Semireplication-competent vesicular stomatitis virus as a novel platform for oncolytic virotherapy
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Among oncolytic viruses, the vesicular stomatitis virus (VSV) is especially potent and a highly promising agent for the treatment of cancer. But, even though effective against multiple tumor entities in preclinical animal models, replication-competent VSV exhibits inherent neurovirulence, which has so far hindered clinical development. To overcome this limitation, replication-defective VSV vectors for cancer gene therapy have been tested and proven to be safe. However, gene delivery was inefficient and only minor antitumor efficacy was observed. Here, we present semireplication-competent vector systems for VSV (srVSV), composed of two trans-complementing, propagation-deficient VSV vectors. The de novo generated deletion mutants of the two VSV polymerase proteins P (phosphoprotein) and L (large catalytic subunit), VSVΔP and VSVΔL respectively, were used mutually or in combination with VSVΔG vectors. These srVSV systems copropagated in vitro and in vivo without recombinatory reversion to replication-competent virus. The srVSV systems were highly lytic for human glioblastoma cell lines, spheroids, and subcutaneous xenografts. Especially the combination of VSVΔG/VSVΔL vectors was as potent as wild-type VSV (VSV-WT) in vitro and induced long-term tumor regression in vivo without any associated adverse effects. In contrast, 90% of VSV-WT-treated animals succumbed to neurological disease shortly after tumor clearance. Most importantly, even when injected into the brain, VSVΔG/VSVΔL did not show any neurotoxicity. In conclusion, srVSV is a promising platform for virotherapeutic approaches and also for VSV-based vector vaccines, combining improved safety with an increased coding capacity for therapeutic transgenes, potentially allowing for multipronged approaches.
KeywordsVesicular stomatitis virus Oncolytic virus Virotherapy Malignant glioma
The use of viruses as targeted cancer therapeutics has shown significant promise in the last few years. Especially the vesicular stomatitis virus (VSV), a relatively new player in the oncolytic virotherapy field, has proven to be effective against a variety of tumor entities such as malignant glioma [1, 2], hepatocellular carcinoma [3, 4], prostate cancer [5, 6], and ovarian carcinoma . However, to date, the inherent neurotoxicity of VSV has hindered clinical development since intracerebral administration causes fatal encephalitis in rodents and nonhuman primates [8, 9]. Thus, replication-competent VSV is associated with an increased risk of systemic dissemination and potentially severe pathology if it enters the CNS. Therefore, attenuated virus variants and propagation-deficient viral vectors were generated. Unfortunately, the reduced toxicity of attenuated replication-competent VSV is invariably accompanied with some reduction of replicative and oncolytic activity [10, 11], whereas the major limitation of propagation-deficient viral vectors has been the inefficient transduction rate of cancer cells in vivo [2, 12].
A new strategy to potentially enhance safety of replication-competent VSV while increasing the capacity for therapeutic transgenes is the use of a semireplication-competent vector system similar to those described for retroviruses and adenoviruses [13, 14]. Here, we successfully developed a semireplication-competent vector system for VSV (srVSV), which is based on two trans-complementing propagation-deficient VSV vectors. The genes essential for viral replication are divided onto two separate packageable vector genomes, so that infectious progeny can only be produced in double-infected host cells. Importantly, the VSV RNA genome does not undergo genetic reassortment or recombination, making it unlikely that the binary system reverts into a replication-competent recombinant VSV . In this study, we used the propagation-deficient, eGFP-expressing VSV*ΔG-vector , which lacks the G gene, in combination with de novo synthesized and rescued deletion mutants VSVΔP-DsRed and VSVΔL-DsRed, lacking the genes P and L, respectively, that encode the components of the viral polymerase complex. Accordingly, three different srVSV combinations were feasible: VSV*ΔG/VSVΔP-DsRed (srVSV(ΔG/ΔP)), VSV*ΔG/VSVΔL-DsRed (srVSV(ΔG/ΔL)), and VSVΔP-DsRed/VSVΔL-DsRed (srVSV(ΔP/ΔL)). All srVSV systems allowed for in vitro reciprocal complementation thus leading to copropagation associated with clear antitumor potency against human glioblastoma cell lines. In addition, the most potent vector combination, srVSV(ΔG/ΔL), was tested in a preclinical subcutaneous (s.c.) glioblastoma mouse model and proved to be only slightly attenuated compared to wild-type VSV (VSV-WT). Tumors regressed in both cohorts, but in contrast to the srVSV-treated group, 90% of VSV-WT-treated animals succumbed to viral neurotoxicity. Most importantly, neither srVSV treatment of tumor-bearing animals nor direct intracranial administration in healthy mice was associated with any sign of neurotoxicity. Eventually, all srVSV systems proved to be safe as we have not been able to detect any sign of recombinatory reversion to the wild-type strain.
