RU2709659C1 - Immunobiological agent and a method for use thereof for inducing specific immunity to the middle eastern respiratory syndrome virus (versions) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, immunology and virology. Described is an immunobiological agent based on human recombinant adenovirus 5-th serotype or recombinant human adenovirus 26-th serotype, containing optimized expression in mammal cells consensus sequence of complete protective antigen S of MERS virus on the basis of sequences of protein S genes of modern strains of virus MERS 2015–2017 years with sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Method for using the immunobiological agent for inducing specific immunity to MERS-CoV. Method of using the immunobiological agent consists in the sequential introduction into the body of mammals of two different immunobiological agents based on human recombinant adenovirus of 5-th serotype or two different immunobiological agents based on human recombinant adenovirus of 26-th serotype with a period of more than 1 week, for inducing specific immunity to MERS-CoV. There is also developed a method for using the immunobiological agent consisting in consecutive introduction into an organism of mammals of any of the immunobiological agents based on human recombinant adenovirus of 5-th serotype and any of the immunobiological preparations based on human recombinant adenovirus 26-th serotype with a period of more than 1 week, for the induction of specific immunity to MERS-CoV, or in consecutive introduction into organism of mammals of any of immunobiological agents on the basis of recombinant human adenovirus of 26-th serotype and any of immunobiological agents on the basis of recombinant human adenovirus of 5-th serotype with interval of more than 1 week, for induction of specific immunity to MERS-CoV. There is also developed a method for using an immunobiological agent consisting in simultaneous introduction into an organism of mammals of any two immunobiological agents on the basis of recombinant human adenovirus of 5-th serotype for induction of specific immunity to BVRS-CoV.

EFFECT: presented agent can be used for prevention of Middle Eastern respiratory syndrome.

9 cl, 7 dwg, 8 tbl, 13 ex

Description

Technical field

The invention relates to biotechnology, immunology and virology. The proposed tool can be used for the prevention of Middle Eastern respiratory syndrome.

The prior art.

Middle Eastern Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Camels are the main source of human infection with MERS-CoV (as well as a natural reservoir). Neutralizing antibodies to MERS-CoV have been found in camels in Oman, the Canary Islands and Egypt. Despite the fact that camels play an important role in the transmission of the virus from animals to humans, the transmission mechanism is not fully understood. In addition to camels, hedgehogs and bats were found infected with coronaviruses phylogenetically close to MERS-CoV in Europe, Thailand, South Africa and China. Analysis of 144 laboratory-confirmed cases (from 9 different countries of the world) and 17 possible cases of infection (Jordan, Italy, etc.) by MERS-CoV virus showed that the majority (59%) was caused by transmission of the virus from person to person. Moreover, infection most often occurred through close contact between people in medical centers (63.2%) and among family members (13.7%) (The WHO MERS-CoV Research Group, State of Knowledge and Data Gaps of Middle East Respiratory Syndrome Coronavirus (MERS- CoV) in Humans, PLOS Currents Outbreaks, 2013, No. 12-1).

The first human case of MERS-CoV was diagnosed in Saudi Arabia in 2012. According to information provided by WHO, at present, cases of the disease are recorded in 27 countries (Algeria, Austria, Bahrain, China, Egypt, France, Germany, etc.), of which the majority (82%) are in Saudi Arabia. The last major outbreak outside of Saudi Arabia occurred in South Korea from May to July 2015, when 36 people died of 186 cases of MERS-CoV infection (Lee SS, Wong NS, Probable transmission chains of Middle East respiratory syndrome coronavirus and the multiple generations of secondary infection in South Korea. International Journal of Infectious Diseases, 2015, No. 38, 65-67). The spread of MERS-CoV infection is of particular concern throughout the world, not only because of its similarity to severe acute respiratory syndrome (SARS), which spread to China and quickly spread to nearly 30 countries in 2002-2003, causing the death of about 800 people ( V. Kumar, YS Jung, R.N. Liang, Anti-SARS coronavirus agents: a patent review (2008 - present), Expert Opinion on Therapeutic Patents, 2013, No. 23, 1337-1348). Like SARS, in most cases, MERS occurs as a lower respiratory disease with fever, cough, shortness of breath and pneumonia, which can progress to acute respiratory syndrome and multiple organ failure. The disease is less likely to occur with mild flu-like illness, and in even rarer cases, it is asymptomatic. To date, according to WHO, 2229 clinically confirmed cases of MERS-CoV infection and 791 fatal cases associated with MERS-CoV infection have been registered, thus, the mortality rate is about 35.5%.

The high mortality rate, the rapid geographical spread of MERS-CoV and a vague etiology of the disease have created an urgent need to create effective means of prevention and treatment of MERS. Regarding the means of treatment, the combination of ribavirin and interferon-α2b has shown good results in the treatment of infected RVRS-CoV rhesus monkeys. In rhesus monkeys, the treatment of which began 8 hours after infection, there were no respiratory anomalies or signs of pneumonia. Moreover, a lower level of systemic and local inflammatory markers, as well as fewer copies of the MERS-CoV genome and less severe histopathological changes in the lungs were detected in the experimental Rhesus monkey group. However, this treatment was started a very short time after infection with MERS-CoV, and the proposed combination was unsuitable for humans (D. Falzarano, E. de Wit, AL Rasmussen, F. Feldmann, A. Okumura, DP Scott, D. Brining , T. Bushmaker, C. Martellaro, L. Baseler, AG Benecke, MG Katze, VJ Munster, H. Feldmann, Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques, Nature Medicine, 2013, No. 19 (10), 1313-1317).

There are currently no registered vaccines against MERS. In contrast to the epidemic caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), which stopped relatively quickly because the reservoir of infection was identified and isolated, MERS-CoV has persisted in the population for 5 years. WHO is currently awaiting new reports of MERS-CoV cases from countries in the Middle East. It is also expected that BVRS-CoV will be brought to other countries by persons infected as a result of contact with animals (for example, camels) or animal products.

Nevertheless, over the years, a lot of efforts have been made to create various vaccines and therapies for coronavirus infections, since the symptoms of MERS-CoV disease are similar to those of SARS-CoV pneumonia. Moreover, since both coronaviruses have crossed the interspecific barrier and infect humans, MERS-CoV is considered a potential threat that, like SARS-CoV, can cause a pandemic. Several approaches were used in the development of SARS-CoV vaccines: a live bacterial vaccine was created (solution according to patent RU 2332457), which contains the SARS-CoV antigen anchored on the cell surface using poly-gamma-glutamic acid synthetase. In another case, for the treatment of SARS, a pharmaceutical composition (patent RU 2411042) is used, comprising a therapeutically effective amount of a recombinant tumor necrosis factor-α receptor (TNF-α) or thalidomide in a pharmaceutically acceptable carrier. And there is also a solution according to the patent RU 2263144, which includes a special strain of the SARS virus CoD, designed to develop means and methods of biological protection.

A technical solution is known according to the patent US 9555094 B2 dated January 31, 2017, in which two variants of the amino acid sequences of the complete protein S of MERS-CoV are proposed. Due to the fact that the authors of the patent US 9555094 B2 filed an application on July 23, 2013, they could not use modern strains of MERS-CoV.

A solution is known where, as a candidate vaccine, a recombinant modified Anpara vaccinia virus was used, capable of expressing the full-size surface S protein of MERS-CoV. The presence of specific antibodies in the blood serum of immunized animals was shown (1: 8000-1: 16000 for the administered doses of 10 ⁷ and 10 ⁸ PFU (plaque-forming unit)), however, boosting at a dose of 10 ⁶ PFU showed significantly higher titers (1: 64000- 1: 128000). The formation of a cellular immune response to the S291 and S823 epitopes of the target antigen S of MERS-CoV was also shown. In animals immunized with a dose of ¹⁰ August pfu, was shown CD8 + level 3 times higher than in animals immunized with doses of ¹⁰ June and ¹⁰ July pfu (A. Volz et al., Protective efficacy of recombinant Modified Vaccinia virus Ankara (MVA) delivering 2 Middle East Respiratory Syndrome coronavirus spike glycoprotein, Virology, 2015, No. 89 (16), 8651-8656).

A technical solution is known in which a candidate vaccine based on a chimeric virus of vesicular stomatitis is proposed, in which the G protein gene is replaced by the full-size S gene of MERS-CoV. The presence of specific IgG in the blood serum of Balb / c mice was shown after a single intramuscular or intranasal immunization (on day 14, 13 μg / ml) and after boosting (after 21 days, 20 μg / ml). The presence of specific antibodies in the serum of rhesus monkeys after a single intramuscular or intranasal immunization was also shown. In addition, virus neutralizing antibodies were detected in the serum of rhesus monkeys, and the titer of antibodies was significantly higher in the group with intramuscular immunization (R. Liu et. Al., A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization, Antiviral Research, 2018, No. 150, 30-38).

