Model Of Herpes Simplex Virus Research Articles

Using Multiphoton Intravital Microscopy to Study Neutrophil Transmigration and Blood-Brain Barrier Permeability in a Mouse Model of Herpes Simplex Virus Encephalitis.

Multiphoton intravital microscopy (MP-IVM) is an imaging technique used for the observation of living organisms at a microscopic resolution. The tissue of interest is exposed through a window allowing imaging of cells in real time. Using MP-IVM, the temporospatial kinetics of leukocyte transendothelial migration can be visualized and quantitated using reporter mice and cell-specific fluorophore-conjugated monoclonal antibodies to track the leukocytes within and outside of vascular beds. Here we describe a method used to study neutrophil transendothelial migration and blood-brain barrier permeability in a mouse model of herpes simplex virus I (HSV) encephalitis.

Investigating the Safety and Efficacy of the Synthetic Drug Herbix on Immune Responses Involved in the Treatment of a Mouse Model of Herpes Simplex Virus.

Herpes simplex virus-1 (HSV-1) infections can cause significant harm to individuals, including blindness, congenital defects, genital herpes, and even cancer, with no definitive cure .so, finding new treatment strategies is crucial. In this study, 25 male BALB/c mice were used to conduct a mouse model of herpes by subcutaneously injecting an HSV-1 suspension (100 µL of 1× PFU/mL). The mice were divided into 5 groups with groups 1 to 3 designated as intervention groups, and groups 4 and 5 serving as positive and negative control groups, respectively. After 2 days of virus inoculation, the mice were treated with different concentrations of Herbix (100, 200, and 300 mg/mL) via subcutaneous injection. Mice Blood samples (0.5 to 1 mL) were taken from the mice before and after the experiments, and after three-week follow-up period, the mice were sacrificed and the spleens were removed for lymphocyte analysis. we found that administration of Herbix at a dose of 300 mg/mL showed the greatest efficacy, characterized by a delay in skin lesion formation, an increment in survival rate and lymphocyte proliferation, upregulation of the gene expression of interferon alpha (IFN-α) and tumor necrosis factor alpha (TNF-α), and an increase in the polarization of cytotoxic and helper T lymphocytes compared to the control group. These results suggest that Herbix at a dose of 300 mg/mL is effective in treating murine herpes and stimulating immune responses, making it a potential candidate for further investigation as an antiherpetic drug.

The R2 non-neuroinvasive HSV-1 vaccine affords protection from genital HSV-2 infections in a guinea pig model

Herpes simplex virus (HSV) infections are common and can cause severe illness but no vaccine is currently available. The recent failure of subunit HSV vaccines has highlighted the need for vaccines that present a diverse array of antigens, including the development of next-generation live-attenuated vaccines. However, most attenuated HSV strains propagate poorly, limiting their ability to elicit protective immune responses. A live-attenuated vaccine that replicates in non-neural tissue but is ablated for transmission into the nervous system may elicit protective immune responses without evoking neurologic complications or establishing life-long infections. Initial studies of R2, a live-attenuated vaccine that is engineered to be unable to invade the nervous system, used the guinea pig genital HSV model to evaluate the ability of R2 to replicate at the site of inoculation, cause disease and infect neural tissues. R2 was then evaluated as a vaccine using three routes of inoculation: intramuscular (IM), intradermal (ID) and intravaginal (IVag) and compared to IM administered gD2+MPL/Alum vaccine in the same model. R2 replicated in the genital tract but did not produce acute or recurrent disease and did not infect the neural tissue. The R2 vaccine-induced neutralizing antibody and decreased the severity of acute and recurrent HSV-2 disease as well as recurrent shedding. The ID route was the most effective. ID administered R2 was more effective than gD2+MPL/Alum at inducing neutralizing antibody, suppressing acute disease, and acute vaginal virus replication. R2 was especially more effective at reducing recurrent virus shedding, the most common source of HSV transmission. The live-attenuated prophylactic HSV vaccine, R2, was effective in the guinea pig model of genital HSV-2 especially when administered by the ID route. The use of live-attenuated HSV vaccines that robustly replicate in mucosal tissues but are ablated for neuroinvasion offers a promising approach for HSV vaccines.

