An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis.

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-ß1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell-derived microglia, HSV-2-induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2-treated microglia exerted IKBKE-dependent type I IFN-mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

CD8+ T cells in the central nervous system of mice with herpes simplex infection are highly activated and express high levels of CCR5 and CXCR3.

Herpes simplex virus type 2 (HSV-2) is a neurotropic virus that can cause meningitis, an inflammation of the meninges in the central nervous system. T cells are key players in viral clearance, and these cells migrate from peripheral blood into the central nervous system upon infection. Several factors contribute to T cell migration, including the expression of chemokines in the inflamed tissue that attract T cells through their expression of chemokine receptors. Here we investigated CD8+ T cell profile in the spinal cord in a mouse model of herpes simplex virus type 2 neuroinflammation. Mice were infected with HSV-2 and sacrificed when showing signs of neuroinflammation. Cells and/or tissue from spinal cord, spleen, and blood were analyzed for expression of activation markers, chemokine receptors, and chemokines. High numbers of CD8+ T cells were present in the spinal cord following genital HSV-2-infection. CD8+ T cells were highly activated and HSV-2 glycoprotein B -specific effector cells, some of which showed signs of recent degranulation. They also expressed high levels of many chemokine receptors, in particular CCR2, CCR4, CCR5, and CXCR3. Investigating corresponding receptor ligands in spinal cord tissue revealed markedly increased expression of the cognate ligands CCL2, CCL5, CCL8, CCL12, and CXCL10. This study shows that during herpesvirus neuroinflammation anti-viral CD8+ T cells accumulate in the CNS. CD8+ T cells in the CNS also express chemotactic receptors cognate to the chemotactic gradients in the spinal cord. This indicates that anti-viral CD8+ T cells may migrate to infected areas in the spinal cord during herpesvirus neuroinflammation in response to chemotactic gradients.

Extracts of Equisetum giganteum L and Copaifera reticulate Ducke show strong antiviral activity against the sexually transmitted pathogen herpes simplex virus type 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Equisetum giganteum L and Copaifera reticulate Ducke have been traditionally used by women of the Tacana tribe in the Bolivian Amazonas for genital hygiene and for treatment of genital infection/inflammation. AIM OF THE STUDY: To assess the ability of extracts from Equisetum giganteum L and Copaifera reticulate Ducke to block genital viral infection by herpes simplex virus type 2. MATERIALS AND METHODS: Equisetum giganteum L and Copaifera reticulate Ducke were collected from the Amazon region of La Paz, Bolivia. Extracts were prepared and screened for anti-viral activity against herpes simplex virus type 2 (HSV-2) using both in vitro and in in vivo models of infection. RESULTS: Equisetum giganteum L and Copaifera reticulate Ducke efficiently blocked HSV-2 infection of cell cultures without major cell cytotoxic effects. Extracts of Equisetum giganteum L and Copaifera reticulate Ducke could prevent HSV-2 disease development when administered together with virus in a mouse model of genital HSV-2 infection. In vitro analyses revealed that both plant extracts exerted their anti-HSV-2 effects by interfering with viral cell attachment and entry, but could not block viral replication post entry. CONCLUSIONS: These studies show that extracts of Equisetum giganteum L and Copaifera reticulate Ducke have potent antiviral activities against HSV-2 comparable to those two previously identified plants, Croton lechleri Müll. Arg. and Uncaria tomentosa (Willd. ex Schult.) DC. These studies confirm that plants used by the Tacana tribe could be explored further for the development of novel topical antiviral microbicides.

Cutting Edge: Genetic Association between IFI16 Single Nucleotide Polymorphisms and Resistance to Genital Herpes Correlates with IFI16 Expression Levels and HSV-2-Induced IFN-ß Expression.

IFN-γ-inducible protein 16 (IFI16) is an immunological DNA sensor proposed to act in the cyclic GMP-AMP synthase-stimulator of IFN genes pathway. Because mice do not have a clear ortholog of IFI16, this system is not suitable for genetic studies of IFI16. In this study, we have compared the dependency on IFI16, cyclic GMP-AMP synthase, and stimulator of IFN genes for type I IFN induction by a panel of pathogenic bacteria and DNA viruses. The IFN response induced by HSV-2 was particularly dependent on IFI16. In a cohort of patients with genital herpes and healthy controls, the minor G allele of the IFI16 single nucleotide polymorphism rs2276404 was associated with resistance to infection. Furthermore, the combination of this allele with the C allele of rs1417806 was significantly overrepresented in uninfected individuals. Cells from individuals with the protective GC haplotype expressed higher levels of IFI16 and induced more IFN-ß upon HSV-2 infection. These data provide genetic evidence for a role for IFI16 in protection against genital herpes.

