Recombinant Herpes Simplex Virus Type Research Articles

Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins.

As obligate intracellular pathogens, viruses activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of salvage nicotinamide adenine dinucleotide (NAD+) synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses through unknown mechanisms. Here, we show that NAMPT restricts herpes simplex virus type 1 (HSV-1) replication by impeding the virion incorporation of viral proteins owing to its phosphoribosyl-hydrolase (phosphoribosylase) activity, which is independent of the role of NAMPT in NAD+ synthesis. Proteomics analysis of HSV-1-infected cells identifies phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins, which are de-phosphoribosylated by NAMPT in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Loss of NAMPT renders mice highly susceptible to HSV-1 infection. Our work describes an additional enzymatic activity of a metabolic enzyme in viral infection and host defence, offering a system to interrogate the roles of protein phosphoribosylation in metazoans.

The anti-tumor efficacy of a recombinant oncolytic herpes simplex virus mediated CRISPR/Cas9 delivery targeting in HPV16-positive cervical cancer

Cervical cancer, often driven by high-risk human papillomavirus (HPV) infections such as HPV16 or HPV18, remains a leading cause of cancer-related deaths. HPV16, found in about 90% of cervical cancer patients, harbors key oncogenic related genes (E6, E7, E2, E5) and an upstream regulatory region (URR) that contribute to cancer progression. This study introduces a novel approach using a recombinant oncolytic herpes simplex virus type 1 (HSV-1) named SONC103, armed with a CRISPR/Cas9 gene editing system. The aim was to target and disrupt integrated HPV16 genes in cervical cancer cells. Results demonstrated SONC103's capability to specifically and effectively knock down HPV16 oncogenes, thereby reducing cell proliferation and promoting apoptosis. Analyses further revealed loss of HPV16 DNA probes in infected cells' chromosomes, significant regulation of cellular processes related to tumor apoptosis, and downregulation of E6/E7 oncoproteins while increasing tumor suppressor proteins P53 and pRB. Notably, SONC103 exhibited substantial inhibition of tumor growth in a murine xenograft cervical cancer model. This study showcases the potential of the recombinant oncolytic HSV-1 virus (SONC103) in combating HPV16-positive cervical cancer by targeting oncogenes and facilitating oncolysis.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

New strategies are needed to improve the treatment of patients with breast cancer (BC). Oncolytic virotherapy is a promising new tool for cancer treatment but still has a limited overall durable antitumor response. A novel replicable recombinant oncolytic herpes simplex virus type 1 called VG161 has been developed and has demonstrated antitumor effects in several cancers. Here, we explored the efficacy and the antitumor immune response of VG161 cotreatment with paclitaxel (PTX) which as a novel oncolytic viral immunotherapy for BC. The antitumor effect of VG161 and PTX was confirmed in a BC xenograft mouse model. The immunostimulatory pathways were tested by RNA-seq and the remodeling of tumor microenvironment was detected by Flow cytometry analysis or Immunohistochemistry. Pulmonary lesions were analyzed by the EMT6-Luc BC model. In this report, we demonstrate that VG161 can significantly represses BC growth and elicit a robust antitumor immune response in a mouse model. The effect is amplified when combined with PTX treatment. The antitumor effect is associated with the infiltration of lymphoid cells, including CD4+ T cells, CD8+ T cells, and NK cells (expressing TNF and IFN-γ), and myeloid cells, including macrophages, myeloid-derived suppressor cells, and dendritic cell cells. Additionally, VG161 cotreatment with PTX showed a significant reduction in BC lung metastasis, which may result from the enhanced CD4+ and CD8+ T cell-mediated responses. The combination of PTX and VG161 is effective for repressing BC growth by inducing proinflammatory changes in the tumor microenvironment and reducing BC pulmonary metastasis. These data will provide a new strategy and valuable insight for oncolytic virus therapy applications in primary solid or metastatic BC tumors.

Response to HIV-1 gp160-carrying recombinant virus HSV-1 and HIV-1 VLP combined vaccine in BALB/c mice.