Rapid diagnostics of genital herpes by loop-mediated isothermal amplification method with fluorescent detection

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

Ocular Glands Become Infected Secondarily to Infectious Keratitis and Play a Role in Corneal Resistance to Infection.

Ocular glands play a critical role in eye health through the secretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea, resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus 1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance.IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated, and treatment options do not address the consequences of ocular gland dysfunction. The present study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa, subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs.

Dual TLR2/9 Recognition of Herpes Simplex Virus Infection Is Required for Recruitment and Activation of Monocytes and NK Cells and Restriction of Viral Dissemination to the Central Nervous System.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

Differentiated Human SH-SY5Y Cells Provide a Reductionist Model of Herpes Simplex Virus 1 Neurotropism

Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses in vitroIMPORTANCE Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus.

Immunomodulation of Host Immunity by Bacteriodes fragillis Polysaccharide A (PSA) Prevents Viral Encephalitis

Abstract A major impact of commensal microbiota on many aspects of host physiology including regulation of host immunity extends beyond the gut to remote sites, including the brain. Studies in mouse models have shown that the commensal Bacteroides fragilis and its capsular PSA can prevent various inflammatory disorders including colitis, experimental autoimmune encephalomyelitis and asthma. However, little is known about the interaction of gut bacteria and their products with the immune system during viral infection. We assessed the immunomodulatory potential of PSA in a murine model of herpes simplex virus (HSV) encephalitis (HSE). Fatal HSE results from inflammation of the brain in susceptible 129 strain of mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils. Naïve 129 mice pretreated with PSA or PBS were then given a 1-week course of the anti-viral drug acyclovir (ACV) from day 4-post HSV infection to simulate a clinical setting. PSA, but not PBS-treated mice survived with dramatically reduced brain inflammation including fewer degranulating IM and altered cytokine and chemokine profiles. Mesenteric lymph node cellularity was increased and IL-10 secreting ICOS+ CD73+ CD4+ T cells and CD73+ CD8+ T cells were induced by PSA treatment. Importantly, IL-10 deficient and Rag knockout mice were not protected, suggesting T/B cells derived IL-10 as the protective mechanism. Interestingly, B cells were required for PSA mediated protection. We infer that the microbiota augment immunity to HSV using pathways distinct from those required for protection in chronic autoimmunity models, and that probiotics in combination with antivirals might act synergistically to limit HSV induced inflammatory diseases.

Virus reactivation is driven by inflammatory monocytes that are controlled by T cells (VIR9P.1163)

Abstract The current dogma is that episodes of transient immunodeficiency triggered by various stressors reactivate herpesviruses that are ubiquitous in the human population. We established a novel model of Herpes simplex virus 1 (HSV) latency in immunodeficient B6.Rag mice inoculated with either a low (LD) or high dose (HD) inoculum by using IVIG and acyclovir to suppress virus replication and promote latency. Remarkably, heat stress induced in vivo reactivation (IVR) of latent HD-Rag mice resulted in uncontrolled HSV replication and fatal encephalitis (HSE) in all mice compared to transient reactivation in LD-Rag mice that all survived. The major difference between the two groups was the massive invasion of inflammatory Ly6Chigh monocytes (IM) into the brainstem (BS) of latent HD-Rag but not LD-Rag mice. T cell transfer prevented IVR induced fatal HSE in HD-Rag recipients by reducing inflammation, especially IM invasion of the BS. Surprisingly, IVR of lethally irradiated latent HD-Rag and wildtype B6 mice induced only transient reactivation resulting in 100% survival; notably, IM were absent in the BS of these mice. Thus, counter intuitively, our data show that IM resident at sites of latency promote rather than suppress HSV reactivation and by modulating IM invasion into the BS, T cells control virus reactivation. This is a novel finding with important clinical implications, especially for treatment of neonates and immunosuppressed cancer patients undergoing transplant therapy.

Differential reliance on autophagy for protection from HSV encephalitis between newborns and adults.

