Phos-tag To counteract innate cell intrinsic host defenses t

To counteract innate, cell intrinsic host defenses that limit Δ34.5 OV replication within tumors, variants were isolated that expressed the HSV-1 Us11 protein, normally produced late in the lifecycle, at immediate-early (IE) times (Mohr and Gluzman, 1996; Mulvey et al., 1999). Remarkably, Δ34.5 OVs expressing IE Us11 remained neuroattenuated (Mohr et al., 2001), but effectively countered interferon-induced, cell-intrinsic anti-viral responses (Mulvey et al., 1999, 2004), replicated substantially better within tumors, and were more effective anti-tumor agents in pre-clinical studies (Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003). Indeed, a related Δ34.5, IE Us11-expressing HSV-1 has completed US phase III trials (Andtbacka et al., 2015; Dolgin, 2015) and a biologics license application recently approved by the FDA (http://www.fda.gov./BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/ucm469411.htm). However, the genetic alteration enabling IE-Us11 expression also deleted the neighboring HSV1 gene encoding the ICP47 immunomodulator (Mohr and Gluzman, 1996; He et al., 1997; Mulvey et al., 1999; Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003). By inhibiting TAP, ICP47 down-regulates cell surface MHC class I expression and allows HSV-1 to complete its productive growth program despite the presence of host anti-HSV-1 CD8+ T-cells (Früh et al., 1995; Hill et al., 1995). Without ICP47, increased clearance of infected cells by CD8+ T-cells could severely restrict OV spread through the tumor impacting both direct oncolysis and anti-tumor immune response development. This limitation is likely critical given the prevalence of HSV-1 seropositive individuals, the rapid seroconversion of sero-negative patients after HSV-1 OV exposure (Hu et al., 2006), and the finding that evading CD8+ T-cells facilitates herpesvirus super-infection of seropositive hosts (Hansen et al., 2010). Surprisingly, both the role of CD8+ T-cell evasion and how viral immuno-modulators impact OV therapy remain unknown in part because human HSV-1 ICP47 has a low affinity for rodent TAP, impairing proper assessment of its biological function in rodent models (Ahn et al., 1996; Tomazin et al., 1996). Moreover, while removing the viral TAP inhibitor was proposed to benefit OV therapy by improving its immune stimulating properties, a direct comparison of how TAP inhibition impacts OV efficacy was never performed (Liu et al., 2003; Dolgin, 2015). Here, we address this problem by isolating an HSV-1 OV armed with the bovine herpesvirus 1 (BHV-1) TAP-inhibitor (UL49.5), which unlike its HSV-1 analog, antagonizes rodent and human TAP (Koppers-Lalic et al., 2005; Verweij et al., 2011a). Significantly, UL49.5-expressing OVs showed superior efficacy treating Phos-tag and breast cancer in murine pre-clinical models that was dependent upon a CD8+ T-cell response. In addition to treating directly injected, subcutaneous (sc) tumors, UL49.5-OV therapy reduced untreated, contralateral sc tumor size and naturally occurring metastasis. This shows that incorporating a TAP inhibitor into an OV induces both local and systemic antitumor responses following intratumoral administration. Moreover, it establishes arming OVs to evade CD8+ T-cells as an effective OV immunotherapy strategy that may applicable across many OV platforms.
Material and Methods
Results To produce a neuroattenuated HSV-1 OV that effectively replicates in tumors and blocks cytosolic peptide display by MHC-I on the surface of infected mouse and human cells, a recombinant deleted for both γ134.5 virulence loci (Δ34.5) was engineered to express HSV-1 Us11 as an IE gene and produce the BHV-1 UL49.5 TAP inhibitor (Fig. 1a, BV49.5). Importantly, while Δ34.5 HSV-1 OVs expressing IE-Us11 counter the limited host defenses in cancer cells and preferentially replicate in tumors, replication and spread of γ134.5-deficient HSV-1 is highly restricted in normal cells and tissues by potent, cell-intrinsic antiviral responses that restricts their growth (Chou et al., 1990; Mohr and Gluzman, 1996; Toda et al., 1999; Rampling et al., 2000; Markert et al., 2000; Mohr et al., 2001; Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003; Mulvey et al., 2004; Hu et al., 2006; Harrington et al., 2010; Senzer et al., 2009; Andtbacka et al., 2015). Furthermore, Δ34.5 OVs expressing IE-Us11 are non-pathogenic in rodents and were also shown to be safe in human trials (Mohr et al., 2001; Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003; Hu et al., 2006; Andtbacka et al., 2015). To avoid introducing additional alterations to the Δ34.5 viral genome, a cassette containing UL49.5 and IE-Us11 genes was targeted by homologous recombination to replace loci formerly occupied by γ134.5-encoding genes. Due to a complex mechanism involving both N and C terminal protein domains, it has not been possible to isolate minimal amino acid substitutions within the UL49.5 ORF that selectively ablate UL49.5 TAP inhibitory activity (Loch et al., 2008; Verweij et al., 2011b; Wei et al., 2011). Instead, an otherwise isogenic variant differing by one additional nucleotide within UL49.5 that results in a frameshift (FS) and is unable to produce functional UL49.5 was constructed (BV49.5-FS). Physical genomic analysis of viral recombinants was performed using stocks grown up following three consecutive limiting dilution steps to purify single plaques. The genome structure was verified by Southern analysis (Fig. 1b) and indicated that the recombinant viruses are genetically stable even after the extensive amplification associated with three sequential plaque purifications and subsequent high-titer stock preparation. Note that while Bsu36l terminal fragments of WT length (Fig. 1b, fragments marked a-WT) are readily observed in DNA isolated from WT HSV-1-infected cells, they were not detected in samples from cells infected with BV49.5 or BV49.5-FS. Instead, Bsu36l terminal fragments in BV49.5 or BV49.5-FS migrate slower (Fig. 1b, fragments marked a-BV49.5), consistent with their greater length due to inclusion of Us11 and UL49.5 ORFs. Likewise, internal BsrGI-Bsu36I fragments of WT length (Fig. 1b, fragments marked c-WT) are only detected in WT virus, but not in samples from cells infected with BV49.5 or BV49.5-FS. Instead, internal BsrGI-Bsu36I fragments in BV49.5 and BV49.5-FS modified to include Us11 and UL49.5 ORFs migrated slower reflecting their larger size (Fig. 1b, fragments marked c-BV49.5). As expected, alterations in the length of terminal BsrGl fragments (Fig. 1b, fragments marked b) were not detected in WT, BV49.5, or BV49.5-FS viruses. This shows that BV49.5 and BV49.5-FS recombinants contain the expected modified terminal and internal HSV-1 genome fragments capable of encoding IE-Us11 and functional or non-functional UL49.5 variants.