Herpes Simplex Virus Type 1 Challenge Research Articles

HSV-1 0∆NLS vaccine elicits a robust B lymphocyte response and preserves vision without HSV-1 glycoprotein M or thymidine kinase recognition

Effective experimental prophylactic vaccines against viral pathogens such as herpes simplex virus type 1 (HSV-1) have been shown to protect the host through T and/or B lymphocyte-driven responses. Previously, we found a live-attenuated HSV-1 mutant, 0ΔNLS used as a prophylactic vaccine, provided significant protection against subsequent ocular HSV-1 challenge aligned with a robust neutralizing antibody response. Yet, how the virus mutant elicited the humoral immune response relative to parental virus was unknown. Herein, we present the characterization of B cell subsets in vaccinated mice at times after primary vaccination and following boost compared to the parental virus, termed GFP105. We found that 0∆NLS-vaccinated mice possessed more CD4+ follicular helper T (TFH) cells, germinal B cells and class-switched B cells within the first 7 days post-vaccination. Moreover, 0∆NLS vaccination resulted in an increase in plasmablasts and plasma cells expressing amino-acid transporter CD98 along with an elevated titer of HSV-1-specific antibody compared to GFP105-vaccinated animals. Furthermore, O∆NLS-vaccine-induced CD4+ (TFH) cells produced significantly more IL-21 compared to mice immunized with the parental HSV-1 strain. In contrast, there were no differences in the number of regulatory B cells comparing the two groups of immunized mice. In comparing sera recognition of HSV-1-encoded proteins, it was noted antiserum from GFP105-vaccinated mice immunoprecipitated HSV-1 thymidine kinase (TK) and glycoprotein M (gM) whereas sera from 0∆NLS-immunized mice did not even though both groups of vaccinated mice displayed similar neutralizing antibody titers to HSV-1 and were highly resistant to ocular HSV-1 challenge. Collectively, the results suggest (1) the live-attenuated HSV-1 mutant 0∆NLS elicits a robust B cell response that drives select B cell responses greater than the parental HSV-1 and (2) HSV-1 TK and gM are likely expendable components in efficacy of a humoral response to ocular HSV-1 infection.

Preventing neonatal herpes: Protection after maternal mRNA-lipid nanoparticle vaccination equals or exceeds that from prior maternal genital infection in murine models

Abstract Neonates are vulnerable to poor outcomes from herpes simplex virus (HSV), but maternal infection eliciting non-sterilizing immunity prior to delivery provides some protection. We are therefore pursuing birthing parent vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNPs) to prevent neonatal herpes. Our trivalent vaccine encodes HSV type 2 (HSV-2) proteins involved in attachment and immune evasion (gC2, gD2, and gE2). We have shown that this vaccine protects against genital HSV and neonatal HSV-2 in mice. Here, we tested how well murine dam vaccination prior to pregnancy protects pups against HSV type 1 (HSV-1) challenge compared to prior maternal intravaginal HSV-1 infection. Dams were immunized intramuscularly with the HSV-2 trivalent mRNA-LNP vaccine or a control immunogen (PolyC RNA-LNP), or were infected intravaginally with HSV-1 (HSV-1 ivag). Pups were challenged intranasally with HSV-1 on the 3rd day of life. Pups born to dams previously immunized or infected survived through 28 days (mRNA 95% vs. HSV-1 ivag 96%, N.S.; vs. PolyC 13%, p<0.0001). Likewise, they were protected from HSV-1 dissemination to the brain, lung, liver, spleen, and kidney by plaque assay (mRNA 2/45 organs affected vs. HSV-1 ivag 6/20, p=0.0083; vs. PolyC 69/80, p<0.0001). Additionally, fewer organs showed necrosis by histopathology of brain, lung, and liver, though not statistically significant, and fewer mRNA and HSV-1 ivag pups had detectable HSV-1 DNA in the trigeminal ganglia by qPCR (mRNA 6/30 vs. HSV-1 ivag 7/26, N.S.; vs. PolyC 17/18, p<0.0001). These studies demonstrate that the mRNA vaccine provides comparable to improved protection against neonatal HSV-1 challenge versus prior maternal HSV-1 intravaginal infection. AD was supported by NIH NIAID T32 AI118684. HMF, SA, GHC, and DW were supported by NIH NIAID R01 AI139618 and an unrestricted grant from BioNTech SE. KPE was supported by NIH T32 NS007180. The funders had no input or influence on the content of this abstract.

