RU2621868C1 - Recombinant stain of vacδ6 vaccinia virus with broken genes of viralence c3l, n1l, j2r, a35r, a56r, b8r for the production of live cultural attenuated vaccine against natural smallpox and other orthopoxviral human infections - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant strain of VACΔ6 vaccinia virus with broken genes C3L, N1L, J2R, A35R, A56R, B8R on the basis of cloned vaccinia virus (L-IVP strain). The proposed strain is deposited in State Collection of causative agents of viral infections, rickettsiosis of the SBSI of the SSC VB Vector with registration No. V-696 and is designed to produce a bladeless, attenuated live culture vaccine against the natural smallpox and other orthopoxviruses with enhanced immunogenic and protective activity.

EFFECT: proposed recombinant strain can be used in medicine to produce a vaccine against natural smallpox and other human orthopoxviral infections.

7 dwg, 2 tbl, 13 ex

Description

The invention relates to a recombinant attenuated vaccinia virus strain (VACΔ6) with violated C3L, N1L, J2R, A35R, A56R and B8R genes based on the cloned vaccinia virus strain L-IVP, which can be used in medicine, biotechnology, in particular in genetic engineering to obtain a live culture attenuated vaccine against smallpox and other human orthopoxvirus infections.

Smallpox virus (VNO), together with species pathogenic for humans, such as monkeypox viruses (SBI), smallpox cows (VOC), buffalo smallpox (PSA) and smallpox vaccine (WWP), belongs to the genus Ortopoxvirus of the family Poxviridae [Moss B. Poxviridae : the viruses and their replication. In Fields Virology / edited by D. M. Knipe. - Philadelphia: Lippincott Williams & Wilkins. - 2007 .-- 5th edn. - P. 2905-2946]. VNO causes smallpox - one of the most dangerous and deadly highly contagious infectious diseases of a person with a mortality rate of 20-30%, which claimed hundreds of millions of lives only in the 20th century and is one of the main causes of blindness in people of that time. At the same time, smallpox is the only human disease that has been eradicated as a result of the global vaccination campaign. This achievement remains one of the greatest triumphs of medical science. Given the serious post-vaccination complications of using the classic live vaccine and confirmation of the eradication of smallpox, in 1980 the World Health Organization recommended no further vaccination against this infection [Wiser I., Balicer RD, Cohena D. An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. // Vaccine. - 2007. - V. 25. - P. 976-984], although the threat of the return of this disease still exists.

As a result of the rejection of universal vaccination against smallpox in the human population more than 30 years ago, the proportion of the population that has lost specific immunity against this disease is increasing every year. This makes humanity more defenseless, not only against the possible infection of VNO, but also with other closely related orthopoxviruses, the natural reservoir of which are various animals, primarily rodents [Monkeypox. Official site of World Health Organization. - 2011. Fact sheet No. 161. http://www.who.int/mediacentre/factsheets/fs161/en/]. Such viruses include SBI, EQA and PSA, which cause smallpox diseases in animals. The spread of these viruses in the human population can potentially lead to their adaptation to the immune system of the human body and the appearance of viral variants that are epidemically dangerous for people. In addition, a great danger is the potential use of UPE as an agent of bioterrorist attacks, which can have disastrous consequences for the entire population of the Earth.

In recent years, unusually numerous outbreaks of orthopoxviral infections among people have begun to be recorded in various regions of the world, for example, in the Democratic Republic of the Congo, the number of cases of infection of SBI has increased 20 times since the 1980s, and outbreaks of this disease are regularly recorded in West Africa, the mortality rate of which is 1-8%. Also, cases of infection of people with PSA have become more frequent in different regions of India [Shchelkunov S. N. An increasing danger of zoonotic orthopoxvirus infections. // PLoS Pathog. - 2013. - V. 9. - e1003756]. In addition, in 2009 in the northwestern region of India, cases of infection of people with a close relative of VNO, camel pox virus, were recorded for the first time during the outbreak of this infection in animals [Bera BC, Shanmugasundaram K., Barua S., Venkatesan G., Virmani N ., Riyesh T., et al. Zoonotic cases of camelpox infection in India. // Vet Microbiol. - 2011. - V. 152. - P. 29-38]. Given the increasing scale of transport links around the world, the probability of the spread of these zoonotic orthopoxviruses becomes extremely high, as evidenced by the case of the importation of smallpox monkeys in the United States [Ninove L., Domart Y., Vervel C., Voinot C., Salez N., Raoult D. , et al. Cowpox Virus Transmission from Pet Rats to Humans, France. // Emerging Infectious Diseases. - 2009. - V. 15 - P. 781-784].

The potential emergence and revival of human diseases caused by orthopoxviruses requires the development of adequate countermeasures. The only method of combating smallpox that has proven effective is vaccination. But the use of classical live vaccines based on the Second World War to vaccinate the population now can lead to an even greater number of adverse reactions and their more severe manifestations than during the campaign to eradicate smallpox. Approximately 25% of the world's population is contraindicated in World War II vaccination. First of all, these are pregnant women and people with a suppressive state of immunity caused by cancer, organ transplantation, HIV infection, etc., as well as people with heart diseases, eczema and atopic dermatitis [Eckart RE, Love SS, Atwood JE, Arness MK, Cassimatis DC, Campbell CL, et al. Incidence and follow-up of inflammatory cardiac complications after smallpox vaccination. // J Am Coll Cardiol. - 2004. - V. 44. - P. 201–205; Kemper A. R., Davis M. M., Freed G. L. Expected adverse events in a mass smallpox vaccination campaign. // Effective Clinical Practice. - 2002. - V. 5. - P. 84-90]. Therefore, one out of every 450 people vaccinated will have a serious side effect leading to hospitalization [Casey C. G., Iskander J. K., Roper M. H., Mast E. E., Wen X. J., Torok T. J., et al. Adverse events associated with smallpox vaccination in the United States, January-October 2003. // JAMA. - 2005. - V. 294. - P. 2734–2743]. Therefore, ideally, modern vaccines should be safer and highly immunogenic, providing quick localization and elimination of the emerging infection. It is also necessary to search for more advanced vaccination regimens to achieve the fastest possible protection. The creation of such modern vaccines is one of the main tasks of modern health care [Verardi P. H., Titong A. and Hagen C. J. A vaccinia virus renaissance. New vaccine and immunotherapeutic uses after smallpox eradication. // Hum Vaccin Immunother. - 2012. - V. 8. - P. 961–970].

Since the structural proteins of orthopoxviruses are highly conservative in nature, immunization of the Second World War provides cross-protection against smallpox and other representatives of this genus, inducing the production of genus-specific antibodies. Therefore, for almost two centuries BOB strains used as vaccines, until the eradication of smallpox in the late ^1970s [Wiser I., Balicer RD, Cohena D. An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. // Vaccine. - 2007. - V. 25. - P. 976-984]. These vaccines, especially the first generation, led to rare but serious post-vaccination complications, the main of which are: post-vaccine encephalitis, progressive and generalized vaccines, vaccine eczema, myopericarditis. Although serious complications were rarely observed, but they became disabled and sometimes died, healthy people were vaccinated [Jacobs BL, Langland JO, Kibler KV, Denzler KL, White SD, Holechek SA, et al. Vaccinia virus vaccines: past, present and future. // Antiviral Res. - 2009. - V. 84. - P. 1-13].

Since the end of the 19th century, a number of modified vaccines based on WWII have been actively developed with improved safety characteristics. Attenuation was achieved using various strategies: sequential passages through alternative hosts, use of genetic engineering: either deletion of immunomodulating and vital genes of the virus, or insertion of host immunomodulating genes into the virus genome [Wiser I., Balicer RD, Cohena D. An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. // Vaccine. - 2007. - V. 25. - P. 976-984].

