Construction of oncolytic reporter influenza viral vectors and assessment of their safety following intracranial administration in rats Текст научной статьи по специальности «Биотехнологии в медицине»

MIRJournal

MicrobioLogy Independent Research JournaL

https://doi.org/10.18527/202411105118 RESEARCH PAPER

Construction of oncolytic reporter influenza viral vectors and assessment of their safety following intracranial administration in rats

Pulkina A. A.1# , Mustafaeva A. S.1 , Romanovskaya-Romanko E. A.1 , Plotnikova M. A.1 , Ozhereleva O. O.1 , Shuklina M. A.1 , Kiseleva L. N.2 , Alekseeva Y. S.3 , Kurmazov N. S.3 , Muslimov A. R.3 , Shurygina A.-P. S.1 , Stukova M. A.1

1 Smorodintsev Research Institute of Influenza, 15/17 Professora Popova Str., St. Petersburg, 197022 Russia

2 A. M. Granov Russian Research Center for Radiology and Surgical Technologies, Pesochny district, 70 Leningradskaya str., St. Petersburg, 197758 Russia

3 Center for Molecular and Cell Technologies, Saint Petersburg State Chemical and Pharmaceutical University, 14 Professora Popova Str., lit. A, St. Petersburg, 197022 Russia

ABSTRACT

INTRODUCTION: Oncolytic viruses are a promising approach for treating malignant brain tumors as part a of combination therapy. OBJECTIVE: To develop reporter influenza A viruses expressing NanoLuc luciferase and evaluate their safety following intracranial administration in rats.

METHODS: Chemiluminescent reporter influenza A virus strains were obtained by reverse genetics. The NS genetic segment of the T_NS124-Luc and E_NS124-Luc strains encoded a fusion protein that combined NS1124 and NanoLuc. In the T_NS124-2A-Luc and E_NS124-2A-Luc strains, the NS1124 and NanoLuc sequences were separated by a 2A co-translational cleavage site. To enhance the tumor specificity of the viruses, the trypsin cleavage site (T) in the hemagglutinin (HA) protein was replaced with an elastase cleavage site (E) by introducing S342^P and R343^I substitutions in the HA region of the E_NS124-Luc and E_NS124-2A-Luc constructs.

RESULTS: The obtained constructs demonstrated comparable reproductive and luminescent activity in MDCK cells. However, vectors containing the 2A site upstream of the transgene infected the glioma cell lines C6, A172, and T98G more effectively. Intracranial administration of a high dose of the virus was safe, causing no neurological or other pathological symptoms in rats. In addition, the luminescent reporter NanoLuc was expressed at the injection site without the formation of active viral progeny. CONCLUSION: This study demonstrated that a chemiluminescent influenza A virus strain can induce transgene expression at the site of intracranial injection without active viral replication.

Keywords: oncolytic virus, influenza vector, luminescent activity, intracranial administration, glioma

# For correspondence: Anastasia Pulkina, Smorodintsev Research Institute of Influenza, 15/17 Professora Popova Str., St. Petersburg, 197022 Russia, e-mail: [email protected]

Citation: Pulkina AA, Mustafaeva AS, Romanovskaya-Romanko EA, Plotnikova MA, Ozhereleva OO, Shuklina MA,

Kiseleva LN, Alekseeva YS, Kurmazov NS, Muslimov AR, Shurygina A-PS, Stukova MA. Construction of oncolytic reporter

influenza viral vectors and assessment of their safety following intracranial administration in rats. MIR J. 2024;11(1):105-18.

https://doi.org/10.18527/202411105118.

Received: December 6, 2024

Accepted: December 16, 2024

Published: December 30, 2024

Copyright: © 2024 Pulkina et al. This is an open access article distributed under the terms of the Creative Commons Attribution International Public License (CC BY), enables reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator.

Funding: The work was carried out as part of a state assignment from the Ministry of Health of the Russian Federation for 20242026: Preclinical studies and development of a finished dosage form of an oncolytic vector based on a modified influenza A virus for the complex therapy of malignant gliomas. Conflict of interests: The authors declare no conflicts of interest.

INTRODUCTION

Glioblastoma is the most common primary malignant brain tumor, characterized by rapid growth of the primary tumor node, high invasiveness, and a pronounced tendency for metastasis and disease relapse [1, 2]. Despite the widespread use of standard triple therapy for malignant gliomas in adults, which includes tumor resection followed by chemotherapy and radiotherapy, patient survival rates remain critically low (only 50%) and an average survival time is approximately 15 months. The challenge lies in the recurrence of tumor growth in the same area, as it is impossible to remove all tumor cells during surgery, coupled with the resistance of some cells to both radio-and chemotherapy [3].