Immunological Identification and Characterization of the Capsid Scaffold Protein Encoded by UL26.5 of Herpes Simplex Virus Type 2.

Herpes simplex virus type 2 (HSV2), a pathogen that causes genital herpes lesions, interferes with the host immune system via various known and unknown mechanisms. This virus has been used to study viral antigenic composition. Convalescent serum from HSV2-infected patients was used to identify viral antigens via 2-D protein electrophoresis and immunoblotting. The serum predominantly recognized several capsid scaffold proteins encoded by gene UL26.5, mainly ICP35. This protein has been primarily reported to function temporarily in viral assembly but is not expressed in mature virus particles. Further immunological studies suggested that this protein elicits specific antibody and cytotoxic T lymphocyte (CTL) responses in mice, but these responses do not result in a clinical protective effect in response to HSV2 challenge. The data suggested that immunodominance of ICP35 might be used to design an integrated antigen with other viral glycoproteins.

Lack of neonatal Fc receptor does not diminish the efficacy of the HSV-1 0ΔNLS vaccine against ocular HSV-1 challenge

The neonatal Fc receptor (FcRn) is constitutively expressed in the cornea and is up-regulated in response to herpes simplex virus type 1 (HSV-1). Previously, we found targeting cornea FcRn expression by small interfering RNA-mediated knockdown reduced the local efficacy of HSV-1 0ΔNLS vaccinated C57BL/6 mice against ocular challenge with HSV-1. The current study was undertaken to evaluate the HSV-1 0ΔNLS vaccine efficacy in FcRn deficient (FcRn KO) mice challenged with HSV-1. Whereas there was little neutralizing antibody detected in the serum of HSV-1 0ΔNLS vaccinated FcRn KO mice, these mice exhibited the same degree of protection against ocular challenge with HSV-1 as wild type (WT) C57BL/6 mice as measured by cumulative survival, infectious virus shed or retained in tissue, and corneal pathology including opacity and neovascularization. Mock-vaccinated FcRn KO mice were found to be more sensitive to ocular HSV-1 infection compared to mock-vaccinated (WT) mice in terms of cumulative survival and virus shedding. In addition, the FcRn KO mice generated significantly fewer effector (CD3+CD44+CD62L-) and central (CD3+CD44+CD62L+) memory CD8+ T cells compared to the WT mice 7 days post infection. Collectively, mock-vaccinated FcRn KO mice are susceptible to ocular HSV-1 infection but HSV-1 0ΔNLS vaccinated FcRn KO mice are resistant suggesting that in addition to the FcRn, other pathways are involved in mediating the protective effect of the HSV-1 0ΔNLS vaccine against subsequent HSV-1 challenge.

Ca2+ -dependent release of ATP from astrocytes affects herpes simplex virus type 1 infection of neurons.

Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play a role in cortical neurons' vulnerability to Herpes simplex virus type-1 (HSV-1) infection through the release of extracellular ATP. We found that the interaction of HSV-1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C-mediated IP3 -dependent intracellular Ca2+ transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca2+ concentration that activates the Glycogen Synthase Kinase (GSK)-3β, whose action is necessary for HSV-1 entry/replication in these cells. Indeed, in co-cultures of neurons and astrocytes HSV-1-infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching-off the HSV-1-induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV-1 challenge, restored the ability of HSV-1 to infect neurons co-cultured with metabolically-inhibited astrocytes. The ATP-activated, P2R-mediated, and GSK-3-dependent molecular pathway underlying HSV-1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK-3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca2+ -dependent release of neuroactive molecules.