During the smallpox eradication campaign, attempts were made to create an attenuated strain of the Great Patriotic War, which would be distinguished by protective efficacy with little side effects [Fenner F., Henderson D. A., Arita I., Jezek Z., Ladnyi I. D. Smallpox and its eradication. Geneva: World Health Organization. - 1988. - 1460 pp.]. The first attenuated strains of the Second World War were obtained as a result of multiple passages of the virus, for example, in the chicken embryo fibroblast culture, the MVA strain [McCurdy L. H., Larkin B. D., Martin J. E., Graham B. S. Modified vaccinia Ankara: potential as an alternative smallpox vaccine. // Clin. Infect. Dis. - 2004. - V. 38. - P. 1749-1753], or on a culture of rabbit kidney epithelial cells at low temperature (30 ° C) - strain LC16m8 [Kenner J., Cameron F., Empig C., Jobes DV, Gurwith M. LC16m8: an attenuated smallpox vaccine. // Vaccine. - 2006. - V. 24. - P. 7009–7022]. In these cases, the attenuation of the virus is due to spontaneous extended deletions and mutations in the viral genome. Moreover, in addition to virulence genes, genes can also be affected whose functions determine the replicative properties of the virus, its sensitive range, etc. For example, MVA is characterized by low reactogenicity, but mutations also affected the host circle genes, which led to the replicative defectiveness of the virus in most mammalian cells, including humans. Therefore, for the formation of a sufficient level of immune defense, it is necessary either repeated administration or the introduction of large doses of viral. Strain LC16m8 is less attenuated than MVA, and due to gene disruption of one of the envelope proteins of the extracellular form of the virus (B5R), it may be less effective in forming immunity against orthopoxviruses, as it is not able to induce the production of antibodies to B5 surface antigen [Jacobs BL , Langland JO, Kibler KV, Denzler KL, White SD, Holechek SA, et al. Vaccinia virus vaccines: past, present and future. // Antiviral Res. - 2009. - V. 84. - P. 1-13].

With the development of molecular biology methods, it has become possible to create modified variants of the Second World War by introducing target sequences into the viral genome, or by removing or disrupting specific genes of the virus itself [Jacobs B. L., Langland J. O., Kibler K. V., Denzler K. L., White S. D., Holechek S. A., et al. Vaccinia virus vaccines: past, present and future. // Antiviral Res. - 2009. - V. 84. - P. 1-13]. Obtaining attenuated BOB can be achieved by disrupting genes encoding various molecular virulence factors, such as inhibitors of the action of interferons, apoptosis of infected cells, inflammatory reactions of the body, as well as genes: the host circle, extracellular virion coat proteins, viral growth factors, and proteins involved in the metabolism of nucleic acids, kelch-like and ankyrin-like proteins of the Second World War, etc. [Marennikova S. S., Schelkunov S. N. Pathogenic human orthopoxviruses. M .: KMK Scientific Press Ltd. - 1988. - 386 s]. An example is the NYVAC BOB strain, one of the most well-characterized genetically attenuated non-replicating BOB mutants. NYVAC obtained from the Copenhagen BOB strain by selective deletion of 18 genes / open translation frames. In an experiment on immunodeficient macaques, NYVAC showed safety, but could not provide protection against subsequent lethal infections of SBI [Edghill-Smith Y., Bray M., Whitehouse C. A., Miller D., Mucker E., Manischewitz J., et al. Smallpox vaccine does not protect macaques with AIDS from a lethal monkeypox virus challenge. // J. Infect. Dis. - 2005. - V. 191. - P. 372-381].

Thus, turning off virulence genes can significantly reduce the pathogenic properties of BOB. One of the most promising areas of such work is the creation by genetic engineering of highly attenuated variants of the Second World War, which are immunogenic and protective at the level of the classical anti-arsenic vaccine, but which are less pathogenic.

Known recombinant strain of Copenhagen BOB (international application No. WO92 / 15672, IPC C12N7 / 00, published on September 17, 1992), developed by Virogenetics Corporation (USA). Deletions in six regions of the pathogen genome were introduced into this BOB strain through recombination of specially created plasmids with the viral genome. However, in the recombinant strain, along with deletions of individual genes, another 12 viral genes were violated, which reduced the ability of BOB to replicate on different lines of human cells, thereby reducing its technological capabilities in vaccine production. As a result, the combination of virulence genes in the recombinant strain differs from the claimed technical solution. In addition, this mutant strain of the Second World War is not certified and is not used in Russia for the production of the classic anti-smallpox vaccine.

The closest analogue (prototype) is strain 1421ABJCN, obtained from the clonal variant of the L-IVP strain BOB (14L-IVP) by a sequential violation of five genes: C3L, N1L, J2R, A56R and B8R, which showed a significant reduction in reactogenicity and neurovirulence compared to the parental strain of L-IVP against the background of preserving the immunogenic and protective properties of the original strain [Yakubitsky S. N., Kolosova I. V., Maksyutov R. A., Schelkunov S. N. Attenuation of the vaccinia virus. // Acta Naturae. - 2015. - No. 27. - S. 125-134].

However, this strain does not have a sufficiently high immunogenic and protective activity.

In order to increase the immunogenic and protective activity of the obtained recombinant BOB strain, the authors of the present application proposed the introduction into the virus genome of a violation of the A35 protein gene having immunosuppressive properties [Rehm KE and Roper RL Deletion of the A35 Gene from Modified Vaccinia Virus Ankara Increases Immunogenicity and Isotype Switching. // Vaccine. - 2011. - V. 29. - P. 3276–3283].

Thus, the technical result of the present invention is the creation using six plasmids of integration of a highly attenuated recombinant strain of BOB to obtain a safer live culture attenuated vaccine against smallpox and other human orthopoxvirus infections with higher immunogenic and protective activity.

The specified technical result is achieved by obtaining a recombinant strain of VACΔ6 BOB with disrupted genes C3L, N1L, J2R, A35R, A56R and B8R, designed to receive a live culture attenuated vaccine against variola virus and other human orthopoxvirus infections and deposited in the State collection of viral infections, ricket infections FBUN SSC VB "Vector" with registration number V-696 (certificate of deposit is attached).

To create a highly attenuated variant of the BOB of the L-IVP strain, the approach of sequential violation of key genes of various molecular virulence factors of this virus was used. Using the published data on the violation of the genes of various strains of the Second World War leading to the attenuation of viruses, we selected the most promising of them for this purpose: C3L, N1L, J2R, A35R, A56R and B8R.

The C3L gene encodes a complement binding protein (KSB) secreted by an infected cell [Kotwal GJ, Isaacs S. N, McKenzie R., Frank MM and Moss B. Inhibition of the complement cascade by the major secretory protein of vaccinia virus. // Science. - 1990. - V. 250. - P. 827-830] and the inhibitory activity of the complement system through the enzymatic inactivation of C3b and C4b by factor I, accelerating their decay and preventing the initiation of the classical and alternative ways of C3 conversion [Sahu A., Isaacs SN, Soulika AM and Lambris JD Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway. // J. Immunol. - 1998. - V. 160. - P. 5596-5604]. Thus, KSB inhibits antibody-dependent, complement-enhanced neutralization of BOB virions. In animal experiments, KSB-free mutants had reduced virulence [Isaacs SN, Argyropoulos E., Sfyroera G., Mohammad S. and Lambris JD Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein. // Journal of Virology. - 2003. - V. 77. - P. 8256-8262].

The N1L gene product is the N1 protein, an intracellular homodimer belonging to the Bcl-2 antiapoptotic family of proteins [Cooray S., Bahar MW, Abrescia NG, McVey CE, Bartlett NW, Chen RA, et al. Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. // J. Gen. Virol. - 2007. - V. 88. - P. 1656-1666] and inhibiting both apoptosis and the signal from the interleukin-1 receptor, leading to activation of the NF-κB pathway (nuclear factor kappa-light-chain-enhancer of activated B cells - a universal transcription factor that controls the expression of genes for the immune response, apoptosis and the cell cycle). Despite the location of this gene in the terminal (variable) region of the genome (close to the left end of the genome of the WR strain), it has been preserved in many representatives of the genus Orthopoxvirus [Bartlett N., Symons JA, Tscharke DC and Smith GL The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. // J. Gen. Virol. - 2002. - V. 83. - P. 1965-1976].

