CD8 T cells and latent herpes simplex virus type 1: keeping the peace in sensory ganglia

Herpes simplex virus type 1 (HSV-1) infections represent a significant worldwide heath problem. The lack of an effective therapy to curtail reactivation of HSV-1 from a state of neuronal latency has lead to significant morbidity and mortality. Effective therapies to prevent reactivation must likely elicit a protective CD8 T-cell response that could act to prevent reactivation from sensory neurons prior to release of infectious virus at the periphery. This review focuses on the present understanding of how CD8 T cells maintain HSV-1 latency and how this knowledge could facilitate the generation of more effective therapeutic modalities.     

CD8 T cells and latent herpes simplex virus type 1: keeping the peace in sensory ganglia

Herpes simplex virus type 1 (HSV-1) infections represent a significant worldwide heath problem. The lack of an effective therapy to curtail reactivation of HSV-1 from a state of neuronal latency has lead to significant morbidity and mortality. Effective therapies to prevent reactivation must likely elicit a protective CD8 T-cell response that could act to prevent reactivation from sensory neurons prior to release of infectious virus at the periphery. This review focuses on the present understanding of how CD8 T cells maintain HSV-1 latency and how this knowledge could facilitate the generation of more effective therapeutic modalities.     

Molecular and biological characterization of a herpes simplex virus type 1 (HSV-1) neuroinvasiveness gene

Pathogenetic studies of herpes simplex virus type 1 (HSV-1) strains ANG and its mouse brain-passaged descendant ANG path revealed no difference in neurovirulence but a significant difference in neuroinvasiveness. Thus, both viruses induced a fatal encephalitis in mice after direct injection into the brain, but only ANG path induced lethal neurologic disease after inoculation on rear footpads. The difference in neuroinvasiveness is not related to the capacity to replicate in mouse neural tissues or mouse cells in general, but is specifically related to virus entry into the peripheral nervous system in the footpad. Marker rescue experiments in which ANG path genes were used to confer neuroinvasiveness on ANG indicated that the gene that codes for glycoprotein D (gD) is responsible for the phenotypic difference. Analyses of the gD genes by dideoxy-sequencing techniques identified a base difference in the coding sequences and predicted that the ANG gD gene codes for alanine (GCC codon) at amino acid position 84 in the open reading frame and the ANG path gD gene codes for glycine (GGC codon) at this site. Using these data, an oligonucleotide probe predicted to be specific for the ANG path gD gene was prepared, and in Southern blot analyses, this probe revealed that neuroinvasiveness-rescued agents had incorporated the base change seen in the ANG path gD gene. We conclude that HSV-1 glycoprotein D functions to effect neuroinvasiveness and we discuss potential mechanisms that may be involved.     

Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation

Cytotoxic CD8(+) T cells play a critical role in controlling herpes simplex virus (HSV) infection and reactivation. However, little is known about the spatiotemporal dynamics of CD8(+) T cells during HSV lesion evolution or about their involvement in immune surveillance after lesion resolution. Using quantum dot-conjugated peptide-major histocompatibility complex multimers, we investigated the in vivo localization of HSV-2-specific CD8(+) T cells in sequential biopsies of human genital skin during acute, resolving, and healed stages of HSV-2 reactivation. Our studies revealed that functionally active CD8(+) T cells selectively infiltrated to the site of viral reactivation. After lesion healing in concert with complete reepithelialization and loss of HSV DNA from skin biopsies, HSV-2-specific CD8(+) T cells persisted for more than two months at the dermal-epidermal junction, adjacent to peripheral nerve endings. In two out of the six sequentially studied individuals, HSV-2 DNA reappeared in clinically and histologically normal-appearing skin. Detection of viral DNA was accompanied by increased numbers of both HSV-specific and total CD8(+) T cells in the dermis. These findings indicate that the frequency and clinical course of HSV-2 reactivation in humans is influenced by virus-specific CD8(+) T cells that persist in peripheral mucosa and genital skin after resolution of herpes lesions.     