Materials and methods
BHK-21 baby hamster kidney and U-87 MG human glioblastoma cells were obtained from the American Type Culture Collection (Manassas, VA). G62 human glioblastoma cells were kindly provided by M. Westphal (University Hospital Eppendorf, Hamburg, Germany). HEK 293-NPeGFPL (clone 206) stably expressing VSV-N, P, and L protein were a gift from A. Pattnaik (University of Nebraska, Lincoln, USA) . All cells were kept in a humidified atmosphere containing 5% CO2 at 37°C. BHK-21, U-87 MG, G62, and 293-NPeGFPL cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Perbio Science). 293-NPeGFPL cells were kept under G418 selection.
Quantitative PCR-based multicycle growth curve analysis
BHK-21 cells were infected in 6-well plates (106 cells/well) with a multiplicity of infection (MOI) of 0.05 of each individual vector of the three potential srVSV vector systems or VSV-WT as positive control. Filtered (0.45 μm) supernatants were collected at the indicated time points, and RNA was extracted from 50 μl supernatant using the RNeasy Mini Kit (Qiagen). RNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). Vector propagation was monitored via real-time RT-PCR to determine the total VSV genomic RNA (gRNA) amount in supernatants . Known plasmid amounts were used to determine the standard curve for real-time RNA quantification. Two independent qPCR primer and probe sets were used, spanning the N-P and the M-G IGR of the VSV genome (see Supplementary Fig. S1c, d). Real-time PCR was carried out with the TaqMan® Gene Expression Master Mix (Applied Biosystems) using a LightCycler® 480 Real-Time PCR System (Roche). For both applied real-time PCRs, the detection limit was 102 gRNA/ml.
In vitro cytotoxicity assay
Human glioblastoma cells were plated in 96-well plates at 104 cells/well in 100 μl medium. Cells were cultured as monolayer or multicellular tumor spheroids. For spheroid cultures, 96-well plates were precoated with 75 μl 1% agar noble (Difco). Cultures were infected with the respective viral system (srVSV or VSV-WT) at an MOI of 0.2 or treated with phosphate-buffered saline (PBS) the following day. Cell viability was assayed in dodecaplicates in n = 3 independent experiments at the indicated time points postinfection using the cell proliferation agent WST-1 (Roche). Results are expressed as percentage of viable cells compared to PBS-treated controls.
For antitumor efficacy testing, 6-week old NOD/SCID mice (Jackson Laboratories) were anesthetized with isoflurane and 106 G62 human glioblastoma cells were subcutaneously injected into the left and right flanks. Tumor growth was monitored with a caliper. At a tumor volume of 0.1 cm3, mice were treated intratumorally with two doses of either 2.8 × 105 TCID50 srVSV(ΔG/ΔL) or 2.8 × 105 TCID50 VSV-WT and PBS as controls. Bilateral tumors were treated alike. When tumor size exceeded 0.8 cm3, mice were sacrificed. In addition, two mice were sacrificed at 3 days post-srVSV treatment and s.c. tumors were prepared for immunofluorescence analysis.
For neurotoxicity analysis, 6-week old CD1 Swiss mice (Charles River) were anesthetized by intraperitoneal injection of ketamine/xylazine (100 and 10 mg/kg of body weight, respectively). 102, 103, and 104 TCID50 srVSV(ΔG/ΔL), as well as 1.4 × 101 and 1.4 × 104 TCID50 VSV-WT or PBS were stereotactically injected into the right frontal lobe of mice brains (1.5 mm lateral, 2 mm rostral to the bregma at 2 mm depth). Animals were monitored for signs of neurological impairment. Two mice of the 104 TCID50 srVSV(ΔG/ΔL)-treated group were sacrificed at 3 days postinjection (dpi), and brains were prepared for immunofluorescence analysis. The brains were sectioned (40 μm) on a Leica VT1000S vibratome (Leica, Bensheim, Germany). Nuclear counterstaining was performed with TO-PRO-3 iodide (Invitrogen). Sections were analyzed by confocal laser scanning microscopy using a Nikon C1S1 microscope (Nikon, Düsseldorf, Germany). All procedures were approved by the governmental board for the care of animal subjects (Regierungspräsidium Darmstadt, Germany).