Oncolytic viral therapy, which targets the direct lysis of tumor cells, modification of the tumor microenvironment, inhibition of tumorigenic vascularization, and activation of specific T cell immunity against tumor antigens, represents a promising approach for the treatment of malignant gliomas [4, 5].

The oncolytic virus can be injected directly into the tumor during angiography before radical surgery and/or act synergistically with chemotherapy and radiotherapy, destroying tumor cells resistant to therapeutic effects and affecting the tumor microenvironment.

Currently, numerous research studies are being conducted to enhance the effectiveness of malignant glioma treatment using various oncolytic viruses with natural or induced oncolytic activity and selective tropism for tumor cells [4-6]. Various strategies are being developed to enhance the effectiveness of viral tumor treatment, in which the virus acts as a vector expressing immunoactive molecules, such as chemokines, cytokines, or nanoantibodies targeting PD-L1, that modulate the tumor microenvironment and/or amplify the virus oncolytic effect [7, 8].

The oncolytic activity of the modified influenza virus has been demonstrated in several preclinical studies [9-14]. General conclusions drawn from these studies, including our own experience, are: (i) the effectiveness of oncolytic therapy directly depends on the administered dose of the virus (viral concentrates with 1010/ml viral particles have maximum activity); (ii) it is safe to administer the oncolytic virus in high titers directly into the tumor tissue; (iii) influenza viruses with a modified ns1 gene have increased oncolytic activity due to enhanced stimulation of the innate immune system in the area of virus application, (iv) high genetic plasticity of the RNA-segmented genome of the influenza virus allows selection of a strain adapted to a specific tumor type.

Elastase-dependent influenza viruses, obtained through targeted mutagenesis or selection, may offer an additional advantage in terms of selective action and enhanced efficiency of viral particle penetration into host cells [15]. Since several human tumors, including glioblastomas, exhibit increased elastase activity due to neutrophil infiltration, elastase-dependent influenza viruses have the potential for multi-cycle replication within the tumor. In contrast, their replication is limited in healthy tissues, where elastase enzymatic activity is typically absent.

In the present study, we developed genetically stable trypsin-dependent and elastase-dependent reporter influenza vectors with high infectious and reporter activity, capable of infecting glial cells. In vivo experiments in rats demonstrated that intracranial administration of the virus at a high dose is safe for the animals and leads to the accumulation of luciferase at the injection site, without the formation of infectious viral progeny.

MATERIALS AND METHODS

Plasmids

The pHW-PR8-NS124-2A-Luc plasmid was obtained based on the previously constructed pHW-PR8-NS124-Luc plasmid [16, 17], in which the protein-coding sequences (NS1124 and NanoLuc) were separated by the P2A peptide (ATNFSLLKOAGDVEENPGP) of Porcine Teschovirus 1 [18], which provides ribosome slippage (Eurogen, Russia). The plasmid encoding the hemagglutinin (HA) of the influenza virus A/Puerto Rico/8/1934 (H1N1) (A/PR/8/34) with mutations S342^P, R343^I [15] was obtained by site-directed mutagenesis (Eurogen, Russia). Plasmids encoding internal and surface proteins of the influenza virus A/PR/8/34 were obtained from the collection of the Laboratory of Vector Vaccines of the Smorodintsev Research Institute of Influenza.

Cell cultures

Vero cells (ATCC #CCL-81, USA) were cultured in OptiPro medium (Gibco, USA) supplemented with 2% GlutaMax (Gibco, USA). MDCK (#FR-58; IRR, USA), A172, T98G and T2 cells (obtained from A. M. Granov Russian Scientific Center of Radiology and Surgical Technologies, Russia) were grown in AlphaMEM medium (Biolot, Russia) supplemented with 10% SC-biol fetal serum (Biolot, Russia). Rat glioma C6 cells (Smorodintsev Research Institute of Influenza) were cultured in DMEM medium (Biolot, Russia) supplemented with 20% SC-biol serum, 1% GlutaMax and 1% sodium pyruvate (Gibco, USA).

Virus construction

Western blot analysis

Recombinant influenza virus A/PR8-NS124-Luc (T_NS124-Luc) was obtained previously [16, 17]. To assemble recombinant viral vectors T_NS124-2A-Luc, E_NS124-Luc, and E_NS124-2A-Luc, Vero cells were transfected with a set of 8 bidirectional plasmids [19] using the Nucleofector II (Amaxa) and the Nucleofection Kit V reagent kit (Lonza #VCA-1003, Switzerland). Trypsin-dependent strains T_NS124-Luc and T_NS124-2A-Luc were propagated in 10-12-day-old developing chicken embryos (CE) (Sinyavinskaya Poultry Farm, Russia), while elastase-dependent strains E_NS124-Luc and E_NS124-2A-Luc - in MDCK cells in the presence of 0.5 pg/ml elastase (Promega, USA).