In a study using an intranasal model of infection of mice with a virus that lost the N1L gene, it was shown that such a BOB mutant induces enhanced local activity of natural killers between 4 and 6 days after infection [Jacobs N., Bartlett NW, Clark RH and Smith GL Vaccinia virus lacking the Bcl-2-like protein N1 induces a stronger natural killer cell response to infection. // Journal of General Virology. - 2008. - V. 89. - P. 2877-2881]. In other studies, in the intradermal model of infection of mice, a deletion mutant for this gene induced lesions of a smaller size than in controls with wild type and revertant [Jacobs N., Chen RA, Gubser C., Najarro P. and Smith GL Intradermal immune response after infection with vaccinia virus. // J. Gen. Virol. - 2006. - V. 87. - P. 1157-1161], as well as less infectious virus was obtained from infected tissue [Bartlett N., Symons JA, Tscharke DC and Smith GL The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. // J. Gen. Virol. - 2002. - V. 83. - P. 1965-1976]. In the study [Ren H., Ferguson BJ, Maluquer de Motes C., Sumner RP, Harman LE, Smith GL Enhancement of CD8 (+) T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor. // Immunology. - 2015. - V. 145. - P. 34-49], it was demonstrated that impaired expression of the N1 protein in the WR BOB strain leads to an increase in CD8 ⁺ T-cell immunogenicity while reducing virulence.

The J2R gene encodes the thymidine kinase (TK) enzyme involved in the regulation of the level of deoxyribonucleoside triphosphate pool, namely, it catalyzes the thymine → thymidine monophosphate reaction [S. Marennikova, S. N. Schelkunov. Human pathogenic orthopoxviruses. M .: KMK Scientific Press Ltd. - 1988. - 386 s]. [Lee MS, Roos JM, McGuigan LC, Smith KA, Cormier N., Cohen LK, et al. Molecular attenuation of vaccinia virus: mutant generation and animal characterization. // Journal of Virology. - 1992. - V. 66. - P. 2617-2630] it was shown that the TK ^- mutant of the strain Wyeth-M BOB has the phenotypic properties of a potential vaccine substrate - intracranial virulence is lower than detection levels (> 8.0 lg), but at this virus is able to grow in mouse skin and cause immunity like a wild-type strain.

In another study, it was found that inactivation of the J2R gene by insertion of a foreign nucleotide sequence into it in the WR BOB strain leads to a significant decrease in neurovirulence and viral titers in various organs of mice infected with this mutant, compared with the wild-type virus [Zhang Q., Liang C., Yu YA, Chen N., Dandekar T., Szalay AA The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation. // Mol. Genet. Genomics - 2009. - V. 282. - P. 417-435]. With an additional violation in the genome of this mutant BOB variant of the F14.5L and A56R genes, tumor-specific replication of the obtained strain was demonstrated, as well as its ability to eliminate tumors coupled with a practical absence of virulence in mice [Chen NG, Yu YA, Zhang Q., Szalay AA Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice. // Journal of Translational Medicine. - 2011. - V. 9. - P. 1-11].

In the structure of the A35R BOB gene, a highly conserved membrane protein A35 is encoded that inhibits antigen presentation limited by MHC class II, immune priming of T lymphocytes and subsequent synthesis of chemokines and cytokines [Rehm KE, Connor RF, Jones GJB, Yimbu K., and Roper RL Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation. // Virology. - 2010. - V. 397. - P. 176]. In [Roper RL Characterization of the Vaccinia Virus A35R Protein and Its Role in Virulence. // Journal of Virology. - 2006. - V. 80. - P. 306–313], the attenuation of the A35 ^- mutant of strain WR BOB was demonstrated in an intranasal infection model of mice compared to wild-type virus and revertant, while maintaining normal growth and morphogenesis in the in vitro system .

Another study also showed the preservation of reproductive abilities by the BOB strain MVA during deletion of the A35R gene against the background of an improvement in its immunogenicity: an increase in the production of BOB-specific immunoglobulins and virus-specific splenocytes secreting γ-interferon, as well as the absence of inhibition of “switching” the class of immunoglobulins to isotype IgG and subclasses of IgG1 and IgG2a. At the same time, the recombinant variant of BOB in the mouse model showed excellent protective efficacy against 500LD ₅₀ WR BOB [Rehm KE and Roper RL Deletion of the A35 Gene from Modified Vaccinia Virus Ankara Increases Immunogenicity and Isotype Switching. // Vaccine. - 2011. - V. 29. - P. 3276–3283]. Similar data were obtained using the A35 ^- mutant strain WR BOB in the model of intraperitoneal infection of mice [Rehm KE, Jones GJB, Tripp AA, Metcalf MW, and Roper RL The Poxvirus A35 Protein Is an Immunoregulator // Journal of Virology. - 2010. - V. 84. - P. 418-425], which also showed an improvement in the cytotoxic response mediated by T-lymphocytes in animals infected with a recombinant strain of BOB compared with wild-type virus.

The A56 transmembrane glycoprotein is encoded by the BOB gene A56R and performs both a hemagglutinating function, ensuring the ability of the virus to attach to the host cell and the function of an “anchor” on the surface of infected cells for two other secreted BOB proteins: serine protease inhibitor (K2) and KSB. The A56-K2 complex reduces superinfection of already infected cells and inhibits the formation of syncytia by infected cells, and the A56-KSB complex protects infected cells from complement attacks [Dehaven BC, Gupta K., Isaacs SN The vaccinia virus A56 protein: a multifunctional transmembrane glycoprotein that anchors two secreted viral proteins. // Journal of General Virology. - 2011. V. 92. - P. 1971-1980].

In a mouse model, deletion of the A56R gene in the NYCBH BOB strain showed a decrease in intracranial and intranasal LD _{50 by} approximately 4 lg compared to the parent strain. This was accompanied by the disappearance of smallpox formations on the scarified skin of mice, despite the level of virus replication close to the wild type [Lee MS, Roos JM, McGuigan LC, Smith KA, Cormier N., Cohen LK, et al. Molecular attenuation of vaccinia virus: mutant generation and animal characterization. // Journal of Virology. - 1992. - V. 66. - P. 2617-2630]. In another study, BOB strain WR was used, changes in the A56R gene of which also led to a strong attenuation of the virus [Zhang Q., Yu YA, Wang E., Chen N., Danner RL, Munson PJ, et al AA Eradication of solid human breast tumors in nude mice with an intravenously injected light-emitting oncolytic vaccinia virus. // Cancer. Res. - 2007. - V. 67. - P. 10038-10046].

The BOB gene BOBR encodes a secreted γ-interferon-binding protein glycoprotein that is similar in amino acid sequence to the extracellular domain of the γ-interferon cell receptor, thereby binding and inhibiting this pleiotropic cytokine in a wide range of species [Alcamı A. and Smith GL Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity. // J. Virol. - 1995. - V. 69. - P. 4633-4639].

Various studies have found that removing the B8R gene leads to attenuation of the virus in a mouse model without a concomitant decrease in immunogenicity [Verardi PH, Jones LA, Aziz FH, Ahmad S. and Yilma TD Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene ( B8R) are attenuated in vivo without a concomitant reduction in immunogenicity. // Journal of Virology. - 2001. - V. 75. - P. 11-18], causing a lower incidence even at high doses [Denes B., Gridley DS, Fodor N., Takatsy Z., Timiryasova TM, Fodor I. Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor. // J. Gene Med. - 2006. - V. 8. - P. 814-823]. In experiments with intradermal infection of rabbits, BOB, devoid of the γ-interferon-binding protein gene, caused lesions with histological differences compared to the wild-type virus (smaller in size and tendency to faster disappearance) [Symons JA, Tscharke DC, Price N. and Smith GL A study of the vaccinia virus interferon-γ receptor and its contribution to virus virulence. // Journal of General Virology. - 2002. - V. 83. - P. 1953–1964; Huang W., Liu Y., Duan D. L., Li H. S., Liu Y., Hong K. X., et al. The construction of attenuated Tiantan recombinant vaccinia virus vector with IFN-gamma receptor gene deletion. // Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. - 2004. - V. 18. - P 43-46].

