Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

Abstract

d Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. Brain tumors are particularly life-threatening due to their sensitive anatomical location. Recently, temozolomide plus radiotherapy has provided a measurable survival benefit to a subset of patients (1), but more effective therapies are still needed. In this regard, oncolytic viruses (OVs) seem particularly promising, as they display higher tumor specificity and possibly fewer side effects than standard therapies (2). One of our OV candidates, attenuated Semliki Forest virus (SFV), was able to fully eradicate orthotopic U87 xenografts in 100% of treated nude mice following a single systemic injection (3). However, in other models, we and others have identified limitations to oncolytic virotherapy; in particular, innate antiviral defenses limit virus replication in tumor cells (4,5).In order to probe further and overcome the mechanisms of glioma resistance to oncolytic SFV, we combined it with oncolytic vaccinia virus (VV), which itself has shown promise in glioma targeting and also has the capacity to facilitate replication of type I interferon (IFN)-sensitive OVs by antagonizing innate antiviral defenses (6)(7)(8). First, we demonstrate efficient killing of DBT mouse glioma cells in vitro but not in vivo by SFV alone, mirroring the results we observed in our previous rat model (4) (Fig. 1A and B). Lack of efficacy could not be explained simply by the immune competence of the animals, as SFV successfully eradicated another type of syngeneic tumor (CT26LacZ) at a similar dose (not shown). Next, we observed that SFV limits its own spread in DBT cells under spatially restrictive conditions (under agarose) and that this limitation could be lifted by coinfecting cells with VV or neutralizing type I IFN using polyclonal antibody (anti-beta interferon [IFN-␤]) or recombinant vaccinia virus-encoded B18R protein (Fig. 1C). Facilitation of SFV spread in DBT cells by VV is dependent on B18R, as we did not see enhancement when B18R-deleted VV was used. While the spread of SFV under agarose was enhanced when SFV was combined with VV, replication of VV itself was strongly inhibited (Fig. 1C), which was also confirmed by quantifying virus output from coinfected DBT cells in free culture (Fig. 1D). DBT cell killing by the virus combinations was synergistic, as calculated by the Chou-Talalay method (Fig. 1E). However, combination of SFV with VV in an orthotopic DBT model did not provide statistically significant improvement in survival compared to the next best therapy, VV alone, by either systemic injection or d...

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma

“…Because under standard cell culture conditions both the virus and secreted proteins, such as IFN-I, may freely diffuse into the supernatant from infected cells, potential low-level para- crine IFN-I signaling between neighboring cells (operating in solid tumors in vivo) may escape detection (10). To test this, we infected monolayers of CT-2A-Fluc cells in 6-well plates with 40 PFU of VA7-EGFP, SFV4, or miRT-124 and overlaid the cells with agarose to assess plaque formation over 3 days.…”

“…Like most alphaviruses, SFV is able to enter the CNS upon systemic delivery, a feature which we have shown can be exploited with a neuroattenuated strain of SFV, VA7, to target brain tumors (9,10). However, in accordance with results showing that SFV infectivity and amplification in nonneuronal CNS cells are regulated by IFN-I (11), both the viral replication and therapeutic efficacy of neuroattenuated SFV vector VA7 were dismal in IFN-I-responsive syngeneic mouse glioma models (10,12).…”

“…Like most alphaviruses, SFV is able to enter the CNS upon systemic delivery, a feature which we have shown can be exploited with a neuroattenuated strain of SFV, VA7, to target brain tumors (9,10). However, in accordance with results showing that SFV infectivity and amplification in nonneuronal CNS cells are regulated by IFN-I (11), both the viral replication and therapeutic efficacy of neuroattenuated SFV vector VA7 were dismal in IFN-I-responsive syngeneic mouse glioma models (10,12). Recent attempts to increase VA7 tumor infectivity and its replication rate by administering to tumor-bearing mice either rapamycin or cyclophosphamide, both of which are known to reduce tumor protection against IFN-I-sensitive vesicular stomatitis virus, were unsuccessful (13), as neither of these drugs led to increased tumor permissiveness to VA7.…”

Bacterial-Mediated Knockdown of Tumor Resistance to an Oncolytic Virus Enhances Therapy

“…1 Previous studies from our lab have identified that localized expression of B18R, a gene from Vaccinia virus which encodes a secreted decoy receptor with a broad antagonizing effect against type 1 IFNs, significantly improved the efficacy of the attenuated VSVΔ51 to grow and kill tumors. 1,5,6 A number of studies have explored combination viral strategies, [7][8][9] but toxicity and safety remain a concern, as knockdown of type 1 IFN in tissues other than tumor presents the risk of susceptibility to unrestricted replication of other infecting viruses. Therefore, a tightly controlled mechanism for tumor-specific B18R production is required to facilitate this strategy.…”

Safe and Effective Treatment of Experimental Neuroblastoma and Glioblastoma Using Systemically Delivered Triple MicroRNA-Detargeted Oncolytic Semliki Forest Virus

“…A7/74 is usually preferred for use as an oncolytic agent due to its natural avirulence in adult rodents. However, type-I IFN a/b (IFNa/b) responses elicited by the host in response to virus infection impede A7/74 efficacy (14,15). The nonstructural proteins nsP3-nsP4 of SFV4 are involved in reducing STAT1 Tyr701 phosphorylation (P-STAT1) upon stimulation of virus infected cells with exogenous IFNb, and it confers its ability to partly resist type-I antiviral defense (16).…”