Measurement of virus infectious activity

The infectious activity of the viruses was assessed using the limiting dilution assay in MDCK and Vero cell cultures, or in the CE. The virus dilutions were prepared in the AlphaMEM or OptiPro medium with the addition of an antibiotic-antimycotic (Gibco, USA) and protease (1 pg/ml TRCK-trypsin or 0.5 pg/ml elastase) and used for infection of MDCK and Vero cells. For infecting CE, the viruses were diluted in DPBS buffer (Biolot, Russia) with the addition of an antibiotic-antimycotic. The 50% tissue culture infectious dose (TCID50) or egg infectious dose (EID50) was calculated according to the Reed and Mench method and expressed in log10 [20].

RT-PCR

Viral RNA was isolated using the RNeasy Mini Kit (Oiagen, Netherlands). The genetic material was amplified using the BioMaster RT-PCR-Extra reagent kit (Biolabmix, Russia) and specific primers to the NS segment [21]. RT-PCR with real-time detection was performed using the BioMaster RT-PCR-Extra (2x) reagent kit (Biolabmix, Russia), InfA-F and InfA-R primers, and the InfA-P oligonucleotide probe.

Measurement of luciferase activity

Luciferase enzymatic activity was measured using the Nano-Glo Luciferase Assay System reagent kit (Promega, USA), dark-walled plates (Thermo Fisher Scientific, USA) and a CLARIOstar multiphotometer (BMG LABTECH, Germany). In vivo chemiluminescence was assessed using the IVIS SpectrumCT In Vivo Imaging System (PerkinElmer, USA).

MDCK cells infected with recombinant strains at a dose of 1.0 log10 TCID50/cell were incubated for 18 h, lysed, and used for electrophoresis in a polyacrylamide gel (Any kD Mini-PROTEAN TGX Stain-Free Protein Gel, Bio Rad, USA). The separated proteins were transferred to a nitrocellulose membrane (Trans-Blot Turbo Transfer Packs, Bio Rad, USA) and stained with monoclonal antibodies 1H7 against the NS1 protein of the influenza virus [22], followed by development with Goat Anti-Mouse IgG H&L (HRP) conjugate (Abcam, UK) and Pierce 1-Step Ultra TMB-Blotting Solution substrate (Thermo Fisher Scientific, USA).

Quantification of infected cells by flow cytometry

The cells were infected with recombinant strains at a dose of 7.0 log10 TCID50/cell and incubated for 18-20 h in a medium containing 2% serum. Staining was performed using the Zombie Aqua Fixable Viability Kit (BioLegend, USA) and FITC-labeled antibodies to the nucleoprotein of the influenza A virus (DDMP, Russia). Analysis was performed using CytoFLEX flow cytometer (Beckman Coulter, USA) and the Kaluza Analysis v2 software (Beckman Coulter, USA).

Laboratory animals

Wistar rats (female, 10-12 weeks, 250-300 g) were obtained from the Stolbovaya nursery (Russia). The studies were conducted in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

Evaluation of virus safety during the intracranial administration to rats

The T_NS124-2A-Luc influenza reporter vector was concentrated and purified by ultrafiltration and tangential flow diafiltration. Purified preparations (10pl) were administered intracranially into the pia mater of rats using a RWD Life Science R480 Nanoliter Injection Pump. The clinical condition of the animals (5 rats per group) was monitored for 10 days, followed by an assessment of neurological deficit [23]. Virus persistence at the injection site was assessed by luminescent activity as well as by the presence of viral genetic material and its infectious activity 24, 48, and 72 h after administration. The group T_NS124-2A-Luc consisted of 3 animals; the intact group included 1 animal.

Statistical analysis

Graphic visualization and statistical data processing were performed in MSExcel and GraphPad Prism v9.5.1. The Shapiro-Wilk criterion was used to test the hypothesis of data distributions normality. If the hypothesis of normality could not be rejected, the significance of differences between groups was determined using variance analysis with Tukey's post hoc test. Otherwise, the Kruskal-Wallis test with Dunn's subsequent criterion was used. The significance level was set up at 0.05.

RESULTS

Construction and characterization of influenza reporter vectors

Reporter strains of influenza virus with chemiluminescent activity were obtained using the reverse genetics method [19]. Plasmids that were used for assembly of viruses contain ns gene modified as follows: the protein-coding sequences of the viral protein NS1124 and NanoLuc luciferase [24] were either fused or separated by the 2A site, which ensures co-translational separation of proteins. A schematic representation of the chimeric genes is presented in Fig. 1A.