A technical solution is known in which two variants of the candidate vaccine against the Middle East respiratory syndrome are proposed, based on recombinant adenoviral vectors 5 and 41 of serotypes expressing the full-size surface S protein of MERS-CoV. The resulting preparations are able to induce the formation of a humoral and cellular immune response to the target antigen S of MERS-CoV in BALB / c mice with the administration of the drug either intramuscularly (i.m.) or intraperitoneally (i.g.). Moreover, in the study of the humoral immune response, the highest titers of antibodies in the blood serum of the test animals were in response to the Ad5-S preparation with the introduction of i.m. (~ 1: 10000). Similarly, the strongest cellular immune response was to Ad5-S (im), for which a cytometric analysis was performed to determine the in vitro production of cytokines (interferon gamma, TNF-α, interleukin-2 and interleukin-10) in the pre-obtained splenocytes of the subjects animals. The experimental values for Ad5-S (im) were: C (interferon gamma) ~ 90 pkg / ml, C (TNF-α) ~ 37 pkg / ml, C (IL-2) ~ 23 pkg / ml, C (IL -10) ~ 4 pc / ml; while the values for Ad41-S i.m were 2-3 times less, and in groups with i.g. administration were approximately zero (X. Guo et al., Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus, Immunology, 2015, No. 145 (4), 476-484).

There is also a technical solution, according to which recombinant adenoviral vectors of the 5th serotype expressing the full-size surface S protein of MERS-CoV (option 1) or expressing the subunit S1 of the surface protein S of MERS-CoV (option 2) were proposed as candidate vaccine variants. After double immunization (priming - intramuscularly; boosting - intranasally), the presence of virus-neutralizing antibodies to MERS-CoV in the serum of the test animals was shown after administration of both option 1 (titer was ~ 1: 1024) and option 2 (titer was ~ 1: 2048). Their neutralizing activity was also shown in vitro, with the strongest neutralizing activity being shown for Ad5-MERS-S1 expressing the secreted protein S1 (E. Kima et al., Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB / c mice, Vaccine, 2014, No. 32, 5975-5982).

Technical solution according to the article (E. Kima et al., Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB / c mice, Vaccine, 2014, No. 32, 5975-5982) as the closest to the claimed composition of the active substance and the method of its use are selected by the authors of the claimed invention for the prototype. A significant drawback of this technical solution is the use of proteins with a consensus sequence with convergence with strains no later than 2014. Also, this technical solution does not provide data on the formation of a cellular immune response with the introduction of antigen, which is an essential characteristic of the candidate vaccine.

Disclosure of the present invention.

The objective of each of the variants of the invention is the creation of an immunobiological agent for the effective induction of an immune response to MERS-CoV.

An immunobiological agent was developed based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing a consensus sequence of the complete protective antigen S of the MERS virus based on the gene sequences of the S protein genes of modern strains of the MERS virus 2015-2017 . with the sequence of SEQ ID NO: 1 (option 1). An immunobiological agent was also developed based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing a consensus sequence of the complete protective antigen S of the MERS virus based on the sequences of the S protein genes of modern strains of the MERS virus 2015-2017 years with the transmembrane domain of the vesicular stomatitis virus glycoprotein with the sequence of SEQ ID NO: 2 (option 2). An immunobiological agent has also been developed based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing the optimized sequence of the receptor-binding domain of protein S based on the sequences of the protein S genes of modern strains of BVRS virus 2015-2017. with the sequence of the leader peptide with the sequence of SEQ ID NO: 3 (option 3). An immunobiological agent was also developed based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing a consensus sequence of protein S receptor-binding domain based on expression of protein S gene sequences of modern BVRS virus strains 2015-2017 for mammalian cells years with the transmembrane domain of the vesicular stomatitis virus glycoprotein with the sequence of SEQ ID NO: 4 (option 4). An immunobiological agent has also been developed based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing the optimized sequence of the receptor-binding domain of protein S based on the sequences of the protein S genes of modern strains of BVRS virus 2015-2017. with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1 with the sequence of SEQ ID NO: 5 (option 5).

The method of using the immunobiological agent according to options 1, or 2, or 3, or 4, or 5 consists in introducing into the mammalian organism this agent in an effective amount to induce specific immunity to MERS-CoV.

The method of using an immunobiological agent consists in the sequential administration of two different immunobiological agents based on a recombinant human adenovirus of the 5th serotype or two different immunobiological agents based on a recombinant human adenovirus of the 26th serotype with an interval of more than 1 week, for induction specific immunity to MERS-CoV. A method has also been developed for using an immunobiological agent, which consists in sequentially introducing into the mammalian organism any of the immunobiological agents based on recombinant human adenovirus of the 5th serotype and any of the immunobiological agents based on recombinant human adenovirus of the 26th serotype with an interval of more than 1 week, for induction of specific immunity to MERS-CoV, or in the sequential administration of any of the immunobiological agents based on recombin to the mammalian organism human adenovirus of the 26th serotype and any of the immunobiological agents based on recombinant human adenovirus of the 5th serotype with an interval of more than 1 week, to induce specific immunity to MERS-CoV. A method has also been developed for using an immunobiological agent, which consists in the simultaneous administration into a mammalian organism of any two immunobiological agents based on recombinant human adenovirus of the 5th serotype for the induction of specific immunity to MERS-CoV.

Brief Description of the Drawings

In FIG. Figure 1 presents the results of studying the expression of various variants of the GVRS-CoV virus glycoprotein S gene in HEK293 cells as part of recombinant adenoviruses Ad5-S, Ad5-S-G, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc by immunoblotting.

1 - cell lysate to which Ad5-S was added;

2 - cell lysate to which Ad5-S-G was added;

3 - medium from cells to which Ad5-RBD was added;

4 - medium from cells to which Ad5-RBD-Fc was added;

5 - cell lysate to which Ad5-RBD-G was added;

6 - cell lysate to which Ad-null was added;

7 — cell lysate to which phosphate-buffered saline was added.

In FIG. Figure 2 presents the results of studying the expression of various variants of the GVRS-CoV virus glycoprotein S gene in HEK293 cells as part of recombinant adenoviruses pAd26-S, pAd26-S-G, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc by immunoblotting.

1 - cell lysate to which Ad26-S was added;

2 - cell lysate to which Ad26-S-G was added;

3 - medium from cells to which Ad26-RBD was added;

4 - medium from cells to which Ad26-RBD-Fc was added;

5 - cell lysate to which Ad26-RBD-G was added;

6 - cell lysate to which Ad-null was added;

7 — cell lysate to which phosphate-buffered saline was added.

In FIG. 3 presents the results of evaluating the effectiveness of immunization with a developed immunological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, RBD, SG, RBD-G, RBD-Fc) of MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 according to the proportion of proliferating CD4 + lymphocytes restimulated with glycoprotein S MERS-CoV, on the 8th day after immunization of the test animals.

The ordinate axis - the number of proliferating cells,%

The abscissa axis - various groups of animals:

1) phosphate buffer (100 μl) - intact animals

2) Ad5-S 10 ⁸ PFU / mouse

3) Ad5-RBD 10 ⁸ PFU / mouse

4) Ad5-SG 10 ⁸ PFU / mouse

5) Ad5-RBD-G 10 ⁸ PFU / mouse

6) Ad5-RBD-Fc 10 ⁸ PFU / mouse.

In FIG. 4 presents the results of evaluating the effectiveness of immunization with a developed immunological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, RBD, SG, RBD-G, RBD-Fc) of MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 according to the proportion of proliferating CD4 + lymphocytes restimulated with glycoprotein S MERS-CoV, on day 15 after immunization of the test animals.

The ordinate axis - the number of proliferating cells,%

The abscissa axis - various groups of animals:

1) phosphate buffer (100 μl) - intact animals

3) Ad5-S 10 ⁸ PFU / mouse

4) Ad5-RBD 10 ⁸ PFU / mouse

5) Ad5-SG 10 ⁸ PFU / mouse

6) Ad5-RBD-G 10 ⁸ PFU / mouse

7) Ad5-RBD-Fc 10 ⁸ PFU / mouse.

In FIG. 5 presents the results of evaluating the effectiveness of immunization with a developed immunological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, RBD, SG, RBD-G, RBD-Fc) of MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 according to the proportion of proliferating CD8 + lymphocytes restimulated with glycoprotein S of MERS-CoV, on day 8 after immunization of test animals.