Maturation-dependent expression of AIM2 in human B-cells.

Intracellular DNA- and RNA-sensing receptors, such as the IFN-inducible protein Absent in Melanoma 2 (AIM2), serve as host sensors against a wide range of infections. Immune sensing and inflammasome activation by AIM2 has been implicated in innate antiviral recognition in many experimental systems using cell-lines and animal models. However, little is known about the expression and function of AIM2 in freshly isolated human cells. In this study we investigated the expression of AIM2 in different cell types derived from human cord and adult peripheral blood, in steady state and following in vitro-activation. Adult but not cord blood B-cells expressed high levels of AIM2 mRNA at steady state. In adults, AIM2 was primarily expressed in mature memory CD27+ B-cells. Both adult and cord blood derived B-cells could induce their transcription of AIM2 mRNA in response to type II IFN but not type I IFN or the AIM2 ligand poly dA:dT. Upon B-cell receptor stimulation, B-cells from adult blood expressed reduced levels of AIM2 mRNA. In addition, we show that adult B-cells were able to release IL-1ß upon stimulation with synthetic DNA. We conclude that functional AIM2 is preferentially expressed in adult human CD27+ B-cells, but is absent in cord blood mononuclear cells.

Identification of adult septic patients in the prehospital setting: a comparison of two screening tools and clinical judgment.

BACKGROUND: Timely identification and treatment of sepsis is crucial for patient outcome. The aim of this study was to compare two previously unvalidated prehospital sepsis screening tools with clinical judgment by emergency medical services (EMS) personnel with respect to identification of septic patients. PATIENTS AND METHODS: We carried out a retrospective cross-sectional study of 353 adult patients, transported by the EMS, with a hospital discharge International Classification of Diseases code consistent with sepsis. We analyzed EMS records for the identification of sepsis according to two screening tools and clinical judgment by EMS providers. The Robson screening tool includes temperature, heart rate, respiratory rate, altered mental status, plasma glucose, and a history suggestive of a new infection. BAS 90-30-90 refers to the vital signs: oxygen saturation, respiratory rate, and systolic blood pressure. McNemar's two related samples test was used to compare the sensitivity of the two screening tools with the sensitivity of clinical judgment. RESULTS: The Robson screening tool had a sensitivity of 75% (18 out of 24 patients for whom all parameters were documented, P<0.001, as compared with clinical judgment). BAS 90-30-90 had a sensitivity of 43% (76 out of 175 patients, P<0.001). EMS personnel documented suspected sepsis in 42 out of 353 (12%) patients with sepsis. CONCLUSION: The Robson screening tool had a sensitivity superior to both BAS 90-30-90 and clinical judgment. This supports our hypothesis that the implementation of a screening tool could lead to increased prehospital identification of sepsis, which may enable a more timely treatment of these patients.

STAT4 regulates antiviral gamma interferon responses and recurrent disease during herpes simplex virus 2 infection.

STAT4 is an important transcription factor that contributes to the incidence and severity of different autoimmune diseases and is implicated in the antiviral immune responses in mice. In this study, we evaluated the role of STAT4 in human and murine herpes simplex virus 2 (HSV-2) infections. We show that STAT4 regulates antiviral gamma interferon (IFN-γ) responses and disease severity during chronic HSV-2 infections in humans and vaccine-induced IFN-γ-mediated protection against HSV-2 infection in mice. In a cohort of 228 HSV-2-infected individuals, representing both patients with recurrent disease and asymptomatic HSV-2 carriers, we found that genetic variations in the STAT4 gene were associated with asymptomatic HSV-2 infection, as well as with increased in vitro secretion of IFN-γ in response to the virus. Mice that lacked STAT4 had impaired HSV-2-specific IFN-γ production and delayed-type hypersensitivity responses following vaccination, which led to impaired viral clearance in the genital tract of vaccinated animals after a genital HSV-2 challenge. We conclude that STAT4 plays an important role in IFN-γ-mediated HSV-2-specific immunity, affecting the severity of genital HSV-2 infection.

