RU2644676C1 - Newcastle disease ndvh-2 virus strain for study of possibility of viral oncolytic preparation development on its basis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: NDVH-2 strain of the Newcastle disease virus is deposited in the State Collection of Viruses of the Virusology Research Institute named after D. I. Ivanovsky of the Ministry of Health of Russia under the registration number 2780. The strain is capable of selectively lysing human tumour cell lines without causing an infectious disease and mammalian death.

EFFECT: study of a possibility of development of a viral oncolytic drug for the therapy of human malignant tumours, with a view to further application in medical virology, biotechnology, microbiology and oncology.

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Description

Technical field

The present invention relates to strain N of the Newcastle disease virus, namely, strain NDVH-2, infecting and lysing human tumor cells in vitro, promising for the development on its basis of a viral oncolytic drug for the treatment of human malignant neoplasms, and can be used in medical virology, biotechnology and microbiology.

State of the art

Today, cancer remains one of the main causes of death among the population of economically developed countries. According to WHO forecasts, the annual number of cancer deaths will only increase and by 2030 will reach 13 million cases per year [World Health Organization (WHO). World Health Statistics 2012. Geneva: WHO. Euro Surveill. - 2012. Vol. 17 (20). Available from: http://www.who.int/gho/publications/world_health_statistics/EN_WHS2012_Full.pdf].

The main methods of treating cancer patients remain surgical removal of the tumor, chemotherapy and radiation therapy, and in the vast majority of cases they have to be combined. The applied treatment methods are often not effective or lead to a significant decrease in the quality of life of patients due to the impact on healthy patient tissue. In recent years, great hopes have been placed on the development of targeted approaches to the treatment of tumors using oncolytic viruses [Gubanova N.V., Gaytan A.S., Razumov I.A., Mordvinov V.A., Krivoshapkin A.L., Netesov S.V., Chumakov P.M. Oncolytic viruses in the therapy of gliomas // MolBiol +. - 2012. - Vol. 46. - R. 780-789]. The feasibility of using virotherapy for the treatment of malignant neoplasms is evidenced by the selectivity of viruses to neoplastic cells with minimal toxicity to normal healthy tissues, as well as the effective replication of viral particles, the destruction of transformed cells and the spread of viral offspring to neighboring tumor cells. In addition to the direct lysis of cancer cells, oncolytic viruses can cause the body's immune response by stimulating adaptive and innate immunity [Yurchenko KS, Gubanova NV, Shestopalova LV, Shchelkanov M.Yu., Shestopalov A.M. Oncolytic properties of the Newcastle disease virus // Pacific Medical Journal. - 2015. - No. 3. - S. 14-19].

Currently, a wide range of viruses has been described used for virotherapy of malignant neoplasms. Currently, in the USA, Canada, China and Europe (Germany, Hungary), a number of VBI strains have been tested in clinical trials for the presence of their antitumor potential (PV701, LaSota, MTN-68, 73-T, HUJ and Ulster). Studies have shown the safety of the strains used, despite the difference in the pathogenic characteristics of the strains. Many studied strains demonstrate an oncolytic effect with intratumoral administration, but are less effective with intravenous injection. It was demonstrated that upon intravenous administration, most of the viruses are almost instantly removed from the bloodstream by the reticuloendothelial system of the liver and spleen [Breitbach CJ, Burke J., Jonker D., Stephenson J., Haas AR, Chow LQM, Nieva J., Hwang T. N. ., Moon A., Part R., Pelusio A., Le Boeuf F., Burns J., Evgin L., De Silva N., Cvancic S., Robertson T., Je JE, Lee YS, Parato K., Diallo JS, Fenster A., Daneshmand M., Bell JC, Kirn DH Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans // Nature. - 2011 .-- Vol. 477. - R. 99-102]. To overcome this barrier, the introduction of huge doses of the virus is required, which increases the cost of the treatment method and creates additional safety problems. In this regard, it became necessary to develop a vaccine capable of infecting and lysing tumor cells with a minimum amount of viral particles. Such viruses must have high replicative activity, tropism for tumor cells and the ability to generate virulent offspring. The patented strain NDVH-2 has the required properties, as confirmed by in vitro studies of cytolytic properties and analysis of the virus genome.

