Next we compared the protease
Next, we compared the protease-specificity of viruses NS116-GFP/AE and NS116-GFP/AT for trypsin, neutrophilic elastase or supernatant of human neutrophils. For later purposes, we determined the viral yield in a co-cultivation system of infected Vero prolyl hydroxylase inhibitor with human neutrophils. The NS116-GFP/elastase-activated influenza virus (AE virus) grew to the same titers in the presence of neutrophils and in the presence of neutrophilic elastase, which were 7.06 log TCID50/ml and 6.8 log TCID50/ml, respectively (Figure 3A). This virus completely lost its reproductive activity in the presence of trypsin. In contrast, the NS116-GFP/AT virus grew to the titer of 7.6 log TCID50/ml in the presence of trypsin, but not in the presence of neutrophilic elastase or supernatants of neutrophils, indicating the specificity of the cleavage site. These titers were in correlation with the GFP expression in infected cells (Figure 3B). Our findings support the hypothesis that tumor-infiltrating immune cells, such as neutrophils, would produce elastase that is sufficient for HA cleavage of the NS116-GFP/AE virus replicating in tumor cells during oncolytic therapy.
To further address the growth properties of the elastase activated virus, we compared multi-cycle growth of NS116-GFP/AE and NS116-GFP/AT in Vero and B16f1 cells in the presence of neutrophilic elastase or trypsin, respectively. Change of the cleavage site in HA protein had no influence on the growth characteristic between the two viruses (Figure 4). Also, the modification of the HA-cleavage site did not lead to the growth disadvantages of NS116-GFP/AE virus in human tumor cell lines, such as PANC-1 or A375 (Figure 4). Trypsin-sensitive NS116-GFP/AT virus showed significantly higher titer in comparison with elastase-sensitive NS116-GFP/AE virus on the CaCo2 cell line. This might be explained by the endogenous production of trypsin-like serine proteases in this cell line allowing HA cleavage (Figure 4). None of the tested cell lines were able to support the replication of NS116-GFP/AE virus without a source of exogenous elastase (Figure S3). As expected the NS116-GFP/AT virus could replicate only in CaCo2 cells without an addition of trypsin (Figure S3).
To test the oncolytic potential of NS116-GFP/AT and NS116-GFP/AE, we intratumorally injected these viruses into a syngeneic murine B16f1 model (Figure 5A). Administration of both, NS116-GFP/AT or NS116-GFP/AE viruses significantly inhibited tumor outgrowth, when compared to the control group (p < 0.05). There was no substantial difference between the two viruses. ΔNS1-H1N1 virus, a corresponding replication-deficient deltaNS1 virus lacking the complete NS1 open reading frame, had no therapeutic effect in this model (Figure S4). This complete NS1 deletion virus did not grow in B16f1 cells, indicating the critical role of virus replication for oncolytic activity.
The pancreatic cell line PANC-1 was chosen to study the oncolytic effect of the NS116-GFP/AE virus in a human tumor xenograft model (Figure 5B). It had been previously shown that influenza virus has an oncolytic activity in pancreatic cell lines. Interestingly, despite both viruses growing to comparable titers in PANC-1 cells in vitro, the NS116-GFP/AE virus had a significantly better therapeutic effect in this model compared to the NS116-GFP/AT virus (p < 0.05). We next determined the presence of influenza virus in tumor tissue by immunohistochemistry (Figure 6). This analysis indicated that both viruses replicated 7 days post-virus injections in the malignant cells as shown by the overlapping staining pattern of HA-specific and tumor-cell-specific signals. We did not observe a positive staining signal for neutrophils. This suggests that virus-activating elastase might have been produced by other cellular sources.
Discussion The use of potentially pathogenic viruses for oncolytic purposes calls for the development of multiple attenuation markers within the virus, which do not inhibit efficient growth in malignant cells. In IAV, we have previously defined such a marker when deleting a nonstructural protein NS1 coded by segment 8. This study examined another attenuation characteristic for conditional growth in tumors. An altered cleavage site of the hemagglutinin coded by segment 4 achieved the attenuation. Similar to the marker “NS1 deletion,” the marker “elastase cleavage site” attenuates the virus in the upper respiratory tissue, as has been previously shown.16, 24 Using two different murine models, we demonstrated that the introduction of the elastase cleavage site into the oncolytic influenza virus does not diminish the therapeutic properties of this oncolytic prototype in vivo. It should be noted that replication defective influenza viruses have little effect in tumor models, such as B16f1 melanoma (Figure S4). Thus, we conclude that the attenuation marker “elastase cleavage site” leads to a conditional replication of the virus in tumor tissue.