To investigate how UL expression
To investigate how UL49.5 expression promoted OV anti-tumor activity, we first determined if UL49.5 expression enabled viral persistence in tumors. After establishing bilateral s.c. MBT2 tumors, left flank tumors were treated with BV49.5 or the FS variant as in Fig. 3a. Two days after the final injection, infectious virus load in OV-treated and untreated (contra-lateral) tumors was determined. BV49.5-treated tumors contained on average 7-fold more virus than BV49.5-FS-treated tumors (Fig. 3c) and infectious virus was not detected in untreated, contralateral tumors (not shown). Thus, functional UL49.5 protein promotes OV persistence in tumors presumably by inhibiting TAP in infected cells. Greater OV persistence likely supports more tumor cell oncolysis and fuels development of CD8+ T-cells that recognize tumor antigens. To determine if UL49.5 TAP-inhibitor expression influenced development of anti-tumor, cell-mediated immunity, antigen-stimulated IFNγ release by CTL isolated from vehicle or OV-treated mice was evaluated. While IFNγ secretion by CD8+-enriched splenocytes from BV49.5-FS or vehicle-treated mice in response to mitomycin C-treated MBT2 cell stimulation was not detectable, IFNγ release was significantly elevated by approximately 5-fold only in splenocytes isolated from BV49.5-treated mice (Fig. 3d). This finding is consistent with the notion that UL49.5-mediated TAP inhibition promotes anti-tumor immunity.
To investigate if a TAP inhibitor-armed OV was advantageous in treating other solid tumors, a mouse mammary cancer model was tested (4T1). This well-characterized model allows primary tumor measurements after direct OV delivery and reliably produces readily quantifiable lung metastases, obviating the need to implant tumors at a distant site and allowing us to take advantage of the more physiologically relevant, natural capacity of murine 4T1 breast cancer serine protease inhibitor to metastasize to the lung. Moreover, therapy-induced regression of metastatic tumors is dependent on CD8+ T-cell responses (Demaria et al., 2005). Fig. 4a shows that BV49.5 was more effective than BV49.5-FS at treating primary 4T1 sc tumors. The enhanced anti-tumor activity of BV49.5 vs BV49.5-FS was significant by 13 d after treatment and improved over time (Fig. 4a; table S2). To determine if the efficacy of BV49.5 compared to the FS variant was due to therapy-induced anti-tumor CD8+ T-cells, OV treatment was evaluated in CD8+ T-cell-depleted mice. Significantly, the greater efficacy of BV49.5 over BV49.5-FS was abrogated by CD8+ T-cell depletion (Fig. 4b,c; table S2). Thus, the superior therapeutic activity of BV49.5 was dependent upon: 1) a single nucleotide difference from BV49.5-FS that enabled TAP inhibitor production; and 2) a host CD8+ T-cell response. Remarkably, BV49.5 was likewise more effective than BV49.5-FS in reducing the number of lung metastases, whereas BV49.5-FS was not detectably better than treatment with vehicle alone (Fig. 5a,b). Significant differences in the number of lung metastases were not detected in BV49.5 vs BV49.5-FS vs vehicle alone-treated mice following CD8+-depletion of BV49.5-treated animals (Fig. 5c). Similar to our findings treating primary 4T1 tumors, the superiority of BV49.5 in reducing lung metastases compared to BV49.5-FS was dependent upon production of the UL49.5 TAP inhibitor and intact CD8+ T-cell responses. Thus, therapy of primary tumors with a TAP inhibitor-armed OV effectively treated tumors at distant sites in two different models, one using a preformed tumor at a contralateral site (MBT2) and another that undergoes more physiological dissemination via natural metastases (4T1).
Discussion By restricting OV replication and spread, host CD8+ T cell responses limit direct tumor oncolysis and the development of a systemic anti-tumor response. Although many herpesviruses, including HSV-1, encode a TAP inhibitor to evade CD8+ T-cells and productively replicate in the presence of CD8+ T-cells, the gene encoding the HSV-1 TAP inhibitor ICP47 has been deleted in many OVs (Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003; Hu et al., 2006; Senzer et al., 2009; Harrington et al., 2010; Andtbacka et al., 2015). Since ICP47 is species specific and not functional in rodents (Ahn et al., 1996; Tomazin et al., 1996), its importance previously went unnoticed in preclinical studies. Furthermore, while removing ICP47 has been widely assumed to benefit OV immunotherapy, a direct comparison of how TAP inhibitor expression might impact OV therapeutic efficacy was not previously performed in a responsive model (Lichty et al., 2014). By engineering an HSV-1 OV expressing the BHV-1 UL49.5 TAP inhibitor, which functions in rodent preclinical models and humans (Verweij et al., 2011a), we show that this OV produces superior therapeutic outcomes compared to three independently isolated OVs unable to produce UL49.5. Not only is a UL49.5-expressing OV a more effective anti-tumor agent following local administration into primary tumors, it is also more potent at reducing tumor growth at distant, untreated sites in two different murine solid tumor cancer models. Moreover, the benefit of UL49.5 expression on OV therapeutic efficacy is dependent upon CD8+ T-cells, which may confer long-lived protection. This establishes inhibiting TAP in infected tumor cells as an effective mechanism to promote OV immunotherapeutic action.