The invention is illustrated by the following graphic materials. The figure 1 shows the General scheme for the construction of plasmids for integration and obtaining recombinant variants of the vaccinia virus with deletion of genes (for example, the A35R gene). In FIG. Figure 2 shows the PCR (polymerase chain reaction) analysis of a recombinant BOB clone with a deletion of six virulence genes - VACΔ6 in comparison with the previously obtained 1421ABJCN and the original 14L-IVP, namely, the electrophoregram of DNA fragments obtained by PCR with primers: A - to the A56R gene , B - for the B8R gene, C - for the C3L gene, N - for the N1L gene, J - for the J2R gene, A '- for the A35R gene, M - 1 kb DNA marker.

In FIG. Figure 3 presents a comparative analysis of the growth dynamics of BOB strains (1421ABJCN and VACΔ6 in comparison with the initial 14L-IVP) on CV-1 cell culture. In FIG. Figure 4 shows a graph of the dynamics of the death of mice twice immunized with VACΔ6, 1421ABJCN and 14L-IVP BOB strains in the amount of 10 ⁷ plaque-forming units (PFU) / mouse after infection with ectromelia virus (VE) at a dose of 100 LD ₅₀ . In FIG. Figure 5 shows the levels of virus-neutralizing antibodies determined by the method of neutralization of BOB on 4647 cell culture. The antibodies were obtained after double subcutaneous immunization of BALB / c mice with strains VACΔ6 and 1421ABJCN along with the parent strain of 14L-IVP BOB at a dose of 10 ⁷ PFU / animal. The data are presented in the form of geometric mean values calculated as –lg from the highest dilution of serum at which 50% neutralization of BOB is achieved, ± standard deviation. In FIG. Figure 6 shows the dynamics of death of sucker mice after intracerebral administration of strains 1421ABJCN and VACΔ6 in comparison with the initial 14L-IVP BOB at a dose of 10 ² PFU / mouse. In FIG. 7 shows a histogram of the arithmetic mean values of the titers of vaccine strains VACΔ6, 1421ABJCN and 14L-IVP in the form of lg PFU / g of organ ± standard deviation determined by the plaque method in the brain homogenate of infected newborn mice after intracerebral infection.

To obtain a technical result, a temporary dominant selection technique is used [Falkner F.G., Moss B. Transient dominant selection of recombinant vaccinia viruses. // Journal of Virology. - 1990. - V. 64. - P. 3108-3111]. To implement this technique, a recombinant plasmid is created that carries both a dominant selectable marker (the E. coli gpt gene under the control of the 7.5K BOB promoter) located outside extended regions of homology with the DNA of the virus and the BOB genome sequences flanking the deleted gene. The bacterial enzyme xanthine-guanine phosphoribosyltransferase synthesized in mammalian cells is able to restore the synthesis of guanosine monophosphate blocked by mycophenolic acid (MPA). As a result of a single crossing-over of the integration plasmid and viral DNA, a recombinant viral genome is formed containing both the gpt gene and sequences representing a fragment of the viral genome with the target gene deletion and the same fragment without deletion. Such a genetic construct is unstable and can exist only under selective pressure. When selective conditions are lifted, intramolecular recombination over homology regions occurs, as a result of which two variants of the virus are formed, with and without deletion, and the entire plasmid part is cleaved (Fig. 1). It should be noted that as a result of intramolecular recombination, all foreign sequences are removed from the genome of the virus, which is very important when creating a directionally attenuated BOB.

Construction of plasmids .

To obtain integration plasmids based on the pMGC20-gpt plasmid, a previously developed scheme was used (Fig. 1). This technical task was achieved using the PCR method, enzymatic hydrolysis, subsequent ligase crosslinking and transformation of E. coli cells strain XL2-blue [Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. - 1989. - 1626 pp.]. As a result, six integration plasmids were obtained - pΔC3L, pΔN1L, pΔJ2R, pΔA35R, pΔA56R and pΔB8R, designed to disrupt selected BOB genes. The constructed plasmids were used at the next stage of work to solve the technical problem of obtaining BOB variants with directionally violated genes of molecular factors of the virulence of the virus.

Construction of a mutant variant of the Second World War.

We used the previously obtained 1421ABJCN BOB strain with five genes sequentially violated (C3L, N1L, J2R, A56R and B8R), which was achieved using integration plasmids pΔC3L, pΔN1L, pΔJ2R, pΔA56R and pΔB8R and using the method of temporary dominant selection [Yaku N., Kolosova I.V., Maksyutov R.A., Schelkunov S.N. Attenuation of the vaccinia virus. // Acta Naturae. - 2015. - No. 27. - S. 125-134].

To obtain a variant of the Second World War with the deletion of the additional sixth A35R gene, the method of temporary dominant selection was also used. For this, a series of experiments was conducted to transfect infected BOB strain 1421ABJCN cells with CV-1 integration plasmid pΔA35R. After four passages under selection, the virus was cloned under agarose plaque. Further, the selected clones of the alleged deletion mutants were grown under non-selective conditions and recloned. After the procedures, the DNA of the clones was isolated and analyzed by PCR using internal primers. Reclons were identified, the fragments of which showed the correspondence of lengths to theoretically calculated ones. According to the results of PCR analysis, stably replicating receptions were selected, worked out, packaged in individual aliquots, titrated by plaque, frozen and used for further work.

Thus, an attenuated vaccinia virus strain L-IVP with six violated virulence genes was obtained: deletion of the B8R, C3L, A35R, A56R, N1L genes and insertion into the J2R gene - VACΔ6 (PCR analysis in Fig. 2). The strain is deposited in the State collection of causative agents of viral infections, rickettsioses FBUN SSC WB "Vector" registration number V-696 from 10.26.2015

Morphological features of the claimed strain.

The authenticity was confirmed in accordance with the requirements of the European Pharmacopoeia 7.0 (p. 1152) by PCR and titration on a cell culture 4647 (transplantable cell culture line; seed and working banks of cells of this line at the 108th and 128th passages are recommended for vaccine production against smallpox [Skarnovich M. O., Radaeva I. F., Vdovichenko G. V., Nechaeva E. A., Sergeev A. A., Petrishchenko V. A., et al. Cell culture 4647 for the production of recombinant bivaccine against smallpox and hepatitis B. // Questions of virology. - 2007. - No. 2. - S. 37-40]). It was determined that when titrating into a monolayer of cell culture 4647, the vaccine strain BOB VACΔ6 produces plaques of the same type with a diameter of 0.5 to 1.0 mm. The lengths of the fragments obtained by PCR analysis of the vaccine strain of BOB correspond to the calculated data given in table 1 and in FIG. 2.

Table 1

The length of the PCR products for the source and recombinant variants of the Second World War

Gene The initial strain of L-IVP of the Second World War, bp Vaccine strain VACΔ6 BOB, bp A35r 1880 1360 A56r 2366 1425 B8r 1555 737 C3l 1542 751 N1L 1784 1431 J2r 512 617

When studying the properties of the obtained strain, its main properties were studied: authenticity, sterility, harmlessness, immunogenic activity, specific safety, neurovirulence, and residual virulence. It was found that when titrating a monolayer of cell culture 4647, the concentration (titer) of the virus is at least 10 ⁷ PFU / ml.

The test results of the replicative properties of the VACΔ6 variant in comparison with 1421ABJCN and the initial 14L-IVP BOB in the cell culture 4647 showed that the virus development curves do not differ significantly (Fig. 3). This indicates that the violation of the selected genes C3L, N1L, J2R, A35R, A56R, and B8R does not affect the reproductive functions of the virus in the studied cell culture, which is important from the point of view of the technology for producing vaccines in cell cultures.

The strain is sterile and harmless when administered subcutaneously for guinea pigs and intraperitoneal infection for white mice.

The 100% protective effect of all three variants of BOB (VACΔ6, 1421ABJCN and 14L-IVP BOB) was shown after double immunization of BALB / c mice at a dose of 10 ⁷ PFU / mouse versus highly virulent for mice VE at a dose of 10 LD ₅₀ / mouse. In order to identify differences between the studied strains of the Second World War, a fundamentally higher resolving dose of SE was selected in the amount of 100 LD ₅₀ / mouse. As a result, for a vaccinating dose of 10 ⁷ PFU / mouse, after double subcutaneous immunization, VACΔ6 BOB showed a 100% protective effect, while for the initial strains 1421ABJCN and 14L-IVP, this indicator was reduced and amounted to only 50 at the end of the experiment (day 14) % and 67% of immunized animals, respectively (Fig. 4). In the future, in the study of protectability, either the WR BOB strain or more homologous to the BOB wok can be used as a resolving agent.

