Tissue-Resident-Memory CD8+ T Cells Bridge Innate Immune Responses in Neighboring Epithelial Cells to Control Human Genital Herpes.

Tissue-resident-memory T cells (TRM) populate the body's barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.

A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection.

Neutrophil responses against pathogens must be balanced between protection and immunopathology. Factors that determine these outcomes are not well-understood. In a mouse model of genital herpes simplex virus-2 (HSV-2) infection, which results in severe genital inflammation, antibody-mediated neutrophil depletion reduced disease. Comparative single-cell RNA-sequencing analysis of vaginal cells against a model of genital HSV-1 infection, which results in mild inflammation, demonstrated sustained expression of interferon-stimulated genes (ISGs) only after HSV-2 infection primarily within the neutrophil population. Both therapeutic blockade of IFNα/ß receptor 1 (IFNAR1) and genetic deletion of IFNAR1 in neutrophils concomitantly decreased HSV-2 genital disease severity and vaginal IL-18 levels. Therapeutic neutralization of IL-18 also diminished genital inflammation, indicating an important role for this cytokine in promoting neutrophil-dependent immunopathology. Our study reveals that sustained type I interferon (IFN) signaling is a driver of pathogenic neutrophil responses and identifies IL-18 as a novel component of disease during genital HSV-2 infection.

Herpes simplex virus (HSV) is a human pathogen that causes genital herpes, an incurable disease that results in recurrent sores and inflammation. Infection with HSV induces a strong antiviral immune response, which results in large numbers of immune cells arriving at these lesions. But while some of these cells help to control viral replication, others might contribute to the inflammation that drives the disease. One of the first immune cells to respond to infection are neutrophils. Although neutrophils are generally protective, especially against bacteria and fungi, they have also been implicated in tissue damage and severe inflammation during viral infections. But what determines whether a neutrophil will help to fight off an infection or increase disease severity is still an open question. To investigate this, Lebratti, Lim et al. studied mice that had been infected with the genital herpes virus HSV-2, which is known to cause significant amounts of inflammation in mice. The experiments revealed that a signaling molecule called type I interferon, which is thought to be antiviral, causes neutrophils at the site of the infection to produce proteins, such as IL-18, which trigger an inflammatory reaction. Lebratti, Lim et al. found that type I interferon and IL-18 had shifting roles during the course of infection. In the early stages, both molecules had a protective effect, confirming results from previous studies. However, as the infection progressed, sustained levels of type I interferon signaling in neutrophils led to excess amounts of IL-18. Lebratti, Lim et al. discovered that blocking interferon signaling or decreasing the levels of IL-18 later during infection unexpectedly reduced the severity of the disease and resulted in less genital tissue damage. Further experiments also showed that mice infected with another genital herpes virus called HSV-1 did not experience sustained levels of type I interferon. This may explain why this virus causes less severe disease in mice. Understanding how the immune system reacts to viruses could reveal new targets for treatments of genital herpes. At the moment, there is little information about IL-18 production during genital herpes in humans. So, the next step is to see whether neutrophils behave in the same way and whether IL-18 can be detected during human disease. It is possible that the same immune components could promote disease in other infections too. If so, this work may help uncover new drug targets for other viral diseases.

T-type calcium channels blockers inhibit HSV-2 infection at the late stage of genome replication.

Herpes simplex virus type 2 (HSV-2) is a highly contagious sexually transmitted virus. The increasing emergence of drug-resistant viral strains has highlighted the crucial need for the development of new anti-HSV-2 drugs with different mechanisms. Ion channels that govern a wide range of cellular functions represent attractive targets for viral manipulation. Here, we tried to identify novel compounds to suppress HSV-2 infection in vitro by screening a small library with ion channels modulators. We found that several T-type calcium channel blockers including benidipine, lercanidipine, lomerizine and mibefradil inhibited HSV-2 infection, while L-type calcium channel blockers nifedipine and nitrendipine showed no significant effect on HSV-2 infection. Furthermore, we found that benidipine exerted the antiviral effect by suppressing the expression of viral genes in the late stage of viral infection. In conclusion, our study suggested that T-type calcium channel blockers, which are clinically wide used, could effectively inhibit HSV-2 infection. These findings could shed light on the mechanism and pharmacological study for HSV-2 infection in the future.

Analysis of ALS-related proteins during herpes simplex virus-2 latent infection.