Stimulation and IFN-α detection
Murine bone marrow (BM)-derived plasmacytoid dendritic cells (pDCs) were generated as previously described . In brief, BM cells were flushed from femur and tibia with RPMI supplemented with 10% FBS (Perbio Science). Erythrocytes were lysed, cells were washed, and single-cell suspensions were cultivated for 8 days in medium supplemented with 100 ng/ml Flt3-L (R&D Systems). As determined by FACS analysis, Flt3-L cultures consisted of ≈20% CD11c+B220+ pDCs (data not shown). For IFN stimulation experiments, 2 × 106 Flt3-L-stimulated BM-pDC bulk culture cells were seeded per 24 well. Cultures were infected with either srVSV, VSV*ΔG, VSVΔL-DsRed, VSV-WT, or VSV*MQ (each n = 2) at an MOI of 2. Supernatants were collected at 24 h postinfection (hpi) and analyzed for IFN-α via ELISA (PBL Biomedical Laboratories).
For comparison of individual time points or columns, statistical difference was determined using unpaired t test. Mice survival curves were plotted as Kaplan–Meier analysis, and statistical significance between treatment groups was compared using the log-rank test.
Novel recombinant viruses were cloned based on the pVSV-XN2 plasmid background and rescued as described previously . A schematic representation of the VSV vector genomes is shown in Fig. 1a, and their identity was confirmed by gene-specific RT-PCR (Supplementary Fig. S1a, b). Both deletion mutants, VSVΔP-DsRed and VSVΔL-DsRed, were unable to propagate and did not generate progeny virions in cell cultures not providing the respective deleted viral gene in trans, as real-time RT-PCR (Supplementary Fig. S1c, d) of supernatants were negative for VSV gRNA (data not shown).
srVSV(ΔG/ΔL) is the most potent srVSV system in terms of vector propagation
In order to assess the replication competence of the three potential srVSV systems, BHK-21 cells were infected with an MOI of 0.05 of each individual vector or VSV-WT as control to generate multicycle growth curves. Vector propagation was monitored on the gRNA level via real-time RT-PCR . In VSV-WT-infected cultures, gRNA associated with secreted progeny virions was first detectable at 6 hpi, reaching a plateau around 12–18 hpi with maximum titers of more than 8 × 108 gRNA/ml (8.77 × 108 ± 9.28 × 107 gRNA/ml, see Fig. 1b). In comparison, all srVSV vector systems showed an earlier onset of replication with first gRNA detectable at 3 hpi and srVSV(ΔP/ΔL) being the most potent in the initial phase with titers of 5.33 × 104 ± 3.05 × 103 gRNA/ml 3 hpi. Both, the srVSV(ΔP/ΔG) and the srVSV(ΔG/ΔL) system lagged behind with titers being about tenfold reduced 3–6 hpi. Consistently, srVSV(ΔP/ΔL) was also the first to reach its plateau at 10–12 hpi with a maximum of 8.44 × 107 ± 3.63 × 106 gRNA/ml before its titer slowly started to regress. In contrast, both srVSV(ΔP/ΔG) and srVSV(ΔG/ΔL) ended up with a more robust replication, reaching titers of 1.19 × 108 ± 1.63 × 106 gRNA/ml for srVSV(ΔP/ΔG) and 7.60 × 108 ± 4.47 × 107 gRNA/ml for srVSV(ΔG/ΔL) at 24 hpi. Thus, the binary system using VSV*ΔG and VSVΔL-DsRed was the most potent srVSV system in terms of vector dissemination even reaching maximum gRNA titers comparable to VSV-WT.
1.58 × 108
4.22 × 106
2.37 × 105
4.22 × 104
srVSV systems are characterized by asymmetric copropagation
As srVSV systems are composed of two vectors with different properties such as gene composition and genome size, the mode of copropagation during the multicycle growth curve was analyzed via two independent qPCRs with amplicons spanning the N-P or M-G IGR of the VSV genome (Supplementary Fig. S1c, d). Combining the obtained qPCR data, single-vector titers were calculated as ratio of the individual vector gRNA per milliliter per total vector gRNA per milliliter for all time points of the multicycle growth curve. As ratios proved to be consistent for each srVSV system throughout the whole observation period of 24 h, time-independent means and standard deviations were calculated. Indeed, the assessment revealed that vector copropagation was not due to symmetric replication of both vector genomes, but could rather be characterized as an asymmetric process (Fig. 1c). Each srVSV system could be defined by a distinct preference of one vector over the other. In case of srVSV(ΔP/ΔG), the VSVΔP vector accounts for 65.49±2.16% and VSVΔG for 34.51 ± 2.16% of the total titer. Even more pronounced is the asymmetry in favor of the VSVΔL vector with a share of 65.99 ± 6.78% (ΔP/ΔL) and 99.55 ± 14.03% (ΔG/ΔL) of the total progeny generated.