The ordinate axis - the number of proliferating cells,%

The abscissa axis - various groups of animals:

1) phosphate buffer (100 μl) - intact animals

3) Ad5-S 10 ⁸ PFU / mouse

4) Ad5-RBD 10 ⁸ PFU / mouse

5) Ad5-SG 10 ⁸ PFU / mouse

6) Ad5-RBD-G 10 ⁸ PFU / mouse

7) Ad5-RBD-Fc 10 ⁸ PFU / mouse.

In FIG. 6 presents the results of evaluating the effectiveness of immunization with a developed immunological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, RBD, SG, RBD-G, RBD-Fc) of MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 according to the proportion of proliferating CD8 + lymphocytes restimulated with glycoprotein S MERS-CoV, on day 15 after immunization of test animals.

The ordinate axis - the number of proliferating cells,%

The abscissa axis - various groups of animals:

1) phosphate buffer (100 μl) - intact animals

3) Ad5-S 10 ⁸ PFU / mouse

4) Ad5-RBD 10 ⁸ PFU / mouse

5) Ad5-SG 10 ⁸ PFU / mouse

6) Ad5-RBD-G 10 ⁸ PFU / mouse

7) Ad5-RBD-Fc 10 ⁸ PFU / mouse.

In FIG. 7 presents the results of evaluating the effectiveness of the developed immunobiological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, SG, RBD, RBD-G, RBD-Fc) of MERS virus optimized for expression in mammalian cells with a sequence selected from SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 according to the increase in the concentration of IFN-gamma in the medium after stimulation of splenocytes immunized with adenovirus constructs of C57 / BL6 mouse recombinant full-size S protein of the MERS-CoV virus.

The ordinate axis is the value of the increase in the concentration of IFN-gamma in the medium of stimulated cells when compared with intact cells (times).

The abscissa axis - the studied groups of animals: intact animals and animals that were administered at 10 ⁸ PFU / mouse Ad5-RBD, Ad5-S, Ad5-RBD-G, Ad5-SG or Ad5-RBD-Fc.

The implementation of the invention.

One of the most promising constructs for creating a candidate vaccine for the prevention of MERS is the receptor-binding domain (RBD) of protein S with the Fc fragment from human IgG1 (Fc). The high immunogenicity of the recombinant RBD-Fc protein has been repeatedly shown by studying the induction of cellular and humoral immune responses in laboratory animals (L. Du et al., Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development, Virology, 2013, No. 87 (17), 9939-9942; (L. Du et al., A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines, PLoS One, 2013, No. 8 (12), e81587; C. Ma et al., Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immu nization: Implication for designing novel mucosal MERS vaccines, Vaccine, 2014, No. 32 (18), 2100-2108; C. Ma et al., Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments - the importance of immunofocusing in subunit vaccine design, Vaccine, 2014, No. 32 (46), 6170-6176). However, this design has a number of drawbacks, the main of which is the insufficient number of epitopes, which can cause a weak protection of this design against MERS-CoV. As a result of the foregoing, it is also rational to use the full-size S BVRS-CoV protein for the highest antigen resistance of the candidate vaccine (K. Modjarrad, MERS-CoV vaccine candidates in development: The current landscape, Vaccine, 2016, No. 34 (26), 2982 -2987). To achieve the most effective induction of immune responses, the authors proposed various variants of modifications of this antigen, as well as the possibility of combining it with the transmembrane domain of the vesicular stomatitis virus glycoprotein to increase the level of expression of the target protein.

Five different variants of nucleotide sequences were obtained (modified S gene of MERS-CoV or receptor binding domain of protein S) by optimizing these sequences for expression in mammalian cells.

Further, several constructs based on recombinant human adenoviruses of the 5th and 26th serotypes were developed for the efficient delivery of modified genes into mammalian cells. Among the genetic vaccines undergoing clinical trials according to November 2017 data, the majority of the vectors used accounted for vaccines and therapeutic drugs based on recombinant adenoviruses - about 21% (The Journal pf Gene Medicine, Gene Therapy Clinical Trials Worldwide, 2017). This preponderance of adenovirus-based genetic vaccines is due to the fact that recombinant adenovirus vectors are among the safest and most effective vectors for humans (KR Van Kampen et. Al., Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. Vaccine, 2005, No. 23 (8), 1029-1036).

Recombinant adenoviruses have the following advantages: they are not able to multiply in human cells, are able to penetrate both dividing and non-dividing cells, induce both a cellular and a humoral immune response, and provide a high level of expression of the target antigen. The introduction of adenoviral vectors into the body is possible in various ways: intranasally, orally, intraperitoneally, intravenously, intramuscularly, subcutaneously. In the vast majority of studies in animal models, recombinant adenoviruses were administered in suspension in saline (APD Souza et. Al., Recombinant viruses as vaccines against viral diseases, Brazilian Journal of Medical and Biological Research, 2005, No. 38, 509- 522).

Recombinant adenoviral vectors are sufficiently stable in suspension, they can also be lyophilized, and in this state do not require freezing, which facilitates the transportation and storage of drugs, especially in developing countries (APD Souza et. Al., Recombinant viruses as vaccines against viral diseases, Brazilian Journal of Medical and Biological Research, 2005, No. 38, 509-522). Of the number of known adenoviruses, the most studied are human adenoviruses of the 5th serotype, so they became the basis for creating vectors for gene therapy. Technologies have been developed for the production of first and second generation vectors, chimeric vectors (containing proteins of viruses of other serotypes) (JN Glasgow et. Al., An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism, Virology, 2004, No. 324 , 103-116) and a number of other vectors. Also, vectors were derived from other serotypes, for example, the 26th (N. Chen et. Al., Adenovirus-Based Vaccines: Comparison of Vectors from Three Species of Adenoviridae, Virology, 2010, No. 84 (20), 10522 -10532).

26th serotype human adenovirus vectors show a high level of immunogenicity in primates, where they are able to induce a potent CD8 ⁺ T-cell response, qualitatively superior to the T-cell response when vectors of the 5th serotype are introduced into the body (J. Liu et al., Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys, Virology, 2008, No. 82, 4844-4852). In this case, recognition of a larger number of epitopes and induction of the production of a wider range of factors, rather than predominantly interferon gamma, occurs (J. Liu et. Al., Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys , Virology, 2008, No. 82, 4844-4852). These data suggest that vectors based on human adenovirus of the 26th serotype have fundamental differences in their ability to induce the formation of an immune response to the target antigen relative to other adenovirus vectors.

To create a consensus amino acid sequence of protein S in the present patent, modern strains of the BVRS virus 2015-2017, current to date, were used.

The invention according to option 1 is a recombinant human adenovirus of the 5th serotype, or a recombinant human adenovirus of the 26th serotype, containing a consensus sequence of the complete protective antigen S of MERS-CoV based on the gene sequences of protein S of modern strains of MERS-CoV (2015-2017) (SEQ ID NO: 1).

The invention according to option 2 is a recombinant human adenovirus of the 5th serotype, or a recombinant human adenovirus of the 26th serotype, containing a consensus sequence of the complete protective antigen S of MERS-CoV based on the gene sequences of protein S of modern strains of MERS-CoV (2015-2017) with the transmembrane domain of the vesicular stomatitis virus glycoprotein (SEQ ID NO: 2).

The invention according to option 3 is a recombinant human adenovirus of the 5th serotype, or a recombinant human adenovirus of the 5th serotype, containing the optimized sequence of the receptor-binding domain of protein S based on the sequences of the protein S genes of modern strains of MERS-CoV (2015-2017). with a leader peptide sequence (SEQ ID NO: 3).

The invention according to option 4 is a recombinant human adenovirus of the 5th serotype, or a recombinant human adenovirus of the 26th serotype containing a consensus sequence of the protein S receptor-binding domain based on expression of protein S sequences of modern strains of MERS-CoV strains for mammalian cells (2015 -2017) with the transmembrane domain of the vesicular stomatitis virus glycoprotein (SEQ ID NO: 4).

The invention according to option 5 is a recombinant human adenovirus of the 5th serotype, or a recombinant human adenovirus of the 26th serotype, containing the optimized sequence of the receptor-binding domain of protein S based on the sequences of the protein S genes of modern strains of MERS-CoV (2015-2017). with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1 (SEQ ID NO: 5).

Several methods have been developed for using these immunobiological agents. Methods of use, presented further in the claims according to options 1-5, include the introduction of the above immunobiological agents for each of the 5 options into the mammalian organism individually in an effective amount to induce specific immunity to MERS-CoV.

The method of using the immunobiological agent according to option 1, or option 2, or option 3, or option 4, or option 5, is to introduce into the mammalian organism this agent in an effective amount to induce specific immunity to the MERS virus.

The method of using the immunobiological agent according to options 1-5 consists in sequentially introducing into the mammalian organism any two immunobiological agents from options 1-5 with an interval of more than 1 week to induce specific immunity to MERS-CoV.

