Herpes Simplex Virus Type 1 Antigen Research Articles

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis.

PurposeHerpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with “unsensing” sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events.MethodsWe examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation.ResultsUV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves.ConclusionsCollectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Qingre Xiaoyanning Capsules alleviate HSV-1 susceptibility induced by emotional stress

In this study, emotional stress-induced herpes simplex virus type 1(HSV-1) susceptibility model was employed to simu-late the pathological state of " depression-induced liver fire", and the protection effect of Qingre Xiaoyanning(QX) in clearing liver fire was investigated. BALB/c mice were randomly divided into a normal group, a HSV-1 group, a restraint stress + HSV-1 group,low-(0. 658 g·kg~(-1)) and high-dose(1. 316 g·kg~(-1)) QX groups, and an acyclovir group. Except for the normal group and the HSV-1 group, the mice in other groups received daily restraint stress for 6 h from day 3 of medication. On day 9 of medication, mice were anesthetized by isoflurane and infected intranasally with HSV-1. Survival rate, weight change, encephalitis symptoms, and eye injury of mice were recorded for 14 d after virus infection. Hematoxylin-eosin(HE) staining and immunohistochemical staining were used to detect pathological changes and HSV-1 antigen distribution. Plaque assay was performed to detect the titer of HSV-1. The protein ex-pression of ICP27 in the mouse brain was detected by Western blot. The experimental results showed that QX could increase the survival rate of HSV-1-infected mice loaded with emotional stress(P<0. 001), reduce the titer of HSV-1 in the mouse brain(P<0. 01), relieve brain inflammation(P<0. 05) and eye injury(P<0. 05), down-regulate the expression of ICP27 related to HSV-1(P<0. 05), and decrease the distribution of HSV-1 antigen in the mouse brain. The results demonstrated that QX significantly reduced the susceptibility to HSV-1 induced by emotional stress, which is expected to provide a theoretical basis for the treatment and preven-tion of HSV-1 infection and promote the clinical development and application of Chinese medicine effective in clearing liver fire.

Inhibition of herpes simplex virus type 1 infection by Sambucus ebulus extract in vitro.

Background: The emergence of drug-resistant strains of herpes simplex virus type 1 (HSV-1) has been increasingly reported. Therefore, attempts to discover new antiviral agents in particular from natural compounds are required. In this study, we evaluated the possible inhibitory effects of hydroalcoholic extract of Sambucus ebulus (S. ebulus ) against HSV-1. Methods: S. ebulus extract was produced by maceration method. MTT assay was used to evaluate the cytotoxicity effects of the S. ebulus extract; also, antiviral effects were measured both by test TCID50 and quantitative real-time PCR methods. To study the inhibitory impact of S. ebulus extract on the expression of HSV-1 antigens, indirect immunofluorescence assay (IFA) was also performed. All analyses were performed using the GraphPad Prism software v. 7.0. Results: In the postexposure assay of HSV-1 with S. ebulus extract at the highest nontoxic concentration (75 μg/mL), S. ebulus extract led to 2.6 log10 TCID50 reduction in infectious virus titer. At the highest nontoxic concentration, the S. ebulus extract led to inhibition rates of 91.2%, based on the quantitative real-time PCR assay results (p<0.001). Also, in the immunofluorescence assay, a significant reduction was observed in fluorescence emission intensity in HSV-1-infected cell treated with S. ebulus extract compared to the control group. Conclusion: S. ebulus extract is a novel and effective natural compound in reducing HSV-1 titer and future studies should be conducted to discover the complete mechanism of antiviral effect of this natural compound.