Polymorphisms in Toll-like receptor 3 confer natural resistance to human herpes simplex virus type 2 infection.

Lack of Toll-like receptor 3 (TLR3) functional activity predisposes children to human herpesvirus 1 (HSV-1) encephalitis. In this study, we have investigated whether there is any link between TLR3 and adult HSV-2 infection by studying genetic variations in TLR3. The frequency of four single-nucleotide polymorphisms (SNPs) in the TLR3 gene in 239 patients with genital HSV-2 infection and 162 healthy controls, as well as the impact of these variants on TLR3 gene-expression levels, were compared. Two SNPs in the TLR3 gene (rs13126816 and rs3775291) were associated with a reduced incidence of HSV-2 infection. The minor allelic variants at both rs13126816 and rs3775291 were more common among healthy HSV-2-seronegative subjects than among HSV-2-infected individuals. This was even more apparent in HSV-1-seronegative individuals. There was, however, no association between any of the four TLR3 SNPs and HSV-2 disease severity, as they were expressed at similar proportions in asymptomatic and symptomatic HSV-2-infected patients alike. Furthermore, when assessing TLR3 mRNA expression in a limited number of HSV-2-infected individuals, we found that individuals carrying the homozygous genotypes for the minor alleles had significantly higher levels of TLR3 mRNA expression in peripheral blood mononuclear cells in response to HSV-2 stimulation than individuals that were homozygous for the major allele variants. Taken together, these results suggest that genetic variations in TLR3 may affect the susceptibility to HSV-2 infection in humans.

Characterization of natural killer cell phenotype and function during recurrent human HSV-2 infection.

Human natural killer (NK) cell differentiation, characterized by a loss of NKG2A in parallel with the acquisition of NKG2C, KIRs, and CD57 is stimulated by a number of virus infections, including infection with human cytomegalovirus (CMV), hantavirus, chikungunya virus, and HIV-1. Here, we addressed if HSV-2 infection in a similar way drives NK cell differentiation towards an NKG2A(-)NKG2C(+)KIR(+)CD57(+) phenotype. In contrast to infection with CMV, hantavirus, chikungunya virus, and HIV-1, recurrent HSV-2 infection did not yield an accumulation of highly differentiated NK cells in human peripheral blood. This outcome indicates that human HSV-2 infection has no significant imprinting effect on the human NK cell repertoire.

Inhibition of γ-secretase cleavage in the notch signaling pathway blocks HSV-2-induced type I and type II interferon production.

We have evaluated the role of γ-secretase, which is a crucial component in the Notch-induced signaling cascade, on herpes simplex virus type 2 (HSV-2)-induced innate and acquired interferon responses in human CD4(+) T cells and plasmacytoid dendritic cells (pDC). We found that blockade of the Notch signaling pathway with a pharmacological γ-secretase inhibitor blocked both HSV-2-induced interferon-γ (IFN-γ) production in CD4(+) T cells, and HSV-2-induced IFN-α production in pDC in a dose-dependent fashion. These effects were not due to an overall suppressive capacity of the γ-secretase inhibitor, as it affected neither phytohemagglutinin (PHA)-induced IFN-γ production in CD4(+) T cells, nor CpG-induced IFN-α production in pDC. Our data suggest that Notch signaling could be involved in HSV-2-induced interferon responses in CD4(+) T-cells and pDC.

A 3'-untranslated region polymorphism in the TBX21 gene encoding T-bet is a risk factor for genital herpes simplex virus type 2 infection in humans.

It was recently shown that the transcription factor T-bet is crucial for adequate innate and acquired immune responses to genital herpes simplex virus type 2 (HSV-2) infection in mice. To test the possible genetic influence of variations in the TBX21 gene encoding T-bet on susceptibility to infection, this study evaluated the frequencies of five different single-nucleotide polymorphisms (SNPs) in the human TBX21 gene in 159 HSV-2-infected individuals and compared them with those in 186 healthy HSV-2-seronegative controls. The data showed that one variation (rs17244587) in the 3'-untranslated region of TBX21 was strongly associated with the incidence of genital HSV-2 infection. The frequency of the A allele at this position was 0.19 in the group of HSV-2-infected individuals compared with 0.05 in the group of uninfected controls (P=9.3x10(-8)). Furthermore, a homozygous AA genotype was found only among HSV-2-infected individuals and not in seronegative controls. These results indicate that the host genetic background may affect susceptibility to HSV-2 infection in humans, with TBX21 as a strong candidate gene.