The features of the method of using the created immunobiological agents is the following:

The method of using the immunobiological agent according to options 1-5 consists in sequentially introducing into the organism of mammals two different immunobiological agents based on recombinant human adenovirus of the 5th serotype or two different immunobiological agents based on recombinant human adenovirus of the 26th serotype with an interval of more than 1 week , for the induction of specific immunity to MERS-CoV.

The method of using the immunobiological agent according to options 1-5 consists in sequentially introducing into the mammalian body any of the immunobiological agents based on recombinant human adenovirus of the 5th serotype and any of the immunobiological agents based on recombinant human adenovirus of the 26th serotype with an interval of more than 1 week , for the induction of specific immunity to MERS-CoV, or in the sequential introduction into the mammalian organism of any of the immunobiological agents based on recombinant of human adenovirus serotype 26, and any of immunobiological means on the basis of recombinant human adenovirus serotype 5 second intervals over a one week for induction of specific immunity to BVRS Cove. The method of using the immunobiological agent according to options 1-5 consists in the simultaneous introduction into the mammalian body of any two immunobiological agents based on recombinant human adenovirus of the 5th serotype to induce specific immunity to MERS-CoV.

Examples.

Example 1

Creating a consensus amino acid sequence of protein S, the most homologous for various modern strains (2015-2017) of MERS-CoV isolated from humans.

The most immunogenic and most promising vaccine antigen among all MERS-CoV proteins is protein S, a trimeric, anchoring glycoprotein of type I, interacting with its related human receptor dipeptidyl peptidase 4 (DPP4), mediating the penetration of the virus. Protein S includes two subunits: S1 (containing RBD and determining the tropism of the virus) and S2 (responsible for cell fusion).

This example shows a method for creating a consensus amino acid sequence of a full-sized protein S, the most homologous for different strains of MERS-CoV isolated from humans for the period from 2015 to 2017.

The original amino acid sequences of the full-length S protein are taken from the official public source NCBI, GenBank, USA (www.ncbi.nlm.nih.gov). An analysis was made of 179 sequences of full-sized protein S from various strains of MERS-CoV isolated from humans. Work with sequences was carried out using computer programs Geneious 10.0.9 and BLAST.

First, the consensus sequence of protein S was selected. This sequence was obtained by aligning all sequences of the full-sized protein S taken from NCBI, GenBank relative to each other in Geneious 10.0.9. A total of 179 sequences were studied, 66 of them showed 100% homology, and the remaining 113 had 99% homology relative to the selected consensus sequence (SEQ ID N0: 1), used for option 1.

Since camels are a reservoir of MERS-CoV and a source of human infection, the obtained consensus sequence of protein S of SEQ ID NO: 1 was examined for the percentage of homology relative to the consensus sequence of the full-length S protein of strains of MERS-CoV isolated from camels and showed 100% homology with it. Alignment was carried out in the BLAST program. The consensus sequence of the full-sized protein S of strains of MERS-CoV isolated from camels was selected by aligning all sequences of the full-sized protein S of strains of MERS-CoV isolated from camels. The sequences were taken from NCBI, GenBank and aligned relative to each other in Geneious 10.0.9. A total of 176 sequences of the protein of MERS-CoV strains isolated from camels were studied, 64 of them showed 100% homology with a consensus sequence, the remaining 112 had 99% homology with respect to the consensus sequence of protein S of MERS-CoV strains isolated from camels.

Example 2

Obtaining various variants of glycoprotein S MERS-CoV.

To study the immunogenicity of various forms of protein S of MERS-CoV - secreted and non-secreted, the sequence of SEQ ID NO: 1 was modified in several ways:

1) To anchor the protein in the membrane and thus obtain an unclassified form of the complete S-protein of MERS-CoV, the sequence of the transmembrane domain of the vesicular stomatitis virus glycoprotein was added to the sequence SEQ ID NO: 1, thus the sequence SEQ ID NO: 2 was created used for option 2.

2) To study the immunogenicity of only the receptor-binding domain (RBD) of the S-protein of MERS-CoV in secreted form, the sequence SEQ ID NO: 3 (used for option 3) was created, containing the sequence of the receptor-binding domain of protein S with the sequence of the leader peptide ( added for protein secretion). According to published data, RBD binds to the human dipeptidyl peptidase 4 receptor (hDPP4) and induces the production of neutralizing antibodies in immunized mice (L. Du et al., Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development, Virology, 2013, No. 87 (17), 9939-9942). The RBD sequence was selected from studies by Lanying Du et. al. (L. Du et al., A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines, PLoS One, 2013, No. 8 (12) , e81587).

3) To study RBD protein S of MERS-CoV in an unclassified form, the sequence SEQ ID NO: 4 (used for option 4) was selected, consisting of RBD protein S of MERS-CoV, to which the sequence of the transmembrane domain of the vesicular stomatitis virus glycoprotein was added.

4) For the study of the secreted form of RBD protein S with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1, the sequence SEQ ID NO: 5 was used (used for option 5). The addition of an Fc fragment from human IgG1 enhances immunogenicity due to the possible binding of the Fc protein fragment to the Fc receptor on antigen-presenting cells (Z. Li et. Al., Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection, Proceedings of the National Academy of Sciences USA, 2011, No. 108, 4388-4393), and also enhance protein stability and extend the in vivo half-life (MY Zhang et. Al., Crossreactive HIV -1-neutralizing activity of serum IgG from a rabbit immunized with gp41 fused to IgG1 Fc: Possible role of the prolonged half-life of the immunogen, Vaccine, 2008, No. 27, 857-63).

Example 3

Obtaining genetic constructs encoding a protein S gene in various variants.

This example provides a method for creating genetic constructs encoding modified variants of the S gene.

To create the final genetic constructs encoding the obtained protein S consensus sequences, the indicated amino acid sequences (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) were converted to nucleotide. Further, the obtained sequences were optimized for expression in mammalian cells by the company CJSC Evrogen (Moscow). All nucleotide sequences were obtained by synthesis by the company CJSC Evrogen (Moscow). As a result, the following designs were obtained:

1) pVax-S containing the nucleotide sequence of the complete S gene of MERS-CoV;

2) pVax-S-G, containing the nucleotide sequence of the complete S gene of MERS-CoV with the G gene of the virus of vesicular stomatitis;

3) pAL2-T-RBD, containing the nucleotide sequence of the receptor-binding domain of protein S with the sequence of the leader peptide gene;

4) pAL2-T-RBD-G, containing the nucleotide sequence of the receptor-binding domain of protein S with the G gene of the virus of vesicular stomatitis;

5) pAL2-T-RBD-Fc, containing the nucleotide sequence of the receptor-binding domain of protein S with the sequence of the leader peptide gene and the nucleotide sequence of the Fc fragment from human IgG1.

After that, using standard genetic engineering methods, the S protein gene sequence from the pVax-S construct was cloned using the Xbal restriction endonuclease into the shuttle plasmid pShuttle-CMV (StrataGen, USA) and the resulting plasmid was named pShuttle-S. Thus, the shuttle plasmid pShuttle-S was created, carrying the nucleotide sequence of the consensus amino acid sequence S (SEQ ID NO: 1) obtained in Example 1 optimized for expression in mammalian cells.

Similarly, the nucleotide sequences of the modified variants of protein S of MERS-CoV were cloned into the shuttle plasmid pShuttle-CMV (StrataGen, USA) and the following shuttle plasmids were obtained:

- pShuttle-S-G (contains the optimized nucleotide sequence of the complete protective antigen S of MERS-CoV with the transmembrane domain of the vesicular stomatitis virus glycoprotein)

- pShuttle-RBD (contains the optimized nucleotide sequence of the receptor-binding domain S of MERS-CoV)

- pShuttle-RBD-G (contains the optimized nucleotide sequence of the receptor-binding domain S of MERS-CoV with the transmembrane domain of the vesicular stomatitis virus glycoprotein)

- pShuttle-RBD-Fc (contains the optimized nucleotide sequence of the receptor binding domain S of MERS-CoV with the optimized sequence of the Fc fragment from human IgG1).

Example 4

Obtaining an immunobiological agent based on recombinant human adenovirus of the 5th serotype, containing:

1) the optimized nucleotide sequence of the receptor binding domain S of MERS-CoV (option 1);

2) the optimized nucleotide sequence of the complete protective antigen S of MERS-CoV with the transmembrane domain of the vesicular stomatitis virus glycoprotein (option 2);

3) the optimized nucleotide sequence of the receptor-binding domain of protein S with the sequence of the leader peptide (option 3),

4) the optimized nucleotide sequence of the receptor-binding domain of protein S with the transmembrane domain of the vesicular stomatitis virus glycoprotein (option 4),

5) the optimized sequence of the receptor-binding domain of protein S with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1 (option 5).