The titers of virus-neutralizing antibodies in the blood serum of mice obtained after double immunization of animals with VACΔ6 and 1421ABJCN strains were also determined in comparison with the initial 14L-IVP BOB at a dose of 10 ⁷ PFU / mouse. For immunization, the method by which the drug is proposed to be administered to a person, i.e. subcutaneous. As a result of the experiments, it was found that subcutaneous immunization of BALB / c mice with all viral preparations causes the production of virus-neutralizing antibodies. Moreover, significantly (p <0.01), the highest titers were observed for the group immunized with the VACΔ6 strain, whereas for groups of animals infected with the original variants 1421ABJCN and 14L-IVP BOB, the titer levels of BOB-neutralizing antibodies were lower and did not differ significantly (Fig. 5 )

In addition, laboratory experiments were conducted to study the residual virulence of the vaccine strain VACΔ6 in comparison with 1421ABJCN and 14L-IVP BOB. For this, BALB / c mice weighing 14-16 g were injected with vaccine strains preparations subcutaneously or intraperitoneally at a dose of 10 ⁷ PFU / mouse, which corresponds to the immunizing dose of the vaccine. Animals of the control group received an injection of saline. Subsequently, on various days after infection, tissues of the brain, lungs, liver, spleen and blood were taken to determine the titer of the virus in the 10% tissue homogenates prepared from them, conducting three consecutive passages on the cell culture 4647. As a result, it was found that with subcutaneous and intraperitoneal immunization of mice on the 3rd, 7th, and 14th day after injection, all the studied viruses do not multiply, do not spread, and do not stay in the animal organism in the studied organs.

A decrease in neurovirulence was shown in the VACΔ6 and 1421ABJCN variants compared to the initial 14L-IVP BOB strain, which was studied after intracerebral infection of newborn mice with the studied BOB strains at a dose of 10 ² PFU / mouse. In the group infected with 14L-IVP of the Second World War, the death of animals began from the 4th day, and the mortality rate on the 8th day of the experiment was 100%. In groups of mice that were injected with strains of VACΔ6 and 1421ABJCN BOB, the death of animals during the experiment (14 days) did not significantly differ, not exceeding 20% (Fig. 6).

The obtained results are confirmed by the levels of viral load detected after titration on a cell culture of 4647 10% of tissue homogenates of brain tissue seized 3 days after intracerebral infection of sucker mice with the studied viruses at a dose of 10 ² PFU / mouse. Comparative indices of neurovirulence were obtained for the VACΔ6 and 1421ABJCN variants, and higher titers of the viral material were shown for the parent strain 14L-IVP BOB (Fig. 7).

A lower reactogenicity of strain VACΔ6 along with variant 1421ABJCN as compared with the initial 14L-IVP BOB was also revealed, which was evaluated by inflammatory necrotic manifestations on the epilated skin of rabbits after intradermal administration of viral preparations at a dose of 10 ² -10 ⁷ PFU / 0.05 ml / injection . The VACΔ6 and 1421ABJCN strains caused an inflammatory reaction only starting with a dose of 10 ⁵ PFU / injection, while the parental variant of 14L-IVP BOB with the lowest dose of 10 ² PFU / injection (Table 2). Inflammatory necrotic manifestations from VACΔ6, as well as from 1421ABJCN, were less pronounced compared to 14L-IVP of the Second World War. At the same time, no differences in inflammatory-necrotic indices were observed between the two studied strains of VACΔ6 and 1421ABJCN of the Second World War.

Table 2.

Comparison of inflammatory and necrotic parameters of WWII strains during intradermal infection of rabbits

Day after infection Virus titer, PFU / injection VACΔ6 1421ABJCN 14LIVP 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ one - - - AT AT AT - - - AT AT AT - - AT AT AT AT - - - AT AT AT - - - AT AT AT - AT AT AT AT AT 3 - - - AT AT AT - - - AT AT B / N AT AT AT AT B / N B / N - - - - AT AT - - - AT AT B / N AT AT AT AT AT B / N 5 - - - - AT B / N - - - - AT B / N AT AT AT AT B / N B / N - - - - AT B / N - - - - AT B / N AT AT AT AT AT B / N 7 - - - - AT B / N - - - - AT B / N AT AT AT AT B / N B / N - - - - AT B / N - - - - AT B / N - AT AT AT B / N B / N 9 - - - - B / N B / N - - - - B / N B / N - - - B / N B / N B / N - - - - B / N B / N - - - - B / N B / N - - AT AT B / N B / N eleven - - - - N B / N - - - - B / N B / N - - - N B / N B / N - - - - N B / N - - - - B / N B / N - - - - B / N B / N 13 - - - - - B / N - - - - - B / N - - - - - B / N - - - - - B / N - - - - - B / N - - - - N B / N fourteen - - - - - B / N - - - - - B / N - - - - - AT - - - - - AT - - - - - B / N - - - - - -

Note: B - inflammation, N - necrosis

Thus, in experiments on animals on a comparative study of harmlessness, it was found that the VACΔ6 strain does not differ from 1421ABJCN, that is, it does not have residual virulence, and is also characterized by reduced neurovirulence and reactogenicity compared to the initial 14L-IVP strain of the Second World War. Moreover, in the in vitro system, the VACΔ6 variant was developed with the same efficiency as the parent 14L-IVP strain.

In addition, in experiments on a comparative study of the specific activity of viruses, it was found that the VACΔ6 variant induces the production of significantly higher titers of BOB-neutralizing antibodies than prototypes: the vaccine strain 14L-IVP and the recombinant variant 1421ABJCN BOB. And also the recombinant variant of VACΔ6 BOB provides complete protection of animals against a lethal dose of 100 LD ₅₀ / mouse of highly pathogenic VE for them, in contrast to the initial strains 1421ABJCN and 14L-IVP.

The storage conditions of the strain:

- storage at temperatures from -70 ° C to -18 ° C.

Strains of bacteria and viruses, cell culture, animals used to obtain the inventive strain VACΔ6 BOB.

We used a clone variant of the L-IVP strain of the Second World War (reg. No. V-401 from the collection of the Federal State Budget Scientific Center of the VB Vektor) - 14L-IVP, an attenuated version of 1421ABJCN based on it, with five violated virulence genes (A56R, B8R, J2R , N1L, C3L) (reg. No. V-653 in the collection of FBSI SSC VB “Vector”) and strain VACΔ6 with deletion of the additional sixth virulence gene A35R based on variant 1421ABJCN (the strain was deposited in the State collection of viral infections, rickettsioses FBSI SSC VB "Vector" reg. No. V-696 of 10.26.2015). In experiments on the study of the protective properties of strains of the Second World War, the strain K-1 VE from the collection of the Federal State Budgetary Scientific Center of the World Bank "Vector" was also used.

In experiments on the construction of the integration plasmid pΔA35R, the strain XL2-blue E. coli {recA1 endA1 gyrA96 thi-1 hsdR17 (r _K- , m _{K +} ) supE44 relA1 lac [F 'proAB lac ^q ZSYMBOL 68 \ f "Symbol" \ s was used 12DM15 Tn10 (Tet ^r ) Amy Cam ^r ] ^{c, d} } from the collection of the State Research Center of the World Bank "Vector".

To obtain a recombinant variant of strain VACΔ6 BOB, a transplantable culture of green monkey kidney cells CV-1 was used. To develop strains of the Second World War and CE, to construct the growth curves of strains of the Second World War, as well as to determine the titers of viruses and levels of virus neutralizing antibodies, we used a cell culture 4647, an African green monkey kidney cell line. Cell culture 4647 certified GISK them. L. A. Tarasevich in accordance with the requirements of RD 42-28-10-89 and is recommended for the production of preventive medical immunobiological preparations (MIBP) (protocol No. 14 dated 10.28.03 of the meeting of the Academic Council of GISK named after L. A. Tarasevich; protocol No. 9 of November 20, 2003 of the MIBP Committee), including for the production of vaccine against smallpox [Skarnovich M.O., Radaeva I.F., Vdovichenko G.V., Nechaeva E.A., Sergeev A. A., Petrishchenko V. A., et al. Cell culture 4647 for the production of recombinant bivaccine against smallpox and hepatitis B. // Questions of virusolo gii. - 2007. - No. 2. - S. 37-40]. Both cell cultures were obtained from the collection of cell cultures FBSI SSC VB "Vector". Cell cultures were cultured on DMEM growth medium supplemented with 10% fetal bovine serum in the presence of 5% CO ₂ at 37 ° C.

