Evaluation of IRES-mediated, cell-type-specific cytotoxicity of poliovirus using a colorimetric cell proliferation assay

Abstract

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and celltype specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

“…Our findings suggest that the intrathalamic virus inoculum remains contained within the CNS of injected animals. The primate neurovirulence assays described here are concordant with the results of previous empirical studies in neuron-like tissue culture models (5,16,17,27,48), in mice transgenic for human Necl-5 (15,16) and in M. fascicularis challenged with intraspinal inoculation of PV1-RIPO (16). Our investigations confirm a dominant role for the IRES and, hence, ribosome recruitment to viral RNAs in PV pathogenesis.…”

Analytical characterization of ch14.18

“…The washed cells were re-suspended in 1 ml PBS. FACS analysis was performed using FACSCalibur from Beckton Dickinson as described earlier by Yang et al 42 Propidium iodide (PI) staining of dead cells and FACS measurement. Cells (10 6 /ml) were incubated with ch14.18 at 37°C for 1 h. The human complement serum was added and incubated at 37°C for another 4 h. Following incubation, cells were transferred to falcon tubes.…”

MAPK Signal-integrating Kinase Controls Cap-independent Translation and Cell Type-specific Cytotoxicity of an Oncolytic Poliovirus

“…HEK-293 cells barely support PVSRIPO growth, 3 block polysome association of viral RNA 25 and resist PVSRIPO cytotoxicity. 4 To establish if this phenotype correlates with a distinct signaling status, we analyzed PI3K/Akt and MAPK signaling in HEK-293 cells (Figure 3a). Compared to established GBM cell lines, e.g., DU54 or U-118, HEK-293 cells show relatively reduced Erk1/2 and p38-MAPK activity resulting in low levels of phospho-eIF4E (Figure 3a).…”

“…In particular, studies with transfection of promoter reporters suggested that active Ras may upregulate the PV receptor, nectin-like molecule 5 (Necl-5). 27 Stimulation of PVSRIPO entry by oncogenic Ras via enhanced receptor expression is unlikely, because Necl-5 is exceedingly abundant in plain HEK-293 cells 4 and, since wild-type PV or PV1S grow efficiently in these cells, 3 is not a factor limiting susceptibility. Accordingly, analyses of Necl-5 levels in mock-and tet-induced HEK-293 Ras cells did not reveal Necl-5 induction upon tet-induced Ras expression (Figure 5a).…”

“…1 This diminishes replication potential in cells of neuronal derivation, e.g., neuroblastoma cell lines 2 or human embryonic kidney 293 (HEK-293) neuroblastic cells 3,4 and eliminates the inherent neuropathogenicity associated with PV infection. The prototype oncolytic PV/ HRV chimera, PVSRIPO, was devoid of pathogenic properties after intracerebral inoculation of 5 × 10 9 tissue culture infectious doses in an investigational new drug-directed dose-range finding, toxicology, and biodistribution study in Macaca fascicularis (personal communication).…”