The virulence of strains is determined by the amino acid sequence of protein F cleavage site. It is known that the efficiency of protein F cleavage of highly virulent strains by cellular proteases is ensured by the presence of basic amino acids at position 115 and 116, phenylalanine (F) at position 117, and arginine at position 113. This fragment is present in the genomic the sequence of strain NDVH-2.

The structure of the HN protein is responsible for the binding of viruses to the surface of tumor cells. Sequencing revealed the presence of substitutions in the amino acid sequence of this protein, which are marker traits that distinguish it from the prototype strain.

Parent for the claimed strain is a virus vaccine against Newcastle disease from strain "N", used for vaccination of birds. Oncolytic properties for the parent strain are not described.

The objective of the invention is to create a strain of Newcastle disease virus that is able to selectively infect and lyse a wide range of human neoplastic cells of different tissue affiliation.

The solution of the technical problem is achieved by carrying out a series of adaptive passages of the parent virus vaccine of strain H on a culture of the Vero cell line (African green monkey kidney epithelium), to obtain a new strain of NDVH-2, which selectively infects and lyses human tumor cells in vitro to create an antitumor cell based on it the drug.

The technical result of the invention is the possibility of creating on the basis of the obtained strain of Newcastle disease virus an oncolytic viral preparation for the treatment of human malignant neoplasms.

The main differences between the proposed strain of NDVH-2 from the above strains of VBI are its ability to selectively infect and actively lyse a wide range of human tumor cells with a small amount of viral particles.

Disclosure of invention

Characterization of the claimed strain

Strain NDVH-2 is a typical representative of paramyxoviruses - the Paramyxoviridae family, the Paramyxovirinae subfamily, the genus Avulavirus, and the species Newcastle Disease Virus (Avian paramixovirus type-1). The strain is deposited in the State virus collection Research Institute of Virology. DI. Ivanovo Ministry of Health of Russia under registration number 2780 (July 7, 2014).

Source of receipt. The inventive strain was obtained by serial passaging of the parent strain H virus vaccine of Newcastle disease birds in a culture of the Vero cell line. After five adaptive passages on a model of developing chicken embryos (RCEs), preparative amounts of viral material were obtained sufficient to characterize the infectious (lytic) properties of the NDVH-2 strain on human tumor cell cultures. Indication of the virus was carried out in a hemagglutination reaction (RGA) with rooster erythrocytes: the hemagglutinating activity index was 256 GAU / 50 μl.

Virus identification. The primary identification of isolated Newcastle disease virus was carried out by RT-PCR with the detection of the results in a closed tube in real time using primers and a probe specific for the conserved regions of the M gene of Newcastle disease virus, according to [Miller P.J., Decanini E.L., Afonso C.L. Newcastle disease: evolution of genotypes and the related diagnostic challenges // Infect Genet Evol. - 2010 .-- Vol. 10 (1). - R. 26-35].

NDVH-2 is deposited in the international GenBank database under the number KU865562 (http://www.ncbi.nlm.nih.gov/nuccore/KU865562.1). The nucleotide sequence of the strain of the Newcastle disease virus NDVH-2 is attached (Appendix 1).

Cultural properties. The NDVH-2 strain is cultured on a monolayer of Vero cell culture. When a cell monolayer is infected, the NDVH-2 strain induces a cytopathic effect (CPD) typical of VBI after 16-48 hours, depending on the multiplicity of infection (at a cultivation temperature of 37 ° C, in an atmosphere with 5% CO ₂ content). The cell line V culture was grown as a monolayer on DMEM medium (Gibco BRL, Germany) containing 10% fetal calf serum (ETF) (Gibco BRL, Germany) with 80 μg / ml gentamicin sulfate. A subconfluent monolayer of Vero cells is infected with an NDVH-2 strain with an optimal infection rate of 0.1-0.01 MOI, after adsorption for 1 h at 37 ° C in an atmosphere with 5% CO ₂ , the infected cell monolayer is incubated in DMEM support medium containing 2% EF before the onset of 75-90% of the CPP. After that, the culture medium together with the cells was selected and the virus-containing suspension was clarified by low-speed centrifugation (5000 rpm, 10 min).