BACKGROUND: Genetics have provided hints on potential molecular pathways involved in neurodegenerative diseases (NDD). However, the number of cases caused exclusively by genetic alterations is low, suggesting an important contribution of environmental factors to NDDs. Among these factors, viruses like herpes simplex viruses (HSV-2), capable of establishing lifelong infections within the nervous system (NS), are being proposed to have a role in NDDs. Despite promising data, there is a significant lack of knowledge on this and an urgent need for more research. METHODS: We have set up a mouse model to study HSV latency and its associated neuroinflammation in the spinal cord. The goal of this model was to observe neuroinflammatory changes caused by HSV latent infections, and if those changes were similar to alterations observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients. RESULTS: In infected spinal cords, we have observed a strong leukocyte infiltration and a severe alteration of microglia close to motor neurons. We have also analyzed ALS-related proteins: we have not found changes in TDP-43 and Fus in neurons, but interestingly, we have found decreased protein levels of C9orf72, of which coding gene is severely altered in some familial forms of ALS and is critical for microglia homeostasis. CONCLUSIONS: Latent infection of HSV in the spinal cord showed altered microglia and leukocyte infiltration. These inflammatory features resembled to those observed in the spinal cord of ALS patients. No changes mimicking ALS neuropathology, such as TDP-43 cytoplasmic inclusions, were found in infected spinal cords, but a decrease in protein levels of C9orf72 was observed. Then, further studies should be required to determine whether HSV-2 has a role in ALS.

Herpes Simplex Virus 2 Counteracts Neurite Outgrowth Repulsion during Infection in a Nerve Growth Factor-Dependent Manner.

During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.

The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway.

ETHNOPHAMACOLOGICAL RELEVANCE: The Chinese herbal prescription JieZe-1 (JZ-1) is based on the modification of Yihuang Tang, which was first described in Fu Qingzhu Nvke by the famous Qing Dynasty doctor Shan Fu as a treatment for leukorrheal diseases. As an in-hospital preparation, JZ-1 has been used in Tongji Hospital for many years to treat various infectious diseases of the lower female genital tract, including cervicitis, vaginitis, genital herpes and condyloma acuminatum. Our previous studies have shown that JZ-1 has curative effects on Candida albicans, Trichomonas vaginalis and Ureaplasma urealyticum infections. AIM OF THE STUDY: Genital herpes is among the most common sexually transmitted diseases (STDs) worldwide and is mainly caused by herpes simplex virus type-2 (HSV-2). Current therapies can relieve symptoms in patients but do not cure or prevent the spread of the virus. This study was designed to investigate the effect of JZ-1 on HSV-2 infection and its mechanism, which is based on autophagy induction, to provide new ideas and a basis for the study of antiviral drugs. MATERIALS AND METHODS: Evaluation of the antiviral activity of JZ-1 was conducted by MTT assay and western blotting. Then, Western blot and immunofluorescence analyses, observations through transmission electron microscopy and experiments with the recombinant lentivirus vector mRFP-GFP-LC3B were used to monitor autophagic flux in VK2/E6E7 cells. To explore the mechanism by which JZ-1 regulates autophagy, western blotting and real-time quantitative PCR (qRT-PCR) were used to determine the expression of phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway proteins and to detect changes in critical molecules in the pathway after the application of a PI3K inhibitor. Additionally, the mRNA expression levels of inflammatory cytokines, namely, IL-6, IFN-α, IFN-ß and TNF-α, were measured with qRT-PCR. RESULTS: HSV-2 infection inhibited autophagy in the VK2/E6E7 cells. Further study revealed that the activation of the PI3K/Akt/mTOR pathway induced by HSV-2 infection may result in the blocked autophagic flux and inhibited autophagosome and autolysosome formation. JZ-1 exhibited significant antiviral activity in the VK2/E6E7 cells, which showed increased cell vitality and reduced viral protein expression, namely, earliest virus-specific infected cell polypeptides 5 (ICP5) and glycoprotein D (gD). We found that JZ-1 treatment inhibited the upregulation of the PI3K/Akt/mTOR pathway proteins and promoted autophagy to combat HSV-2 infection, while PI3K inhibitor pretreatment prevented the enhanced autophagy induced by JZ-1. Moreover, JZ-1 attenuated the increase in inflammatory cytokines that had been induced HSV-2 infection. CONCLUSION: Our results showed that JZ-1 protects against HSV-2 infection, and this beneficial effect may be mediated by inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling axis.

Peculiar vegetative tumor-like genital herpes simplex nodules with brisk tissue eosinophilia in patients with human immunodeficiency virus infection.