The preparation of the pAd5-S recombinant adenovirus plasmid containing the consensus sequence of the complete protective antigen S of MERS-CoV (SEQ ID NO: 1) (variant 1) was optimized for expression in mammalian cells by homologous recombination between the pAd-Easy plasmid (AdEasy ™ Adenoviral Vector System , StrataGen, USA) containing the genomic part of human adenovirus 5 serotype with deleted E1 and E3 regions, and the shuttle plasmid pShuttle-S (obtained in example 3), carrying homologous parts of the adenovirus genome and an expression cassette with the target gene m (protein S). For this, the shuttle plasmid pShuttle-S obtained in Example 3 was linearized with Pmel restriction endonuclease.

Homologous recombination was carried out in E. coli cells of strain BJ5183. Ad-Easy plasmid was mixed with pShuttle-S plasmid, and then E. coli cells were transformed with the resulting mixture by electroporation according to the MicroPulser ™ Electroporation Apparatus Operating Instructions and Applications Guide (Bio-Rad, USA). After transformation, E. coli cells of strain BJ5183 were plated on plates with LB agar containing a selective antibiotic and grown for 18 hours at a temperature of + 37 ° C. The transformation efficiency was 10 ¹⁰ -10 ¹¹ transformed clones per 1 μg of plasmid pBluescript II SK (-).

As a result of homologous recombination, a cassette with the target transgene (protein S) appeared in the pAd-Easy plasmid, and the antibiotic resistance gene changed.

Thus, a recombinant adenovirus plasmid pAd5-S was constructed containing the full-size genome of recombinant human adenovirus of the 5th serotype (with deleted E1 and E3 regions of the genome) with the integrated genetic construct obtained in Example 3. Next, the plasmid pAd5-S was digested with restriction endonuclease I and transfected it with a permissive culture of cells of the human embryonic kidney of the HEK 293 line. Cells of the HEK 293 line contain in their genome an integrated E1 region of the 5th serotype human adenovirus genome, As a result, reproduction of recombinant replicatively defective human adenoviruses of the 5th serotype can occur in them. On the sixth day after transfection, the first blind passages were performed to more efficiently obtain recombinant adenovirus. After the onset of the cytopathic effect of the virus (microscopic data), cells with the culture medium were thawed three times to destroy the cells and exit the virus. As a result, material was obtained, which was then used to accumulate preparative amounts of recombinant adenoviruses.

The activity of the pAd5-S preparation was evaluated hereinafter by the standard titration method on a culture of sensitive 293 HEK cells in plaque formation reactions.

To confirm the creation of the design of the proposed recombinant pseudo-adenovirus particle based on human adenovirus 5 serotype expressing the S BVRS-CoV gene S, a polymerase chain reaction (PCR) was carried out by a known standard method.

Similarly, four more recombinant adenoviruses were obtained: Ad5-S-G, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc.

Example 5

Obtaining an immunobiological agent based on a recombinant human adenovirus of the 26th serotype, containing:

1. the optimized nucleotide sequence of the receptor binding domain S of MERS-CoV (option 1);

2. The optimized nucleotide sequence of the complete protective antigen S of MERS-CoV with the transmembrane domain of the vesicular stomatitis virus glycoprotein (option 2);

3. the optimized nucleotide sequence of the receptor-binding domain of protein S with the sequence of the leader peptide (option 3),

4. the optimized nucleotide sequence of the receptor-binding domain of protein S with the transmembrane domain of the vesicular stomatitis virus glycoprotein (option 4),

5. the optimized sequence of the receptor-binding domain of protein S with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1 (option 5).

In the recombinant vector pAd26-ORF6-Ad5, an expression cassette with the S gene of MERS-CoV was placed. For this, the pAd26-ORF6-Ad5 vector was linearized using Pmel restriction endonuclease, and the pShuttle-S plasmid construct obtained in Example 3 was processed with Pmel restriction endonuclease. The hydrolysis products were ligated, after which the plasmid pAd26-S was obtained using standard methods.

In the next step, the plasmid pAd26-S was hydrolyzed with Pacl and Swal restriction endonucleases and transfected with it in a constant culture of HEK 293 cells. On the third day after transfection, the first blind passages were performed to more efficiently obtain recombinant adenovirus. After the onset of the cytopathic effect of the virus (microscopic data), cells with the culture medium were thawed three times to destroy the cells and exit the virus. As a result, material was obtained, which was then used to accumulate preparative amounts of recombinant adenoviruses. The activity of the pAd26-S preparation was evaluated hereinafter by the standard titration method on 293 HEK cell culture in the plaque formation reaction.

To confirm the construction of the proposed recombinant pseudo-adenovirus particle based on the recombinant human adenovirus of the 26th serotype expressing the MERS-CoV S gene, polymerase chain reaction (PCR) was carried out by a known standard method.

Similarly, four more recombinant adenoviruses were obtained: pAd26-S-G, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc.

The ability of engineered recombinant adenoviruses to express different variants of the S protein gene in cells was further shown.

Example 6

Verification of the expression of different variants of the glycoprotein S gene of MERS-CoV in HEK293 cells as part of recombinant adenoviruses Ad5-S, Ad5-S-G, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc by immunoblotting.

HEK293 cells were cultured in DMEM supplemented with 10% fetal calf serum in an incubator at 37 ° C and 5% CO ₂ . Cells were placed on 35 mm ² Petri dishes and incubated for one day until 70% confluency was achieved. Then, the studied preparations of recombinant adenoviruses (Ad5-S, Ad5-SG, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc) were added to the cells, the control preparation (Ad-null - recombinant adenovirus without inserts) at the rate 100 PFU / cell and phosphate buffered saline as a negative control. After a day, the expression of MERS-CoV antigens was checked by immunoblotting. For this medium, cells were taken into separate tubes for analysis of secreted forms of protein, and the cells were washed with sterile phosphate-saline buffer and then lysed using a Cell Culture Lysis Reagent lysis buffer (Promega, USA) containing 25 mM Tris- phosphate (pH 7.8), 2 mM DTT, 2 mM trans-1,2-diaminocyclohexane, 10% glycerol and 1% Triton® X-100 for analysis of transmembrane proteins.

After media selection and cell lysis, the total protein concentration in the samples was measured using Bradford reagent (Sigma) according to the manufacturer's instructions. An aliquot of protein-aligned samples was mixed with Laemmli application buffer (Sample Buffer, Laemmli, Sigma, USA) and incubated for 10 minutes at 98 ° C.

Next, denaturing electrophoresis was performed using a Mini-PROTEAN ® TGX ™ Precast Protein Gels polyacrylamide gel (Bio-Rad, USA) in Tris / glycine / SDS Running Buffer 10X (Bio-Rad, USA) using the Mini-PROTEAN® Tetra Cell system (Bio-Rad, USA) for 20 minutes at 250V. After electrophoresis, proteins were transferred onto a Trans-Blot® Turbo ™ Mini Nitrocellulose Transfer Packs (Bio-Rad) nitrocellulose membrane in Tris-CAPS buffer (Bio-Rad, USA) using the Trans-Blot® SD Semi-Dry Transfer Cell transfer system ( Bio-Rad, USA) for 10 minutes at 25V. Then blocking of a nonspecific signal was carried out. For this, the membrane was incubated in a solution of 5% skim milk in phosphate-buffered saline with 0.05% Tween20 (TFSB) for one hour at room temperature. After that, the membrane was incubated for 1 hour at 37 ° C with antibodies to protein S (40069-RP02) in a solution of 5% skim milk in TFSB. Then, the membrane was washed with a TFSB solution and the membrane was treated with a solution of secondary antibodies conjugated with horseradish peroxidase in a solution of 5% skim milk in TFSB at 37 ° C on a shaker for 1 hour. Then the membrane was thoroughly washed with a solution of TFSB. Further detection was performed using the Clarity ™ Western ECL Substrate kit (Bio-Rad, USA) and developed on Amersham Hyperfilm® ECL ™ film (GE Healthcare, USA).

The results are presented in FIG. 1. As can be seen from the data obtained, in all cells transduced with recombinant adenoviruses Ad5-S, Ad5-SG, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc, the expression of various variants of the target protein S gene was observed, while the proteins S, SG, RBD-G were detected in lysates (transmembrane form), and proteins RBD and RBD-Fc were found in media (secreted form). All detected fragments coincide in molecular weight with theoretical values. In control samples, expression was not observed.

Example 7

Verification of the expression of different variants of the glycoprotein S gene of MERS-CoV in HEK293 cells as part of recombinant adenoviruses pAd26-S, pAd26-S-G, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc by immunoblotting.