A HSV1 mutant leads to an attenuated phenotype and induces immunity with a protective effect.

Herpes simplex virus type 1 (HSV1) is a complicated structural agent with a sophisticated transcription process and a high infection rate. A vaccine against HSV1 is urgently needed. As multiple viral-encoded proteins, including structural and nonstructural proteins, contribute to immune response stimulation, an attenuated or deficient HSV1 vaccine may be relatively reliable. Advances in genomic modification technologies provide reliable means of constructing various HSV vaccine candidates. Based on our previous work, an M6 mutant with mutations in the UL7, UL41, LAT, Us3, Us11 and Us12 genes was established. The mutant exhibited low proliferation in cells and an attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant can induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.

Prior inhibition of AKT phosphorylation by BX795 can define a safer strategy to prevent herpes simplex virus-1 infection of the eye

PurposeTo evaluate the prophylactic antiviral efficacy, corneal tolerance and toxicity of topically dosed BX795, a non-nucleoside small-molecule inhibitor of herpes simplex virus type-1 (HSV-1). MethodsProphylactic treatment with BX795 was performed both in-vitro on human corneal epithelial cells and in-vivo on mice prior to HSV-1 challenge. Viral burden was evaluated using a standard plaque assay. In a separate experiment, mice were treated topically 3-times daily for 4-weeks with BX795 to evaluate corneal tolerance and toxicity. Phenol-red thread measurements, fluorescein staining and optical coherence tomography (OCT) were used to evaluate tear production, dryness and corneal structural changes. Corneal sensitivity and intraocular pressure were measured using esthesiometery and tonometery respectively. ResultsBoth in-vitro and in-vivo results showed a robust suppression of HSV-1 infection when treated prophylactically with BX795. The fluorescein stain and phenol-red results for the BX795-treated eyes did not show signs of corneal surface dryness when compared to trifluridine (TFT), an FDA-approved topical antiviral. The OCT measurements showed no signs of structural changes to the cornea suggesting that BX795 treatment was well tolerated without any apparent signs of toxicity or inflammation. The corneal sensitivity of BX795-treated eyes was not significantly different from TFT-treated eyes. No significant increase in the intraocular pressure of BX795-treated mice was observed. ConclusionsProphylactic treatment with BX795 protects corneal cells from HSV-1 infection. The antiviral is well-tolerated on murine corneas without any detectable toxicity.

Assessment of a new arbidol derivative against herpes simplex virus II in human cervical epithelial cells and in BALB/c mice

As one of the highly contagious forms, herpes simplex virus type 2 (HSV-2) commonly caused severe genital diseases and closely referred to the HIV infection. The lack of effective vaccines and drug-resistance proclaimed the preoccupation for alternative antiviral agents against HSV-2. Molecules bearing indole nucleus presented diverse biological properties involving antiviral and anti-inflammatory activities. In this study, one of the indole molecules, arbidol derivative (ARD) was designed and synthesized prior to the evaluation of its anti-HSV-2 activity. Our data showed that the ARD effectively suppressed HSV-2-induced cytopathic effects and the generation of progeny virus, with 50% effective concentrations of 3.386 and 1.717 μg/mL, respectively. The results of the time-course assay suggested that the ARD operated in a dual antiviral way by interfering virus entry and impairing the earlier period of viral cycle during viral DNA synthesis. The ARD-mediated HSV-2 inhibition was partially attained by blocking NF-κB pathways and down-regulating the expressions of several inflammatory cytokines. Furthermore, in vivo studies showed that oral administration of ARD protected BALB/c mice from intravaginal HSV-2 challenge by alleviating serious vulval lesions and histopathological changes in the target organs. Besides, the treatment with ARD also made the levels of viral protein, NF-κB protein and inflammatory cytokines lower, in consistent with the in-vitro studies. Collectively, ARD unveiled therapeutic potential for the prevention and treatment of HSV-2 infections.

