WO2014011072A1

WO2014011072A1 - Producer of newcastle disease virus strain ndv/anas crecca/omsk/9/2010 usable for producing a candidate anti-cancer preparation on the basis thereof

Info

Publication number: WO2014011072A1
Application number: PCT/RU2012/000560
Authority: WO
Inventors: Владимир Аркадьевич ЗАБЕЛИН; Александр Михайлович ШЕСТОПАЛОВ
Original assignee: Zabelin Vladimir Arkad Evich; Shestopalov Aleksandr Mihailovich
Priority date: 2012-07-11
Filing date: 2012-07-11
Publication date: 2014-01-16

Abstract

The invention relates to veterinary virology and microbiology, and specifically to a strain of Newcastle disease virus, and can be used for studying oncolytic properties and oncolysis mechanisms, and also for developing candidate anti-cancer preparations on the basis of viruses capable of selectively lysing neoplasms in mammals. The technical result of the proposed invention is the production of a novel Newcastle disease virus strain NDV/Anas crecca/Omsk/9/2010 capable of selectively lysing cancer cells in mammals without causing an infectious disease in, and death of, the mammals. The strain has been deposited in the Collection of Microorganism Cultures at the State Research Centre of Virology and Biotechnology "Vector", under registration number № V-539. The strain has been used to produce a model for studying the anti-tumour activity of Newcastle disease viruses on the basis of transplanted tumours in mice.

Description

The strain producer of the Newcastle disease virus NDV / Anas crecca / Omsk / 9/2010, used to create on its basis

candidate anticancer drug

Technical field

The invention relates to veterinary virology and microbiology, namely, a strain of the Newcastle disease virus, and can be used to study the oncolytic properties and mechanisms of oncolysis, as well as to develop candidate anticancer drugs based on viruses that can selectively lyse mammalian neoplasms.

State of the art

According to the World Health Organization, more than five million people die from malignant tumors every year in the world, and more than three million cases of new diseases are recorded. To date, the cure rate of cancer is only 26%. Oncology mortality rates in the Novosibirsk region are among the highest in Siberia and exceed the average level in Russia. In the dynamics of the detected ™ and mortality from cancer in Novosibirsk, there has been a steady increase.

The development of highly effective cancer treatment methods is one of the most urgent tasks of modern medicine. Despite some progress in this area, our ability to detect tumors in the early stages and selectively affect them remains extremely limited.

The main disadvantage of the currently used methods of chemotherapy and radiotherapy is their low therapeutic index (TI). This indicator characterizes the difference in the degree of effect of the corresponding therapeutic agent on tumor and normal cells of the body (in a simplified form, the ratio of the number of killed cancer cells to the number of killed normal cells). The therapeutic index of modern chemotherapeutic agents does not exceed 6: 1. According to the opinion accepted in the international scientific community, the TI of drugs directed by bacterial vectors and oncolytic viruses can reach 100,000: 1, which is due to their high specificity for tumor cells. Currently, evidence has been obtained that the Newcastle disease virus (hereinafter - VBI) has oncolytic activity and is able to selectively lyse cancer cells, while remaining safe for normal mammalian tissue cells. Several studies have demonstrated the ability of this virus to specific oncolysis. A significant difference was found in the rate of virus replication in tumor and normal mammalian cells (Zhan X, Slobod KS, Krishnamurthy S, Luque LE, Takimoto T, Jones B, Surman S, Russell CJ, Portner A, Hurwitz JL. (2008) / Sendai virus recombinant vaccine expressing hPIV-3 H or Flicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections.// Vaccine. 2008 Jun 25; 26 (27-28): 3480-8. Epub 2008 May 1).

There is an assumption that the high tropism of VBI to tumor cells is due to impaired synthesis of proteins responsible for the elimination of VBI (Elankumaran S, Rockemann D, Samal SK. (2006) / Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death .// J Virol. 2006 Aug; 80 (15): 7522-34). It is known that antitumor drugs based on the VBI line PV1703 and Cassell73 are currently undergoing the first stage of clinical trials (Ravindra PV, Tiwari AK, Sharma B, Chauhan RS./ Newcastle disease virus as an oncolytic agent.// Indian J Med Res. 2009 Nov; 130 (5): 507-13).