Human immunodeficiency virus (HIV) induced AIDS causes a large number of infections and deaths worldwide every year, still no vaccines are available to prevent infection. Recombinant herpes simplex virus type 1 (HSV-1) vector-based vaccines coding the target proteins of other pathogens have been widely used for disease control. Here, a recombinant virus with HIV-1 gp160 gene integration into the internal reverse (IR) region-deleted HSV-1 vector (HSV-BAC), was obtained by bacterial artificial chromosome (BAC) technology, and its immunogenicity investigated in BALB/c mice. The result showed similar replication ability of the HSV-BAC-based recombinant virus and wild type. Furthermore, humoral and cellular immune response showed superiority of intraperitoneal (IP) administration, compared to intranasally (IN), subcutaneous (SC) and intramuscularly (IM), that evidenced by production of significant antibody and T cell responses. More importantly, in a prime-boost combination study murine model, the recombinant viruses prime followed by HIV-1 VLP boost induced stronger and broader immune responses than single virus or protein vaccination in a similar vaccination regimen. Antibody production was sufficient with huge potential for viral clearance, along with efficient T-cell activation, which were evaluated by the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC). Overall, these findings expose the value of combining different vaccine vectors and modalities to improve immunogenicity and breadth against different HIV-1 antigens.

Targeting carcinoembryonic antigen-expressing tumors using a novel transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1

VG2025 is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) that uses transcriptional and translational dual regulation (TTDR) of critical viral genes to enhance virus safety and promote tumor-specific virus replication without reducing virulence. The TTDR platform is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor-specific carcinoembryonic antigen (CEA) promoter, coupled with translational control of the neurovirulence factor ICP34.5 using multiple microRNA (miR)-binding sites. VG2025 further incorporates IL-12 and the IL-15/IL-15 receptor alpha subunit complex to enhance the antitumor and immune stimulatory properties of oncolytic HSVs. The TTDR strategy was verified invitro and shown to be highly selective. Strong invivo antitumor efficacy was observed following both intratumoral and intravenous administration. Clear abscopal and immune memory effects were also evident, indicating a robust antitumor immune response. Gene expression profiling of treated tumors revealed increased immune cell infiltration and activation of multiple immune-signaling pathways when compared with the backbone virus. Absence of neurotoxicity was verified in mice and in rhesus monkeys. Taken together, the enhanced tumor clearance, excellent safety profile, and positive correlation between CEA levels and viral replication efficiency may provide an opportunity for using biomarker-based precision medicine in oncolytic virotherapy.

VG161 activates systemic antitumor immunity in pancreatic cancer models as a novel oncolytic herpesvirus expressing multiple immunomodulatory transgenes.

The VG161 represents the first recombinant oncolytic herpes simplex virus type 1 carrying multiple synergistic antitumor immuno-modulating factors. Here, we report its antitumor mechanisms and thus provide firm theoretical foundation for the upcoming clinical application in pancreatic cancer. Generally, the VG161-mediated antitumor outcomes were analyzed by a collaboration of techniques, namely the single-cell sequencing, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADSI-MSI) and nanostring techniques. In vitro, the efficacy of VG161 together with immune checkpoint inhibitors (ICIs) has been successfully shown to grant a long-term antitumor effect by altering tumor immunity and remodeling tumor microenvironment (TME) metabolisms. Cellular functional pathways and cell subtypes detected from patient samples before and after the treatment had undergone distinctive changes including upregulated CD8+ Tand natural killer cells. More importantly, significant antitumor signals have emerged since the administration of VG161 injection. In conclusion, VG161 can systematically activate acquired and innate immunity in pancreatic models, as well as improve the tumor immune microenvironment, indicative of strong antitumor potential. The more robusting antitumor outcome for VG161 monotherapy or in combination with other therapies on pancreatic cancer is worth of being explored in further clinical trials.

Teserpaturev/G47Δ: First Approval.