Newborns are more susceptible to severe disease from infection than adults, with maturation of immune responses implicated as a major factor. The type I interferon response delays mortality and limits viral replication in adult mice in a model of herpes simplex virus (HSV) encephalitis. We found that intact type I interferon signaling did not control HSV disease in the neonatal brain. However, the multifunctional HSV protein γ34.5 involved in countering type I interferon responses was important for virulence in the brain in both age groups. To investigate this observation further, we studied a specific function of γ34.5 which contributes to HSV pathogenesis in the adult brain, inhibition of the cellular process of autophagy. Surprisingly, we found that the beclin binding domain of γ34.5 responsible for inhibiting autophagy was dispensable for HSV disease in the neonatal brain, as infection of newborns with the deletion mutant decreased time to mortality compared to the rescue virus. Additionally, a functional beclin binding domain in HSV γ34.5 did not effectively inhibit autophagy in the neonate, unlike in the adult. Type I IFN responses promote autophagy in adult, a finding we confirmed in the adult brain after HSV infection; however, in the newborn brain we observed that autophagy was activated through a type I IFN-independent mechanism. Furthermore, autophagy in the wild-type neonatal mouse was associated with increased apoptosis in infected regions of the brain. Observations in the mouse model were consistent with those in a human case of neonatal HSV encephalitis. Our findings reveal age-dependent differences in autophagy for protection from HSV encephalitis, indicating developmental differences in induction and regulation of this innate defense mechanism after HSV infection in the neonatal brain.

Ocular Inflammation and Infection

The eye is a unique and necessary organ that is constantly exposed to the environment. In an immune-privileged environment such as the eye, a delicate balance exists between immune responses that limit damage and those responses that can result in irreversible damage. Ocular inflammation and infection are therefore potentially blinding events. Efforts to analyze the delicate balance between helpful and harmful immune responses in the eye have involved a variety of animal studies and models which analyze not only the specific etiologic agents of infection and the contributions of their products in inflammation and vision loss but also the underlying host factors responsible for those responses. Other studies have probed the development of novel therapeutics based on endogenous host factors as well as pathogen-specific targets and have tested these with routinely used therapeutics in improved regimens in an effort to improve visual outcome. The ultimate goal is to provide patients with the appropriate treatment that rescues vision regardless of disease. The scope of this special issue involves a wide variety of areas in ocular infection and inflammation highlighting recent developments in the epidemiology, pathogenesis, and treatment of various types of ocular infections and inflammation. The first paper of this special issue reviews the challenges of cataract surgery and visual rehabilitation in patients with uveitis. This discussion also includes management pearls in combating complications that arise in this particular group of patients. The second and third papers continue the theme of inflammation, analyzing chemotactic cytokine production in the cornea. The second paper reports a time course effect in the synthesis of IL-8 and MCP by stromal cells stimulated with LPS. The third paper uses an ex vivo model of herpes simplex virus 1 (HSV1) corneal infection to demonstrate that neutrophils are important in T-cell recruitment and control of HSV1 replication via synthesis of IP-10. The following three papers address epidemiological issues with studies on microbiological profiles and treatment outcomes of scleritis, chronic postoperative endophthalmitis, and fungal ocular infections. The key message of these studies is the importance of early identification and determination of drug susceptibility and proper therapeutic and/or surgical intervention in saving useful vision. The seventh and eight papers discuss the use of corticosteroid therapy in endophthalmitis and other types of ocular inflammation. The seventh paper reviews the visual outcome of cases of filtering bleb-associated endophthalmitis treated with or without intravitreal dexamethasone, while the eighth paper reviews the clinical use of loteprednol etabonate in a variety of ocular inflammatory conditions. The final paper of the special issue discusses hyperactivation of the renin-angiotensin system in inflammation and retinal neural dysfunction. The authors suggest that inhibition of this system may be a novel therapeutic approach to preventing or treating inflammation-based ocular diseases. This special issue includes the following papers: “Cataract surgery in uveitis,” “IL-8 and MCP gene expression and production by LPS-stimulated human corneal stromal cells,” “Resident corneal cells communicate with neutrophils leading to the production of IP-10 during the primary inflammatory response to HSV-1 infection,” “Clinico-microbiological profile and treatment outcome of infectious scleritis: experience from a tertiary eye care center of India,” “Chronic postoperative endophthalmitis: a review of clinical characteristics, microbiology, treatment strategies, and outcomes,” “Support of the laboratory in the diagnosis of fungal ocular infections,” “Intravitreal dexamethasone in the management of delayed-onset bleb-associated endophthalmitis,” “Advances in corticosteroid therapy for ocular inflammation: loteprednol etabonate,” and “Renin-angiotensin system hyperactivation can induce inflammation and retinal neural dysfunction.” Michelle Callegan Meredith Gregory-Ksander Mark Willcox Susan Lightman