Genital herpes simplex virus (HSV) infection in a human immunodeficiency virus (HIV) patient can present as a vegetative nodule. Clinical differential diagnoses of the nodule include condyloma latum, condyloma acuminatum, viral or fungal infection, and cutaneous neoplasms. Histological examination of herpetic nodules has been reported to show thick pseudoepitheliomatous hyperplasia with dense dermal lymphoplasmacytic infiltrate and multifocal multinucleated cells with herpetic viral cytopathic changes. We report two patients with HIV presenting with vegetative tumor-like HSV nodules with distinctive histopathologic pattern of inflammation that has not been described in the literature before. All samples displayed slightly acanthotic epidermis with focal ulceration, dense dermal sclerosis, scattered plasma cells, and a brisk lymphoeosinophilic infiltrate found dissecting between dense collagen bundles. This pattern of inflammation is an important clue that can guide the pathologist to look for focal herpetic viral changes in the epidermis, as patients with HIV possibly tend to amount a predominantly eosinophilic immune response in inflammatory skin conditions.

Poly(dA:dT) Suppresses HSV-2 Infection of Human Cervical Epithelial Cells Through RIG-I Activation.

Epithelial cells of the female reproductive tract (FRT) participate in the initial innate immunity against viral infections. Poly(dA:dT) is a synthetic analog of B form double-stranded (ds) DNA which can activate the interferon (IFN) signaling pathway-mediated antiviral immunity through DNA-dependent RNA Polymerase III. Here we investigated whether poly(dA:dT) could inhibit herpes simplex virus type 2 (HSV-2) infection of human cervical epithelial cells (End1/E6E7). We demonstrated that poly(dA:dT) treatment of End1/E6E7 cells could significantly inhibit HSV-2 infection. Mechanistically, poly(dA:dT) treatment of the cells induced the expression of the intracellular IFNs and the multiple antiviral IFN-stimulated genes (ISGs), including IFN-stimulated gene 15 (ISG15), IFN-stimulated gene 56 (ISG56), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 2 (OAS2), myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (Viperin), and guanylate binding protein 5 (GBP5). Further investigation showed that the activation of RIG-I was largely responsible for poly(dA:dT)-mediated HSV-2 inhibition and IFN/ISGs induction in the cervical epithelial cells, as RIG-I knockout abolished the poly(dA:dT) actions. These observations demonstrate the importance for design and development of AT-rich dsDNA-based intervention strategies to control HSV-2 mucosal transmission in FRT.

HSV-2 Cellular Programming Enables Productive HIV Infection in Dendritic Cells.

Genital herpes is a common sexually transmitted infection caused by herpes simplex virus type 2 (HSV-2). Genital herpes significantly enhances the acquisition and transmission of HIV-1 by creating a microenvironment that supports HIV infection in the host. Dendritic cells (DCs) represent one of the first innate cell types that encounter HIV-1 and HSV-2 in the genital mucosa. HSV-2 infection has been shown to modulate DCs, rendering them more receptive to HIV infection. Here, we investigated the potential mechanisms underlying HSV-2-mediated augmentation of HIV-1 infection. We demonstrated that the presence of HSV-2 enhanced productive HIV-1 infection of DCs and boosted inflammatory and antiviral responses. The HSV-2 augmented HIV-1 infection required intact HSV-2 DNA, but not active HSV-2 DNA replication. Furthermore, the augmented HIV infection of DCs involved the cGAS-STING pathway. Interestingly, we could not see any involvement of TLR2 or TLR3 nor suppression of infection by IFN-ß production. The conditioning by HSV-2 in dual exposed DCs decreased protein expression of IFI16, cGAS, STING, and TBK1, which is associated with signaling through the STING pathway. Dual exposure to HSV-2 and HIV-1 gave decreased levels of several HIV-1 restriction factors, especially SAMHD1, TREX1, and APOBEC3G. Activation of the STING pathway in DCs by exposure to both HSV-2 and HIV-1 most likely led to the proteolytic degradation of the HIV-1 restriction factors SAMHD1, TREX1, and APOBEC3G, which should release their normal restriction of HIV infection in DCs. This released their normal restriction of HIV infection in DCs. We showed that HSV-2 reprogramming of cellular signaling pathways and protein expression levels in the DCs provided a setting where HIV-1 can establish a higher productive infection in the DCs. In conclusion, HSV-2 reprogramming opens up DCs for HIV-1 infection and creates a microenvironment favoring HIV-1 transmission.

A role for the CCR5-CCL5 interaction in the preferential migration of HSV-2-specific effector cells to the vaginal mucosa upon nasal immunization.