Vaccine-induced antibodies target sequestered viral antigens to prevent ocular HSV-1 pathogenesis, preserve vision, and preempt productive neuronal infection

The cornea is essential for vision yet highly sensitive to immune-mediated damage following infection. Generating vaccines that provide sterile immunity against ocular surface pathogens without evoking vision loss is therefore clinically challenging. Here, we tested a prophylactic live-attenuated vaccine against herpes simplex virus type 1 (HSV-1), a widespread human pathogen that can cause corneal blindness. Parenteral vaccination of mice resulted in sterile immunity to subsequent HSV-1 challenge in the cornea and suppressed productive infection of the nervous system. This protection was unmatched by a relevant glycoprotein subunit vaccine. Efficacy of the live-attenuated vaccine involved a T-dependent humoral immune response and complement C3 but not Fcγ-receptor 3 or interferon-α/β signaling. Proteomic analysis of viral proteins recognized by antiserum revealed an unexpected repertoire dominated by sequestered antigens rather than surface-exposed envelope glycoproteins. Ocular HSV-1 challenge in naive and subunit-vaccinated mice triggered vision loss and severe ocular pathologies including corneal opacification, scar formation, neovascularization, and sensation loss. However, corneal pathology was absent in mice receiving the live-attenuated vaccine concomitant with complete preservation of visual acuity. Collectively, this is the first comprehensive report of a prophylactic vaccine candidate that elicits resistance to ocular HSV-1 infection while fully preserving the cornea and visual acuity.

Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection.

The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar-/- and Irf9-/- mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-γ. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18-/- mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response.

Topical Administration Is a Promising Inoculating Route versus Intramuscular Inoculation for the Nanoparticle-Carried DNA Vaccine to Prevent Corneal Infections

Purpose: To evaluate the comparative effect of topical versus intramuscular administration of nanoparticle-carried DNA vaccine in preventing corneal herpes simplex virus type 1 (HSV-1) infection. Methods: Nanoparticle [polyethylenimine (PEI)-Fe₃O₄]-carried DNA vaccine (PEI-Fe₃O₄-pRSC-gD-IL-21) or DNA vaccine (pRSC-gD-IL-21) alone were topically versus intramuscularly inoculated into one eye each of mice on days 0, 14 and 28. Three weeks after the final immunization, the specific immune responses and clinical degrees of primary herpes simplex keratitis were evaluated. Results: Topical inoculation of nanoparticle-carried DNA vaccine induced mice to generate similar levels of specific HSV-1-neutralizing antibody, IFN-γ and IL-4 in serum and specific killing (cytotoxicity) and proliferative activities of the splenic lymphocytes, but a significantly higher level of secretory IgA in tears compared to those of intramuscular inoculation. More importantly, the mice inoculated topically showed a significantly decreased herpes simplex keratitis severity than the mice inoculated intramuscularly after HSV-1 challenge on the corneas of the mice. Conclusions: Topical inoculation of nanoparticle-carried DNA vaccine elicits a stronger specific local immune response and more effectively inhibits herpes simplex keratitis as compared to intramuscular inoculation in an HSV-1 ocular challenge mouse model. Thus, topical administration may be a promising inoculating route for the nanoparticle-carried DNA vaccine to prevent corneal infections.

A single intramuscular vaccination of mice with the HSV-1 VC2 virus with mutations in the glycoprotein K and the membrane protein UL20 confers full protection against lethal intravaginal challenge with virulent HSV-1 and HSV-2 strains.

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 107 VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.