Pathogenicity for humans. Newcastle disease virus strains are not pathogenic for humans [Cassel W.A., Garrett R.E. Newcastle disease virus as an antineoplastic agent // Cancer. - 1965. - Vol. 18. - P. 863-868; Laurie S.A., Bell J.C., Atkins H.L., Roach J., Bamat M.K., O'Neil J.D., Roberts M.S., Groene W.S., Lorence R.M. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization // Clin Cancer Res. - 2006. - Vol. 12 (8). - R. 2555-2562].

Pathogenicity for poultry. Newcastle disease virus strains are classified into lentogenic (slightly pathogenic), mesogenic, and velogenic (highly pathogenic) (OIE, 2015). Strain NDVH-2 refers to a pathogenic variant of the Newcastle disease virus. Infectious activity of the isolated virus was 7.4 ± 0.07 lg ELD ₅₀ / ml.

The pathogenicity of the strain was established according to standard methods in the tests for determining the average life time (MDT) of developing chicken embryos and the intracerebral index of pathogenicity of daily chickens (ICPI) [OIE, World Organization for Animal Health, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Paris, France , 7th edition. 2012. Vol. 1-2. Ch.2.03.14. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.14_NEWCASTLE_DIS.pdf.].

The average life time of MDT = 48 hours when the virus is diluted 10 ^-7 , which corresponds to the value for highly pathogenic (velogenic) VBI strains. The index of intracerebral pathogenicity index for chickens is ICPI = 1.25, which corresponds to the mesogenic pathotype.

Pathogenicity for mammals. The strain is not pathogenic for mammals [Alexander D.J. Newcastle disease virus and other avian paramyxoviruses // Rev. Sci. Tech. - 2000. - Vol. 19 (2). - R. 443-462].

For long-term storage, the strain must be lyophilized using a solution of gelatin and sucrose (SAG) as a protective medium. Lyophilization with the addition of SAGE in a ratio of 1: 4 allows you to maintain a stable infectious activity of the virus for at least 2 years when stored at -70 ° C.

The following culture media are used to cultivate the strain in cell culture: MEM, DMEM containing 1% ETS, 100 μg / ml penicillin and 100 IU / ml streptomycin.

Examples of using the Newcastle disease virus strain NDVH-2 to study the possibility of developing a viral oncolytic drug for the treatment of human malignancies

When studying the infectious (cytolytic) properties of the strain NDVH-2 on a panel of tumor and normal human cells (see Example 1), it was shown that it has a high degree of selective lytic activity in vitro with respect to the used tumor cell cultures. When infected with the NDVH-2 strain of normal human diploid fibroblasts, the cell culture viability is reduced by 20%, while in relation to tumor cells this indicator is from 70 to 40%. It should be noted that to achieve the indicated cytolytic effect, 10 viral particles per cell are sufficient, which demonstrates the high virulence and replicative characteristics of the strain.

The greatest cytolytic effect was observed for cell lines: U87 (glioblastoma) - 29% of viable cells on the 3rd day after infection, H1299 (cancer of the lung) - 35% and for breast adenocarcinoma cells SkBr-3 and MCF7 - 50% and 67%, respectively , which is significantly lower than that for normal human diploid cells.