Bupropion (Zyban, Wellbutrin) inhibits nicotine-induced viral reactivation in herpes simplex virus type 1 latent rabbits

We reported that nicotine applied via a transdermal patch (21 mg/day) induced viral reactivation and ocular shedding in herpes simplex virus type 1 (HSV-1) latent rabbits. One possible mechanism of action involves the release of catecholamines and other similar agents, triggering HSV reactivation. Bupropion (Zyban, Wellbutrin), a non-nicotine aid to smoking cessation, inhibits neuronal uptake of norepinephrine, serotonin, and dopamine. To determine whether bupropion inhibits HSV reactivation, rabbits latent with HSV-1 were grouped (at least 10 rabbits/group) and treated as follows: nicotine patch (transdermal delivery) and bupropion [Zyban sustained-release tablets (150 mg) twice a day (oral)], nicotine patch only, Zyban tablets only [twice a day (oral)], nicotine patch with oral placebo [twice a day (oral)], or no drug treatment. Eyes were swabbed for 22 consecutive days. The appearance of HSV-1 in the tear film was significantly less frequent in the bupropion-treated rabbits, in terms of positive rabbits/total rabbits, positive eyes/total eyes, and positive swabs/total swabs. Nicotine-treated rabbits had 78/440 (17.7%) positive/total swabs, and nicotine/placebo-treated rabbits had 149/792 (18.8%) positive/total swabs, whereas bupropion-treated rabbits had 23/440 (5.2%), and nicotine/bupropion-treated rabbits had 47/792 (5.9%) positive/total swabs. Thus, bupropion significantly reduces nicotine-induced HSV reactivation in latent rabbits.     

Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus.

The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate inhibited HSV-1 and HSV-2 DNA polymerase competitively with dGTP, the Ki values being 8.5 and 5.8 microM, respectively, whereas for acyclovir-triphosphate, the Ki value was 0.07 microM for the two enzymes. Both compounds had relatively low levels of activity against the cellular DNA polymerase alpha, with Ki values of 175 and 3.8 microM, respectively. (S)-Penciclovir-triphosphate did inhibit DNA synthesis by HSV-2 DNA polymerase with a defined template-primer, although it was not an obligate chain terminator like acyclovir-triphosphate. These results provide a biochemical rationale for the highly selective and effective inhibition of HSV-2 and VZV DNA synthesis by penciclovir and for the greater activity of penciclovir than that of acyclovir when HSV-2-infected cells were treated for a short time.     

Genotypic characterization of the DNA polymerase and sensitivity to antiviral compounds of foscarnet-resistant herpes simplex virus type 1 (HSV-1) derived from a foscarnet-sensitive HSV-1 strain

Foscarnet is widely used for the treatment of acyclovir-resistant herpesvirus infections, and foscarnet-resistant herpesvirus infections are a serious concern in immunocompromised patients. Twenty-seven single-plaque isolates of herpes simplex virus type 1 (HSV-1) resistant to foscarnet were selected from foscarnet- and acyclovir-sensitive HSV-1 strain TAS by exposure to foscarnet, and the DNA polymerase genes were analyzed. The sensitivities of these mutants to foscarnet, cidofovir, S2242, acyclovir, ganciclovir, and penciclovir were determined. A single amino acid substitution, double amino acid substitutions, and a combination of a single amino acid substitution with a deletion or insertion of amino acid residues in the viral DNA polymerase were demonstrated in 21, 4, and 2 isolates, respectively. Of the 27 isolates, an amino acid substitution of serine for asparagine at amino acid position 724 in the DNA polymerase (724 S-N) was detected in 8 isolates. An amino acid substitution in conserved region II was demonstrated in these eight isolates as well as four other isolates. The mutation in the DNA polymerase responsible for resistance to foscarnet was located between the pre-IV region and conserved region V, especially within conserved region II. All the isolates were sensitive or hypersensitive to cidofovir and ganciclovir. Seven, 5, and 15 of the 27 isolates were also sensitive to S2242, acyclovir, and penciclovir, respectively. Thus, most of the foscarnet-resistant HSV-1 isolates were sensitive or hypersensitive to cidofovir and ganciclovir.     

In vivo gene transfer to the rat retina using herpes simplex virus type 1 (HSV-1)-based amplicon vectors

The purpose of our study was to evaluate the transduction profiles of herpes simplex virus type 1 (HSV-1)-based amplicon vectors following subretinal injection in the rat. Two amplicon vectors were tested, pHy-CMVGFP and pHy-RPEGFP, both carrying the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) ubiquitous promoter or the RPE65-specific promoter, respectively. For the two amplicon vectors, the GFP reporter gene was efficiently expressed in retinal pigment epithelial (RPE) cells but not in the adjacent photoreceptors. GFP expression was maximum as early as 2 days post-administration but decreased over time to become almost undetectable at 6 weeks postinjection. Super-transduction with a second amplicon vector, pHSVlac, reactivated expression of GFP in approximately 10% of the cells initially transduced at 2 days postinjection of pHy-CMVGFP or pHy-RPEGFP. Reactivation of transgene expression was transient, no GFP signal was detected 8 days after pHSVlac injection. In conclusion, HSV-1 amplicon vectors allow rapid and efficient, but transient, gene transfer in RPE cells following subretinal injection.     

CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo

Bone marrow-derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8(+) T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded beta2-microglobulin-deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8(-) and CD8(+) DCs. Only the CD8(+), and not the CD8(-), DC subset demonstrates cross-priming ability. FACS((R)) studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8(+) and the CD8(-) DC subsets, respectively, had one or more associated beads. These results indicate that CD8(+) DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.     

Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.     

Fusion from without induced by herpes simplex virus type 1

Strains ANG and ANG path of herpes simplex virus type 1 (HSV1) produced fusion from without (FFWO) of cells in culture. FFWO required 45 min to become complete. In contrast, fusion from within (FFWI) was not detected until 3-4 h after infection, depending on the cell type. FFWO was temperature dependent: at 0 degrees no fusion could be observed, but increase of temperature increased the degree of fusion. The pH optimum for FFWO was 7.8-8.5. The FFWO activity of the virus was found to be slightly more heat stable at 46 degrees than was infectivity. FFWO was produced in Vero, CV-1 and BSC1 cells, but not in BHK clone 13 or in primary or secondary rabbit kidney cells. FFWO was linked to the presence of virus particles and perhaps to other sedimentable, infected-cell material but not to soluble factors. Actinomycin D, cycloheximide, and UV irradiation did not block this activity, indicating no direct activity of the HSV1 genome for FFWO.     

Suppression of herpes simplex virus type 1 reactivation from latency by (+-)-9-([(Z)-2-(hydroxymethyl)cyclohexyl]methyl) guanine (L-653,180) in vitro.

Latent herpes simplex virus type 1 (HSV-1) infection was induced in human embryonic lung cells in vitro by using a combination of viral replication inhibitors and elevated temperature. Under reactivating conditions (superinfection by human cytomegalovirus or temperature manipulation), a nonantiviral thymidine kinase inhibitor (L-653,180) was found to suppress or delay reactivation of HSV-1 from latently infected human embryonic lung cells. L-653,180 alone or in combination with interferon was ineffective as a primary or acute viral replication inhibitor and was unable to induce latent HSV-1 infection in cell culture. These data suggest that initial or acute virus replication and replication resulting from reactivation from latency are separate events.     

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) can regulate dendritic cell-induced activation and cytotoxicity of CD8(+) T cells independently of CD4(+) T cell help

The mechanisms that regulate the strength and duration of CD8(+) cytotoxic T cell activity determine the effectiveness of an antitumor immune response. To better understand the antitumor effects of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody treatment, we analyzed the effect of CTLA-4 signaling on CD8(+) T cells in vitro and in vivo. In vitro, cross-linking of CTLA-4 on purified CD8(+) T cells caused decreased proliferative responses to anti-CD3 stimulation and rapid loss of activation marker expression. In vivo, blockade of CTLA-4 by neutralizing anti-CTLA-4 mAb greatly enhanced the accumulation, activation, and cytotoxic activity of CD8(+) T cells induced by immunization with Ag on dendritic cells (DC). This enhanced response did not require the expression of MHC class II molecules on DC or the presence of CD4(+) T cells. These results demonstrate that CTLA-4 blockade is able to directly enhance the proliferation and activation of specific CD8(+) T cells, indicating its potential for tumor immunotherapy even in situations in which CD4(+) T cell help is limited or absent.     

Delivery of RNA interference triggers to sensory neurons in vivo using herpes simplex virus

Importance of the field: Pain is a hugely important area of research attracting considerable academic and commercial interest. However, the application of RNA interference (RNAi) to the study of nociceptive processes and the development of new analgesics has been limited by the specific challenges associated with the delivery of RNAi triggers to the cell bodies of sensory neurons in the dorsal root ganglia (DRG).Areas covered in this review: In the past five years, delivery of small-interfering RNA (siRNA) to the DRG and spinal cord has achieved effective and specific silencing of targeted genes in various animal models of pain. However, delivery of short-hairpin RNA (shRNA) or artificial microRNA (miRNA) to sensory neurons in vivo has not been feasible using most delivery systems currently available. What the reader will gain: Replication-defective vectors based on herpes simplex virus (HSV), which are particularly efficient at targeting DRG neurons, have been recently engineered to express shRNA and artificial miRNA. Whilst silencing induced by siRNA is transient and requires relatively high doses of silencing triggers, HSV-mediated expression of shRNA/miRNA in sensory neurons allows silencing of targeted genes for at least one week following a single injection.Take home message: The potential to use inducible or tissue-specific promoters and to simultaneously silence multiple gene targets, in addition to recent studies suggesting that artificial miRNAs may have improved safety profiles, hold clear advantages for the use of miRNA-based vectors for gene silencing in sensory neurons.     

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.