Evaluation of ocean biomass products that activate cell-medicated immunity in mice cutaneously infected with herpes simplex virus type 1

We screened and evaluated the activation of cell-medicated immunity by ocean biomass products using a cutaneous herpes simplex virus type 1 (HSV-1) infection model. In this model, a delayed-type hypersensitivity (DTH) associated with cell-mediated immunity is an important immune defense systems. An aqueous fraction of ovary of Seriola quinqueradiata (A-SQ) or a lipophilic fraction of head and internal organs of Hymenocephalus lethonemus (L-HL) was administered orally to mice infected with HSV-1 three times daily during 8 days (days 0 to 7) after infection. They delayed the progress of herpetic skin lesions without toxicity and significantly increased the DTH reaction to inactivated HSV-1 antigen on the footpads of infected mice. Further, A-SQ and L-HL increased the production of interferon-γ from splenocytes of HSV-1-infected mice by the inactivated-HSV-1. Thus, the efficacies of A-SQ and L-HL against HSV-1 infection may be due to the augmentation of cell-mediated immunity through DTH reaction.

IMMUNOGLOBULIN INDICATORS TO VIRUSES CYTOMEGAL AND GENITAL HERPES IN THE BLOOD SERUM OF WOMEN WITH NON-ATIPICAL ENDOMETRIAL HYPERPROLIFETATIVE PATHOLOGY

The aim of the study was to analyze the level of Ig M- and Ig G-antibody (Ab) for cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) in serum of women with non-atypical endometrial hyperproliferative pathology. Materials and methods: The analysis of immunoglobulin indices to CMV and HSV-2 in serum of women with non-atypical endometrial hyperproliferative pathology. In women with uterine body polyps the presence of CMV in the uterine cavity was found in 54.8% of cases, in women with non-atypical endometrial hyperplasia in 38.3% of cases. The levels of Ig G-Ab and Ig M-Ab to CMV in serum have a clear dependence on the degree of antigen expression in endometrial tissue. HSV-2 antigens were determined in 22.58 ± 5.31% of women with uterine body polyps and in 8.33 ± 3.57% of patients with non-atypical endometrial hyperplasia while increasing serum specific antibodies to HSV-2. Results: The results indicate that there is a clear link between viral infection of hyperproliferatively altered endometrium and the determination of positive immunoglobulin levels in peripheral blood, which may be a reliable marker of chronic persistent viral infection in a woman's body. Conclusions: In women with uterine body polyps, the presence of CMV in the uterine cavity was found in 54.84 ± 6.32% of cases, in women with non-atypical endometrial hyperplasia in 38.33 ± 6.28% of cases. The levels of Ig G-Ab and and Ig M-Ab to CMV in serum have a clear dependence on the degree of antigen expression in endometrial tissue. HSV-2 antigens were determined in 22.58 ± 5.31% of women with uterine body polyps and in 8.33 ± 3.57% of patients with non-atypical endometrial hyperplasia while increasing serum specific antibodies to HSV-2. The results indicate that there is a clear link between viral infection of the target tissue (hyperproliferatively altered endometrium) and the determination of a positive level of peripheral blood immunoglobulin, which may be a reliable marker of chronic persistent viral infection in a woman.

Polyethylene glycol-coated Zinc Oxide Nanoparticle: an Efficient Nanoweapon to Fight Against Herpes Simplex Virus Type 1

We aimed to determine the possible inhibitory effects of zinc oxide nanoparticles (ZnO-NPs) and polyethylene glycol (PEG)-coated ZnO-NPs (ZnO-PEG-NPs) on herpes simplex virus type 1 (HSV-1). PEGylated ZnO-NPs were synthesized by the mechanical method. Antiviral activity was assessed by 50% tissue culture infectious dose (TCID50) and real-time PCR assays. To confirm the antiviral activity of ZnO-NPs on expression of HSV-1 antigens, indirect immunofluorescence assay was also conducted. 200μg/ml ZnO-PEG-NPs could result in 2.5 log10 TCID50 reduction in virus titer, with inhibition rate of approximately 92% in copy number of HSV-1 genomic DNA. ZnO-PEG-NPs could be proposed as a new agent for efficient HSV-1 inhibition. Our results indicated that PEGylation is effective in reducing cytotoxicity and increasing antiviral activity of nanoparticles.

Photodynamic Inactivation of Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract OrthoquinTM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or premature release of viral genomes, as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that the broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins.