Disclosure of invention

The technical result of the invention is the use of strain NDV / Anas crecca / Omsk / 9/2010 of the Newcastle disease virus to create, on its basis, a candidate anti-cancer drug that can selectively lyse cancer cells in mammals without causing an infectious disease and death of mammals.

To identify the oncolytic properties of the strain NDV / Anas crecca / Omsk / 9/2010, experiments were carried out to infect mice with tumor cells previously incubated with the strain NDV / Anas crecca / Omsk / 9/2010. The results of this experiment revealed the presence of the proposed VBI strain the ability to neutralize tumor cells, preventing the formation of the tumor. This can occur under the influence of the immune system induced by the presence of the virus, or, more likely, by the direct lysis mechanism of the tumor NDV / Anas crecca Omsk 9/2010 tumor that we propose. cells. The second hypothesis is also supported by the fact that when the strain NDV / Anas crecca / Omsk / 9/2010 was introduced into an already formed tumor, a decrease in the dynamics of tumor growth was observed. Morphological examination revealed the destruction of the tumor tissue by the studied strain, and at the ultrastructural level, viral particles were detected inside the cancer cells. When the killed virus was introduced to the animals, no changes in the growth dynamics of the neoplasms were observed. Thus, the presence of live viral particles is necessary for the manifestation of oncolytic activity, and one of the probable markers of oncolytic activity is the structure of the fusion protein cleavage site corresponding to highly pathogenic VBI variants and the ability to lyse cells. The proposed strain has these markers.

The obtained data reveal the presence of the ability of the strain NDV / Anas crecca / Omsk / 9/2010 to specifically destroy mammalian neoplasms in the in vivo system without prior adaptation to the tumor cell culture, which is not currently described in the literature. This makes this strain a promising agent for creating anticancer drugs based on it through preliminary adaptation with a wide spectrum of interaction and a high therapeutic index.

Brief Description of the Drawings

Figure 1 presents graphs of the dynamics of the growth of tumors KREBS-2, incubated with highly and low pathogenic viruses in mice of BALB / C. The abscissa axis shows the time of measuring the size of the tumor (days), the ordinate axis represents the tumor volume in mm ³ , where: 1 - control option; 2 - low pathogenic virus; 3 - highly pathogenic virus.

Figure 2 presents graphs of the growth dynamics of KREBS tumors incubated with dead and live VBI strains in BALB / C mice two weeks after inoculation of tumor cells. The abscissa axis shows the time of measuring the size of the tumor (days), the ordinate axis represents the tumor volume in mm, where: 4 - control; 5 - a dead strain of VBN; 6 - live strains of VBN.

Fig. 3 shows the central part of the tumor with active macrophages 7.

Figure 4 shows the field of necrosis 8. Figure 5 shows the collection of viral particles 9 on the endoplasmic reticulum (EPR) 10.

Figure 6 shows a completely destroyed tumor cell 1 1, the space of which is filled with viroplasm 12.

The best embodiment of the invention

Example 1. Identification of the oncolytic properties of the strain NDV / Anas crecca Omsk / 9/2010 upon infection of mice with cells of the transplanted tumor line KREBS 2, previously incubated with the strain NDV / Anas crecca / Omsk / 9/2010.

To identify neutralizing activity, tumor cells incubated with NDV / Anas crecca / Omsk / 9/2010 (highly pathogenic), tumor cells incubated with NDV / Chukotka / goose / 7/2008 (low pathogenic, obtained from the collection) were introduced into three groups of 10 mice Department of Zoonotic Infections and Influenza, Federal State Institution Scientific Research Center of WB “Vector”), tumor cells incubated with saline solution (control group). The measurement of tumors began from the 15th day of the experiment, when the tumor in the control group was clearly determined visually and tactilely. Tumor growth dynamics was observed for 28 days. There were no significant differences in the growth dynamics of the neoplasms in the control group and in the group infected with the NDV / goose / Chukotka / 7/2008 strain. In the group infected with NDV / Anas crecca / Omsk / 9/2010, tumor formation was not observed.