Time lapse fluorescence microscopy of srVSV(ΔG/ΔL) copropagation
srVSV shows no sign of recombinatory reversion to replication competence
Next, we also looked directly for potential recombination events by RT-PCR as a more sensitive means. cDNA of the 20th passage of the srVSV systems and the respective spiking controls was prepared and used to perform a nested PCR. The outer PCR selectively amplified the genome of one recombinant vector out of the binary system as the reverse primer binding site is constituted in the gene deletion of the other vector genome (Supplementary Fig. S2). For srVSV(ΔG/ΔL) the VSV*ΔG genome was amplified, whereas for srVSV(ΔP/ΔG) and srVSV(ΔP/ΔL) the VSVΔP-DsRed genome was amplified. The inner VSV gene-specific PCRs then allowed us to check for potential recombination events at the locus of the actual gene deletion. Consistent with the phenotypic analysis (Fig. 3a), we have not been able to detect any recombination event for the srVSV systems. The VSV-G (for srVSV(ΔG/ΔL)) and VSV-P gene (for srVSV(ΔP/ΔG) and srVSV(ΔP/ΔL)) were not detectable, whereas the according amplicons were detected for the spiking control (see Fig. 3b). These data were corroborated by sequence analysis of the outer PCR amplicons, which clearly evidenced presence of the respective fluorescence marker gene (data not shown). Thus, in both, the phenotypic and genotypic analysis, recombination among the vector genomes was not detectable while the respective spiking controls were positive, the latter validating the applicability of the applied assays.
srVSV exhibits antitumor activity in vitro and in vivo
srVSV(ΔG/ΔL) exhibits reduced neurotoxicity compared to VSV-WT
srVSV(ΔG/ΔL) is a potent type I IFN inducer
As it was previously shown that type I IFN-inducing strains of VSV were strongly attenuated regarding their toxicity , the capacity of srVSV(ΔG/ΔL) to induce IFN was evaluated in murine pDC cultures. pDCs were infected with either srVSV(ΔG/ΔL), VSV*ΔG, VSVΔL-DsRed, or VSV-WT as negative and VSV*MQ as positive control at an MOI of 2. Culture supernatants were collected at 24 hpi and analyzed for IFN-α via ELISA. Unsurprisingly, the VSV-M multimutated positive control VSV*MQ induced the strongest type I IFN response, with 3,200.11 ± 57.02 pg IFN-α per ml supernatant, whereas VSV-WT treatment induced 18-fold lower amounts (174.47 ± 16.70 pg/ml; p < 0.0001), being consistent with Waibler et al. . In striking contrast to VSV-WT, srVSV(ΔG/ΔL) proved to be a very potent type I IFN inducer with significantly elevated IFN-α levels of 1,035.52 ± 50.57 pg/ml (p < 0.0001). However, single-vector treatment with propagation-deficient VSV*ΔG and VSVΔL-DsRed induced only basal IFN-α levels.
Here, we developed a semireplication-competent VSV vector system composed of two separate propagation-incompetent viral vectors that shows significant anticancer activity without the neurotoxicity usually found in VSV infection of mice and nonhuman primates. Three different vector pairs of three separate propagation-incompetent vectors VSVΔP-DsRed, VSV*ΔG, and VSVΔL-DsRed were feasible, and all three combinations were able to effectively trans-complement each other and generate progeny virions (Figs. 1b, c and 2 and Table 1).
