S. Moira Brown

Intralesional injection of herpes simplex virus 1716 in metastatic melanoma

We have previously shown that avirulent but replication-competent herpes simplex virus (HSV) 1716 causes cell death in human melanoma cell lines in vitro and selectively replicates in melanoma tissue in nude mice. We now present a pilot study of intratumoral injection of HSV1716 into subcutaneous nodules of metastatic melanoma in five patients with stage 4 melanoma. Two patients each received one injection, two received two injections, and one received four injections of 10(3) plaque-forming units HSV1716. In one patient, flattening of previously palpable tumour nodules was seen 21 days after two direct injections of HSV1716, and in injected nodules from all three patients who received two or more injections there was microscopic evidence of tumour necrosis. Immunohistochemical staining of injected nodules revealed evidence of virus replication confined to tumour cells. These findings suggest that HSV1716 is non-toxic and could be of therapeutic benefit in patients with metastatic melanoma.

ISOLATION OF HERPES SIMPLEX VIRUS FROM HUMAN TRIGEMINAL GANGLIA, INCLUDING GANGLIA FROM ONE PATIENT WITH MULTIPLE SCLEROSIS

Herpes-simplex virus (H.S.V.) was isolated from 18 of 39 trigeminal ganglia (T.G.) obtained within 12 h of death. The virus was isolated from ten persons who had died of trauma, from one case of lymphoma, and from one case of multiple sclerosis. In the cadaver with histologically confirmed multiple sclerosis, large bilateral areas of demyelination were present near the points of entry of the nerve root, and the possibility that H.S.V. migration to the root entry zone caused demyelination cannot be excluded.

The Herpes Simplex Virus (HSV) Protein ICP34.5 is a Virion Component that Forms a DNA-Binding Complex with Proliferating Cell Nuclear Antigen and HSV Replication Proteins

The replicative ability of ICP34.5-null herpes simplex virus (HSV) is cell type and state dependent. In certain cells, ICP34.5 interacts with protein phosphatase 1 to preclude host cell protein synthesis shutoff by dephosphorylation of the eukaryotic initiation factor eIF-2α. However, host cell shutoff is not induced by ICP34.5-null HSV in most cells, irrespective of type and state. In general, dividing cells support replication of ICP34.5-null HSV; nondividing cells cannot. Previously the authors showed that ICP34.5 binds to proliferating cell nuclear antigen (PCNA), a protein necessary for cellular DNA replication and repair. Here the authors demonstrate that (1) the interaction between ICP34.5 and PCNA involves two regions of the virus protein; (2) ICP34.5 forms a complex with HSV replication proteins that is DNA binding; (3) at early times in infection, ICP34.5 colocalizes with PCNA and HSV replication proteins in cell nuclei, before accumulating in the cytoplasm; and (4) ICP34.5 is a virion protein. In light of ongoing clinical trials assessing the safety and efficacy of ICP34.5-null HSV, it is vital that the roles of ICP34.5 in HSV replication are understood. The authors propose that in nondividing cells, ICP34.5 is required to switch PCNA from repair to replication mode, a prerequisite for the initiation of HSV replication.

Proliferative activity and in vitro replication of HSV1716 in human metastatic brain tumours

The neurotropic herpes simplex virus mutant HSV1716 lacks the gene encoding the virulence factor ICP34.5 and cannot replicate in non‐dividing cells where proliferating cell nuclear antigen (PCNA) is not actively engaged in cellular DNA synthesis. In the brain, tumoral expression of PCNA therefore confers on it oncolytic specificity and may determine its efficacy. Three phase I trials in glioma patients and one in metastatic melanoma patients have established that HSV1716 is safe and replicates selectively in tumour tissue. Here we examine the in situ PCNA profiles of common human metastatic brain tumours and determine their in vitro permissivity for HSV1716 replication to ascertain their suitability for HSV1716 therapy.

HSV Growth, Preparation, and Assay

Whether herpes simplex virus (HSV) is viewed as a pathogen or as a model eukaryotic system, it is virtually certain that any experimental work will require the virus to be grown and assayed. The following chapter is therefore seen as the fundamental first step before embarking on more intellectually and technically challenging technology. Its importance should not however be underestimated. It never fails to surprize us that people who describe themselves as virologists have little understanding of the basic requirements needed to attain a contamination-free, high-titer, low particle:plaque-forming units (PFU) ratio, genetically pure virus stock.

