Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients.

BACKGROUND: DISC-hGMCSF is a gH-deleted HSV-2 based vector expressing human GM-CSF that is being developed for cancer immunotherapy. To support first clinical use, a range of preclinical safety studies were performed using DISC-hGMCSF in addition to DISC-murine-GMCSF and the backbone vector, TA-HSV. METHODS: The toxicity of the DISC vectors was assessed by repeated dose, neurovirulence and neuroinvasiveness studies in mice, and by safety studies in rabbits, guinea pigs and athymic nude mice. Studies were also conducted to determine whether the vector could establish latency in local ganglia in mice following intradermal injection, and whether it could reactivate from the latent state. The vector biodistribution following intravenous administration was also investigated in mice, using PCR to detect vector DNA. RESULTS: The DISC vectors were essentially non-toxic in all the systems studied. No adverse reactions were seen in mice receiving four intravenous doses of DISC-mGMCSF and the results from studies of neurovirulence, neuroinvasiveness, local tolerance in rabbit, general safety in mice and guinea pigs and safety in athymic nude mice were consistent with DISC being unable to replicate and cause disease. The vector could establish latency in local ganglia in mice, but at low efficiency, and could not reactivate infectious virions. Following intravenous administration, vector DNA was widely distributed up to Day 28, but by Day 56 had disappeared from gonads and brain and was only found in blood and liver. CONCLUSION: The panel of safety studies provided evidence that DISC-hGMCSF will be unable to replicate and cause disease, and has low toxicity in man. These data were presented to the Medicines Control Agency and the Gene Therapy Advisory Committee as part of the regulatory submissions for a clinical trial in melanoma patients. These submissions have been approved, and DISC-hGMCSF has now entered a phase I clinical trial in the UK by direct intratumoural injection.

Interaction of nucleic acids with core-like and subcore-like particles of bluetongue virus.

Bluetongue virus (BTV) core-like particles (CLPs) were synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus. Purified CLPs were shown to bind single-stranded RNA in three different assay systems: gel retardation, nitrocellulose binding, and sucrose gradient sedimentation. CLPs showed equal affinity for BTV-specific and non-BTV RNA and also bound DNA. RNAase protection experiments demonstrated that bound RNA was accessible to immobilized ribonuclease, suggesting that the RNA was predominantly present on the outside of the CLPs. By using individually purified VP7 and VP3 in separate assays, the binding activity was shown to reside on VP3. These results indicate further functional homologies between BTV VP3 and the rotavirus inner-core VP2 protein.

Three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy.

When the viral proteins VP3 and VP7 of bluetongue virus (BTV) are expressed simultaneously in the baculovirus system, core-like particles form spontaneously. The 3-D structure of these core-like particles, determined from cryo-electron micrographs, reveals an icosahedral structure 72.5 nm in diameter with 200 triangular spikes arranged on a T = 13,I lattice; The five spikes around each of the fivefold axes are absent. This is in contrast to the native BTV core particles which have a complete T = 13,I lattice of 260 spikes. The spikes, attributed to VP7 trimers appear as triangular columns 8.0 nm in height with distinct inner and outer domains. The inner shell of the core-like particles, or subcore-like particle, has a T = 1 lattice composed of 60 copies of VP3. The subcore-like particle is noticeably thicker around the fivefold positions. Pores in the subcore-like particle are situated near each of the local sixfold axes, below each six-membered ring of spikes. These pores could allow the passage of metabolites and RNA to and from the core for RNA transcription during infection. It is possible that the synthetic core-like particles have an incomplete complement of VP7 spikes because the ratio of VP7 to VP3 produced in the dual expression system is less than the 13:1 required for complete core-like particles. Only the VP7 spikes which have the strongest affinity for the VP3 inner core and are involved in maintaining the structural integrity of the core-like particle are incorporated. The BTV core-like particle shows greater morphological similarity to the rotavirus than to the reovirus core particle.

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.

We have previously reported the assembly of virus-like particles (VLPs), consisting of the four major structural proteins of bluetongue virus (BTV), in Spodoptera frugiperda cells coinfected with recombinant baculoviruses (French et al. (1990). J. Virol. 64, 5695-5700). In this paper we report further studies using this system to assemble heterologous VLPs containing the outer capsid proteins (VP2 and VP5) of a range of different BTV serotypes. S. frugiperda cells were coinfected with three recombinant baculoviruses; a dual recombinant expressing VP3 and VP7 (of BTV-17 and -10, respectively) in combination with a single recombinant expressing VP2 of BTV-1, -2, -10, -11, 13, or -17 and an additional single recombinant expressing VP5 of BTV-2, BTV-10, or BTV-13. The resultant VLPs were purified and analyzed by electronmicroscopy, Western immunoblotting, and hemagglutination assays to determine whether double-shelled VLPs had been assembled. In the course of these experiments the VP2 proteins of all six available serotypes were successfully incorporated into VLPs. Particles from two different combinations of chimeric VLPs (having VP2 derived from BTV-1 or that of BTV-17) were used to raise antisera in guinea pigs. Both of these sera showed high neutralizing antibody titers against live BTV, indicating that heterologous VLPs may have potential for use in anti-BTV vaccines.

Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins.

Bluetongue virus (BTV) VP1 protein, a component of the viral RNA-directed RNA polymerase, but not the VP4 or VP6 proteins, was specifically incorporated into baculovirus expressed BTV core-like particles (composed of VP3 and VP7) and BTV virus-like particles (composed of VP2, VP3, VP5, and VP7). The VP1 protein has been shown to be associated with subcore particles composed of VP3. The data suggest that the VP1 protein of BTV has both enzymatic and structural roles in the virus life cycle.