Depending on the objectives of the experiment, to study the various biological properties of the VACΔ6 BOB, we used laboratory animals from the nursery FBUN SSC VB "Vector": mice of the Balb / c line, females weighing 20-22 g (age 10-12 weeks) or weighing 14- 16 g (age 4-5 weeks), as well as 1-2 daily suckers of the same line weighing 5-6 g; Chinchilla rabbits weighing 1.5-2.0 kg. All experiments with animals were carried out in compliance with the "Rules for the work using experimental animals."

The nucleotide sequence of DNA of BOB strain L-IVP was obtained from the database with identification number DQ121394 (http://www.poxvirus.org).

The invention is illustrated by the following examples of specific performance.

Example 1. Obtaining plasmids integration. Two fragments of the BOB genome were obtained by PCR, flanking each broken gene on the left (L-flank) and on the right (R-flank). The oligonucleotide primers for PCR were designed so that deletion of the target gene did not lead to disruption of the adjacent genes. The calculation of primers for PCR and the selection of reaction conditions was performed using the program "Vector NTI Suite 7" company InforMax, USA. Primers were synthesized on an ABI-394 automatic synthesizer (Applied Biosystems, USA) at the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk):

For C3L gene deletion:

L-flank:

5'-TAGTTTATCGAGATCTGATCTG 3 '- (BglII),

5'-GGGGGGGTGACCAAAATTATACAATGGTTTATGTC-3 '(BstEII);

R-flank:

5'-GGGGGGGTCTCCATTTATTTATCCGTAAAAATGTT-3 '(BstEII),

5'-GGGGAAGCTTATATAATGCGTCGATGTCAG-3 '(HindIII).

For deletion of the N1L gene:

L-flank:

5'-GGGTTGGATCCTTTACACATAGATCTACTACAGGCGGAACA-3 '(BamHI),

5'-CCGAAGGTGACCTCTTTAGGATGATTGAAACATATTTTGATGA-3 '(BstEII);

R-flank:

5'-GCCAAGGTCACCGATCGTTGTCATTTCTCCAAAGAATATATCTA-3 '(BstEII),

5'-GGGAAAGCTTAATTTGTGAAGATGCCATGTACTACGCT-3 '(HindIII).

For a J2R gene deletion:

L-flank:

5'-GAGCTCATGGATCACAACCAGTATCTCT-3 '(SacI),

5'-AGATCTGGATCCAACAATGTCTGGAAAGAACTGT-3 '(BglII);

R-flank:

5'-AGATCTGTCGACGAATTCTGTGAGCGTATGGC-3 '(BglII),

5'-AAGCTTTGGATCTTACATCAGAAATTAAAA-3 '(HindIII).

For deletion of the A35R gene:

L-flank:

5'-GGGGAAGCTTTCATGATGACACCAGAAAACGACGAA-3 '(HindIII),

5'-GCAACTGCAGATGGCGGCGTACGTTAACGACTTATT-3 '(PstI);

R-flank:

5'-GGGGCTGCAGATCATAAAAGTTGTAAAGTAAATAATAAAA-3 '(PstI),

5'-GGCAAAGGATCCTCAGATATAATTTTTTGATGTTCTGT-3 '(BamHI).

For deletion of the A56R gene:

L-flank:

5'-GGGAAAGCTTATAAAAAATGAGGATTTCGCCCCA-3 '(HindIII),

5'-CCGAAGGTGACCTTAGATGTCTGAGGAAAAGGTGTAGCGT-3 '(BstEII);

R-flank:

5'-GCCAAGGTCACCGTTCACGTAAATACAAAACAGAGAACAA-3 '(BstEII),

5'-GGGAAGGATCCAAATGGAACATGTTCGCCATTAGACA-3 '(BamHI).

For deletion of the B8R gene:

L-flank:

5'-CCCAGATCTTGCTACCGTGAATATAAATCCGTT-3 '(BglII),

5'-ConverterCGGTGACCATCTCATGGTGTTGTTTGTTATTTGACT-3 '(BstEII);

R-flank:

5'-ConverterCGGTCACCTGATACAAAAAACGAAATAAAACTGCATA-3 '(BstEII),

5 ' -AppAAGCTTCGAACTAGTGAAATTTTTTCTGACCTTA-3 '(HindIII).

The recognition sites of restriction endonucleases, the names of which are indicated in brackets on the right, are underlined.

Both flanking regions of each gene were cloned simultaneously into the vector plasmid pMGC20-gpt carrying the ampicillin resistance gene for selection in the prokaryotic system. For this, the PCR fragments and the vector plasmid were processed with the necessary restriction enzymes indicated above, in accordance with the recommendations of the enzyme manufacturer (SibEnzyme, Russia). Next, DNA fragments and plasmid were isolated and purified from 1% agarose gel (Medigen, Russia) using the Gel Extraction Kit (QIAGEN, Germany) in accordance with the manufacturer's instructions. Then, two fragments of viral DNA and vector plasmid were ligated, taken in the ratio 5: 5: 1, respectively, following the recommendations of the manufacturer of the T4 phage DNA ligase enzyme (SibEnzyme, Russia). The obtained ligase mixtures were used for chemical transformation of competent cells of E. coli strain XL2-blue [Methods of genetic engineering. Molecular Cloning: Per. from English / Maniatis T., Fritsch E., Sambrook J. - M .: World. - 1984. - 480 s]. Then, biomass from grown individual colonies in the presence of ampicillin was accumulated. Plasmid DNA from cell biomass was isolated by alkaline lysis [Lee S. Y., Rasheed S. A simple procedure for maximum yield of high-quality plasmid DNA. // Biotechniques. - 1990. - V. 9. - P. 676-679] and purified using the PCR Purification Kit (QIAGEN, Germany). Samples of unhydrolyzed plasmid DNA were analyzed on a 1% agarose gel on Tris acetate buffer. According to the results of the analysis, the necessary options were selected and further analyzed by hydrolysis with the corresponding restriction endonucleases, as well as by PCR analysis, using “external” primers for each gene, for example, for the A35R gene:

5'-GGGGAAGCTTTCATGATGACACCAGAAAACGACGAA-3 ',

5'-GGCAAAGGATCCTCAGATATAATTTTTTGATGTTCTGT-3 '.

Example 2. Obtaining recombinant variants of WWII.

Recombinant BOB was obtained on CV-1 cell culture using cationic lipid-mediated transfection using Lipofectin Reagent (Invitrogen, USA) and selective medium containing IFC, xanthine and hypoxanthin (Sigma, USA). For this, the daily monolayer of CV-1 cells was infected with BOB strain 1421ABJCN with a multiplicity of infection of 0.1 PFU / cell, followed by incubation for 1.5 h at 37 ° C. Then, the cell monolayer was washed with serum-free medium, and a mixture of 2 μg recombinant plasmid pΔA35R with 20 μg transfection agent in DMEM medium (BioloT, Russia) containing 25 μg / ml IFC, 250 μg / ml xanthine and 15 μg / ml was added. hypoxanthine, followed by incubation at 37 ° C and 5% CO ₂ . After 24 hours, the medium was replaced by selective support (with 2% fetal bovine serum (ESC) (HyClone, USA)), which contained IFC, xanthine and hypoxanthine, and incubation continued under the same conditions for another day. After four to five passages under selection conditions (until cytopathic action (CPP)), the virus was cloned.