The combination of VSV*ΔG and VSVΔL-DsRed proved to be the most potent in terms of vector propagation (Fig. 1b, Table 1) and in vitro antitumor efficacy (Fig. 4), being only slightly attenuated compared to VSV-WT. That this combination outperforms the other srVSV systems is most likely due to the small genome size of the VSV-L gene deleted VSVΔL-DsRed vector (5.6 kB, Fig. 1a). Compared to full-length VSV, the smaller sized VSVΔL-DsRed genome is statistically favored for replication, packaging, and viral shedding. This is consistent with VSV-defective interfering particles, as apart from the 5′–3′ terminal complementarity as major determinant, genome size was also shown to impact their replicative dominance over VSV-WT [15, 27]. Moreover, the asymmetric contributions of either vector genome and particularly the overrepresentation of VSVΔL-DsRed in the srVSV(ΔG/ΔL) and the srVSV(ΔP/ΔL) system (Fig. 1c) as well as the fact that both VSVΔL-DsRed containing srVSV systems exhibit superior antitumor efficacy compared to srVSV(ΔP/ΔG) (Fig. 4) can also be explained by the genome size-dependent replicative advantage. In fact, despite the low initial MOI of 0.2, coamplification led to a nearly complete tumor cell killing in vitro at 96 hpi for glioma monolayer and spheroid cultures, underscoring the strong oncolytic potential of both srVSV(ΔG/ΔL) and srVSV(ΔP/ΔL). However, compared to srVSV(ΔP/ΔL), the (ΔG/ΔL) combination proved to be somewhat more potent in terms of vector propagation. The VSV*ΔG vector, which lacks the ability to produce infectious virus but still exhibits functional replication and transcription, provides the L polymerase for immediate VSVΔL-DsRed replication and transcription upon coinfection. This is opposed to srVSV(ΔP/ΔL), as here only double-infected cells support efficient genome replication and transcription, which might explain for the marginal reduced replication competence of the latter system. Accordingly, the most potent srVSV system, srVSV(ΔG/ΔL), was assessed for its antitumor potency in a s.c. G62 human glioblastoma xenograft model. All srVSV-treated tumors showed a clear response starting at 2 dpi. Tumors regressed and viral dissemination could be detected throughout the neoplastic tissue by immunohistochemistry (Fig. 5a, b). Although, compared to VSV-WT-treated mice tumor regression was significantly slower, srVSV treatment was not associated with any severe adverse effects (Fig. 5c). Whereas 90% of mostly (70%) tumor-free VSV-WT-treated animals had to be euthanized due to neurotoxicity, srVSV treatment resulted in long-term survival of all animals with 80% tumor clearance at 100 dpt compared to both control cohorts. Thus, it can be assumed that replication of srVSV is self-contained to the injection site and adjacent areas of the topically treated tumor, reducing the risk of a systemic infection or dissemination.
On the other hand, this intrinsic safety of binary srVSV systems may reduce therapeutic efficacy upon systemic application, as cells within target tissue need to be double infected to trigger oncolytic copropagation in the tumor. However, when delivered locoregionally, the srVSV system indeed exhibits a vastly improved therapeutic index when compared to VSV-WT, as even after direct intracerebral administration of escalating viral doses into mouse brains no toxicity could be observed. All srVSV-treated mice (low- and high-dose cohorts) showed 100% event-free survival up to the end point of the study (40 days, Fig. 6a). Again, immunohistochemistry of mice sacrificed at 3 dpi displayed locally restricted srVSV copropagation at the needle track and its close proximity (Fig. 6b). In contrast, both low- and high-dose cohorts of VSV-WT-treated mice developed neurotoxicity with a median survival of 7.5 dpi (1.4 × 101 TCID50) or even 4.5 dpi for the high-dose cohort (1.4 × 104 TCID50, Fig. 6a). Hence, in direct comparison with VSV-WT, the srVSV-associated neurotoxicity is at least >700-fold reduced while retaining its potent oncolytic activity.
In addition, the srVSV systems were also safe with respect to potential recombinatory reversion to a replication-competent phenotype, as after 20 consecutive passages at limiting dilutions, phenotypic and genotypic analyses were negative for recombinant replication-competent virus, with VSV-WT spiked positive controls validating the assays (Fig. 3). This observation is also consistent with earlier studies on the potential recombination between temperature-sensitive VSV mutants .
In summary, relative to VSV-WT, srVSV systems present a promising platform for virotherapeutic approaches, as they are genetically stable and exhibit considerably reduced neurotoxicity while retaining their antitumor potency. Furthermore, srVSV systems offer a strongly increased coding capacity so that both viral vectors can be “armed” to express therapeutic transgenes allowing for multipronged approaches, combining their inherent oncolytic effect with a tumor microenvironment modulating suicide and/or immunostimulatory “payload” to boost antitumor potency. Eventually, with respect to both biosafety and coding capacity, srVSV systems may not only prove valuable for oncolytic virotherapy but also represent an attractive vector vaccine platform.
This work was supported by grants from the Wilhelm-Sander-Foundation and the Schering foundation Deutsche Forschungsgemeinschaft (Graduate College 1172). We thank Stefan Momma and Anna Kraft for CLSM assistance.
Disclosure of potential conflict of interests
The authors declare no conflict of interests related to this study.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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