The results of this experiment (see FIG. 1) show the presence of a VBI strain pathogenic for birds to neutralize mammalian tumor cells and prevent tumor formation. This process can occur under the influence of the immune system induced by the presence of the virus, or by the mechanism of direct lysis of tumor cells by viral particles. If the second assumption is considered correct, the presence of oncolytic activity in a strain pathogenic for birds and the absence of it on a non-pathogenic one is explained by the fact that apatogenic strains of VBI are not able to lyse cells, while the studied strain, on the contrary, has this ability, which allows it to lyse cancerous mammalian cells, preventing tumor formation. Example 2. The study of the oncolytic properties of the strain NDV / Anas crecca / Omsk / 9/2010 on a model of a solid tumor of KREBS 2 mice with the introduction of the virus into the formed tumor in vivo.

To evaluate the effect of VBI on an already formed tumor, the following experiment was performed: three groups of 13 mice were intramuscularly infected with a tumor. After 2 weeks, 0.2 ml of live NDV / Anas crecca Omsk / 9/2010 virus, killed NDV / Anas crecca Omsk virus 9/2010 and nat. solution accordingly. The dynamics of tumor growth was observed for 28 days (see figure 2). There were no significant differences in growth dynamics between the group infected with the killed NDV / Anas crecca / Omsk / 9/2010 and the control group. In the group infected with live NDV / Anas crecca / Omsk 9/2010, a decrease in the tumor growth rate was observed. Moreover, on average, the size of the tumor in mice subjected to a single exposure to VBI was 88% of the size of the tumor in the control group. Thus, a single injection of VBN caused a decrease in the tumor by 12% compared with the control group. The results of this experiment demonstrate the ability of the claimed strain to influence the growth dynamics of mammalian tumors, this property can be used in anti-cancer therapy.

Example 3. Morphological study of the interaction of VBN with mammalian tumor cells in vivo. 12 hours after infection, 3 mice from each group were opened. Structural analysis at the light-optical level revealed the accumulation of active macrophages (7) in the tumor area, which indicates the activation of the immune response, in addition, necrosis regions (8) of tumor cells were detected in the area of the virus (Fig. 3, 4), this indicates that a single injection of the virus initiated the destruction of tumor cells. At the ultrastructural level using electron microscopy (Fig. 5), the collection of viral particles (9) by EPR (10) was revealed. This indicates an intense virus – cell interaction; we are witnessing a phase of active infection of tumor cells with VBI particles, which is a direct sign of oncolytic activity.

A morphological study of the material taken 1 1 day after infection at the light-optical level revealed almost complete destruction of the tumor tissue, in fact, active mitoses were preserved only in peripheral region of the tumor. Macrophage activity was also noted. Completely destroyed cells (1 1) were found (Fig. 6) filled with viroplasm (12). All this suggests that a single exposure to the virus caused irreversible changes in the structure of the tumor, almost destroying the tumor cells, and the presence of macrophages indicates that the immune processes were also started. Such a complex effect, when direct lysis of tumor cells by the virus is accompanied by activation of the immune system, is an undoubted advantage of the claimed drug and makes the strain very promising from the point of view of application in anti-cancer therapy.

The data obtained indicate the unique ability of strain NDV / Anas crecca / Omsk / 9/2010 to exhibit selective lytic activity against mammalian tumor cells, as well as actively replicate and persist in these cells for a long time without preliminary adaptation, destroying the neoplasm.

Thus, the unique properties of the proposed strain allow it to be used to study the mechanisms of destruction of mammalian cancer cells at the ultrastructural level using light-optical and electron microscopy methods. These properties are promising for basic and applied medical research.

These properties make the strain NDV / Anas crecca / Omsk 9/2010 very promising for further studies of oncolytic activity and the development of candidate antitumor drugs.