The complete nucleotide sequence of the genome of the NDVH-2 strain is deposited in the international GenBank database under the number KU865562 (http://www.ncbi.nlm.nih.gov/nuccore/KU865562.1). When analyzing the complete nucleotide sequence of the genome of the NDVH-2 strain in comparison with the published sequences of various strains of the Newcastle disease virus, it was shown that the strains closest in nucleotide sequence to the present strain (99% homology) are Mukteswar strain (Newcastle disease virus strain Mukteswar, GenBank JF950509 .1, EF201805.1) - 48 nucleotide differences, and strains JS / 9/05 / Go (GenBank FJ430160.1) and JS / 7/05 / Ch (GenBank FJ430159.1) - 51 and 55 nucleotide differences, respectively. At the same time, with other VBI strains, the percentage of homology in the nucleotide sequences is 92% or lower.

Comparison of the nucleotide sequence of the coding proteins F (Y16170.1) and HN (Y17149.1) of the parent strain and strain NDVH-2 presented in the GenBank database revealed 3 nucleotide substitutions for the F gene and 15 for the HN gene (see Table 1) . Some of these substitutions result in amino acid substitution. So for protein F, glutamic acid Glu at the 15th position in the parent strain is replaced by Asp aspartic acid in the strain NDVH-2. Three amino acid substitutions were found for the HN protein: Val ₂₄ → Gly, Ser ₃₆₈ → Arg and Ile ₄₅₃ → Val. These amino acid substitutions are marker features for the NDVH-2 strain, distinguishing it from the prototype strain.

Example 1. The study of the lytic activity of the strain NDVH-2 on the panel of normal (non-tumorigenic) diploid and tumor cultures of human cells.

Infectious (lytic) activity of the NDVH-2 strain was determined in vitro on cultures of human tumor cells of various tissue affiliation: line H1299 (non-small cell lung cancer), U87 (glioblastoma), SkBr-3 and MCF7 (breast adenocarcinoma) and HFF10 (normal extreme fibroblasts) human flesh). Cells were plated on a 96-well plate a day before infection in an amount of 20,000 per well. Infection was carried out by suspension of the virus in 25 μl of phosphate buffer, followed by incubation for 1 hour at a temperature of 37 ° C and in an atmosphere with 5% CO ₂ content. The number of viral particles was 10 viral particles per cell. Determination of physical titer was carried out according to [Malenovska N. Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: A case study of three animal viruses // J Virol Methods. - 2013 .-- Vol. 191 (2). - P. 136-140] and was determined based on the ratio of viral particles to latex grains of known concentration. Viral particles and latex grains were counted using a Libra210 electron microscope (CarlZeiss).

The lytic activity of the studied strains for the respective cell types was determined by the method of colorimetric analysis using CellTiter 96®AQueous OneSolutionCellProliferationAssay reagent (Promega, USA) according to the attached manufacturer's instructions. Cell viability of all human tumor lines and normal human diploid fibroblasts on the third day after infection with strain NDVH-2 was assessed by the relative values of the proportion of living cells to control untreated cells. The results of the study are presented in FIG. 1 in the form of a histogram as the average relative values of the proportion of living cells on the third day after treatment with virus strains to the fraction of control untreated cells taking into account the standard deviation (average relative value ± standard deviation).

In FIG. 1: Cytolytic effect of strain NDVH-2 on the human tumor cell line and normal human diploid fibroblasts in vitro on the third day after infection. H1299 (non-small cell lung cancer), U87 (glioblastoma), SkBr-3 and MCF7 (breast adenocarcinoma), HFF10 (normal human foreskin fibroblasts).

It was shown that the strain NDVH-2 has a low cytolytic effect on normal human diploid cells (cell viability is 80% ± 5) and effectively lyses tumor cultures of H1299 (32% ± 7), U87 (29 ± 10), SkBr-3 (50 % ± 9), MCF7 (67% ± 7). Thus, the strain NDVH-2 has a pronounced ability for selective infection and lysis of tumor cells in a culture of various tissue affiliation.

Example 2. The definition and comparative analysis of the complete nucleotide sequence of the genome of strain NDVH-2.