Our current study focused on elucidating the role of specific chemokine-receptor interactions in antigen (Ag)-specific immune cell migration from nasal to genital mucosal tissues. This cellular migration is critical to induce effective Ag-specific immune responses against sexually transmitted genital infections. In this study, nasal immunization with live attenuated HSV-2 TK- induced the upregulation of CCR5 expression in effector immune cells, including CD4+ T cells, in Ag-priming sites and vaginal tissue. The CCR5 ligands CCL3, CCL4, and CCL5 all showed upregulated expression in vaginal tissue; in particular, CCL5 expression was highly enhanced in the stromal cells of vaginal tissue after nasal immunization. Intravaginal blockade of CCL5 by using neutralizing antibody diminished the number of HSV-2-specific effector cells in the vagina. Furthermore, loss of CCR5, a receptor for CCL5, impaired the migration of nasally primed Ag-specific effector cells from the airway to vagina. Effector cells adoptively transferred from CCR5-deficient mice failed to migrate into vaginal tissue, consequently increasing recipient mice's susceptibility to HSV-2 vaginal infection. These results indicate that the CCR5-CCL5 chemokine pathway is required for the migration and retention of nasally primed Ag-specific effector cells in vagina for providing protective immunity against HSV-2 infection.

IL-22 suppresses HSV-2 replication in human cervical epithelial cells.

Interleukin (IL)-22, a member of the IL-10 family, plays a role in antiviral immune responses to a number of viral infections. However, it is unclear whether IL-22 is involved in the mucosal immunity against herpes simplex virus 2 (HSV-2) infection in the female reproductive tract (FRT). In this study, we studied whether IL-22 could inhibit HSV-2 infection of human cervical epithelial cells (End1/E6E7 cells). We showed that End1/E6E7 cells express the functional IL-22 receptor complex (IL-22R1 and IL-10R2). When treated with IL-22, End1/E6E7 cells expressed the higher levels of IFN-stimulated genes (ISGs: ISG15, ISG56, OAS-1, OAS-2, and Mx2) than untreated cells. In addition, IL-22-treated cells produced higher levels of the tight junction proteins (ZO-1 and Occludin) than untreated cells. Mechanistically, IL-22 could activate the JAK/STAT signaling pathway by inducing the phosphorylation of STAT1 and STAT3. These observations indicate the potential of IL-22 as an anti-HSV-2 agent in the FRT mucosal innate immunity against HSV-2 infection.

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice.

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2'3'-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2'3'-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2'3'-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo.

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

Prevalence of Herpes Simplex Virus 2 (HSV-2) infection and associated risk factors in a cohort of HIV negative women in Durban, South Africa.

BACKGROUND: Herpes Simplex Virus 2 (HSV-2) is one of the most common sexually transmitted infections (STIs) worldwide and is a risk factor for the acquisition and transmission of other STIs, including HIV. We determined the prevalence and predictors of HSV-2 infection among women screened for a HIV prevention trial in Durban, South Africa. Univariate and multivariate logistic and Cox regression models were used to determine the correlates and predictors of HSV-2 infection at enrolment and seroconversion during the study respectively. RESULTS: Prevalence of HSV-2 at screening was 65% and crude incidence was 22.3 per 100 person-years (PY) (95% CI 20.4-24.3). The HIV seroconversion was significantly higher among those testing positive for HSV-2 at baseline compared to women who were negative [8.7 per 100 person years (PY) versus 5.2 per 100 PY; (p < 0.001)]. In univariate analysis, age was determined to be the most significant predictor for HSV-2 diagnosis, while co-infection with syphilis was also a significant predictor, while age and co-infection with syphilis remained the two most significant predictors of having HSV-2 in multivariate analysis at baseline. Consistent with these results, along with HIV seroconversion, age was also identified as a significant predictor for incidence of HSV-2. CONCLUSION: Given the unacceptably high prevalence and incidence rates of HSV-2 infection reported here, HSV-2 and general STI education needs to be reinforced in these communities, with a focus on condom education for prevention. HSV-2 has emerged as the most prevalent STI which is most often asymptomatic and unrecognized, and which increases women's risk of acquiring other STIs, including HIV.

Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection.

Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency.

Integrative pharmacokinetic-pharmacodynamic modeling and simulation of amenamevir (ASP2151) for treatment of recurrent genital herpes.

Amenamevir is a novel drug that targets the viral helicase-primase complex. While dose-dependent efficacy had been observed in non-clinical studies, no clear dose dependence has been observed in humans. We therefore developed a pharmacokinetic/pharmacodynamic (PK/PD) model to explain this inconsistency between species and to clarify the immune-related healing of amenamevir in humans. The model consisted of a non-linear kinetic model for a virtual number of virus plaques as a built-in biomarker. Lesion score was defined as an endpoint of antiviral efficacy, and logit model analysis was applied to the ordered-categorical lesion score. The modeling results suggested the time course profiles of lesion score could be explained with the efficacy terms in the logit model, using change in number of virus plaques as an indicator of the effects of amenamevir and time elapsed as an indicator of the healing of the immune response. In humans, the PD effect was almost dose-independent, and immune-related healing may have been the driving force behind the reduction in lesion scores. Drug efficacy is occasionally masked in diseases healed by the immune response, such as genital herpes. The PK/PD model proposed in the present study must be useful for explanation the PK/PD relationship of such drugs.