Use of Transcriptional Profiling to Delineate the Initial Response of Mice to Intravaginal Herpes Simplex Virus Type 2 Infection

Intravaginal (ivag) infection of mice with herpes simplex virus type 2 (HSV-2) causes genital tissue damage, quickly followed by development of fatal encephalopathy. To delineate initial host responses generated by HSV-2 infection, here oligonucleotide microarrays compared gene expression in vaginal tissue from uninfected mice and mice 1, 2, 3, 4, 5, 6, or 7 days after ivag infection with 10(4) pfu HSV-2. While comparison of mRNA expression in uninfected and HSV-infected vaginal tissue detected few changes during the first 2 days post infection (dpi), there were 156 genes whose expression was first significantly altered 3 dpi that remained significantly modified at all later time points examined. These 156 genes were significantly enriched in canonical pathways associated with interferon (IFN) signaling, activation of IFN elements by intracellular pattern recognition receptors, and antiviral immunity induced by cytosolic RIG-like receptors. Evaluation of this gene set with the National Center for Biotechnology Information Gene and INTERFEROME databases corroborated pathway analysis, as function of most (53%) were linked to IFN-mediated host immunity. In the final set of experiments, ivag administration of the Toll-like receptor 3 agonist polyinosinic: polycytidylic acid (poly I:C) 24 h before ivag HSV-2 infection reduced the incidence of genital pathology and encephalopathy, while these poly I:C-treated mice were subsequently protected from ocular HSV-2 challenge lethal to uninfected controls. The latter results imply that the exuberant antiviral immunity produced in our experimental model is simply formed too late to prevent viral replication and dissemination, and that poly I:C-induced formation of an antiviral state protecting against primary ivag infection also permits development of HSV-specific protective immunity.

Immunogenicity and Efficacy of Intramuscular Replication-Defective and Subunit Vaccines against Herpes Simplex Virus Type 2 in the Mouse Genital Model

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus that is highly prevalent worldwide, causing a range of symptoms that result in significant healthcare costs and human suffering. ACAM529 is a replication-defective vaccine candidate prepared by growing the previously described dl5-29 on a cell line appropriate for GMP manufacturing. This vaccine, when administered subcutaneously, was previously shown to protect mice from a lethal vaginal HSV-2 challenge and to afford better protection than adjuvanted glycoprotein D (gD) in guinea pigs. Here we show that ACAM529 given via the intramuscular route affords significantly greater immunogenicity and protection in comparison with subcutaneous administration in the mouse vaginal HSV-2 challenge model. Further, we describe a side-by-side comparison of intramuscular ACAM529 with a gD vaccine across a range of challenge virus doses. While differences in protection against death are not significant, ACAM529 protects significantly better against mucosal infection, reducing peak challenge virus shedding at the highest challenge dose by over 500-fold versus 5-fold for gD. Over 27% (11/40) of ACAM529-immunized animals were protected from viral shedding while 2.5% (1/40) were protected by the gD vaccine. Similarly, 35% (7/20) of mice vaccinated with ACAM529 were protected from infection of their dorsal root ganglia while none of the gD-vaccinated mice were protected. These results indicate that measuring infection of the vaginal mucosa and of dorsal root ganglia over a range of challenge doses is more sensitive than evaluating survival at a single challenge dose as a means of directly comparing vaccine efficacy in the mouse vaginal challenge model. The data also support further investigation of ACAM529 for prophylaxis in human subjects.

Delayed but effective induction of mucosal memory immune responses against genital HSV-2 in the absence of secondary lymphoid organs.

To examine whether local immunization in the absence of secondary lymphoid organs (SLOs) could establish effective antiviral memory responses in the female genital tract, we examined immunity in the vaginal tracts of LTα-/- mice, LTα-/- SPL (splenectomized), and control C57BL/6 (WT) mice. All three groups of mice were immunized intravaginally (IVAG) with attenuated thymidine kinase-negative (TK(-)) Herpes simplex virus type 2 (HSV-2) and challenged 4-6 weeks later with wild-type (WT) HSV-2. Both groups of LTα-/- mice exhibited delayed viral clearance and prolonged genital pathology after immunization. Following IVAG WT HSV-2 challenge, LTα-/- and LTα-/- SPL mice had significantly lower levels of HSV-2-specific IgG and IgA in the vaginal secretions. Although the frequency of B and T cells in the vaginal mucosa was comparable or higher in both groups of LTα-/- mice, lower frequency of HSV-2-specific interferon-γ (IFNγ)-producing CD3+ T cells was seen after immunization and after challenge, compared with WT group. Despite this, immunized mice in all three groups showed complete sterile protection against IVAG WT HSV-2 challenge. These results show that even in the absence of SLOs, IVAG immunization generates effector memory immune responses at genital mucosa that can provide antiviral protection against subsequent viral exposures. This will inform new strategies to design mucosal vaccines against sexually transmitted infections.

