Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells

Iris J C Dautzenberg,Pierre Boulanger,Sanne K Van Den Hengel,Steve J Cramer,Rob C Hoeben,Saw-See Hong,Diana J M Van Den Wollenberg,Lars Ravesloot,Jeroen De Vrij

doi:10.1038/s41598-017-17709-z

Abstract

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus’ tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two- and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus’ capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.

Highlights

The mechanism of RV T3D oncolysis can be primarily attributed to its unrestricted replication and killing of tumour cells bearing genomic mutations that lead to active RAS signalling via RalGEF/p38 downstream pathways, while in untransformed, healthy cells the reovirus’ infectious cycle is limited by the dsRNA-activated protein kinase R (PKR)-mediated antiviral responses[4,5]
We show that the baculovirus AcMNPV expressing JAM-A (BVJAM)-RV complexes facilitate infection and replication of RV and increase cell killing in U-118 MG glioblastoma cells which are refractory to RV under normal cell culture conditions[17]
The full-length JAM-A gene including a human influenza hemagglutinin (HA) tag was inserted in the pBlueBac4.5/V5-His vector

Summary

Introduction

The mechanism of RV T3D oncolysis can be primarily attributed to its unrestricted replication and killing of tumour cells bearing genomic mutations that lead to active RAS signalling via RalGEF/p38 downstream pathways, while in untransformed, healthy cells the reovirus’ infectious cycle is limited by the dsRNA-activated protein kinase R (PKR)-mediated antiviral responses[4,5]. Other cellular determinants as receptor availability and virus uncoating efficiency have been shown to influence RV’s ability to kill cancer cells[6,7,8,9,10]. Replication of the oncolytic-virus increases anti-tumour immunity, thereby enhancing the therapeutic efficacy of RV11,12. RV demonstrates an outstanding safety profile and anti-tumour efficacy has been witnessed in several cancer types. In these studies RV is used either as monotherapy or in combination with conventional treatment[13]. RV’s ability to enter tumour cells may be negatively affected by the scarcity and inaccessibility of its cellular receptor JAM-A, it remains to be established how important this factor is, taking into account the existence of alternative, e.g. JAM-A-independent, entry mechanisms[17,18]

Methods

Results

Conclusion