Teserpaturev/G47Δ (Delytact®) is a third-generation (triple-mutated) recombinant oncolytic herpes simplex virus type 1 being developed by Daiichi Sankyo Co., Ltd. for the treatment of certain solid cancers. Teserpaturev/G47Δ has been approved for the treatment of malignant glioma in Japan and is currently in clinical development for the treatment of prostate cancer (phase II), malignant pleural mesothelioma (phase I) and recurrent olfactory neuroblastoma (phase I). This article summarizes the milestones in the development of teserpaturev/G47Δ leading to this first approval for the treatment of malignant glioma.

Utility of a Recombinant HSV-1 Vaccine Vector for Personalized Cancer Vaccines.

Current approaches to cancer immunotherapy include immune checkpoint inhibitors, cancer vaccines, and adoptive cellular therapy. These therapies have produced significant clinical success for specific cancers, but their efficacy has been limited. Oncolytic virotherapy (OVT) has emerged as a promising immunotherapy for a variety of cancers. Furthermore, the unique characteristics of OVs make them a good choice for delivering tumor peptides/antigens to induce enhanced tumor-specific immune responses. The first oncolytic virus (OV) approved for human use is the attenuated herpes simplex virus type 1 (HSV-1), Talimogene laherparepvec (T-VEC) which has been FDA approved for the treatment of melanoma in humans. In this study, we engineered the recombinant oncolytic HSV-1 (oHSV) VC2-OVA expressing a fragment of ovalbumin (OVA) as a fusion protein with VP26 virion capsid protein. We tested the ability of VC2-OVA to act as a vector capable of stimulating strong, specific antitumor immunity in a syngeneic murine melanoma model. Therapeutic vaccination with VC2-OVA led to a significant reduction in colonization of tumor cells in the lungs of mice intravenously challenged B16cOVA cells. In addition, VC2-OVA induced a potent prophylactic antitumor response and extended survival of mice that were intradermally engrafted with B16cOVA tumors compared with mice immunized with control virus.

Enhanced anti-tumor response elicited by a novel oncolytic HSV-1 engineered with an anti-PD-1 antibody.

Oncolytic viruses as cancer vaccines modulate the tumor microenvironment and act synergistically with immune checkpoint inhibitors to overcome resistance. Taking advantage of the loading capacity for exogenous genes, we generated a recombinant herpes simplex virus type 1 (HSV-1), HSV-aPD-1, carrying a full-length humanized anti-PD-1 monoclonal antibody (anti-PD-1 mAb) that replicates and expresses anti-PD-1 mAbs in tumor cells in vitro and in vivo. Its anti-tumor effect was assessed in human PD-1 knock-in mice by analyzing tumor inhibition, cell populations and RNA expression in tumors, and serum cytokine levels. Enhanced anti-tumor immune responses and T-cell infiltration were induced by HSV-aPD-1 compared with unloaded virus or anti-PD-1 therapy in both MC38 and B16-F10 models, resulting in improved treatment efficacy in the latter. Moreover, compared with unloaded HSV-1 or HSV-1 loaded with GM-CSF/IL-2 combined with anti-PD-1 mAbs, HSV-aPD-1 displayed similar therapeutic control of tumor growth. Finally, tumor RNAseq analysis in the B16-F10 model showed upregulated IFN pathway and antigen processing and presentation genes, and downregulated angiogenesis and cell adhesion genes, which all contribute to tumor inhibition. These findings indicate the clinical potential of HSV-aPD-1 as monotherapy or combination therapy, especially in tumors resistant to immune checkpoint inhibitors.

Suppression of CD80 Expression by ICP22 Affects Herpes Simplex Virus Type 1 Replication and CD8+IFN-γ+ Infiltrates in the Eyes of Infected Mice but Not Latency Reactivation.