Nasal and skin delivery of IC31®-adjuvanted recombinant HSV-2 gD protein confers protection against genital herpes

Genital herpes caused by herpes simplex virus type 2 (HSV-2) remains the leading cause of genital ulcers worldwide. Given the disappointing results of the recent genital herpes vaccine trials in humans, development of novel vaccine strategies capable of eliciting protective mucosal and systemic immune responses to HSV-2 is urgently required. Here we tested the ability of the adjuvant IC31® in combination with HSV-2 glycoprotein D (gD) used through intranasal (i.n.), intradermal (i.d.), or subcutaneous (s.c.) immunization routes for induction of protective immunity against genital herpes infection in C57BL/6 mice. Immunization with gD plus IC31® through all three routes of immunization developed elevated gD-specific serum antibody responses with HSV-2 neutralizing activity. Whereas the skin routes promoted the induction of a mixed IgG2c/IgG1 isotype profile, the i.n. route only elicited IgG1 antibodies. All immunization routes were able to induce gD-specific IgG antibody responses in the vaginas of mice immunized with IC31®-adjuvanted gD. Although specific lymphoproliferative responses were observed in splenocytes from mice of most groups vaccinated with IC31®-adjuvanted gD, only i.d. immunization resulted in a significant splenic IFN-γ response. Further, immunization with gD plus IC31® conferred 80–100% protection against an otherwise lethal vaginal HSV-2 challenge with amelioration of viral replication and disease severity in the vagina. These results warrant further exploration of IC31® for induction of protective immunity against genital herpes and other sexually transmitted infections.

The impact of methamphetamine use on host immune response to herpes simplex virus type 2 (105.15)

Abstract Methamphetamine (METH) use is associated with increased risk for sexually transmitted infections (STIs) that is not believed to be due to increased behavioral risk factors alone. However, the impact of METH at the time of exposure to an STI has not been explored. We hypothesized that METH would impact normal host immune responses to an STI pathogen, herpes simplex virus type 2 (HSV-2), resulting in alterations in disease. Female C57BL6 mice were treated with METH (10 mg/kg) or saline daily for 5 days, and inoculated intravaginally with HSV-2 on treatment day 3. METH treated mice experienced significantly earlier onset of disease and enhanced disease progression compared to controls. Unexpectedly, METH treated mice had increased IFNγ levels in the genital tract early after challenge, but this did not correlate with decreased vaginal virus titers. Subsequently, later after virus challenge there were significantly less IFNγ secreting CD4+ T cells in the genital tract of METH treated mice, but an increased number of these cells in the iliac lymph nodes. Our studies confirm our hypothesis that METH alters immune responses to intravaginal HSV-2 challenge with a corresponding impact on disease progression. The alterations in IFNγ production in the genital tract are of particular interest because of the important role of this cytokine in HSV-2 control. These findings have important public health implications and may be more broadly applicable to other important STI’s such as HIV.

Withdrawal from Morphine Reduces Cell-Mediated Immunity against Herpes Simplex Virus Generated by Natural Immunization

In a previous study, the authors have shown that herpes simplex virus type 1 (HSV-1) glycoprotein B DNA vaccine but not live vaccine (non-virulent KOS strain) failed to induce protective immunity against acute HSV-1 challenge in morphine-dependent mice. The present study reports the effect of morphine withdrawal on protective immunity induced by live HSV-1 immunization. BALB/c mice were vaccinated with KOS strain as a live vaccine. Three weeks later, they were exposed to morphine for 14 days. On day 14, withdrawal was induced by administration of normal saline instead of morphine. One day later, immune responses against HSV-1 were assessed by measuring cytotoxicity, lymphocyte proliferation and interferon-γ production. Protection against HSV-1 was assessed by measuring the mortality rate after acute HSV-1 challenge. The results showed that withdrawal from morphine reduces protective immunity against acute HSV-1 challenge. These findings raise the possibility that withdrawal from morphine may increase the susceptibility of drug addicts to infectious diseases.