Herpes simplex virus induces neural oxidative damage via microglial cell Toll-like receptor-2

BackgroundUsing a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis.MethodsPurified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2-/- mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from β-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia.ResultsStimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2-/- microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2-/- microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2-/- microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2-/- mice.ConclusionsThese studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.

Subconjunctival antisense oligonucleotides targeting TNF-α influence immunopathology and viral replication in murine HSV-1 retinitis

To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in immunopathology and viral replication in the contralateral eye in the von Szily model of herpes simplex virus (HSV)-1 acute retinitis. In vivo distribution was analyzed after subconjunctival injection of FITC-labeled antisense oligonucleotides (ASON). After HSV-1 (KOS) was injected in the right anterior chamber (AC) in BALB/c mice, the course of the contralateral retinitis was evaluated. The left eyes were treated with either TNF-alpha ASON, sequence-unspecific control (CON), or buffer. The ocular TNF-alpha content was quantified by ELISA. The delayed-type hypersensitivity (DTH) reaction, uptake of [3H]thymidine from regional lymph nodes (rln)- and spleen cells, serum-neutralizing antibodies, and viral titer in the eyes were evaluated. After subconjunctival injection, FITC-labeled ASON were found in the choroid and retina. In the TNF-alpha ASON-treated eyes, TNF-alpha expression and the incidence and severity of retinitis were reduced on day 8 postinfection (PI) (p < 0.05). On day 10 PI, higher viral titers were only seen in the eyes of the TNF-alpha ASON group (p < 0.05), and retinitis was slightly more severe on day 12 PI. While the HSV-1 specific [3H]thymidine uptake from rln cells was higher in the TNF-alpha ASON mice (p < 0.05), the [3H]thymidine uptake from spleen cells, the DTH response, and the neutralizing-antibody titers did not differ between the groups. After regional blockade of TNF-alpha in experimental HSV-1 retinitis TNF-alpha seems to possess an antiviral capacity against HSV-1 in the contralateral eye and participates in the immunopathology of HSV-1-induced acute retinitis.

Age-related CD8 T Cell Clonal Expansions Constrict CD8 T Cell Repertoire and Have the Potential to Impair Immune Defense

Peripheral T cell diversity is virtually constant in the young, but is invariably reduced in aged mice and humans. CD8+ T cell clonal expansions (TCE) are the most drastic manifestation of, and possible contributors to, this reduced diversity. We show that the presence of TCE results in reduced CD8+, but not CD4+, T cell diversity, and in functional inability to mobilize parts of the CD8+ T cell repertoire affected by TCE. In the model of herpes simplex virus (HSV)-1 infection of B6 mice, >90% of the responding CD8+ T cells use Vβ10 or Vβ8 and are directed against a single glycoprotein B (gB498-505) epitope, gB-8p. We found that old animals bearing CD8+ TCE within Vβ10 or Vβ8 families failed to mount an effective immune response against HSV-1, as judged by reduced numbers of peptide-major histocompatibility complex tetramer+ CD8 T cells and an absence of antiviral lytic function. Furthermore, Vβ8 TCE experimentally introduced into young mice resulted in lower resistance to viral challenge, whereas Vβ5+ TCE induced in a similar fashion did not impact viral resistance. These results demonstrate that age-related TCE functionally impair the efficacy of antiviral CD8+ T cell immunity in an antigen-specific manner, strongly suggesting that TCE are not the mere manifestation of, but are also a contributing factor to, the immunodeficiency of senescence.

