Pathogenesis of herpes simplex virus-induced ocular immunoinflammatory lesions in B-cell-deficient mice

The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin mu chain gene-targeted B-cell-deficient mice (muK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of muK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the muK/O mice. First, the lack of antibody responses in muK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4(+) T cells, further contributing to the severity of HSK lesion expression in muK/O mice. Second, muK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1-mediated HSK lesions.     

In vitro-generated antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells control the severity of herpes simplex virus-induced ocular immunoinflammatory lesions

Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor beta (TGF-beta) and interleukin-2 could induce up to 90% of CD4(+) T cells to become Foxp3(+) and able to mediate suppression in vitro. CD11c(+) dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-beta, could convert Foxp3(-) CD4(+) cells into Foxp3(+) CD4(+)cells by producing TGF-beta. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.     

Physical mapping of herpes simplex virus-induced polypeptides.

Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

IL-10 and natural regulatory T cells: two independent anti-inflammatory mechanisms in herpes simplex virus-induced ocular immunopathology

Two prominent anti-inflammatory mechanisms involved in controlling HSV-1-induced corneal immunopathology (stromal keratitis or SK) are the production of the cytokine IL-10 and the activity of natural regulatory T cells (nTregs). It is not known whether, under in vivo conditions, IL-10 and nTregs influence the corneal pathology independently or in concert. In the current study using wild-type and IL-10(-/-) animals, we have assessed the activity of nTregs in the absence of IL-10 both under in vitro and in vivo conditions. The IL-10(-/-) animals depleted of nTregs before ocular infection showed more severe SK lesions as compared with the undepleted IL-10(-/-) animals. In addition, nTregs purified from naive WT and IL-10(-/-) animals were equally able to suppress the proliferation and the cytokine production from anti-CD3-stimulated CD4(+)CD25(-) T cells in vitro. Furthermore, intracellular cytokine staining results indicated that nonregulatory cells expressing B220 and CD25 markers were the major IL-10-producing cell types in the lymphoid tissues of HSV-infected mice. In contrast, in the infected corneas, cells with the CD11b(+)Gr1(+) phenotype along with a minor population of Foxp3(-)CD4(+) and a few F4/80(+) cells produced IL-10. Our current investigations indicate that at least two independent anti-inflammatory mechanisms are involved in limiting the corneal lesions in SK, both of which may need to be modulated to control SK therapeutically.     

Activation of endothelial roundabout receptor 4 reduces the severity of virus-induced keratitis

Antiangiogenic molecules exert a feedback control to restrain pathological angiogenesis, which includes physical binding or inhibition of angiogenic signaling in blood vessel endothelial cells. The latter is the case in which Slit2 ligand-dependent activation of the blood vessel endothelial cell receptor roundabout 4 (Robo4) occurs. In this study, we demonstrate that Robo4 receptors are upregulated following HSV infection of the eye on the majority of the new blood vessel endothelial cells that occur in the corneal stroma. However, expression levels of the ligand for Robo4 receptors, Slit2, was not significantly increased during the disease process, and the knockdown of Slit2 gene expression using lentiviral short hairpin RNAs had no effect on the extent of pathological angiogenesis. In contrast, providing additional Slit2 protein by subconjunctival administration resulted in significantly reduced angiogenesis. The Slit2 binding to Robo4 was shown to block the downstream vascular endothelial growth factor signaling molecules Arf 6 and Rac 1 and reduce the antiapoptotic molecule Bcl-xL in blood vessel endothelial cells. Our results indicate that augmenting the host Robo4/Slit2 system could provide a useful therapeutic approach to control pathological angiogenesis associated with HSV induced stromal keratitis.     

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

IL-12 reduces the severity of Sendai virus-induced bronchiolar inflammation and remodeling

The goal of this research was to determine whether differential pulmonary IL-12 gene expression controls susceptibility to Sendai virus-induced chronic airway inflammation and fibrosis in inbred rat strains. Sendai virus-resistant F344 rats and susceptible BN rats were studied from 1 to 14 days following virus inoculation. F344 rats had 3.4-fold higher IL-12 mRNA levels detected by real-time PCR in lung than BN rats as early as two days following inoculation. This increase in mRNA was associated at two days with increased total IL-12 protein and with a 2-fold increase in numbers of bronchiolar, OX-6-positive dendritic cells and an increased number of IL-12 p40-positive, bronchiolar macrophages and dendritic cells (p<0.05). Virus-susceptible BN rats treated with 3 mug of recombinant, mouse IL-12 intraperitoneally at the time of virus inoculation had a 22.1% decrease in severity of chronic bronchiolar inflammation and a 23.8% decrease in fibrosis compared to virus-inoculated BN rats treated with saline. IL-12 treatment induced increased IFN-gamma mRNA and protein expression after virus inoculation (p<0.05). The results demonstrate that there is differential pulmonary IL-12 gene expression between virus-susceptible and resistant rat strains and that IL-12 treatment can provide significant protection from virus-induced chronic airway inflammation and remodeling during early life.     

Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase

The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or dATP) is not required to initiate or sustain highly processive synthesis. The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.     

Vaccine therapy for ocular herpes simplex virus (HSV) infection: periocular vaccination reduces spontaneous ocular HSV type 1 shedding in latently infected rabbits.

Periocular vaccination of rabbits with preexisting herpes simplex virus type 1 (HSV-1) latent infection with recombinant HSV-2 glycoproteins B and D (gB2 and gD2) plus adjuvant significantly reduced ocular viral shedding. Rabbits were infected in both eyes with HSV-1 strain McKrae. Following HSV-1 infection and the establishment of latency (28 days postinfection), rabbits were given a periocular subconjunctival vaccination three times at 3-week intervals. Beginning 3 weeks after the final vaccination, tear films were collected daily and cultured to detect the presence of HSV-1 and determine the spontaneous HSV-1 ocular shedding rates. Periocular vaccination increased the mean HSV-1 serum neutralizing antibody titer to fivefold above that seen in mock-vaccinated latently infected rabbits. gB enzyme-linked immunosorbent assay (ELISA) antibody titers were increased approximately 8-fold, and gD ELISA antibody titers were increased 60-fold. These increases were all statistically significant (P < 0.0001). In two independent experiments, vaccination reduced the spontaneous shedding rate by approximately 2.5-fold (P < 0.0004). In addition, the percentage of eyes that never shed virus during the 6 week postvaccination test period increased threefold (20% in controls versus 60% in vaccinated animals; P < 0.007). These results show that spontaneous ocular shedding of HSV-1 in latently infected rabbits can be significantly reduced by local periocular vaccination. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1 ocular shedding. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans is possible.     

Inhibition of herpes simplex virus-induced cytopathic effect by modified hen egg-white lysozymes

Hen egg-white lysozyme and three of its basic derivatives obtained by chemical modification were tested for their activity in vitro against a wild strain of herpes simplex virus type 1. Marked inhibition of the cytopathic effect was exhibited by the three chemical derivatives and the heat-inactivated lysozyme, whereas the native enzyme displayed only modest anticytopathic activity. Enzymatic activity did not appear to be necessary for the antiherpes activity of the lysozyme compounds. Instead, other properties such as their basic nature seemed to be relevant to their antiherpes effectiveness in vitro. At the concentrations used, all compounds but one had no significant effect on cell viability and growth. Some of the compounds tested caused formation of deposits on the surface of the cells. Some correlation between deposit formation and antiherpes cytopathic activity was found. The antiherpes efficacy in vitro and toxicity of the modified lysozymes were compared with those of known antiviral agents. The lysozymes were less toxic than the reference antiviral agents, and some of them were also more active.     

Role of suppressor T cells in herpes simplex virus-induced immune deviation.

Herpes simplex virus type 1 inoculated into the anterior chamber of the mouse eye induces suppression of anti-herpes simplex virus T-cell-mediated delayed hypersensitivity. This suppression is virus-specific, and mediated by splenic T lymphocytes, and it can be adoptively transferred to naive recipients.     

The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification.

Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification.     

Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice

Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly, Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality.     

Role of peptidergic neurons in ocular herpes simplex infection in mice

Peptide-containing nerve fibers (peptidergic fibers) abundantly innervate the mammalian cornea. We investigated their role in ocular herpes simplex infection in mice by using capsaicin, which causes degeneration and permanent loss of peptidergic neurons in neonates and temporary peptide depletion in adult animals. The corneas of neonatally denervated BALB/c mice were observed for capsaicin-induced keratitis at 11-14 wk of age and were then infected bilaterally with herpes simplex virus 1 (HSV-1); trigeminal (TG) ganglia were cocultivated 6 wk later to establish the rate of latent infection. We also applied capsaicin eye drops to adult BALB/c mice that had been infected with HSV-1 6 wk earlier, and measured viral shedding before, and 3 days and 2 months after, administration of capsaicin drops; TG ganglia of these animals were cocultivated at 3 days and 2 months after capsaicin application. Neurotrophic keratitis was found in 81% of neonatally denervated animals; mortality rate due to HSV-1 infection was reduced from 80% in the controls to 24% in the capsaicin-treated group, and recovery of latent virus by cocultivation was reduced by 50%. Viral shedding could not be produced by capsaicin eye drops in adult animals infected with HSV-1. However, recovery of latent virus was significantly reduced in TG ganglia sampled 3 days and 2 months after capsaicin drops were instilled. Our findings suggest 1) that peptidergic fibers play a crucial role in the establishment of trigeminal HSV-1 latency and 2) that reactivation of latently infected ganglia can be inhibited by topical capsaicin.