The specificity to proteolytic enzymes was changed by introduction of point mutations S342^P and R343^I into the plasmid encoding HA of the influenza virus strain A/PR/8/34, leading to the replacement of the trypsin proteolytic cleavage site to elastase (Fig. 1A). Laboratory strain A/PR/8/34 was used as the source of the remaining genes for plasmids construction. As a result, a set of four reporter vectors was generated: the T_NS124-Luc and T_NS124-2A-Luc viruses containing HA cleavable by trypsin-like serine proteases, and the E_NS124-Luc and E_NS124-2A-Luc strains, containing modified HA that is activated by elastase.

Genetic stability and reproductive activity of constructed vectors

All constructed vectors were genetically stable over the five consecutive passages. Fig. 1B shows the RT-PCR results, demonstrating that the length of the NS genomic segments of the strains after the fifth passage corresponds to that of the control plasmids. The amplification products of the NS genomic segments of the clones from previous passages also matched the corresponding segments of the control plasmids (data not shown). The bioluminescence signal for all strains reached 105 relative light units (RLU). The expression of the chimeric NS1 protein was confirmed by Western blot analysis of the cell's lysate (Fig. 1C). According to the data obtained by

Western blotting done with antibodies against the NS1 protein (Fig. 1D) molecular weights of the synthesized chimeric proteins corresponded to the theoretically predicted values. In addition, partial co-translational separation of the chimeric protein at ribosome skip site 2A was demonstrated (Fig. 1B, lanes 3 and 4).

The reproductive activity of the trypsin-dependent and elastase-dependent reporter strains reached 8.0 log10 TCID50/ml when cultivated in Vero and MDCK cell lines. The recombinant viruses in the absence of exogenous proteases in the CE had different growth characteristics, as expected. The infectious activity of trypsin-dependent strains T_NS124-Luc and T_NS124-2A-Luc in CE reached 9.0 log10 EID50/ml. At the same time, the elastase-dependent viruses E_NS124-Luc and E_NS124-2A-Luc did not replicate in CE without the addition of exogenous elastase. The presence of the co-translational cleavage site 2A did not significantly affect the infectious activity of the strains, proving that their functional activity and structure were preserved.

To confirm the specificity of proteolytic cleavage of HA by trypsin and elastase, the reproductive and reporter activity of the constructed strains was assessed when culturing viruses in media containing both homologous and heterologous proteases (Fig. 2).

Incubation of cells infected with reporter virus strains in a medium containing a homogeneous protease initiated a multi-cycle infection. Peak reproductive activity was recorded at 48 h post infection and reached 7.7 log10 TCID50/ml for trypsin-dependent strains and 7.0 log10 TCID50/ml for elastase-dependent viruses. The presence of the 2A site between the NS1124 protein and NanoLuc luciferase sequences did not affect the rate of viral reproduction, confirming the versatility of this construct.

The luciferase activity of the strains growing in a medium containing heterologous protease reached a peak after 24 h and did not exceed 103 RLU. Among all the strains, the highest level of luminescent signal under these conditions was registered for the trypsin-dependent virus with a 2A site insert.

Incubation of infected cells in a medium with homologous protease led to intense luminescence reaching 105 RLU, which confirms the specificity of the proteolytic cleavage site of HA. The peak of luciferase activity for trypsin-dependent viruses was detected at 24 h post infection, and for elastase-dependent strains -at 48 h.

Thus, genetically stable influenza reporter vectors, reproduction of which depended on the presence of trypsin (T_NS124-Luc and T_NS124-2A-Luc) or elastase (E_NS124-Luc and E_NS124-2A-Luc), were constructed

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Fig. 1. Influenza reporter vectors with luminescent activity. (A) Schematic representation of the genomic fragments HA and NS of the obtained reporter strains. The proteolytic cleavage site is highlighted in red bold. NS1124 is a sequence encoding the NS protein of the influenza A/PR/8/34 virus, truncated to 124 amino acid residues; Luc is a sequence encoding the NanoLuc luciferase of the deep-sea shrimp Oplophorusgracilirostris; STOP is a stop codon; NEP is a sequence encoding the nuclear export protein of the influenza A/PR/8/34 virus; 2A is a sequence encoding the P2A autoproteolysis site of Porcine Teschovirus 1. The gene segments were designed using the Unipro UGENE program [25]. (B) Electropherogram of the amplification products of the NS genomic segment of reporter influenza vectors. M -molecular weight marker; 1 - reporter strain T_NS124-2A-Luc; 2 - strain E_NS124-2A-Luc; 3 - control plasmid pHW-PR8/NS124-2A-Luc; 4 - strain E_NS124-Luc; 5 - control plasmid pHW-PR8/NS124-Luc. The viral material obtained after the fifth passage was used for RT-PCR for all strains. (C) Results of Western blot staining of lysates of cells infected at multiplicity of infection of 1 TCID50/cell with antibodies against NS1 (1H7); lanes 1-4 - cells infected with influenza reporter vectors T_NS124-Luc, E_NS124-Luc, T_NS124-2A-Luc, and E_NS124-2A-Luc, respectively; M - molecular weight marker, kDa; the synthesized chimeric proteins are highlighted by arrows. (D) Calculated molecular weight of NS1 chimeric proteins expressed upon infection of cells with the constructed reporter vectors (calculations performed using the ExPASy ProtParam server [26]).