ABSTRACTPreviously, we reported that herpes simplex virus type 1 (HSV-1) ICP22 binds to the CD80 promoter and suppresses its expression in vitro and in vivo. To better understand the impact of ICP22 binding to CD80 on HSV-1 infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40-amino-acid (aa) region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, which does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. The replication of this recombinant virus in vitro and in vivo was higher than that of the ICP22-null virus, but virus replication kinetics were lower than those of the wild-type (WT) control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in dendritic cells (DCs) and interferon gamma (IFN-γ) expression in CD8+ T cells but not CD4+ T cells in infected mouse corneas. In contrast to the significantly reduced virus replication in the eyes of ocularly infected mice, the levels of latency reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding appears to reduce virus replication and enhance CD8+IFN-γ+ infiltrates in corneas of infected mice, with no effect on latency reactivation.IMPORTANCE Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates the expression of the costimulatory molecule CD80 but not CD86. In this study, we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to the CD80 promoter. This recombinant virus replicated less efficiently in vitro and in vivo than did the WT control virus, although CD80-expressing CD11c+ cells and IFN-γ-expressing CD8+ T cells were increased. Interestingly, the levels of latency and reactivation in the two viruses were similar despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 with the CD80 promoter could be used to temper the immune response.

Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes.

Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.

Antitumor efficacy of oncolytic HSV-1 expressing cytosine deaminase is synergistically enhanced by DPD down-regulation and EMT inhibition in uveal melanoma xenograft.

Uveal melanoma (UM) is the most common intraocular tumor in adults and has a high incidence of metastases. Possible treatments remain limited in UM with enucleation and radiation, leading to poor prognosis in this chemo-resistant carcinoma. Thus, urging demand for novel treatment is needed. We examined the antitumor efficacy of a new recombinant oncolytic herpes simplex virus type 1 (oHSV-1) armed with E.coli cytosine deaminase (CD). We determined the efficacy of the oncolytic virus in UM cell lines. In vivo experiments showed that oHSV-CD/5-fluorocytosine (5-FC) treatment reduce tumor volume and prolonged survival. We further demonstrated the molecular mechanisms of oHSV-CD/5-FC treatment. The oncolytic virus down-regulated IL-6 expression and thereby reversed the epithelial-mesenchymal transition (EMT) phenotype. Dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-fluorouracil (5-FU) metabolism, was also down-regulated. Therefore, the efficacy of oHSV-CD/5-FC was synergistically enhanced by DPD down-regulation and EMT inhibition. This study provides solid evidence for the antitumor efficacy of oHSV-CD/5-FC treatment in vitro and in vivo. The molecular mechanisms of this treatment may bring a new therapeutic approach for future treatment of UM.

Loss of ICP22 in HSV-1 Elicits Immune Infiltration and Maintains Stromal Keratitis Despite Reduced Primary and Latent Virus Infectivity.

PurposeWe previously have reported that ICP22, an immediate early gene of herpes simplex virus type 1 (HSV-1), binds to the CD80 promoter to suppress CD80 expression in antigen-presenting cells, leading to reduced T-cell function and protection. In contrast, overexpression of CD80 exacerbates corneal scarring (CS) in ocularly infected mice. In this study we tested the hypothesis that the absence of ICP22 could increase disease severity.MethodsTo test our hypothesis, BALB/c mice were ocularly infected after corneal scarification with a recombinant HSV-1 lacking the ICP22 gene with its parental wild-type (WT) virus (KOS) as a control. Virus replication in the eye, CS, angiogenesis, latency, and reactivation between ICP22 null virus and WT KOS were determined. In addition, expression of IL-2, IL-4, IFN-γ, IFN-α, granzyme A, granzyme B, and perforin by CD4 and CD8 T cells in corneas of infected mice on days 3, 5, 7, 10, 14, 21, and 28 postinfection were determined by flow cytometry.ResultsWe found similar levels of eye disease and angiogenesis in mice following corneal scarification and ocular infection with the ICP22 null virus or parental WT virus despite reduced virus replication in the eye and reduced latency and reactivation in mice ocularly infected with ICP22 null virus. The similar level of eye disease in ICP22 null virus– and WT virus–infected mice correlated with expression of various proinflammatory cytokines that infiltrated the eye after HSV-1 infection.ConclusionsOur study identified a critical role for ICP22 in HSV-1 pathogenicity and suggests that HSV-1–associated CS is more dependent on host immune responses to infection than to virus replication in the eye. Thus, HSV-1 as means of survival uses ICP22 as a mechanism of immune escape that protects the host from increased pathology.

