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  • br Funding This work was partly

    2020-10-22


    Funding This work was partly supported by the grant ‘Maria Grazia Cuccoli’ from Fondazione Cassa di Risparmio di Bologna.
    Declarations of interest
    Acknowledgements
    Introduction The main strategy of oncolytic viral therapy is based on the fact that naturally occurring or engineered viruses are able to conditionally infect tumor PAC-1 and replicate in them, thereby inducing cell lysis. The viral tropism to malignant cells is usually dependent on molecular alterations in cancer cells acquired during carcinogenesis, which leads to the loss of viral defense mechanisms, thus enabling viral amplification. These alterations of cancer cells involve defects in the interferon (IFN), p53, and pRb pathways along with an activation of the Ras/Raf1/MEK/ERK pathway3, 4, 5 or alterations in vessel walls. A number of phase II and phase III clinical trials using vaccinia, herpes virus, or adenoviruses have indicated the beneficial results of virotherapy, calling for the development of less explored virus families to generate clinically applicable prototypes of oncolytic viruses. We have generated an oncolytic influenza A virus (IAV) using the above-mentioned principles of molecular targeting of the virus to cancer cells. Specifically, we have shown that an attenuated vaccine prototype virus which lacks full-length NS1 protein, the viral antagonist of PKR, efficiently replicates in vitro in IFN-defective and RAS mutant cancer cells. Correspondingly, this prototype was effective as an oncolytic agent in different models in vivo. Continuous passaging of the oncolytic IAV resulted in adaptive mutations throughout the viral genome, which led to enhanced stability and growth for both tumor and producer cell lines without affecting pathogenicity. We hypothesized that the modification of the cleavage site of the hemagglutinin (HA), the viral entry protein, might further promote a conditionally replicative growth in tumors. The cleavage of HA by host proteases is an essential step in the life cycle of IAV, allowing multi-cycle replication and viral spread within infected tissue. All IAVs containing a monobasic cleavage site (a single Arg or Lys residues) require the presence of trypsin or trypsin-like serine proteases for activation.11, 12 Such proteases are typically found in the upper respiratory tract as well as in the gastrointestinal tract. For this reason, IAV infection is primarily limited to these sites. It was demonstrated that an IAV containing an elastase cleavage site in its HA is attenuated in mice and pigs.13, 14, 15, 16, 17 Thus, an alteration of HA protease susceptibility can attenuate viral spread. Importantly, malignant tissue is known to express proteases which promote tumor invasion. These proteases are either expressed by invading immune cells, such as neutrophils and macrophages, or by tumor cells themselves. They form a protease-rich environment around those malignant cells facilitating activation of oncolytic viruses. In this study, we investigated whether an attenuated IAV with an elastase-sensitive HA cleavage site is able to replicate in tumor cells and exerts an oncolytic effect in vivo. For this, we modified the HA of a previously generated GFP-expressing tumor adapted influenza A NS116-GFP/A virus. This alteration did not change viral growth characteristics in vitro when the suitable protease was added and multi-cycle viral replication remained when infected cells were co-cultivated with isolated neutrophils. Intratumoral application of the generated virus had an efficient therapeutic effect when different tumor models were used.
    Results To obtain an elastase-dependent viral vector, we took advantage of the high mutability of influenza viruses and their ability to adapt. A trypsin-dependent influenza ΔNS1-H1N1 virus expressing an HA from influenza A/New Caledonia/20/99 virus was serially passaged on Vero cells in the presence of porcine pancreatic elastase. After 6 passages, we obtained a virus ΔNS1-H1N1-E, which could grow to a titer of 7 log 50% tissue culture infective dose (TCID50/ml) on Vero cells in the presence of porcine pancreatic elastase. Interestingly, the resulting virus was also sensitive to human neutrophil elastase activation (Figure S1). Sequence analysis of the HA gene revealed three nucleotide changes at positions 1056, 1060, and 1061 from T, G, A to C, T, G, respectively. Those changes corresponded to two amino acid substitutions at the cleavage site of the HA molecule: serine and arginine at positions 342 and 343 were replaced by proline and isoleucine, respectively (Figure 1). These mutations were then introduced into the genome of the previously described NS116-GFP/A virus by side-directed mutagenesis of the HA coding plasmid using standard reverse genetics methods developed for influenza virus.21, 22 Obtained mutant was named NS116-GFP/AE and was sensitive to activation by porcine pancreatic elastase and human neutrophil elastase (Figure 2). The parental virus which is activated by trypsin was named “NS116-GFP/AT.” We used this virus, as it has previously been shown to serve as a stable viral vector. Moreover, NS116-GFP/AT did not show any toxicity when systemically administrated by the intravenous route at a titer of 108 TCID50 in mice (Figure S2). No infectious virus was isolated from brain, lungs, liver, gut, muscle, or kidney, indicating the lack of virus dissemination after systemic administration. Since a significant attenuation of elastase-dependent influenza virus in comparison with trypsin-dependent virus in mice model has been already shown,15, 17 we did not perform further biodistribution assays.