Role of IFN-alpha/beta signaling in the prevention of genital herpes virus type 2 infection.

This study has shown that IFN-alpha/beta signaling is crucial for combating primary herpes simplex virus type 2 (HSV-2) infection and for responding to immunotherapy using ligands to TLR3, 7 and 9, but not for vaccine-induced immunity. Both genital viral replication and the disease progression were enhanced in HSV-2-infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR-/-). IFN-alpha/betaR-/- mice were, however, able to mount a normal HSV-2-specific Th1 response and acquired sterilizing immunity following vaccination. Anti-viral treatments using agonists to TLR3, 7 and 9 by administration of synthetic dsRNA, imiquimod and oligonucleotides containing unmethylated CpG motifs, respectively, were strongly dependent on IFN-alpha/beta receptor signaling for their efficacy. Even though all treatments had a weak impact on local vaginal viral replication in infected IFN-alpha/betaR-/- animals, they did not affect disease progression or mortality in these animals as opposed to wild type controls where all three treatments reduced viral replication as well as disease severity and mortality. Lack of IFN-alpha/betaR signaling also blocked production of IFN-gamma and TNF-alpha in response to TLR9 activation. These studies have shown that IFN-alpha/beta receptor signaling is important for multiple events in the anti-viral defense.

Neurokinin 1 receptor signaling affects the local innate immune defense against genital herpes virus infection.

We show that genital infection with neurotropic HSV type 2 (HSV-2) induced a significant increase of the neuropeptide substance P (SP) within the genital tract of mice. SP was shown to weakly interfere with the HSV-2 replication. Furthermore, lack of SP signaling through the use of mice deficient in the SP receptor, neurokinin 1 receptor (NK1R), revealed an important role for SP in the innate defense against HSV-2. NK1R-deficient mice had significantly enhanced levels of HSV-2 in the genital tract and in the CNS following infection and a significantly accelerated disease progression, which was associated with an impaired NK cell activity locally in the vagina. Lack of NK1R signaling did, however, not impair the animals' ability to mount a protective immune response to HSV-2 following vaccination with an attenuated virus. Both NK1R+/+ and NK1R-/- mice developed strong HSV-2-specific Th1 T cell responses following vaccination. No genital viral replication was observed in either vaccinated NK1R-deficient or NK1R+/+ control animals following a genital HSV-2 challenge, and all of these animals survived without any symptoms of disease. In conclusion, the present results indicate that SP and NK1R signaling contributes to the innate resistance against HSV-2 infection in mice.

Protective immunity to genital herpes simplex [correction of simpex] virus type 2 infection is mediated by T-bet.

We show, for the first time, that the transcription factor T-bet, which is implicated in IFN-gamma production, is required for the induction of vaccine-induced antiviral immune protection. T-bet was found to be important in both the innate and acquired immune protection against genital HSV-2 infection. T-bet(-/-) and T-bet(+/+) mice were infected vaginally with HSV-2 and examined daily for disease and mortality. T-bet(-/-) mice had significantly higher virus titers than T-bet(+/+) mice following a primary HSV-2 infection, and succumbed significantly earlier to the infection. This result was associated with an impaired NK cell cytotoxic capacity and NK cell-mediated IFN-gamma production in the T-bet(-/-) mice. To assess the induction of acquired antiviral immune protection, mice were vaccinated with an attenuated virus before infection. Vaccinated T-bet(-/-) mice could not control viral replication following an HSV-2 challenge and had significantly higher virus titers and mortality rates than vaccinated T-bet(+/+) mice that remained healthy. The impaired acquired immune protection in T-bet(-/-) mice was associated with a significantly decreased HSV-2-specific delayed-type hypersensitivity response and a significantly reduced HSV-2-specific IFN-gamma production from CD4(+) T cells. However, T-bet deficiency did not impair either the IFN-gamma production or the cytotoxic capacity of HSV-2-specific CD8(+) T cells. We conclude that T-bet plays a crucial role in both the innate defense and the generation of vaccine-induced immunity against genital HSV-2 infection in mice.