and characterized. All viruses demonstrated high luminescence activity, reaching 105 RLU in the presence of the corresponding proteolytic enzyme. The reporter and infectious activities of the strains did not depend on the presence of the 2A site before the NanoLuc luciferase sequence.

Efficiency of infection of glioma cell lines with the constructed strains

To assess the oncolytic potential of the developed reporter strains, their ability to infect tumor cell cultures was evaluated. The rat glioma cell line C6, the primary culture T2 [27], and transplantable human glioblastoma cell lines A172 and T98G were used as tumor models. The permissive MDCK cells were used for comparison.

The percent of infected cells was determined by flow cytometry using fluorescently labeled monoclonal antibodies against the nucleoprotein (NP) of the influenza virus. The obtained data allowed us to determine the infection efficiency of different cell lines by the studied viruses (Fig. 3).

The highest efficiency of cell infection by the constructed reporter vectors was recorded for the rat glioma cell line C6 and the primary culture of glioblastoma T2 [27]. In the human glioblastoma cell lines A172 and T98G, the percentage of infected cells was lower, reaching about 50% of the infection level in the permissive MDCK cells.

Notably, T_NS124-2A-Luc and E_NS124-2A-Luc vectors containing the 2A site in the chimeric ns1 gene

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Fig. 2. Reproductive and luminescent activity of influenza reporter vectors. (A) Growth curve of influenza reporter vectors in the presence of TPCK-trypsin or elastase. (B) Luminescent activity of elastase-dependent or TPCK-trypsin-dependent strains with modified NS1 protein. A diurnal monolayer of MDCK cells was infected at a multiplicity of infection of 0.001 TCID50/cell and analyzed continuesly during 72 h. Experiments were performed in triplicates for each virus. The reproduction rate of strains during multi-cycle infection was estimated in the supernatant of infected cells by titration of the resulting viral progeny in MDCK cells in the presence of homologous protease. The dotted line indicates the detection limit. Luciferase activity was measured in the cell lysate; RLU - relative light units.

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demonstrated a significantly higher infection activity compared to T_NS124-Luc and E_NS124-Luc viruses lacking the 2A site (p<0.05, Fig. 3). This data indicates a potential role for the 2A site in improving the ability of vectors to infect glioma cells.

The safety of the studied vectors upon intracranial administration

A key step in the development of oncolytic therapy for glioblastomas is the safety assessment of the studied preparation upon intracranial administration into the pia mater of healthy animals. The T_NS124-2A-Luc strain, which was purified and concentrated to 9.0 log10 EID50/ml, was selected for the safety study. The scheme of the experiment is shown in Fig. 4A. The animals were monitored daily for 10 days. No decrease in body weight or lethal outcomes were recorded during this period (Fig. 4B).

To study the persistence of the virus in the rat brain, the luminescent activity of the vector, the presence of viral genetic material, and its infectious activity were

assessed during the first 72 h after administration of the virus (Fig. 4B-D).

In all examined brain tissue samples at all time points a high level of luminescent signal (up to 105 RLU) was observed, that is sufficient for intravital visualization of NanoLuc as a part of the influenza vector (Fig. 4E).

It is noteworthy that infectious virus as well as viral RNA were not detected in brain samples 72 h after vector administration. These data confirm limited viral replication and its rapid excretion from brain tissue, which indicates the safety of the reporter vector.

No statistically significant differences were found between the experimental and control groups when studying the general motor activity and exploratory behavior of animals (Fig. 5). The data obtained indicate the absence of neurological disorders in laboratory animals after intracranial administration of the reporter vector.