Acyclovir Sensitivity and Neurovirulence of Herpes Simplex Virus Type 1 with Amino Acid Substitutions in the Viral Thymidine Kinase Gene, Which Were Detected in the Patients with Intractable Herpes Simplex Encephalitis Previously Reported.

Several cases of herpes simplex encephalitis (HSE) caused by acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) have been reported. Amino acid substitutions of R41H, Q125H, and A156V in the viral thymidine kinase (vTK) gene have been reported to confer ACV resistance. Recombinant HSV-1 clones, containing each amino acid substitution in the vTK gene, were generated using the bacterial artificial chromosome system. A recombinant HSV-1 with the Q125H substitution showed ACV resistance while the R41H or A156V substitutions were ACV-sensitive. Furthermore, the Q125H recombinant HSV-1 was less virulent than the repaired virus, but it maintained neurovirulence in mice at relatively high levels. Substitution of Q125H, which was detected in the neonatal HSE patient, conferred ACV resistance, but the substitutions of R41H and A156V, which were detected in immunocompetent adult HSE patients, did not. This suggests that HSE caused by ACV-resistant HSV-1 might be a very rare event to occur during the course of ACV treatment in immunocompetent patients. Showing resistance to ACV treatment does not always indicate emergence of ACV-resistant HSV-1 in HSE patients.

Overexpression of µ-Opioid Receptors in Peripheral Afferents, but Not in Combination with Enkephalin, Decreases Neuropathic Pain Behavior and Enhances Opioid Analgesia in Mouse

The current study used recombinant herpes simplex virus type I to increase expression of µ-opiate receptors and the opioid ligand preproenkephalin in peripheral nerve fibers in a mouse model of neuropathic pain. It was predicted that viral vector delivery of a combination of genes encoding the µ-opioid receptor and preproenkephalin would attenuate neuropathic pain and enhance opioid analgesia. The behavioral effects would be paralleled by changes in response properties of primary afferent neurons. Recombinant herpes simplex virus type 1 containing cDNA sequences of the µ-opioid receptor, human preproenkephalin, a combination, or Escherichia coli lacZ gene marker (as a control) was used to investigate the role of peripheral opioids in neuropathic pain behaviors. Inoculation with the µ-opioid receptor viral vector (n = 13) reversed mechanical allodynia and thermal hyperalgesia and produced leftward shifts in loperamide (ED50 = 0.6 ± 0.2 mg/kg vs. ED50 = 0.9 ± 0.2 mg/kg for control group, n = 8, means ± SD) and morphine dose-response curves (ED50 = 0.3 ± 0.5 mg/kg vs. ED50 = 1.1 ± 0.1 mg/kg for control group). In µ-opioid receptor viral vector inoculated C-fibers, heat-evoked responses (n = 12) and ongoing spontaneous activity (n = 18) were decreased after morphine application. Inoculation with both µ-opioid receptor and preproenkephalin viral vectors did not alter mechanical and thermal responses. Increasing primary afferent expression of opioid receptors can decrease neuropathic pain-associated behaviors and increase systemic opioid analgesia through inhibition of peripheral afferent fiber activity.

Corneal Epithelial Cells Exhibit Myeloid Characteristics and Present Antigen via MHC Class II.

PurposeTo explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial–mesenchymal transition (EMT).MethodsCorneas were harvested from mice 6 days following scratch injury, ragweed pollen–induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells.ResultsEctopic expression of classic “myeloid” antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell–cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro.ConclusionsThis study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple “myeloid” antigens warrants careful evaluation of flow cytometry data involving corneal digests.