Suzanne Thomas

A Protein Encoded by the Herpes Simplex Virus (HSV) Type 1 2-Kilobase Latency-Associated Transcript Is Phosphorylated, Localized to the Nucleus, and Overcomes the Repression of Expression from Exogenous Promoters When Inserted into the Quiescent HSV Genome

ABSTRACT Herpes simplex virus (HSV) is characterized by its ability to establish a latent infection in sensory neurons, from which it can periodically reactivate. The mechanisms of latency, however, remain unclear. The HSV genome is quiescent during latency except for the expression of the latency-associated transcripts (LATs). Although the exact function of the LATs remains obscure, current evidence suggests they are multifunctional and are involved in both establishment of latency and reactivation from latency. The LATs contain several open reading frames (ORFs). One or more of the functions of the LATs could therefore be protein mediated. We have previously reported that deregulated expression of the largest of the HSV type 1 (HSV-1) LAT ORFs (∼274 amino acids) greatly enhances virus growth in cell types that are normally relatively nonpermissive for HSV replication and also that it complements mutations to the immediate-early (IE) gene ICP0 (S. K. Thomas, G. Gough, D. S. Latchman, and R. S. Coffin, J. Virol. 73:6618-6625, 1999). Here we show that LAT ORF expression overcomes the repression of expression from exogenous promoters introduced into the HSV-1 genome which normally occurs in the absence of IE gene expression. To further explore LAT ORF function, we have generated an epitope-tagged LAT ORF, LATmycHis, which forms punctate structures in the infected-cell nucleus reminiscent of the structures formed by ICP0. These are associated with the appearance of a phosphorylated form of the protein and are formed adjacent to, or around the edges of, viral replication compartments. These results provide further evidence that the HSV-1 LAT ORF protein is biologically functional and that the tightly regulated expression of this protein may be important in the wild-type latency phenotype in vivo.

166. OncoVEX: A Family of Oncolytic Herpes Simplex Viruses Optimised for Therapeutic Use

2601 Background: HSV in which ICP34.5 is inactivated directs tumour selective cell lysis in vitro and in vivo. Such viruses have proven safe in Phase I clinical trials by intra-tumoral injection in glioma and melanoma patients. Oncolytic HSV containing an active gene has not previously been tested in man. METHODS Previous work has used serially passaged isolates of HSV-1 which are attenuated in human tumour cells compared to clinical isolates. To produce improved viruses, we have deleted ICP34.5 from clinical isolates of HSV-1. This greatly enhanced tumour cell killing. One of these viruses was further deleted for ICP47 (which blocks antigen presentation) and GM-CSF was inserted. This aimed to maximize anti-tumour immune responses following injection and provide an in situ, patient-specific, anti-tumour vaccine, combined with oncolysis. RESULTS The anti-tumour properties of the virus were greatly improved by each of the modifications made. In vivo, both injected and non-injected tumours were cured and animals were protected against further tumour cell challenge. Following this promising data a Phase I clinical trial with the virus (OncoVEXGM-CSF) is underway including patients with a number of tumour types. This has given promising results so far. In addition, further versions of the OncoVEX virus expressing other active genes have been constructed and tested in pre-clinical models. These include viruses expressing TNFproportion and TNFproportion combined with GM-CSF, intended for use with radiotherapy, and versions of the virus expressing pro-drug activating genes combined with the delivery of a fusogenic glycoprotein to maximize the properties of the virus for local tumour control. Each of these have shown promising results in pre-clinical tumour models, including in combination with chemotherapy where synergism has been demonstrated. CONCLUSIONS In summary, continued development of the OncoVEX technology has provided a family of viruses incorporating a range of active genes intended to maximize the effects of the virus under the different circumstances for which oncolytic virus therapy might be considered for clinical use. [Table: see text].

Examination of the potential interactions between herpes simplex virus vectors and replication-competent virus in vitro and in vivo

In this paper, we have studied the potential interactions of replication incompetent herpes virus vectors with replication-competent virus both in vitro and in vivo. This might be thought to be particularly important for the use of HSV as compared to other virus vectors, as humans often harbour latent HSV. A vector virus carrying a transgene could interact with endogenous wild-type virus when used in gene therapy procedures. For this study we constructed four recombinant viruses containing marker genes to allow interactions between replication competent and disabled vector viruses to be explored. Recombination of viruses under replicating conditions was assessed in vitro and in vivo in the peripheral nervous system following the inoculation of combinations of viruses into the hindpaw of BALB/c mice and the examination of virus content in the dorsal root ganglia. Recombination between the viruses, when co-administered was found to occur such that transgene-bearing replication-competent viruses were generated only when the transgene was inserted at a non-essential site in the HSV genome. When the transgene was inserted into an essential site in the disabled virus, or when disabled and non-disabled virus were administered separately to the same site, transgene-bearing replication-competent recombinants were not observed. In the central nervous system, the ability of disabled, LacZ containing virus to reactivate latent replication-competent virus was examined. CNS latency was established by infecting BALB/c mice with a replication competent, GFP containing virus by the intra nasal route. After the establishment of latency, disabled virus was injected intra-cerebrally. Reactivation could not be detected as evidenced by a lack of GFP expression and replicating virus even though robust LacZ expression from the incoming vector virus could be detected. A similar lack of reactivation occurred when vector virus was inoculated into the footpad following the establishment of latency in the PNS by a replication competent virus. These experiments show (i) that insertion of the transgene in an essential site of the viral genome prevents its incorporation into an hazardous replication-competent recombinant derivative, indicating that non-homologous recombination between disabled and replication competent viruses does not occur at the level of sensitivity of the in vitro/in vivo assays used here, (ii) even homologous recombination in vivo only occurs at detectable levels when vector and replication competent virus are co-administered, and (iii) inoculation of vector HSV into the nervous system is very unlikely to reactivate latent wild-type virus that may be present.