Thus, intracranial administration of the influenza reporter vector was safe for the rats, did not cause neurological deficit, and was accompanied by the

Fig. 3. Efficiency of infection by reporter vectors. Diurnal monolayer of (A) MDCK, (B) rat glioma C6, (C) A172, (D) T98G, and (E) T2 cells were infected at a dose of 7.0 log10 TCID50/cell in three independent replicates for each virus and incubated for 17 h in a medium containing 2% serum. Uninfected cells (cell control - CC) were used as a negative control; infected cells were stained with the Zombie Aqua Fixable Viability Kit and FITC-labeled antibodies against influenza A virus NP and counted using a flow cytometer. * - p<0.05; ** - p<0.01, *** -p<0.001 (two-way ANOVA analysis followed by Tukey's post hoc test).

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DISCUSSION

Currently, the use of oncolytic viruses is considered a promising method of immunotherapy of glioblastomas. This is confirmed by many clinical studies performed with various oncolytic viral candidates, including herpes viruses (HSV-1), reovirus (Reovirus, Reolysin), adenoviruses, vesicular stomatitis virus (VSV),

paramyxoviruses (measles virus, Newcastle disease virus), cowpox virus, Sendai virus and polyomavirus [1-6, 28, 29]. These viruses have the ability not only to directly destroy tumor cells, but also to modify the tumor microenvironment, enhancing the antitumor immune response [28, 30]. Influenza viruses are promising candidates for oncolytic therapy because of their ability to stimulate innate immunity and cytotoxic T cell response [31-33]. However, their use is associated with several limitations and challenges.

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Fig. 4. Safety assessment of the reporter influenza strain upon intracranial administration. Purified reporter vector T_NS124-2A-Luc was administered intracranially to Wistar rats at a dose of 7.0 log10 TCID50/animal in 10 pl. The SPGN buffer was administered as a control preparation in the same volume. No preparations were administered to the intact animals. (A) Experimental scheme; hpi - hours post infection, dpi - days post infection. (B) Body weight dynamics of animals (n=5 per group) during 10 days after intracranial administration of the reporter vector T_NS124-2A-Luc. (C) Luminescent activity. (D) Presence of viral RNA measured by PCR. (E) Infectious virus in the brain tissue homogenate of animals of the control (n=1) and experimental groups in 24 (n=2), 48 (n=3), and 72 h (n=2) after administration. (F) Intravital visualization of the reporter vector 2-24 h after intracranial administration of the preparation.

Fig. 5. Results of physiological tests. The T_NS124-2A-Luc reporter vector was administered to rats intracranially at a dose of 7.0 log10 TCID50/ animal (n=5). Intact rats (without administration of preparations, n=5) and rats administered SPGN buffer (n=5) were used as control groups. Physiological tests to assess neurological deficit were performed on day 10 after administration of the preparations.

One of the key challenges is the presence of neutralizing antibodies against modern strains of influenza A virus, a consequence of frequent seasonal epidemics and widespread human vaccination [34]. This can significantly reduce the effectiveness of influenza vectors as oncolytic agents. An alternative approach involves using 'old' influenza viruses to which the population lacks immunity; however, these viruses pose risks related to virulence, genetic instability, and potential epidemic spread. Additionally, some influenza virus strains exhibit neurotropism, which can result in neurological complications, necessitating a thorough assessment of their safety [35, 36].

Despite these limitations, influenza viruses possess unique characteristics that enable the development of hyperattenuated vectors for the targeted expression of transgenes, such as cytokines and chemokines, at the tumor site [7, 8]. These transgenes can amplify the antitumor immune response by activating T cells, NK cells, and macrophages, as well as facilitating immune cell infiltration into the tumor [7].

In this study, we developed influenza virus reporter vectors activated by trypsin-like proteases (T_NS124-Luc and T_NS124-2A-Luc) or tumor-associated elastase (E_NS124-Luc and E_NS124-2A-Luc). For this purpose, the mutations S342^P and R343^I were introduced into the HA cleavage site. All vectors demonstrated a high efficiency of infection in glioma cell lines (C6, T2,

REFERENCES

1. Wu W, Klockow JL, Zhang M, Lafortune F, Chang E, Jin L, et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance, Pharmacological Research. 2021;171:105780. https:// doi.org/10.1016/j.phrs.2021.105780.

2. Kotecha R, Odia Y, Khosla AA, Ahluwalia MS. Key Clinical Principles in the Management of Glioblastoma. JCO Oncol Pract. 2023;19(4):180-9. https://doi.org/10.1200/0P.22.00476.

3. Bernstock JD, Blitz SE, Hoffman SE, Gerstl JVE, Chiocca EA, Friedman GK. Recent oncolytic virotherapy clinical trials outline a roadmap for the treatment of high-grade glioma. Neurooncol Adv. 2023;5(1):081. http://www.doi.org/10.1093/noajnl/ vdad081.