HEK293 cells were cultured in DMEM supplemented with 10% fetal calf serum in an incubator at 37 ° C and 5% CO ₂ . Cells were placed on 35 mm ² Petri dishes and incubated for one day until 70% confluency was achieved. Then, the studied preparations of recombinant adenoviruses (pAd26-S, pAd26-SG, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc) were added to the cells, the control preparation (Ad26-null - recombinant adenovirus without inserts) at the rate 100 PFU / cell and phosphate buffered saline as a negative control. After a day, the expression of MERS-CoV antigens was checked by immunoblotting. For this medium, cells were taken into separate tubes for analysis of secreted forms of protein, and the cells were washed with sterile phosphate-saline buffer and then lysed using a Cell Culture Lysis Reagenb lysis buffer (Promega, USA) containing 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM trans-1,2-diaminocyclohexane, 10% glycerol and 1% Triton® X-100 for analysis of transmembrane proteins.

After media selection and cell lysis, the total protein concentration in the samples was measured using Bradford reagent (Sigma) according to the manufacturer's instructions. An aliquot of protein-aligned samples was mixed with Laemmli application buffer (Sample Buffer, Laemmli, Sigma, USA) and incubated for 10 minutes at 98 ° C.

Next, denaturing electrophoresis was performed using a Mini-PROTEAN ® TGX ™ Precast Protein Gels polyacrylamide gel (Bio-Rad, USA) in Tris / glycine / SDS Running Buffer 10X (Bio-Rad, USA) using the Mini-PROTEAN® Tetra Cell system (Bio-Rad, USA) for 20 minutes at 250V. After electrophoresis, proteins were transferred onto a Trans-Blot® Turbo ™ Mini Nitrocellulose Transfer Packs nitrocellulose membrane (Bio-Rad, USA) in Tris-CAPS buffer (Bio-Rad, USA) using a Trans-Blot® SD Semi-Dry Transfer transfer system Cell (Bio-Rad, USA) for 10 minutes at 25V. Then blocking of a nonspecific signal was carried out. For this, the membrane was incubated in a solution of 5% skim milk in phosphate-buffered saline with 0.05% Tween20 (TFSB) for one hour at room temperature. After that, the membrane was incubated for 1 hour at 37 ° C with antibodies to protein S (40069-RP02) in a solution of 5% skim milk in TFSB. Then, the membrane was washed with a TFSB solution and the membrane was treated with a solution of secondary antibodies conjugated with horseradish peroxidase in a solution of 5% skim milk in TFSB at 37 ° C on a shaker for 1 hour. Then the membrane was thoroughly washed with a solution of TFSB. Further detection was performed using the Clarity ™ Western ECL Substrate kit (Bio-Rad, USA) and developed on Amersham Hyperfilm® ECL ™ film (GE Healthcare, USA).

The results are presented in FIG. 2. As can be seen from the obtained data, in all cells transduced with recombinant adenoviruses pAd26-S, pAd26-SG, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc, the expression of different variants of the target protein S gene was observed, with proteins S, SG, RBD-G were detected in lysates (transmembrane form), and proteins RBD and RBD-Fc were found in media (secreted form). All detected fragments coincide in molecular weight with theoretical values. In control samples, expression was not observed.

Example 8

The method of using an immunobiological agent based on recombinant human adenoviruses of the 5th and 26th serotypes (Ad5-S, Ad5-SG, Ad5-RBD, Ad5-RBD-G, Ad5-RBD-Fc, pAd26-S, pAd26-SG, pAd26-RBD, pAd26-RBD-G, pAd26-RBD-Fc) by introducing into the body of a mammal in an effective amount to induce specific immunity to MERS-CoV.

The developed immunobiological agent based on recombinant human adenoviruses of the 5th and 26th serotypes containing a consensus sequence of protective antigen (S, SG, RBD, RBD-G, RBD-Fc proteins) MERS-CoV with the sequence optimized for expression in mammalian cells, selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 is used by introducing into the body of a mammal any of the methods of administration known for this viral vector (subcutaneously, intramuscularly , intravenously, intranasally). At the same time, an immune response develops in the mammalian organism to the target protein of glycoprotein BVRS-CoV.

One of the main characteristics of the effectiveness of immunization is the titer of antibodies. The example presents data on changes in the titer of antibodies against glycoprotein BVRS-CoV 21 days after a single intramuscular immunization of animals with an immunobiological agent, including recombinant human adenovirus of the 5th or 26th serotype, containing a consensus sequence of protective antigen optimized for expression in mammalian cells ( proteins S, SG, RBD, RBD-G, RBD-Fc) MERS-CoV with the sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4 : 5.

In the experiment, mammals were used - mice of the C57BL / 6 line, females of 18 g. All animals were divided into 37 groups of 5 animals, which were administered intramuscularly:

1) Ad5-S 10 ⁷ PFU / mouse

2) Ad5-RBD 10 ⁷ PFU / mouse

3) Ad5-SG 10 ⁷ PFU / mouse

4) Ad5-RBD-G 10 ⁷ PFU / mouse

5) Ad5-RBD-Fc 10 ⁷ PFU / mouse

6) Ad5-null 10 ⁷ PFU / mouse

7) Ad5-S 10 ⁸ PFU / mouse

8) Ad5-RBD 10 ⁸ PFU / mouse

9) Ad5-SG 10 ⁸ PFU / mouse

10) Ad5-RBD-G 10 ⁸ PFU / mouse

11) Ad5-RBD-Fc 10 ⁸ PFU / mouse

12) Ad5-null 10 ⁸ PFU / mouse

13) Ad5-S 10 ⁹ PFU / mouse

14) Ad5-RBD 10 ⁹ PFU / mouse

15) Ad5-SG 10 ⁹ PFU / mouse

16) Ad5-RBD-G 10 ⁹ PFU / mouse

17) Ad5-RBD-Fc 10 ⁹ PFU / mouse

18) Ad5-null 10 ⁹ PFU / mouse

19) Ad26-S 10 ⁷ PFU / mouse

20) Ad26-RBD 10 ⁷ PFU / mouse

21) Ad26-SG 10 ⁷ PFU / mouse

22) Ad26-RBD-G 10 ⁷ PFU / mouse

23) Ad26-RBD-Fc 10 ⁷ PFU / mouse

24) Ad26-null 10 ⁷ PFU / mouse

25) Ad26-S 10 ⁸ PFU / mouse

26) Ad26-RBD 10 ⁸ PFU / mouse

27) Ad26-SG 10 ⁸ PFU / mouse

28) Ad26-RBD-G 10 ⁸ PFU / mouse

29) Ad26-RBD-Fc 10 ⁸ PFU / mouse

30) Ad26-null 10 ⁸ PFU / mouse

31) Ad26-S 10 ⁹ PFU / mouse

32) Ad26-RBD 10 ⁹ PFU / mouse

33) Ad26-SG 10 ⁹ PFU / mouse

34) Ad26-RBD-G 10 ⁹ PFU / mouse

35) Ad26-RBD-Fc 10 ⁹ PFU / mouse

36) Ad26-null 10 ⁹ PFU / mouse

37) phosphate buffered saline

Three weeks later, animals were sampled from the tail vein and blood serum was isolated. The antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) according to the following protocol:

1) Protein (S) was adsorbed on the wells of a 96-well ELISA plate for 16 hours at a temperature of + 4 ° C.

2) Next, to get rid of nonspecific binding, the plate was "clogged" with 5% milk dissolved in TPBS in a volume of 100 μl per well. Incubated on a shaker at a temperature of 37 ° C for an hour.

3) Serum samples of immunized mice were bred using a 2-fold dilution method. A total of 12 dilutions of each sample were prepared.

4) 50 μl of each diluted serum sample was added to the wells of the plate.

5) Next, incubation was carried out for 1 hour at 37 ° C.

6) After incubation, the wells were washed three times with phosphate buffer.

7) Secondary antibodies against mouse immunoglobulins conjugated to horseradish peroxidase were then added.

8) Next, incubation was carried out for 1 hour at 37 ° C.

9) After incubation, the wells were washed three times with phosphate buffer

10) Then, a solution of tetramethylbenzidine (TMB) was added, which is a substrate of horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 minutes by the addition of sulfuric acid. Next, the optical density of the solution (OD) in each well was measured using a spectrophotometer at a wavelength of 450 nm.

The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results obtained (geometric mean value) are presented in table 1.

The results of the experiment showed that the developed immunobiological agent according to p. 1-10, introduced into the body of a mammal, induces a humoral immune response to the BVRS-CoV glycoprotein in the entire range of selected doses. At the same time, it is obvious that an increase in doses will lead to an increase in the titer of antibodies in the blood of mammals before the onset of a toxic effect.