Recovery of Herpes Simplex Virus Genetic Information from Human Trigeminal Ganglion Cells following Superinfection with Herpes Simplex Virus Type 2 Temperature-sensitive Mutants

Explant cultures of human trigeminal ganglia were derived from 36 individuals. Those cultures which failed to release herpes simplex virus (HSV) spontaneously were superinfected at various times after establishment in vitro with a range of HSV-2 temperature-sensitive (ts) mutants. Eight cultures from six individuals contained HSV-specific genetic information which could be detected or rescued following superinfection. Restriction enzyme analysis of ts+ virus recovered from the ganglia of two individuals following superinfection was identical to that of endogenous HSV-1 spontaneously released from parallel cultures. Retrieval of ts+ virus by this technique suggests products of the superinfecting virus activated expression of whole genomes or that spontaneous virus expression occurred unrelated to the act of superinfection.

Evolution of GADD34 expression after focal cerebral ischaemia.

GADD34, a stress response protein associated with cell rescue, DNA repair and apoptosis, is expressed in the ischaemic brain. The C-terminal region of GADD34 has homology with the Herpes Simplex Virus protein, ICP34.5, which overcomes the protein synthesis block after viral infection by actively dephosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). The carboxy terminus of GADD34 is also capable of dephosphorylating eIF2alpha and therefore has the capacity to restore the protein synthesis shutoff associated with ischaemia. This study examines the distribution and time course of GADD34 expression after focal cerebral ischaemia. Focal ischaemia or sham procedure was carried out on Sprague-Dawley rats with survival times of 4, 12, 24 h, 7 and 30 days. Brains were processed for histology and immunohistochemistry. Ischaemic damage was mapped onto line diagrams and GADD34 positive cells counted in selected regions of cortex and caudate. GADD34 immunopositive cells (mainly neurones), expressed as cells/mm2, were present in ischaemic brains at 4 h (e.g., peri-infarct cortex 20 +/- 5; contralateral cortex 3 +/- 1, P < 0.05). Of the time points examined, numbers of GADD34 positive cells were highest 24 h after ischaemia (peri-infarct cortex 31 +/- 7.3, contralateral cortex 0.1 +/- 0.1, P < 0.05). Immunopositive cells, following a similar time course, were identified within the peri-infarct zone in the caudate nucleus and in ipsilateral cingulate cortex (possibly as a consequence of cortical spreading depression). GADD34 positive cells did not co-localise with a marker of irreversible cell death (TUNEL). Taken together, GADD34 positive cells in key neuroanatomical locations pertinent to the evolving ischaemic lesion, the lack of co-localisation with TUNEL and the proteins known effects on restoring protein synthesis, repairing DNA and involvement in ischaemic pre-conditioning suggests that it has the potential to influence cell survival in ischaemically compromised tissue.

Genetic studies with herpes simplex virus type 1. Analysis of mixed plaque-forming virus and its bearing on genetic recombination.

Abstract The progeny from genetic crosses between syn (syncytial plaque morphology) and syn + (non-syncytial plaque morphology) herpes simplex type 1 virus (HSV-1) produce three types of plaque; pure syn , pure syn + and, at low frequency, mixed plaques that are partially of syn and partially syn + morphology. The progeny found in mixed plaques consist mostly of pure syn and pure syn + segregants with again a low frequency of mixed syn-syn + plaque-forming virus. The sedimentation velocities and inactivation kinetics of mixed plaque-forming virus and total infectious virus are very similar and suggest that mixed plaques are not produced by aggregates of pure syn and pure syn + virions. Genetic analysis of the segregants from mixed plaques produced from two-factor crosses of the syn alleles and various temperature-sensitive ( ts ) mutants reveal that the syn and syn + segregants from a mixed plaque are in many cases true breeding for only one allele at the unselected ts locus. Taken together, these results indicate that a significant proportion of the mixed plaque-forming virus exists as single virions that are partially heterozygous at the syn locus. The bearing of these results on the recombination process of HSV-1 is discussed.

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.

Genetic studies with herpes simplex virus type 1: quantitative analysis of the products from two-factor crosses.

Abstract The syn mutant of herpes simplex type 1 virus has been used in conjunction with ts mutants to identify the genetic products of two-factor crosses. All four possible genotypes were observed in the progeny virus from the seven crosses analysed.