4. Lin D, Shen Y, Liang T. Oncolytic virotherapy: basic principles, recent advances and future directions. Sig Transduct Target Ther. 2023;8:156. https://doi. org/10.1038/s41392-023-01407-6.

A172, and T98G). It should be noted that constructs with 2A site, which ensures co-translational separation of proteins, showed increased infectious activity. This may be due to improved functional activity of the NS1 protein when it is separated from the transgene.

In vivo experiments showed that intracranial administration of the trypsin-dependent vector T_ NS124-2A-Luc at a high dose is safe for rats and does not lead to the development of neurological deficit. Simultaneously, the luminescent reporter NanoLuc was expressed at the site of vector administration, which allowed visualization of the virus administration zone. Importantly, transgene expression is observed in the absence of active viral replication.

The obtained results open prospects for further optimization of viral vectors, studying the pharmacokinetics of transgenes and developing oncolytic preparations based on the influenza viruses expressing cytokines and chemokines. In addition, vectors expressing NanoLuc may have their own therapeutic effect owing to the immunomodulatory action of the modified NS1 protein. Replacing NanoLuc with chemokines such as CXCL-9/10/11, can enhance the therapeutic effect of vectors, by activating of antitumor immunity and modifiying the tumor microenvironment [7, 37]. This will become the main direction for further research aimed at creating safe and effective oncolytic preparations for the treatment of malignant gliomas.

5. Bartlett DL, Liu Z, Sathaiah M, Ravindranathan R, Guo Z, He Y, Guo ZS. Oncolytic viruses as therapeutic cancer vaccines. Mol Cancer. 2013;12(1):103. https:// doi.org/10.1186/1476-4598-12-103.

6. Vorobjeva IV, Zhirnov OP. Modern approaches to treating cancer with oncolytic viruses. MIR J. 2022;9(1):91-112. https://doi.org/10.18527/2500-2236-2022-9-1-91-112.

7. Pol JG, Workenhe ST, Konda P, Gujar S, Kroemer G. Cytokines in oncolytic virotherapy. Cytokine Growth Factor Rev. 2020;56:4-27. https://doi.org/10.1016/)'. cytogfr.2020.10.007.

8. Tokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S, et al. CXCL9, CXCL10, CXCL11/ CXCR3 axis for immune activation - A target for novel cancer therapy. Cancer Treat Rev. 2018;63:40-7. https://doi.org/10.1016/j.ctrv.2017.11.007.

9. Kabiljo J, Laengle J, Bergmann M. From threat to cure: understanding of virus-induced cell death leads to

highly immunogenic oncolytic influenza viruses. Cell Death Discov. 2020;6:48. https://doi.org/10.1038/ s41420-020-0284-1.

10. Ogbomo H, Michaelis M, Geiler J, van Rikxoort M, Muster T, Egorov A, et al. Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells. Med Microbiol Immunol. 2010;199(2):93-101. https://doi. org/10.1007/s00430-009-0139-0.

11. Sturlan S, Stremitzer S, Bauman S, Sachet M, Wolschek M, Ruthsatz T, et al. Endogenous expression of proteases in colon cancer cells facilitate influenza A viruses mediated oncolysis. Cancer Biol Ther. 2010;10(6):592-9. https://doi.org/10.4161/ cbt.10.6.12565.

12. Hock K, Laengle J, Kuznetsova I, Egorov A, Hegedus B, Dome B, et al. Oncolytic influenza A virus expressing interleukin-15 decreases tumor growth in vivo. Surgery. 2017;161(3):735-6. https://doi. org/10.1016/j.surg.2016.08.045.

13. Shurygina APS, Kartashev AV, Kovanko EG, Kiseleva LN, Pustovalov YI, Slita AK, et al. Oncolytic potential of recombinant influenza A virus vectors on a model of malignant glioma in vivo. Vopr Onkol. 2016;62(1):138-45. (In Russian). PMID: 30444592.

14. Kabiljo J, Kuznetsova I, Homola J, Prodinger S, Laengle J, Sachet M, et al. P09.04 Oncolytic H5N1 influenza strain displays superior therapeutic properties independent of immuno-stimulatory interleukin-2 transgene expression J Immunother Cancer. 2020;8(Suppl 2). https://doi.org/10.1136/jitc-2020-ITOC7.104.

15. Kuznetsova I, Arnold T, Aschacher T, Schwager C, Hegedus B, Garay T, et al. Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase. Mol Ther Oncolytics. 2017;7:37-44. http://www.doi. org/10.1016/j.omto.2017.09.002.