Infectivity and growth kinetics of Herpes Simplex Virus type-2 in MOLT4 CCR5+ and CEM CCR5+ T cell lines

Herpes simplex virus type-2 (HSV-2) is an important sexually transmitted pathogen that infects the genital mucosal epithelial cells causing ulcerative lesions at the site of entry, facilitating HIV infection. The infection of epithelial cells and skin resident dendritic cells with HSV-2 causes a release of chemokine and retinoic acid which attracts CD4+ T-cells to the genital mucosa. In this study, we investigated whether HSV-2 (ATCC VR734) could infect and replicate in two T-cell lines (CEM CCR5+ and MOLT4 CCR5+). The growth of HSV-2 was assessed by plaque assay while the intracellular HSV-2 was identified using infectious center and indirect immunofluorescence assays. The replication of HSV-2 in T-cell lines was compared to a cell line (Vero) which is routinely used for growing HSV-2. Analysis indicated that a low level of infection was detected in the two T-cells lines and was dependent on the infectious dose as well as the time of adsorption. Indirect immunofluorescence showed presence of HSV-2 antigens in the CEM CCR5+ and Vero cell lines but not in MOLT4 CCR5+. The data suggests that T-cells can support growth of HSV-2 which might contribute to changes in gene expression of T-cells. This is an important aspect that needs to be further investigated in relation of HIV-1/HSV-2 viral synergy.

NK cells require antigen-specific memory CD4+ T cells to mediate superior effector functions during HSV-2 recall responses in vitro.

Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4+ T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4+ T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4+ T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4+ T cells to produce IFN-γ.

Augmentation of T helper type 1 immune response through intestinal immunity in murine cutaneous herpes simplex virus type 1 infection by probiotic Lactobacillus plantarum strain 06CC2

We previously found that Lactobacillus plantarum strain 06CC2 showed probiotic potential, and its oral administration effectively induced Th1 cytokine production and activated the Th1 immune response associated with intestinal immunity in mice. In this study, to evaluate its potential as a versatile oral adjuvant for treatment of viral infection, we assessed the immunomodulatory activity of 06CC2 on murine cutaneous herpes simplex virus type 1 (HSV-1) infection, in which a major immune defense system is a delayed-type hypersensitivity (DTH) reaction based on activation of the Th1 immune response, in relation to its oral efficacy for alleviation of herpetic symptoms. In the HSV-1 infection model, oral administration of 06CC2 (20mg/mouse) twice daily for seven days starting two days before infection was significantly effective in delaying the development of skin lesions in the early phase of infection and reducing virus yields in the brain on day 4 after infection. In addition, 06CC2 significantly augmented the DTH reaction to inactivated HSV-1 antigen and elevated interferon (IFN)-γ production by HSV-1 antigen from splenocytes. On day 2, natural killer (NK) cell activity was significantly elevated, and the elevation was still observed on day 4. Furthermore, gene expressions of interleukin-12 receptor β2 and IFN-γ in Peyer's patches were augmented on day 4 by 06CC2 administration. Thus, 06CC2 was suggested to alleviate herpetic symptoms in mice in correlation with augmentation of the Th1 immune responses associated with NK cell activity through intestinal immunity. Strain 06CC2 may be a versatile oral adjuvant to activate Th1 immune response.

Activation of Cellular Immunity in Herpes Simplex Virus Type 1-Infected Mice by the Oral Administration of Aqueous Extract of Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. is used as a nutritive vegetable and spice. Its ethanol extract has been previously shown to be significantly effective in alleviating herpetic skin lesions in mice. In this study, we evaluated the alleviation by the aqueous extract (AqMOL) and assessed the mode of its anti-herpetic action in a murine cutaneous herpes simplex virus type 1 (HSV-1) infection model. AqMOL (300 mg/kg) was administered orally to HSV-1-infected mice three times daily on days 0 to 5 after infection. AqMOL significantly limited the development of herpetic skin lesions and reduced virus titers in the brain on day 4 without toxicity. Delayed-type hypersensitivity (DTH) reaction to inactivated HSV-1 antigen was significantly stronger in infected mice administered AqMOL and AqMOL augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice at 4 days post-infection. AqMOL administration was effective in elevating the ratio of CD11b(+) and CD49b(+) subpopulations of splenocytes in infected mice. As DTH is a major host defense mechanism for intradermal HSV infection, augmentation of the DTH response by AqMOL may contribute to their efficacies against HSV-1 infection. These results provided an important insights into the mechanism by which AqMOL activates cellular immunity. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract 4287: Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity

Abstract Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) immunotherapy designed to selectively replicate in tumors and to initiate an anti-tumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. The talimogene laherparepvec-induced immune mediated mechanism of action in both the virus-injected and distant tumors have yet to be fully understood therefore, we evaluated viral replication, tumor cell lysis, and the changes in immune cell populations in both the injected and distant tumors using syngeneic contralateral tumor models. Animals received a single intratumoral dose of the murine surrogate of talimogene laherparepvec (OncoVEXmuGM-CSF), which induced regression in the majority of virus-injected tumors and tumor growth inhibition/regression in the contralateral (uninjected) tumors. HSV-1 antigen was only detected by IHC in the virus-injected tumor and not the contralateral tumor (and no other tissues). Virally-mediated tumor destruction (oncolysis) was also localized to only the virus-injected tumor. In preliminary experiments, morphometric analysis of the tumor tissue revealed that the percent area of CD3+ and CD8+ lymphocytes were significantly increased in both the virus-injected and contralateral tumors compared to the formulation control mice. In addition, the percent area occupied by CD103+ cells (a marker found on potent cytotoxic T cell stimulating dendritic cells) was increased in the virus-injected tumor compared to the contralateral tumor and the tumors in the control animals. Although cellular infiltration was increased in both virus-injected and contralateral tumors and was inversely correlated with tumor volume, the decreased volume of injected tumors was attributed to viral oncolysis. Taken together these data supports that talimogene laheparepvec activates a systemic T cell mediated anti-tumor immune response. Citation Format: Julia Piasecki, Jim Rottman, Tiep Le, Rafael Ponce, Courtney Beers. Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2015-4287

Granulocytes in Ocular HSV-1 Infection: Opposing Roles of Mast Cells and Neutrophils

The contributions of mast cells (MCs) to immunologic defense against pathogens in the eye are unknown. We have characterized pericorneal MCs as tissue-resident innate sentinels and determined their impact on the immune response to herpes simplex virus type-1 (HSV-1), a common ocular pathogen. The impact of mast cells on the immune response to HSV-1 infection was investigated using MC-deficient Kit(W-sh) mice. Virus titers, inflammatory cytokine production, eicosanoid profiles, cellular immune responses, and ocular pathology were evaluated and compared with C57BL/6J mice during an acute corneal HSV-1 infection. Corneas of Kit(W-sh) mice have higher viral titers, increased edema, and greater leukocyte infiltration following HSV-1 infection. Following infection, cytokine profiles were slightly elevated overall in Kit(W-sh) mice. Eicosanoid profiles were remarkably different only when comparing uninfected corneas from both groups. Neutrophils within infected corneas expressed HSV-1 antigen, lytic genes, and served as a disease-causing vector when adoptively transferred into immunocompromised animals. Myeloid-derived suppressor cells did not infiltrate into the cornea or suppress the expansion, recruitment, or cytokine production by CD8+ T cells following acute HSV-1 infection. Collectively, these findings provide new insight into host defense in the cornea and the pathogenesis of HSV-1 infection by identifying previously unacknowledged MCs as protective innate sentinels for infection of the ocular surface and reinforcing that neutrophils are detrimental to corneal infection.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

Ultrasound as a method to enhance antitumor ability of oncolytic herpes simplex virus for head and neck cancer.