To carry out full genome sequencing, RNA was isolated from the virus suspension, which was used to prepare the DNA library using the TruSeq RNA Library kit. The quality of the library was evaluated at Bioanalyzer. The library was sequenced on the MiSeq platform with a 2 × 300 set, and the resulting paired reads were used for de novo genome assembly (CLC GW). Open reading frames were found in the genome and gene annotation was performed. The complete nucleotide sequence of the genome of the NDVH-2 strain is deposited in the international GenBank database under the number KU865562 (http://www.ncbi.nlm.nih.gov/nuccore/KU865562.1). Analysis of the complete nucleotide sequences showed that the strain NDVH-2 belong to class 2 of genotype 3. Compared to the parent strain, the strain NDVH-2 has 18 nucleotide substitutions in the F and NH proteins, four of which lead to amino acid substitutions (see Table 1) . So for protein F, glutamic acid Glu at the 15th position in the parent strain is replaced by Asp aspartic acid in the strain NDVH-2. Three amino acid substitutions were found for the HN protein: Val ₂₄ → Gly, Ser ₃₆₈ → Arg and Ile ₄₅₃ → Val. These amino acid substitutions are marker features for the NDVH-2 strain, distinguishing it from the prototype strain.

List of sources used

1. Yurchenko K.S., Gubanova N.V., Shestopalova L.V., Shchelkanov M.Yu., Shestopalov A.M. 2015. Oncolytic properties of the Newcastle disease virus // Pacific Medical Journal - 2015. - No. 3. - S. 14-19.

2. Alexander D.J. Newcastle disease virus and other avian paramyxoviruses // Rev Sci Tech. - 2000. - Vol. 19 (2). - P. 443-462.

3. Breitbach CJ, Burke J., Jonker D., Stephenson J., Haas AR, Chow LQM, Nieva J., Hwang T.N., Moon A., Patt R., Pelusio A., Le Boeuf F., Burns J., Evgin L., De Silva N., Cvancic S., Robertson T., Je JE, Lee YS, Parato K., Diallo JS, Fenster A., Daneshmand M., Bell JC, Kirn DH Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans // Nature. - 2011 .-- Vol. 477. - P. 99-102.

4. Cassel W.A., Garrett R.E. Newcastle disease virus as an antineoplastic agent // Cancer. - 1965. - Vol. 18. - P. 863-868.

5. Gubanova N.V., Gaytan A.S., Razumov I.A., Mordvinov V.A., Krivoshapkin A.L., Netesov S.V., Chumakov P.M. 2012. Oncolytic viruses in the therapy of gliomas // MolBiol +. - 2012. - Vol. 46. - P. 780-789.

6. Laurie S.A., Bell J.C., Atkins H.L., Roach J., Bamat M.K., O'Neil J.D., Roberts M.S., Groene W.S., Lorence R.M. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization // Clin Cancer Res - 2006. - Vol. 12 (8). - P. 2555-2562.

7. Malenovska H. Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: A case study of three animal viruses // J Virol Methods. - 2013 .-- Vol. 191 (2). - P. 136-140.

8. Miller P.J., Decanini E.L., Afonso C.L. Newcastle disease: evolution of genotypes and the related diagnostic challenges // Infect Genet Evol. - 2010 .-- Vol. 10 (1). - P. 26-35.

9. OIE, World Organization for Animal Health, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Paris, France, 7th edition. 2012. Vol. 1-2. Ch.2.03.14. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.14_NEWCASTLE_DIS.pdf.

10. World Health Organization (WHO). World Health Statistics 2012. Geneva: WHO. Euro Surveill. - 2012. Vol. 17 (20). Available from: http://www.who.int/gho/publications/world_health_statistics/EN_WHS2012_Full.pdf.

Claims (1)

The strain of the virus of Newcastle disease NDVH-2, deposited in the State virus collection Research Institute of Virology. DI. Ivanovo Ministry of Health of Russia under registration number 2780, to study the possibility of creating an antitumor drug based on it.

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