Recombinant oncolytic poliovirus eliminates glioma in vivo without genetic adaptation to a pathogenic phenotype

Abstract

Many viruses, either naturally occurring or as a result of genetic manipulation, exhibit conditional replication in transformed cells. This principle is the basis for experimental therapeutic approaches exploiting the oncolytic potential of such agents without the danger of collateral damage to resistant normal tissues. One of the potential obstacles to these approaches is the possibility of genetic adaptation of oncolytic viruses upon replication in susceptible tumor tissues. Genetic variation can reverse genetic manipulations of parental viral genomes that determine attenuation of virulence, selective tumor cell tropism or other desirable traits. Alternatively, it may convey new properties not originally associated with parental strains, e.g., adaptation to a human host range. We examined genetic stability of an oncolytic nonpathogenic poliovirus recombinant considered for therapy of recurrent glioblastoma multiforme (GBM). This was done by serial passage experiments in glioma xenografts in vivo and investigation of phenotypic and genotypic markers of attenuation. Intratumoral inoculation of oncolytic poliovirus produced efficient tumor regress and elimination without altering temperature-sensitive growth, selective cytotoxicity, or genetic markers of attenuation of virus recovered from inoculated animals. Our studies demonstrate that active viral oncolysis of malignant glioma does not alter the conditional replication properties of oncolytic nonpathogenic poliovirus recombinants.

Figure 1

HTB-15 xenograft growth after vehicle- or PVS-RIPO treatment. All tumor sizes were determined by caliper measurement. Open squares represent average sizes of 20 xenografts (bilateral tumors in 10 animals) and crossed open squares are the median sizes of 8 xenografts (bilateral tumors in 4 animals observed for 40 days post xenograft implantation). Closed squares are the median sizes of 4 bilateral xenografts in 2 vehicle-treated animals. The size range of all xenografts at any given interval is indicated. Virus/vehicle inoculation occurred on day 12, after measuring of the tumors.

Figure 2

Histopathological analysis of PVS-RIPO induced oncolysis of HTB-15 xenografts in athymic mice. The bar shown with low-magnification images represents 1 mm. (a) Cross-section of a vehicle-injected tumor in an animal sacrificed 40 days after tumor implantation. The xenograft displays typical dense, hyper-cellular morphology. (b) Representative xenografts 10 days post PVS-RIPO inoculation. Only a minor, central portion of the xenograft retains the characteristic appearance of tumor (arrows). The boxed region is shown at higher magnification in (c). (c) Detail of (b) (bottom panel). Dense infiltrates surrounding and invading the central part of the xenograft can be distinguished at higher magnification. (d) Overview of a HTB-15 xenograft 28 days after PVS-RIPO inoculation. A scar had replaced the tumor, but a minor focus of remaining active tissue re-arrangement is visible (yellow arrow). A large patent vessel (black arrow) and surrounding collapsed minor vessels may represent the former tumor’s vascular supply. The boxed region is shown at higher magnification in (e). (e) Detail of (d) showing the area of active tissue re-modeling at higher magnification. (f) Two out of 4 tumors had entirely been eliminated 28 days post PVS-RIPO and replaced by a scar. The remnants of several large vessels are visible at the center of the scar’s base (arrow).

Figure 3

Recovery of PVS-RIPO from HTB-15 xenografts 10 days post virus inoculation. Numbering refers to the animal and location of the xenograft the virus originated from. Estimates of PFU/mg xenograft tissue and total PFU/xenograft recovered are shown below for each tumor. The plaque assay of tumor 015R shows an artifact due to detachment of the cell layer during staining. The numbers shown were calculated from a repeat assay.

Figure 4

Differential cell killing assay of PVS-RIPO. (a) Assays measuring differential cell killing of the control lot (L0603006; top panel) and xenograft passaged lot (022208; bottom panel) in HTB-14 cells (●) and HEK-293 cells () yielded similar results. (b) C values refer to the virus titer at half maximal cell killing; ED₅₀ values represent virus titers at 50% cell killing. C and ED₅₀ values were unchanged upon PVS-RIPO xenograft passage.

Figure 5

Comparison of representative sequence chromatograms of the polymorphic base positions 97 (a) and 1824 (b) identified through PVS-RIPO sequencing. Sequencing results for xenograft passaged PVS-RIPO (lot 022208) were compared with those for the GMP reference plasmid, the master virus seed and the toxicology lot L0603006 The nucleotide numbers refer to the genome of PVS-RIPO, the polymorphic bases are arrowed.