Before cloning, the viral suspension was sonicated and titrated on a CV-1 cell culture. The virus was cloned by plaque under an agarose coating. For this, the monolayer of CV-1 cells was infected with a virus at a dilution of 10-20 PFU / well, followed by adsorption for 1 h at 37 ° C. Then, the unsorbed virus was removed, and a mixture of DMEM medium with 2% ESC and 1% low-melting agarose was added to the monolayer (Sigma, USA). After the agarose solidified, the monolayer of cells was incubated in an incubator at 37 ° C and 5% CO ₂ for 48 hours. Next, an aqueous solution of neutral red diluted in DMEM containing 1% low-melting agarose was added to the well, followed by incubation for 5 hours at 37 ° C. After that, individual plaques were carefully transferred to the monolayer of CV-1 cells. Subsequently, these clones were grown under non-selective conditions and recloned as described above. According to the results of PCR analysis, stably replicating receptions were selected, worked out, packaged in individual aliquots, titrated by plaque, and frozen for use in future work.

Example 3. Isolation of viral DNA.

To isolate viral DNA, the CV-1 monolayer of cells was infected with viral clones with a multiplicity of 0.1 PFU / cell, followed by incubation under DMEM with 2% ESC, for 48 h at 37 ° C and 5% CO ₂ . Upon reaching 90% of the CPD, the virus was released from the cells by freezing and thawing twice. Viral DNA was then isolated using the QIAamp DNA Mini Kit (QIAGEN, Germany) in accordance with the manufacturer's recommendations.

Example 4. PCR analysis of DNA of mutant strains of the Second World War.

Clones were analyzed by PCR for abnormalities in their DNA genes using the corresponding pairs of primers that were synthesized on an ABI-394 automatic synthesizer (Applied Biosystems, USA) at the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk):

For C3L gene deletion:

5'-TCGCGCTTTACATTCTCGAATCT-3 ',

5'-TGTTCGTGTGTTCTTGCGGTGA-3 ';

For deletion of the N1L gene:

5'-GGGTTGGATCCTTTACACATAGATCTACTACAGGCGGAACA-3 ',

5'-GGGAAAGCTTAATTTGTGAAGATGCCATGTACTACGCT-3 ';

To embed in the J2R gene:

5'-ATATGTTCTTCATGCCTAAACGA-3 ',

5'-ATGAAGGAGCAAAAGGTTGTAAC-3 ';

For deletion of the A35R gene:

5'-ACGACGGATGCTGAAGCGTGTTATA-3 ',

5'-AAACGATGTTACCAATCGTTTGCTAGGT-3 ';

For deletion of the A56R gene:

5'-GTGGTATGGGACACCACAAATATAAA-3 ',

5'-ATTAAACATTCCTAGAATTAATCCCGCTC-3 ';

For deletion of the B8R gene:

5'-TCACAAATATGATGGTGATGAGCGA-3 ',

5'-CGTGATATACCCTAGCCATAGGCAT-3 '.

PCR was performed in thin-walled Eppendorf type microtubes in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, USA). Reagents for the reaction manufactured by SibEnzyme (Russia). A mixture of test system components per sample:

- SE-buffer for Taq DNA polymerase (pH 8.5 at 25 ° С: 60 mM Tris-HCl, 25 mM KCl, 1.5 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 0.1% Triton X-100);

- a mixture of triphosphates (0.2 mm each dNTP);

- 0.7 μM forward and reverse primers;

- 2.5 units Taq DNA polymerase;

- about 2-10 ng of the DNA template;

- sterile deionized water (H ₂ O _di ) up to 25 μl.

Then the tubes were transferred to an amplifier and PCR was performed according to a program that included the following steps:

- preliminary DNA denaturation at 94 ° C, 1 min 30 s;

- 20 cycles consisting of:

1. DNA denaturation at a temperature of 94 ° C, 20 s,

2. annealing the primers at 60 ° C for 30 s,

3. synthesis of a complementary chain at 72 ° C, 1 min.

- after the last cycle, the tubes were heated for 3 min at 72 ° C.

Amplification products were stored at 4 ° C until electrophoretic analysis.

Fragment analysis was performed by electrophoresis in 1.5% agarose gel in a Tris-acetate buffer with ethidium bromide at a concentration of 0.2 μg / ml. To control the size of the obtained amplified fragment, a 1 kb DNA marker was introduced (SibEnzyme, Russia). Electrophoresis was performed with a voltage gradient of 10 V / cm for 60 minutes. For photographing the gel, the Image Station 440CF image visualization system (Kodak, USA) was used.

Example 5. Titration of the virus.

The viral suspension was pretreated twice with an ultrasonic disintegrator. Titration of the virus was carried out at a 90-100% daily monolayer of cell culture 4647 using dilutions of the virus 10 ^-1 -10 ^-6 and sorbing it for 1 h at 37 ° C. The viral suspension was then selected and DMEM medium with 2% ESC was added. After incubation for 72 hours at 37 ° C and 5% CO _{2, the} support medium was removed and the cell monolayer was stained using 0.2% crystalline violet dye diluted in a mixture of 10% ethanol and 2% formaldehyde. The number of plaques was counted and the titer of the virus per ml was calculated.

Example 6. The accumulation and purification of viruses:

- a monolayer of cell culture 4647 grown on culture bottles was infected with the studied BOB variants using a multiplicity of 1.0 PFU / cell and DMEM medium with 2% ESC;

- the monolayer was incubated for 48 hours at 37 ^° C until a complete CPD was formed and a cryolysate of infected cells was obtained by two freeze-thaw cycles;

- the cryolysate was treated twice with an ultrasonic disintegrator and the cell debris was precipitated by centrifugation at 5000 rpm and 4 ° C for 15 min;

- the supernatant was retained, and the precipitate was dissolved in DMEM medium and treated twice with an ultrasonic disintegrator, then the cell debris was again precipitated by centrifugation at 5000 rpm and 4 ° C for 15 min - two more cycles were performed according to this scheme;

- the virus was besieged from the combined supernatant by centrifugation at 14000 rpm and 4 ° C for 120 minutes;

- the precipitate was resuspended in physiological saline, processed three times using an ultrasonic disintegrator, packaged and stored at -70 ° C.

The titer of all samples was determined by the method of plaques in the monolayer of cells 4647. It was shown that all selected variants of the Second World War are produced using the method described above in sufficient quantities for immunizing animals.

Example 7. Determination of the growth dynamics of mutant variants of the Second World War.

To study the dynamics of the development of the initial 14L-IVP BOB and mutant variants 1421ABJCN and VACΔ6, we used a 90-100% daily monolayer of cell culture 4647 infected with viruses with a multiplicity of infection of 0.1 PFU / cell, followed by adsorption for 1 h at 37 ° C. After sorption, unsorbed viral particles were removed, and incubation was carried out under DMEM with 2% ESC at 37 ° C and 5% CO ₂ . The time after infection was 24, 48 and 72 hours. At each time step, the plaque method, as described above, was used to determine the titer of the virus in the culture suspension after freezing and thawing twice.

Example 8. Evaluation of a protective immune response.

To determine the protective immune response, groups of BALB / c mice, females weighing 14-16 g, were infected subcutaneously with the parental strain 14L-IVP BOB and recombinant variants 1421ABJCN and VACΔ6 at a dose of 10 ⁷ PFU / 0.1 ml / mouse. The animals of the control group were injected with physiological saline (BioloT, Russia) in the same volume. Immunization was carried out twice with an interval of 28 days. 28 days after the second immunization, the animals were intranasally inoculated with highly pathogenic VE mice for infection through the respiratory tract. For this, the animals were introduced into a state of mild anesthesia with diethyl ether (Kuzbassorgkhim, Russia). In this state, mice were injected with 15 μl of a suspension of EB in each nostril using a micropipette according to the method of [Martinez MJ, Bray MP, Huggins JW A mouse model of aerosol-transmitted orthopoxviral disease: morphology of experimental aerosol-transmitted orthopoxviral disease in a cowpox virus-BALB / c mouse system. // Arch. Pathol. Lab. Med. - 2000. - V. 124. - P. 362-377]. The total titer of the virus administered to the animal was 10 or 100 LD ₅₀ /0.02 ml / mouse. Mice were monitored for 14 days, recording the number of surviving and dead animals.