Interleukin 29 enhances expression of toll receptor 3 and mediates antiviral signals in human keratinocytes

Interleukin 29 (IL-29) is a class II cytokine and displays numerous immune functions other than its anti-viral and antiproliferation activities. This study is focused on the effect of IL-29 on human keratinocytes (KCs). Primary KCs were stimulated by various concentrations of IL-29 for different time periods, and antiviral proteins and TLR3 gene expression were then analyzed by real-time PCR. The signal pathways activated by IL-29 in KCs were detected by western blot. The antiviral activity of IL-29 was determined by methylthiazolyldiphenyl-tetrazolium bromide, and small interfering RNA knockdown was used to analyze the role of toll receptor 3 (TLR3) in the antiviral activity of IL-29. IL-29 was able to induce expression of antiviral proteins and TLR3 gene expression in KCs. IL-29 pretreatment strongly enhanced herpes simplex virus type 1 (HSV-1)-induced expression of the interferon β (IFN-β) gene and protected the KCs from HSV-1 challenge. The IL-29 antiviral activity was partially dependent on TLR3 expression induced by this cytokine, and mechanistic studies demonstrated that the regulation of TLR3 expression by IL-29 might be partially dependent on Janus kinase /signal transducer and activator of transcription (JAK-STATs) activation. IL-29-induced TLR3 expression is involved in antiviral activity of IL-29 in KCs, which suggests a feasible method to cure certain viral infections of the skin.

A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells.

BackgroundThe rapid onset of potentially lethal neuroinflammation is a defining feature of viral encephalitis. Microglia and astrocytes are likely to play a significant role in viral encephalitis pathophysiology as they are ideally positioned to respond to invading central nervous system (CNS) pathogens by producing key inflammatory mediators. Recently, DNA-dependent activator of IFN regulatory factor (DAI) has been reported to function as an intracellular sensor for DNA viruses. To date, the expression and functional role of DAI in the inflammatory responses of resident CNS cells to neurotropic DNA viruses has not been reported.MethodsExpression of DAI and its downstream effector molecules was determined in C57BL/6-derived microglia and astrocytes, either at rest or following exposure to herpes simplex virus type 1 (HSV-1) and/or murine gammaherpesvirus-68 (MHV-68), by immunoblot analysis. In addition, such expression was studied in ex vivo microglia/macrophages and astrocytes from uninfected animals or mice infected with HSV-1. Inflammatory cytokine production by glial cultures following transfection with a DAI specific ligand (B-DNA), or following HSV-1 challenge in the absence or presence of siRNA directed against DAI, was assessed by specific capture ELISA. The production of soluble neurotoxic mediators by HSV-1 infected glia following DAI knockdown was assessed by analysis of the susceptibility of neuron-like cells to conditioned glial media.ResultsWe show that isolated microglia and astrocytes constitutively express DAI and its effector molecules, and show that such expression is upregulated following DNA virus challenge. We demonstrate that these resident CNS cells express DAI in situ, and show that its expression is similarly elevated in a murine model of HSV-1 encephalitis. Importantly, we show B-DNA transfection can elicit inflammatory cytokine production by isolated glial cells and DAI knockdown can significantly reduce microglial and astrocyte responses to HSV-1. Finally, we demonstrate that HSV-1 challenged microglia and astrocytes release neurotoxic mediators and show that such production is significantly attenuated following DAI knockdown.ConclusionsThe functional expression of DAI by microglia and astrocytes may represent an important innate immune mechanism underlying the rapid and potentially lethal inflammation associated with neurotropic DNA virus infection.