Quantitative Analysis of Herpes Simplex Virus Reactivation In Vivo Demonstrates that Reactivation in the Nervous System Is Not Inhibited at Early Times Postinoculation

Recent studies utilizing an ex vivo mouse model of herpes simplex virus (HSV) reactivation have led to the hypothesis that, under physiologic conditions inducing viral reactivation, the immune cells within the infected ganglion block the viral replication cycle and maintain the viral genome in a latent state. One prediction from the ex vivo study is that reactivation in ganglia in vivo would be inhibited at early times postinoculation, when the numbers of inflammatory cells in the ganglia are greatest. To distinguish between an effect of the immune infiltrates on (i) infectious virus produced and/or recovered in the ganglion and (ii) the number of neurons undergoing lytic transcriptional activity (reactivating), an assay to quantify the number of neurons expressing lytic viral protein in ganglia in vivo was developed. Infectious virus and HSV protein-positive neurons were quantified from days 9 through 240 postinoculation in latently infected trigeminal ganglia before and at 22 h after hyperthermic-stress-induced reactivation. Significant increases in the amount of virus and the number of positive neurons were detected poststress in ganglia at all times examined. Unexpectedly, the greatest levels of reactivation occurred at the times examined most proximal to inoculation. Acyclovir was utilized to stop residual acute-phase virus production, and this treatment did not reduce the level of reactivation on day 14. Thus, the virus measured after induction was a product of reactivation. These data indicate that, in contrast to observations in the ex vivo model, immune cells in the ganglia during the resolution of acute infection do not inhibit reactivation of the virus in ganglia in vivo.

The correlation of MHC haplotype and development of Behçet's disease-like symptoms induced by herpes simplex virus in several inbred mouse strains

It has been postulated that human leukocyte antigen (HLA)-B51 is associated with Behçet's disease. In previous study, we induced Behçet's disease-like symptoms in ICR mice inoculated with herpes simplex virus. In this study, several inbred mouse strains — B10.BR (H-2 k), B10.RIII (H-2 r), C57BL/6 (H-2 b), C3H/He (H-2 k), Balb/c (H-2 d) — which had different types of major histocompatibility complex (MHC), were inoculated with herpes simplex virus type 1 (KOS strain) in order to demonstrate the role of histocompatibility antigen in the development of Behçet's disease-like symptoms. Behçet's disease-like symptoms developed in 40–50% of B10.BR, B10.RIII and C57BL/6 strains, but in only 2% of C3H/He and Balb/c. B10.BR and C3H/He strains had a common haplotype (H-2 k) but the rate of manifestation was different. So, we conclude that MHC is not directly correlated with development of Behçet's disease-like symptoms in the animal model of herpes simplex virus induced Behçet's disease-like symptoms.

Biological Properties of Herpes Simplex Virus 2 Replication-Defective Mutant Strains in a Murine Nasal Infection Model

We used a mouse nasal model of herpes simplex virus 2 (HSV-2) infection to examine the biological properties of HSV-2 wild-type (wt), TK-negative, and replication-defective strains in vivo. Nasal septa tissue is the major site of wt viral replication post intranasal (i.n.) inoculation. The HSV-2 strain 186 syn+-1 wt virus caused lethal encephalitis at doses of 104 PFU and above per nostril, and at lower doses no neurons in the trigeminal ganglia were positive for the latency-associated transcript, indicating a lack of latent infection. The 186ΔKpn TK-negative mutant virus replicated in nasal septa tissue but showed low-level replication in trigeminal ganglia at only one timepoint. In situ hybridization of trigeminal ganglia showed that the number of LAT-positive neurons was proportional to the inoculum dose from 103 to 106 PFU per nare. The replication-defective mutant virus 5BlacZ showed no replication in nasal septa tissue and no persistence of viral DNA at the inoculation site or the trigeminal ganglia. Nevertheless, inoculation of 5BlacZ or the double-mutant dl5-29 at distal sites reduced acute replication and latent infection of 186ΔKpn following intranasal challenge. This infection model provides a biological system to test the properties of HSV-2 strains and shows that replication-defective mutant strains do not persist at sites of inoculation or in sensory ganglia but can induce immune protection that reduces the latent viral load of a challenge virus.