Example 9

The method of using the developed immunobiological agent based on recombinant human adenovirus of the 5th serotype containing a consensus sequence of protective antigen (proteins S, SG, RBD, RBD-G, RBD-Fc) MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, by sequentially introducing them into mammals with an interval of 1 week to induce specific immunity to MERS-CoV .

The experiment was performed according to the protocol described in example 7.

All animals were divided into 29 groups (3 animals each), which were administered intramuscularly:

1. phosphate buffer (100 μl), and after a week phosphate buffer (100 μl)

2. phosphate buffer (100 μl), and after a week Ad5-null 10 ⁸ PFU / mouse

3. Ad5-null 10 ⁸ PFU / mouse, and after a week phosphate buffer (100 μl)

4. Ad5-null 10 ⁸ PFU / mouse, and after a week Ad5-null 10 ⁸ PFU / mouse

5. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

6. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad5-RBD 10 ⁸ PFU / mouse

7. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

8. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

9. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad5-RBD-Fc 10 ⁸ PFU / mouse

10. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad5-RBD 10 ⁸ PFU / mouse

11. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

12. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

13. Ad5-RBD-10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

14. Ad5-RBD-10 ⁸ PFU / mouse, and after a week Ad5-RBD-Fc 10 ⁸ PFU / mouse

15. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

16. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

17. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad5-SGD 10 ⁸ PFU / mouse

18. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

19. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad5-RBD-Fc 10 ⁸ PFU / mouse

20. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

21. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

22. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-RBD-10 ⁸ PFU / mouse

23. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

24. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-RBD-Fc 10 ⁸ PFU / mouse

25. Ad5-RBD-Fc 10 ⁸ PFU / mouse, and after a week Ad5-RBD-Fc 10 ⁸ PFU / mouse

26. Ad5-RBD-Fc 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

27. Ad5-RBD-Fc 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

28. Ad5-RBD-Fc August ¹⁰ pfu / mouse, and a week Ad5-RBD August ¹⁰ pfu / mouse

29. Ad5-RBD-Fc 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

The results are presented in tables 2 and 3.

Thus, the results of the experiment fully confirmed that sequential immunization with the developed immunobiological agents in various combinations including various forms of the S-MERS-CoV protein S will cause a more powerful induction of the immune response than immunization according to a similar scheme with the same antigen.

Example 10

Determination of the effectiveness of immunization with a developed immunobiological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, SG, RBD, RBD-G, RBD-Fc) MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, according to the proportion of proliferating lymphocytes.

Proliferative analysis allows you to evaluate the ability of lymphocytes to intensively divide after meeting with an antigen. In order to evaluate proliferation, the authors used the staining of lymphocytes with CFSE fluorescent dye. This dye binds to cellular proteins, and persists for a long time and is never transmitted to neighboring cells in the population. However, the fluorescent label is passed on to daughter cells. The concentration of the label, and, consequently, the fluorescence intensity, decrease exactly two times. Therefore, dividing cells are easy to track by decreasing their fluorescence.

In the experiment, C57BL / 6 mice were used. All animals were divided into 7 groups (3 animals each), which were administered intramuscularly:

1) phosphate buffer (100 μl)

2) Ad5-null 10 ⁸ PFU / mouse

3) Ad5-S 10 ⁸ PFU / mouse

4) Ad5-RBD 10 ⁸ PFU / mouse

5) Ad5-SG 10 ⁸ PFU / mouse

6) Ad5-RBD-G 10 ⁸ PFU / mouse

7) Ad5-RBD-Fc 10 ⁸ PFU / mouse

Doses of recombinant adenoviruses were selected based on data obtained in determining the titer of antibodies. After two weeks, the animals were euthanized. Lymphocytes were isolated from the spleen by centrifugation in a ficoll-urographin gradient. Then, the isolated cells were stained with CFSE according to the procedure (BJ Quah et. Al., Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester, Nature Protocols, 2007, No. 2 (9), 2049-2056) and cultured in the presence of antigen. Next, the cells were analyzed by flow cytometry. The results obtained are presented in FIG. 3, 4, 5, 6. Thus, it can be concluded that the obtained adenoviral constructs induce the formation of an antigen-specific immune response (both CD4 + and CD8 +).

As can be seen from the results of the experiment (Figs. 3, 4, 5, 6), the developed immunobiological agent according to Claim 1, Clause 2, Clause 3, Clause 4, Clause 5 at this dose effectively stimulate the proliferation of lymphocytes.

Example 11

The method of using the developed immunobiological agents based on recombinant human adenoviruses of the 5th and 26th serotypes containing a consensus sequence of protective antigen (S, SG, RBD, RBD-G, RBD-Fc proteins) MERS-CoV with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, by sequentially introducing them into the body of mammals with an interval of 1 week, to induce a specific immunity to MERS-CoV.

The experiment was performed according to the protocol described in example 7. Combinations of immunobiological agents were selected based on examples 7 and 8.

All animals were divided into 31 groups (3 animals each), which were administered intramuscularly:

1. phosphate buffer (100 μl), and after a week phosphate buffer (100 μl)

2. Ad26-null 10 ⁸ PFU / mouse, and after a week phosphate buffer (100 μl)

3. phosphate buffer (100 μl), and after a week Ad26-null 10 ⁸ PFU / mouse

4. Ad26-null 10 ⁸ PFU / mouse, and after a week Ad26-null 10 ⁸ PFU / mouse

5. Ad5-null 10 ⁸ PFU / mouse, and after a week phosphate buffer (100 μl)

6. phosphate buffer (100 μl), and after a week Ad5-null 10 ⁸ PFU / mouse

7. Ad5-null 10 ⁸ PFU / mouse, and after a week Ad5-null 10 ⁸ PFU / mouse

8. Ad5-null 10 ⁸ PFU / mouse, and after a week Ad26-null 10 ⁸ PFU / mouse

9. Ad26-null 10 ⁸ PFU / mouse, and after a week Ad5-null 10 ⁸ PFU / mouse

10. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad26-RBD-G 10 ⁸ PFU / mouse

11. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad26-S 10 ⁸ PFU / mouse

12. Ad26-S 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

13. Ad26-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

14. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad26-RBD 10 ⁸ PFU / mouse

15. Ad26-SG 10 ⁸ PFU / mouse, and after a week Ad5-RBD 10 ⁸ PFU / mouse

16. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad26-SG 10 ⁸ PFU / mouse

17. Ad26-SG 10 ⁸ PFU / mouse, and after a week Ad5-RBD 10 ⁸ PFU / mouse

18. Ad26-RBD 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

19. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad26-SG 10 ⁸ PFU / mouse

20. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad26-RBD-G 10 ⁸ PFU / mouse

21. Ad5-RBD-G 10 ⁸ PFU / mouse, and after a week Ad26-SG 10 ⁸ PFU / mouse

22. Ad26-SG 10 ⁸ PFU / mouse, and after a week Ad5-RBD-G 10 ⁸ PFU / mouse

23. Ad26-RBD-G 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

24. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad26-RBD 10 ⁸ PFU / mouse

25. Ad26-S 10 ⁸ PFU / mouse, and after a week Ad5-RBD 10 ⁸ PFU / mouse

26. Ad5-RBD 10 ⁸ PFU / mouse, and after a week Ad26-S 10 ⁸ PFU / mouse

27. Ad26-RBD 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

28. Ad5-SG 10 ⁸ PFU / mouse, and after a week Ad26-S 10 ⁸ PFU / mouse

29. Ad26-SG 10 ⁸ PFU / mouse, and after a week Ad5-S 10 ⁸ PFU / mouse

30. Ad5-S 10 ⁸ PFU / mouse, and after a week Ad26-SG 10 ⁸ PFU / mouse

31. Ad26-S 10 ⁸ PFU / mouse, and after a week Ad5-SG 10 ⁸ PFU / mouse

The results are presented in tables 4 and 5.

Thus, the experimental results fully confirmed that sequential immunization with the developed immunobiological agents according to p. 1, 2, 3, 4 and p. 6, 7, 8, 9, including various adenoviral vectors (based on human adenoviruses 5th and 26th serotypes) will cause a more powerful induction of the immune response than immunization according to a similar scheme with the same vector.

Example 12

Determination of the effectiveness of immunization with a developed immunobiological agent based on a recombinant adenovirus containing a consensus sequence of protective antigen (proteins S, SG, RBD, RBD-G, RBD-Fc) of MERS virus optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, according to the increase in the concentration of IFN-gamma in the medium after stimulation of splenocytes immunized with adenovirus constructs of C57 / BL6 mouse recombinant full-sized protein m S virus BVRS Cove.

To determine the level of IFN gamma, the Mouse IFN gamma Platinum ELISA kit (Affymetrix eBioscience, USA) was used.

