Immunotherapy with oncolytic viruses (OVs) becomes a full-fledged neoadjuvant therapy method in the paradigm of evidence-based medicine for the growing number of cancers. The use of OVs for immunologically "cold" tumors causing minimal immune response and having the clearly immunosuppressive tumor microenvironment is especially relevant. Recombinant OVs carrying the sequences of proteins activating the immune system can be used to stimulate antitumor response. The study aimed to assess oncoselectivity and antitumor activity of the recombinant OV designed based on the LIVP vaccinia virus strain showing expression of human and murine interpheron alpha sequences (hIFN alpha and mIFN alpha, respectively). The in vitro experiments showed that the recombinant OVs designed showed oncoselectivity in relation to tumor cell lines of appropriate species. The ability to effectively infect human adenocarcinoma and glioblastoma cell lines was reported for LIVP-hIFN alpha. LIVP-mIFN alpha showed selectivity in relation to glioma Gl261 and melanoma B16 in vitro. The in vivo experiment involving the C57Bl/6 mice with subcutaneous melanoma & Vcy;16 showed the ability of the intravenously administered LIVP-mIFN alpha to reduce the size of the subcutaneous tumor allograft and increase tumor infiltration with the CD8+ and NK cells. The recombinant virus designed can be a potential platform for the development of oncolytic virotherapy of human melanoma and glioblastoma.

Among oncolytic viruses, modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia virus (VV) is a well-studied variant with promising results in preclinical and clinical trials. The Lister VV strain from the Moscow Institute of Viral Preparations (LIVP) has been studied to a lesser extent than MVA and has a different oncolytic property from MVA. The aim of this work was to compare the oncolytic efficacy of LIVP and MVA strains against solid tumors. We developed recombinant variants LIVP-RFP and MVA-RFP; to enhance onco-selectivity thymidine kinase (TK) gene was inactivated by insertion of red fluorescent protein (RFP) gene to the TK locus. The replication kinetics and oncolytic activity of the obtained recombinant strains were evaluated in vitro and in vivo on tumor cell lines and mouse syngeneic tumor models of metastatic mouse 4T1 mammary adenocarcinoma, CT26 colon adenocarcinoma, and B16 melanoma. Both MVA-RFP and LIVP-RFP showed high replication efficiency in tumor cells and pronounced oncolytic activity against B16 melanoma and 4T1 breast adenocarcinoma allografts. In relation to 4T1, which is a model of triple negative human breast cancer, LIVP-RFP showed more than 50% increased cytotoxicity in in vitro tests compared to MVA-RFP, as well as a significant slowdown in the progression of 4T1 allografts and an increase in animal survival in experiments in vivo. Thus, the LIVP strain may be more promising than MVA as a platform for the development of recombinant oncolytic viruses for the breast cancer treatment.

Diffuse gliomas are incurable, prevalent, and aggressive central nervous system tumors. Therefore, the development of selective oncolytic viral strains for malignant neoplasms is highly relevant. This study aimed to create an oncolytic virus based on a vaccine strain of poliovirus type 3 with natural antitumor activity. To achieve this goal, we replaced the internal ribosome entry site (IRES) of poliovirus with the corresponding fragment of human rhinovirus 30. The resulting recombinant oncolytic strain RVP3 retained the serotype of poliovirus type 3, as confirmed by virus neutralization micro-test with specific antiserum. In addition, the oncolytic efficacy of RVP3 was assessed in vitro on a broad panel of cell cultures. According to the results, RVP3 has changed its tropism, losing the ability to replicate in conditionally normal cell lines of embryonic astrocytes and embryonic fibroblasts while retaining the ability to replicate in tumor cells.

INTRODUCTION. Developing novel medicines based on non-pathogenic enterovirus strains exhibiting oncotropic and oncolytic properties represents an up-to-date and safe approach to complex cancer treatment and postoperative metastasis prevention. Safety pharmacology studies are a necessary step in the preclinical development of medicinal products.AIM. The study aimed to investigate the single and repeated-dose general toxicity, local tolerance, safety pharmacology, and pyrogenicity of medicinal products based on non-pathogenic LEV4, LEV7, LEV8, LEV14, and Russo enterovirus strains as part of preclinical safety studies.MATERIALS AND METHODS. The study used medicinal products of highly purified group A, B, and C enteroviruses at a titre of 2×107–5×108 CPD50/mL (CPD50 is a cytopathogenic dose of the virus causing 50% cell lysis) and normal saline as a diluent. The viruses were propagated in Vero cells. The safety study used 220 male and female BALB/c mice, 440 male and female Wistar rats, and 18 male Soviet chinchilla rabbits. The study animals received an intravenous dose of 1×105 or 1×106 CPD50/animal once (single-dose toxicity) or weekly for 90 days (repeated-dose toxicity). Clinical examination, laboratory testing, and necropsy were performed on days 45 and 91 of the experiment. Statistical data processing was performed using Prism 8.0 software (GraphPad Software, Inc., USA).RESULTS. Upon single administration of each of the five enterovirus medicinal products to mice and rats, the authors observed complete survival, upward trends in body weight gain, and no gross or histopathological changes in the brain, spleen, liver, kidneys, lungs, or at the injection site. Upon repeated administration at the study doses, the medicinal products caused no functional changes in the organs and systems. All the studied parameters were within the normal physiological ranges for male and female rats. Histopathological examination revealed no pathological changes or specific cytolytic and/or cytopathic effects. No local irritation was observed. None of the investigational medicinal products showed pyrogenicity.CONCLUSIONS. The obtained preclinical results demonstrate the safety of antineoplastic agents based on live non-pathogenic LEV4, LEV7, LEV8, LEV14, and Russo enteroviruses.

Transient gene expression is one of the most common methods in molecular biology, equally relevant for basic research projects and biotechnological industries. Despite the existence of commercial transfection systems, which afford high transfection efficiency and high expression levels of reporter genes, expanding such systems to industrial scales is often problematic due to high costs of the reagents. The well-described methods of cationic and calcium-phosphate transfection are accessible and ensure reproducible results at much lower costs. This study is aimed at comparative validation of calcium phosphate and cationic (polyethylenimine-based) transfection protocols along with the commercially available TurboFect reagent for mono- and cotransfections on a panel of commonly used cell lines including HEK293T, Huh7, BHK-21, CHO and MRC5. The efficiency of transfection with plasmid constructs encoding different fluorescent proteins was measured by flow cytometry. Of all the tested methods, calcium phosphate transfection afforded the highest efficiency of plasmid DNA delivery in all the cell lines except BHK21, for which the PEI method turned out to be more efficient than calcium phosphate transfection, and CHO, for which both methods showed comparable efficiency.