Technical applicability

Characterization of the claimed strain. Strain NDV / Anas crecca Omsk / 9/2010 is a typical representative of the Newcastle disease virus of the genus Avulavirus family Paromyxoviridae. The inventive strain isolated from dead birds during an outbreak in the Omsk region. The strain was deposited in the collection of cultures of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "(FGUN SSC WB" Vector ") Rospotrebnadzor under registration number N ° V-539. Source of receipt. An isolate of the Newcastle disease virus, which was called NDV / Anas crecca / Omsk / 9/2010, was isolated from pathological material obtained from a bird that died during an outbreak in the Omsk region (spleen, lung, small intestine, brain). The virus was isolated on developing chicken embryos (RE) in the first passage, the death of RSC was observed after 1, 5 - 2 days from the moment of infection. Indication of the virus was carried out in a hemagglutination reaction (RGA) with rooster erythrocytes, hemagglutination with horse erythrocytes was not observed: hemagglutinating activity indices were 1: 64-1: 128 GAU / 0.2 ml. The infectious activity of the isolated virus was 5.2 lg EID ₅₀ / ml.

Virus identification. The primary identification of isolated influenza A virus was carried out by the method of polymerase chain reaction with the stage of reverse transcription (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 PJ, Decanini EL, Afonso CL./ Newcastle disease: evolution of genotypes and the related diagnostic challenges./ nfect Genet Evol. 2010 Jan; 10 (l): 26-35. Epub 2009 Sep 30).

Genetic and phylogenetic analysis. RNA was extracted from the virus suspension using the Ribo-sorb reagent kit (FGUN TsNIIE), the reverse transcription reaction was performed from the terminal, universal primer Random dN6 using the enzyme M-MuLV reverse transcriptase (FGUN TsNIIE). For sequencing, a section of the Newcastle disease virus genome was selected containing the C-terminal segment of the M gene, the Ν-terminal segment of the F gene, and the intergenic region. Using the nucleotide sequence of this site, the amino acid sequence of the site of proteolytic cleavage of the fusion protein, which is the main marker of pathogenicity for the Newcastle disease virus, can be recreated. For sequencing, primers based on SEPRL were used (Kim LM, King DJ, Curry PE, Suarez DL, Swayne DE, Stallknecht DE, Slemons RD, Pedersen JC, Senne DA, Winker K, Afonso CL./ Phylogenetic diversity among low- virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates.// J Virol. 2007 Nov; 81 (22): 12641 -53. Epub 2007 Sep 12). PCR was performed on DNA-Engine devices (Bio Rad, USA) using reagents manufactured by the Federal State Institution of Research and Development. Amplification fragments were sequenced using the cycle sequence method with ABI PRISM Big Dye ™ v.1.1 kit (Applied Biosystems, USA), according to the manufacturer's instructions using an ABI-3100 PRISM ™ Genetic Analyzer capillary automatic sequencer (Applied Biosystems, USA). Alignment of nucleotide sequences was performed using the DNASTAR program block according to the Clustal algorithm.

Phylogenetic analysis was performed using the MEGA 3.1 program using the Neighbor Joining method using the Kimura 2-parameter algorithm with the Bootstrap Test of Phylogeny (1000 repetitions) [Kumar S.].

Phylogenetic analysis of the nucleotide sequence of the genes showed that the strain NDV / Anas crecca / Omsk / 9/2010 belongs to class 2, genotype 7. The strain is closest to the strains previously described in Southeast Asia.

Cultural properties. Strain NDV / Anas crecca / Omsk / 9/2010, when cultured on the monolayer of the primary culture of chicken embryo fibroblast cells (FEC), causes a cytopathic effect (CPD) on days 2-4 (cultivation temperature 37 ° C, in the atmosphere 5% C0 ₂ ).

Pathogenicity for humans. Newcastle disease virus strains are not pathogenic for humans.

Pathogenicity for poultry. Strain NDV / Anas crecca / Omsk / 9/2010 belongs to the highly pathogenic variants of the Newcastle disease virus: intracerebral pathogenicity index = 2.

Pathogenicity for mammals. The strain does not exhibit pathogenicity in mammals

Duration of storage. 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 stable infectious activity of the virus for at least 2 years when stored at -70 ° C.

Claims

Claim

A strain producer of the Newcastle disease virus NDV / Anas crecca / Omsk / 9/2010, used to create a candidate anti-cancer drug based on it, deposited in the Vector State Center for Virology and Biotechnology of the Federal State Institution of Science under the registration number JV ° V-539.