The procedure for conducting ELISA. The wells of the plate were washed with a single wash buffer twice with 200 μl per well, and then 100 μl of standards and 100 μl of sample dilution solution were added as a negative control. 50 μl of sample dilution solution was added to the sample wells, and then 50 μl of the samples themselves (medium from stimulated splenocytes). A solution of antibodies conjugated with biotin was prepared. To do this, dilute the conjugate with a volume of 60 μl in 5.94 ml of assay buffer. Then, 50 μl of a solution of antibodies conjugated with biotin were added to all wells, the plate was covered with a lid and incubated at room temperature on a shaker at 400 rpm for 2 hours. Next, a solution of streptavidin conjugated with horseradish peroxidase was prepared. To do this, dilute the conjugate with a volume of 60 μl in 5.94 ml of assay buffer. The wells of the plate were washed twice with a single washing buffer with a volume of 200 μl per well and 100 μl of a solution of streptavidin conjugated with horseradish peroxidase were added to all wells of the tablet. The plate was incubated at room temperature on a shaker at 400 rpm for 1 hour. Then, the wells of the plate were washed twice with a single washing buffer with a volume of 200 μl per well and 100 μl of TMB substrate were added to all wells of the tablet and incubated in the dark at room temperature for 10 minutes, and then 100 μl of stopping solution was added to all wells. The optical density value was determined by measuring on a tablet spectrophotometer (Multiskan FC, Thermo) at a wavelength of 450 nm.

The results of measuring IFN-gamma production on the 15th day after immunization of test animals with adenoviral constructs are presented graphically in FIG. 8 and table 6 in the form of an increase in the concentration of IFN-gamma (times) when comparing cells stimulated with recombinant full-sized protein S BVRS-CoV, with intact cells.

According to the results of the study, it was shown that the introduction of the obtained constructs to animals leads to a high level of induction of IFN-gamma expression by splenocytes upon stimulation with the recombinant protein S of the MERS-CoV virus, which indicates the formation of specific T-cell immunity.

Example 13

The method of using the developed immunobiological agents based on recombinant human adenoviruses of the 5th serotype, containing a consensus sequence of protective antigen (RBD-G and S protein) MERS-CoV optimized for expression in mammalian cells with a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 4 by their simultaneous introduction into the body of mammals, to induce specific immunity to MERS-CoV.

The experiment was performed according to the protocol described in example 7. The combination of immunobiological agents was selected based on examples 8 and 11.

All animals were divided into 17 groups (5 animals each), which were administered intramuscularly:

1. phosphate buffer (100 μl)

2. Ad5-null 10 ⁵ virus particles / mouse

3. Ad5-null 10 ⁶ virus particles / mouse

4. Ad5-null 10 ⁷ virus particles / mouse

5. Ad5-null 10 ⁸ virus particles / mouse

6. Ad5-null 10 ⁹ virus particles / mouse

7. Ad5-null 10 ¹⁰ viral particles / mouse

8. Ad5-null 5 * 10 ¹⁰ viral particles / mouse

9. Ad5-null 10 ¹¹ virus particles / mouse

10. Ad5-S + Ad5-RBD-G 10 ⁵ viral particles / mouse

11. Ad5-S + Ad5-RBD-G 10 ⁶ viral particles / mouse

12. Ad5-S + Ad5-RBD-G 10 ⁷ virus particles / mouse

13. Ad5-S + Ad5-RBD-G 10 ⁸ viral particles / mouse

14. Ad5-S + Ad5-RBD-G 10 ⁹ virus particles / mouse

15. Ad5-S + Ad5-RBD-G 10 ¹⁰ viral particles / mouse

16. Ad5-S + Ad5-RBD-G 5 * 10 ¹⁰ viral particles / mouse

17. Ad5-S + Ad5-RBD-G 10 ¹¹ virus particles / mouse

The results are presented in tables 7 and 8.

Thus, the experimental results fully confirmed that the simultaneous immunization with the developed immunobiological agents according to p. 1 and 4 induces a humoral immune response to the MERS-CoV glycoprotein in the dose range from 10 ⁶ virus particles / mouse to 10 ¹¹ virus particles / mouse. At the same time, it is obvious that an increase in doses will lead to an increase in the titer of antibodies in the blood of mammals before the onset of toxic effects.

Industrial applicability

An advantage of the claimed technical solution is the use of such doses of recombinant adenoviruses expressing the gene for a full-sized protein, which can increase immunogenicity, but at which toxic effects on animals are not yet observed. The advantages include an additional increase in the immunogenicity of the receptor-binding domain of the S-gene of MERS-CoV due to the addition of a leader sequence for secretion of the protein from the cell into the external environment. One of the advantages of the claimed technical solution is the availability of an adequate T-cell response (both CD4 + and CD8 +) to the introduction of antigen.

Thus, an immunobiological agent was created based on recombinant human adenovirus of the 5th serotype, containing human adenovirus of the 5th serotype with deleted E1 / E3 regions and an integrated genetic construct encoding the optimal consensus amino acid sequences of the protective antigen S that are most homologous to different strains of MERS -KoV (from 2015-2017). Also, an immunobiological agent based on recombinant human adenovirus of the 26th serotype was created, containing human adenovirus of the 26th serotype with a deleted E1 region, replaced by an open reading frame 6 by an open reading frame of a human adenovirus of the 5th serotype and with an integrated genetic construct encoding the developed optimal consensus amino acid sequences of the protective S antigen, the most homologous for different strains of MERS-CoV (from 2015-2017). In this case, the coding sequences of various forms of protein S of MERS-CoV are expressed by recombinant pseudo-adenovirus particles directly in the body of the subject. The developed immunobiological agent can be considered as a candidate drug for preclinical studies, as a broad-spectrum antiviral vaccine that can simultaneously effectively protect a person from infection with various strains of MERS-CoV. A technology for the production of such a vaccine is proposed.

Claims (9)

1. An immunobiological agent based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing a consensus sequence of the complete protective antigen S of the MERS virus based on the gene sequences of the S protein genes of modern strains of the MERS virus 2015-2017 gg with the sequence of SEQ ID NO: 1 (option 1). 2. An immunobiological agent based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing the consensus sequence of the complete protective antigen S of the MERS virus with the transmembrane domain of the vesicular stomatitis SE Glycoprotein NO sequence with stenosis: 2 (option 2). 3. An immunobiological agent based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing the optimized sequence of the receptor-binding domain of protein S with the sequence of the leader peptide with the sequence of SEQ ID NO: 3 (option 3). 4. An immunobiological agent based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing a consensus sequence of protein S receptor-binding domain with mammalian transmembrane domain of the vesicular stomatitis virus glycoprotein ID NO: SEQ sequence 4 (option 4). 5. An immunobiological agent based on recombinant human adenovirus of the 5th serotype or recombinant human adenovirus of the 26th serotype, containing the optimized sequence of the receptor-binding domain of protein S with the sequence of the leader peptide and the sequence of the Fc fragment from human IgG1 with the sequence SEQ ID NO: 5 (option 5). 6. The method of using the immunobiological agent according to Claim 1, or Clause 2, or Clause 3, or Clause 4, or Clause 5, comprising administering to the mammalian organism this agent in an effective amount to induce specific immunity to the MERS virus. 7. The method of using an immunobiological agent in an effective amount for the induction of specific immunity to the MERS virus according to claims. and / or 1, and / or 2, and / or 3, and / or 4, and / or 5, which consists in the sequential administration of two different immunobiological agents to the mammalian organism based on recombinant human adenovirus of the 5th serotype or two different immunobiological agents based on recombinant human adenovirus of the 26th serotype with an interval of more than 1 week, for the induction of specific immunity to MERS-CoV. 8. The method of using an immunobiological agent in an effective amount for the induction of specific immunity to the MERS virus according to paragraphs. and / or 1, and / or 2, and / or 3, and / or 4, and / or 5, which consists in the sequential introduction into the mammalian body of any of the immunobiological agents based on recombinant human adenovirus of the 5th serotype and any of the immunobiological agents based on a recombinant human adenovirus of the 26th serotype with an interval of more than 1 week to induce specific immunity to MERS-CoV, or in the sequential administration of any of the immunobiological agents based on the recombinant human adenovirus of the 26th se of the type and any of the immunobiological agents based on recombinant human adenovirus of the 5th serotype with an interval of more than 1 week, for the induction of specific immunity to MERS-CoV. 9. The method of using an immunobiological agent in an effective amount for the induction of specific immunity to the MERS virus according to claims. and / or 1, and / or 2, and / or 3, and / or 4, and / or 5, which consists in the simultaneous introduction into the organism of mammals of any two immunobiological agents based on recombinant human adenovirus of the 5th or 26th serotype for induction of specific immunity to MERS-CoV.

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