Effects of a traditional Chinese medicine, Longdanxiegan formula granule, on Toll-like receptor pathway in female guinea pigs with recurrent genital herpes.

OBJECTIVE: The aim of the present study was to investigate the effects of Longdanxiegan formula granule (LDXGFG), a Chinese traditional medicine on Toll-like receptor (TLR) pathway in recurrent genital herpes. MATERIALS AND METHODS: An experimental recurrent genital herpes model was constructed using herpes guinea pig model. The effect of LDXGFG on expression levels of TLR pathway genes were detected using real-time polymerase chain reaction. Furthermore, the dendritic cells and Langerhans cells were isolated and the TLR pathway genes of these cells were assayed after LDXGFG treatment. RESULTS: The result suggested two different expression patterns of TLR pathway genes in genital herpes and recurrent genital herpes, including upregulated genes and downregulated genes. TLR1, TLR4, TLR6, TLR7, TLR8, TLR9, and TLR10 showed a significant decrease while, TLR2, TLR3, and TLR5 increased in genital herpes and recurrent genital herpes guinea pigs. Meanwhile, the downregulated genes in genital herpes and recurrent genital herpes were stimulated by LDXGFG. By contrast, the upregulated genes decreased significantly after LDXGFG treatment. In both dendritic cells and Langerhans cells, the TLR pathway genes exhibited same pattern: the LDXGFG corrected the abnormal expression of TLR pathway genes. CONCLUSION: The present results suggest that LDXGFG is an alternative, inexpensive, and lasting-effect medicine for herpes simplex virus 2 infection.

Regulatory T cells are essential to promote proper CD4 T-cell priming upon mucosal infection.

Regulatory T cells (Tregs) limit autoimmunity and immunopathology using a variety of suppressive mechanisms, but their roles during pathogen-directed immune responses remain unclear. Following herpes simplex virus-2 (HSV-2) infection, mice lacking Tregs fail to control viral replication, pointing to a role for Tregs in facilitating productive immune responses. Using adoptive transfer of T-cell receptor transgenic CD4 T cells into Treg-sufficient or Treg-depleted mice prior to HSV-2 infection, we found that Tregs are required for timely accumulation of HSV-2-specific CD4 T cells within the infected tissues. Further, Tregs are critical for appropriate trafficking of dendritic cells (DCs) from the vaginal mucosa to the draining lymph nodes, which results in fully effective CD4 T-cell priming, activation, and ultimately migration to the infected tissues. Using CTLA-4 conditional knockout mice, we demonstrate that Tregs impact DC migration through a CTLA-4-mediated mechanism. Together, our data highlight the critical role of Tregs in proper potentiation of adaptive immune responses to microbial infection.

[Efficacy of polyprenyl phosphates in the experimental genital herpes model].

An experimental model of the primary genital herpes (herpes simplex type 2, HSV-2) in the female guinea pigs was suggested to study the infectious process activity of polyprenyl phosphates (PPP) and PPP+acyclovir (AC) complex treatment. The morphofunctional features of the guinea pig ovaries were studied in the control and experimental groups (the latter were inoculated with PPP and/or AC as a primary infection treatment) at the stage of the recurrent genital herpes aggravation. It was shown that in the case of combined PPP +AC use significant changes in the disease symptoms were observed, as well as a decrease in the infectious process activity and duration, and positive remote effect on the ovarian morphophysiology.

Basal Autophagy Is Required for Herpes simplex Virus-2 Infection.

Autophagy is a conserved catabolic process of the cell, which plays an important role in regulating plethora of infections. The role of autophagy in Herpes simplex virus-2 (HSV-2) infection is unknown. Here, we found that HSV-2 does not allow induction of an autophagic response to infection, but maintains basal autophagy levels mostly unchanged during productive infection. Thus, we investigated the importance of basal autophagy for HSV-2 infection, using pharmacological autophagy suppression or cells genetically deficient in an autophagy-essential gene (ATG5). Interference with basal autophagy flux in cells significantly reduced viral replication and diminished the infection. These results indicate that basal autophagy plays an indispensable role required for a productive infection. Importantly, this study draws a sharp distinction between induced and basal autophagy, where the former acts as a viral clearance mechanism abrogating infection, while the latter supports infection.