16. Sergeeva MV, Pulkina AA, Romanovskaya-Romanko EA, Mustafaeva AS, Egorov AY, Stukova MA. Rapid Assessment of Neutralizing Antibodies Using Influenza Viruses with a Luciferase Reporter. Appl Biochem Microbiol. 2022;58(7):878-86. https://doi. org/10.1134/S0003683822070067.

17. Pulkina AA, Sergeeva MV, Krokhin A, Stukova MA, Egorov A. Evidence for the extracellular delivery of influenza NS1 protein. MIR J. 2021;8(1):7-37. https:// doi.org/10.18527/2500-2236-2021-8-1-27-37.

18. Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011;6(4):e18556. https://doi. org/10.1371/journal.pone.0018556.

19. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. USA. 2000;97:6108-13. https:// doi.org/10.1073/pnas.100133697.

20. Reed LJ, Muench H.A simple method of estimating fifty per cent endpoints. Am J Epidemiol. 1938;27:493-7. https://doi.org/10.1093/oxfordjournals.aje.a118408.

21. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol. 2001;146(12):2275-89. https://doi.org/10.1007/ s007050170002.

22. Krivitskaya VZ, Sorokin EV, Tsareva TR, Sergeeva MV, Kadyrova RA, Romanovskaya-Roman'ko EA, et al. Generation and Characterization of the Monoclonal Antibody Panel Specific to the NS1 Protein of the Influenza A Virus. Appl Biochem Microbiol. 2018;54:756. https://doi.org/10.1134/ S0003683818070049.

23. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12:1-21. https://doi. org/10.1089/neu.1995.12.1.

24. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012;7(11):1848-57. http:// www.doi.org/10.1021/cb3002478.

25. Okonechnikov K, Golosova O, Fursov M; UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28(8):1166-7. http://www.doi. org/10.1093/bioinformatics/bts091.

26. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999;112:531-52. https:// doi.org/10.1385/1-59259-584-7:531.

27. Kiseleva LN, Kartashev AV, Vartanyan NL, Pinevich AA, Filatov MV, Samoilovich MP. Characterization of New Human Glioblastoma Cell Lines. Cell Tiss. Biol. 2018;12:1-6. https://doi.org/10.1134/ S1990519X18010108.

28. Hamad A, Yusubalieva GM, Baklaushev VP, Chumakov PM, Lipatova AV. Recent Developments in Glioblastoma Therapy: Oncolytic Viruses and Emerging Future Strategies. Viruses. 2023,15:547. https://doi.org/10.3390/v15020547.

29. Macedo N, Miller DM, Haq R, Kaufman HL. Clinical landscape of oncolytic virus research in 2020. J Immunother Cancer. 2020;8(2):e001486. http://www. doi.org/10.1136/jitc-2020-001486.

30. Berkey SE, Thorne SH, Bartlett DL. Oncolytic Virotherapy and the Tumor Microenvironment. Adv Exp Med Biol. 2017;1036:157-72. http://www.doi. org/10.1007/978-3-319-67577-0_11.

31. Muster T, Rajtarova J, Sachet M, Unger H, Fleischhacker R, Romirer I, et al. Interferon resistance promotes oncolysis by influenza virus NS1-deletion mutants. Int J Cancer. 2004;110(1):15-21. http:// www.doi.org/10.1002/ijc.20078.

32. Sun F, Xu Y, Deng Z, Yang P. A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8+ T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma. Int Immunopharmacol. 2023;120:110323. https://doi. org/10.1016/j.intimp.2023.110323.

33. Li C, Tian Y, Sun F, Lei G, Cheng J, Tian C, et al. A Recombinant Oncolytic Influenza Virus Carrying GV1001 Triggers an Antitumor Immune Response. Hum Gene Ther. 2024;35(1-2):48-58. https://doi. org/10.1089/hum.2022.206.

34. Gerlach T, Elbahesh H, Saletti G, Rimmelzwaan GF. Recombinant influenza A viruses as vaccine vectors. Expert Rev Vaccines. 2019;18(4):379-92. https://doi. org/10.1080/14760584.2019.1582338.

35. Ekstrand JJ. Neurologic complications of influenza. Semin Pediatr Neurol. 2012;19(3): 96-100. https:// doi.org/10.1016/j.spen.2012.02.004.

36. Khandaker G, Zurynski Y, Buttery J, Marshall H, Richmond PC, Dale RC, et al. Neurologic complications of influenza A(H1N1)pdm09: surveillance in 6 pediatric hospitals. Neurology. 2012;79(14):1474-81. https://doi.org/10.1212/WNL.0b013e31826d5ea7.

37. Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol. 2017;17(9):559-72. http://www.doi.org/10.1038/ nri.2017.49.