To determine the LD ₅₀ VE, female BALB / c mice weighing 20–22 g were infected intranasally, as described above, with four ten-fold dilutions of VE at a dose of 10–10 ⁴ PFU / 0.02 ml / mouse. The control group was injected with an equal volume of saline, on which viral dilutions were prepared. Observation of the animals was carried out for two weeks, given the number of survivors and deaths.

Example 9. Measurement of the titer of neutralizing antibodies.

Virus neutralizing antibody titers were determined in the blood sera of female BALB / c mice weighing 14-16 g infected subcutaneously with mutant variants 1421ABJCN and VACΔ6 in comparison with the original strain 14L-IVP BOB at a dose of 10 ⁷ PFU / 0.1 ml / mouse. An equal volume of saline was administered to the control group. Immunization was carried out twice with an interval of 28 days. 28 days after the second immunization in animals pre-anesthetized with dimethyl ether, blood samples were taken from the retrobulbar venous plexus and incubated at 4 ° C for 24 hours to form a fibrin clot. Serum was obtained by subsequent centrifugation for 10 min at 5000 g. Serum preparations were inactivated at 56 ° C for 30 minutes. The titer of neutralizing antibodies was determined on 90-100% monolayer of cell culture 4647 according to the standard method [Leparc-Goffart I., Poirier B., Garin D., Tissier MH, Fuchs F., Crance JM. Standardization of a neutralizing anti-vaccinia antibodies titration method: an essential step for titration of vaccinia immunoglobulins and smallpox vaccines evaluation. // Journal of Clinical Virology. - 2005.– V. 32. - P. 47-52], using dilutions of the studied sera 1: 5, 1:25, 1: 125. The neutralization efficiency was calculated relative to the number of plaques in the wells without serum (working dilution of the virus was considered such a dilution that, when counting PFU, gave from 40-60 plaques / well).

Example 10. The study of residual virulence of strains of the Second World War.

The study of the residual virulence of the vaccine strain VACΔ6 in comparison with 1421ABJCN and 14L-IVP BOB was carried out on female BALB / c mice weighing 14-16 g, which were administered BOB preparations subcutaneously or intraperitoneally at a dose of 10 ⁷ PFU / 0.1 ml / mouse . Animals of the control group were injected with saline in a volume of 0.1 ml. After 3, 7, and 14 days after infection, samples of blood and tissues of the brain, lungs, liver, and spleen were sterilely extracted from mice previously euthanized by cervical dislocation, from which 10% tissue homogenates were prepared on DMEM medium according to the scheme described in [Vijaysri S., Jentarra G., Heck MC, Mercer AA, McInnes CJ, Jacobs BL Vaccinia viruses with mutations in the E3L gene as potential replicationcompetent, attenuated vaccines: intra-nasal vaccination. // Vaccine. - 2008. - V. 26. - P. 664-676]. In the prepared tissue homogenates, as well as in the samples obtained after three consecutive passages of these suspensions in a 4647 cell culture, virus concentrations were determined by the plaque method described above using a 4647 monolayer of cell culture.

Example 11. Research methods for the neurovirulence of WWII variants.

To study the neurovirulence of the studied strains of the Second World War, we used 1-2 daily BALB / c sucker mice, intracerebrally infected with viruses at a dose of 102 PFU / 0.01 ml / mouse. Animals from the control group were injected with an equal volume of saline. Mice were monitored for 14 days, recording the number of surviving and dead animals. In addition, in some groups in some animals pre-euthanized by cervical dislocation, brain samples were sterilized 3 days after infection. From the samples obtained, 10% tissue suspensions were prepared on DMEM medium according to the procedure described in [Vijaysri S., Jentarra G., Heck MC, Mercer AA, McInnes CJ, Jacobs BL Vaccinia viruses with mutations in the E3L gene as potential replicationcompetent, attenuated vaccines: intra-nasal vaccination. // Vaccine. - 2008. - V. 26. - P. 664-676], for further determination of virus titers in them by plaque in a monolayer of cell culture 4647.

Example 12. Comparison of the reactogenicity of strains of WWII.

The study of the reactogenicity of the recombinant variant of VACΔ6 in comparison with the original strains 1421ABJCN and 14L-IVP BOB was carried out on Chinchilla rabbits weighing 1.5-2.0 kg, which previously epilated the sides. To carry out the experiment, six ten-fold dilutions of BOB strains in physiological saline were made to a content of 10 ² -10 ⁷ PFU / 0.05 ml. Dilutions were introduced intradermally into the animals in the above volume, each dilution was doubled. Observation of the animals was carried out for 14 days, recording the time of appearance and healing of inflammatory necrotic infiltrates, depending on the titer and the studied viral strain.

Example 13. Data analysis.

Statistical processing of experimental data was carried out using Excel from Microsoft Office 2010 (Microsoft Corp., USA). Using t-student criterion, the revealed intergroup differences were evaluated. Statistically significant experimental results were considered values at a significance level of P <0.05 [Ashmarin I.P., Vorobyov A.A. Statistical methods in microbiological studies. L .: State. ed. honey. lit. - 1962. - 186 p.].

The calculation of the lethal dose of RE causing the death of 50% of animals (LD ₅₀ ) was carried out according to the Kerber method. Antibody titers were calculated as geometric mean under the assumption that the sample elements are distributed according to the lognormal law, and are defined as -lg from the highest dilution of serum, at which 50% neutralization of BOB is achieved, ± standard deviation.

Thus, the results of the above studies demonstrate the possibility of improving the specific activity (in particular, immunogenic and protective activities) of the previously obtained recombinant variant 1421ABJCN BOB. As well as the possibility of creating a safe third-generation vaccine against smallpox, predicting the high probability of protecting the population from possible acts of bioterrorism associated with the use of variola virus, as well as from outbreaks of diseases caused by zoonotic orthopoxviruses, using the candidate live vaccine based on the claimed attenuated vaccinia virus strain L-IVP with six violated virulence genes (VACΔ6).

The application materials show that:

- the strain can be used as a live culture attenuated vaccine against smallpox and other human orthopoxvirus infections;

- the strain was obtained using six integration plasmids constructed by the authors — pΔC3L, pΔN1L, pΔJ2R, pΔA35R, pΔA56R and pΔB8R using the method of temporary dominant selection;

- in studies for comparison used strains of the Second World War:

1421ABJCN - previously obtained by the authors of the prototype strain of the Second World War with five violated virulence genes,

14L-IVP - a clone of the initial strain of L-IVP of the Second World War, adopted in Russia for the production of the classic anti-pox vaccine of the first generation;

- the replicative properties of the obtained WWII variant were studied in the cell culture 4647 by determining the dynamics of the virus growth. The results allow us to conclude that the violation of the selected genes C3L, N1L, J2R, A35R, A56R and B8R does not affect the productive properties of BOB in this cell culture, which is important from the point of view of the technology for producing a highly attenuated new generation anti-arterial vaccine;

- in experiments on animals, the authors found that:

the preparation of the recombinant strain VACΔ6 induces significantly higher titers of BOB-neutralizing antibodies compared to the original variant 1421ABJCN and the clone of the vaccine strain L-IVP BOB;

the mutant variant of the Great Patriotic War VACΔ6 along with the initial strains 1421ABJCN and 14L-IVP provide complete protection of mice against 10 LD ₅₀ highly pathogenic for them VE. And with a tenfold increase in the number of REs per animal (100 LD ₅₀ ), VACΔ6 shows a 100% protective effect compared to 50% for 1421ABJCN and 67% for 14L-IVP WWII;

VACΔ6 BOB is absent in the studied organs of infected mice on days 3, 7, and 14 after both subcutaneous and intraperitoneal injections;

the neurovirulent and reactogenic properties of the recombinant strain VACΔ6 BOB do not significantly differ from those of the original variant 1421ABJCN, and is lower in comparison with the parameters of the clone of the vaccine strain L-IVP BOB.

Claims (1)

Recombinant vaccinia virus strain VACΔ6 with violated C3L, N1L, J2R, A35R, A56R and B8R genes, designed to receive a live culture attenuated vaccine against variola virus and other human orthopoxvirus infections and deposited in the National collection of viral infections WB "Vector" with registration number V-696.

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