Low-intensity ultrasound is a useful method to enhance the delivery of drugs to target cells via a range of mechanisms including the transient formation of micropores in the cell membrane, a process known as sonoporation. The effect of ultrasound on oncolytic herpes simplex virus type-1 (HSV-1) infection in oral squamous cell carcinoma (SCC) was examined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the neurovirulent γ134.5 gene and exhibited cell fusion actions, were used. Cells grown in multi-well plates were infected with HSV-1 and exposed to ultrasound in the presence or absence of microbubbles after an adsorption period. The number of plaques was significantly greater than that of the untreated control. SAS cells were inoculated subcutaneously into nude mice and tumors were produced. Tumors were injected with HSV-1 RH2 with or without microbubbles and then exposed to ultrasound through the covering skin. The amount of the virus in tumor tissues 3 days after the injection was higher in tumors treated with HSV-1 RH2 and ultrasound than in tumors treated with RH2 only. The expression of the HSV-1 antigen was also increased by ultrasound and microbubbles. Tumor growth was suppressed with HSV-1 RH2 in combination with ultrasound, especially with microbubbles. These results indicated that ultrasound increased the efficiency of the HSV-1 infection in SAS cells and nude mouse tumors. This method can potentially be useful to enhance the antitumor effects of oncolytic HSV-1 on head and neck cancer treatment.

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 107–108M−1 proving their high-affinity.The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available “HSV-Type 2 IgG-ELISA” (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection.

Association of HSV-1 Antigen Distribution in the Cornea with Clinical Characteristics of Herpetic Stromal Keratitis

To investigate whether the clinical characteristics of stromal herpetic simplex keratitis (HSK) are associated with herpes simplex virus type 1 (HSV-1) antigens distribution in the pathologic cornea. Pathologic corneal buttons from 8 eyes were obtained during keratoplasty at the Shandong Eye Institute from 2006 to 2009. Immunohistochemical examination was performed to detect the distribution of HSV-1 antigens, including position, depth, and load in the cornea. Each of the 8 pathologic corneal buttons was positive for HSV-1 antigen by immunohistochemical staining, and HSV-1 antigen was detected in the corneal stroma but not in the corneal epithelium or endothelium. Combined with the clinical characteristics, it was found that the distribution depth of HSV-1 antigen in the cornea was partly related to the disease course, and the load was related to the delay time for treatment. Furthermore, HSK could be effectively cured by deep anterior lamellar keratoplasty (DALK). The distribution characteristics of HSV-1 in HSK corneal stroma supports the theory of HSV-1 latency in the cornea and guides the selection of DALK, rather than penetrating keratoplasty (PKP), for clinical stromal HSK treatment.

Therapeutic effect of deep anterior lamellar keratoplasty for active or quiescent herpetic stromal keratitis

To evaluate the therapeutic effect of deep anterior lamellar keratoplasty (DALK) in patients with herpetic stromal keratitis (HSK). Forty-three eyes belonging to 42 patients with HSK, including 22 eyes in the active phase and 21 eyes in the quiescent phase, underwent DALK at the Shandong Eye Institute from January 2006 to December 2009. All patients with active disease had received intravenous acyclovir and amniotic membrane implants prior to DALK. Herpes simplex virus type 1 (HSV-1) antigens from excised corneal buttons were detected by immunohistochemistry. The follow-up ranged from 1 to 4years (mean, 29.1months). Graft rejection occurred in one eye (2.3%) and was reversed. Among the other 42 survived grafts (97.7%), 37 remained clear at the last visit. The best spectacle-corrected visual acuity was 20/200 or better in 95.2% of eyes and 20/40 or better in 38.1% of eyes. Six eyes (14.0%) developed recurrent HSK, one of which received a second keratoplasty due to ineffective antiviral medication. There were no significant differences in endothelial cell density between 6months and 12months after the surgery. By immunohistochemistry, HSV-1 antigens were observed in the stroma of 18 of 32 corneal buttons. DALK can not only remove the corneal lesions of HSK but also reduce latent or persistent viral loads in the cornea. In eyes with active or quiescent HSK but otherwise healthy endothelia, DALK seems to be safe and promising for its favorable visual outcome, graft survival rate, and low endothelial cell loss.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.