LFA-3 Plasmid DNA Enhances Ag-Specific Humoral- and Cellular-Mediated Protective Immunity against Herpes Simplex Virus-2 in Vivo: Involvement of CD4+ T Cells in Protection

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses. To investigate specific roles of adhesion molecules in immune induction we coimmunized LFA-3 and ICAM-1 cDNAs with a gD plasmid vaccine and then analyzed immune modulatory effects and protection against lethal herpes simplex virus (HSV)-2 challenge. We observed that gD-specific IgG production was enhanced by LFA-3 coinjection. However, little change in IgG production was observed by ICAM-1 coinjection. Furthermore, both Th1 and Th2 IgG isotype production was driven by LFA-3. LFA-3 also enhanced Th cell proliferative responses and production of interleukin (IL)-2, interferon-γ, IL-4, and IL-10 from splenocytes. In contrast, ICAM-1 showed slightly increasing effects on T-cell proliferation responses and cytokine production. β-Chemokine production (RANTES, MIP-1α, and MCP-1) was also influenced by LFA-3 or ICAM-1. When animals were challenged with a lethal dose of HSV-2, LFA-3-coimmunized animals exhibited an enhanced survival rate, as compared to animals given ICAM-1 or gD DNA vaccine alone. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. These studies demonstrate that adhesion molecule LFA-3 can play an important role in generating protective antigen-specific immunity in the HSV model system through increased induction of CD4+ Th1 T-cell subset.

Use of herpes simplex virus (HSV) type 1 ISCOMS 703 vaccine for prophylactic and therapeutic treatment of primary and recurrent HSV-2 infection in guinea pigs.

The effect of subunit vaccination on the incidence and severity of primary and recurrent genital herpes was investigated in the female guinea pig model of herpes simplex virus (HSV) type 2 genital infection. After prophylactic immunization with zwitterionic detergent-solubilized HSV-1 glycoproteins formulated with alhydrogel or as immunostimulating complex particles, significant reductions in the incidence and severity of primary herpetic illness were observed in both vaccinated groups compared with immunization-naive controls. There was a significant reduction in the incidence of spontaneous herpetic recurrences after administration of HSV-1 antigens formulated as immunostimulatory complexes to guinea pigs in a prophylactic mode (P<.01). Increased levels of both postimmunization and postchallenge ELISA and neutralizing antibodies were significant correlates of protection against primary herpetic disease in a prophylactic scenario. However, no correlation was observed between elevated ELISA or neutralizing antibody levels and protection against recurrent disease following prophylactic or therapeutic administration of HSV-1 subunit vaccines.

Treatment of primary herpes simplex virus infection in guinea pigs by imiquimod

Imiquimod (also known as R-837 and S-26308) is an imidazoquinoline immune response modifier and is available in the US and several other countries for the treatment of external genital warts. Imiquimod has no direct antiviral activity but demonstrates efficacy in several animal models of virus infection. The drug is recognized by antigen presenting cells including monocytes, macrophages, B-cells and dendritic cells and induces these cells to produce cytokines including interferon-α (IFN-α) and others. Imiquimod’s ability to inhibit primary lesion development in the guinea pig model of Herpes simplex virus (HSV) intravaginal infection was studied. Imiquimod given intravaginally reduced primary lesions, reduced virus shedding and reduced virus content of spinal cords from HSV infected guinea pigs. A single drug application of 0.5 mg/kg reduced lesion frequency when given between 24 h before inoculation to 16 h after inoculation. A single drug application of 5 mg/kg reduced lesion frequency and severity when administered between 72 h before inoculation to 24 h after inoculation. The antiviral effect resulting from interferon induction in the animal lasts much longer than the drug itself, thus imiquimod is different than drugs having direct antiviral activity. Twice daily drug application for 4 days was effective when initiated up to 72 h after inoculation, however, once lesions began to appear, imiquimod treatment was not able to stop lesion development. Imiquimod treatment inhibited lesion development and/or virus shedding in guinea pigs inoculated with HSV-1, HSV-2 or virus isolates resistant to acyclovir. Imiquimod is currently in clinical trials for treating human HSV infections.