WO2021206581A1 - Genetic construct-based vaccine against coronavirus infection - Google Patents

Abstract

The invention relates to molecular biology, biotechnology, medicine and can be used to prevent and treat corona virus infection, mainly caused by 2019-nCoV. A vaccine is proposed based on a genetic construct encoding a fusion protein, including fragments of M, S, N, E new coronavirus proteins. The speed and simplicity of preparation (from 2-3 hours to 4-5 days), safety are the advantages of the developed vaccine, due to the nature of the molecule- of the active substance, as well as to the lack of an ACE2 binding site in the fusion protein synthesized from the genetic construct, and the lack of its homology with proteins of the organism, induction of the immune response profile, to a large extent represented by the cytotoxic immune response, in addition to the humoral one. The vaccine can be administered as standard or by electroporation.

Description

Description

Title of Invention:

GENETIC CONSTRUCT-BASED VACCINE AGAINST CORONAVIRUS INFECTION

Technical Field

[0001] The invention relates to molecular biology, biotechnology, medicine and can be used to prevent or treat coronavirus infection.

Background Art

[0002] Coronaviruses (CoV) are a large family of viruses that cause illness from a common cold to more serious illnesses, such as the Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV). The new coronavirus (nCoV, COVID-19, SARS-CoV-2) is a new strain that has never been detected in humans before

[https://www.who.int/ru/health-topics/coronavirus/coronavirus].

[0003] A feature of this strain was the rapid spread from person to person, as well as the complex course of the disease.

[0004] The first cases of COVID-19 infection were recorded in China in December 2019. However, as of March 4, 2020, a total of 93,090 cases of COVID-19 and 3198 deaths were registered in the world. Most cases and deaths are in China (80422 and 2984, respectively), but cases of coronavirus and deaths due to it are also recorded in 76 countries of the world

[https://www.who.int/docs/default-source/coronaviruse/situation- reports / 20200304-sitrep-44-covid-19.pdf? sfvrsn = 783b4c9d_2], WHO has recognized coronavirus infection as a pandemic.

[0005] On April 3, 2020, a total of 1,095,904 cases of COVID-19 and 58,814 deaths were registered in the world. Most cases and deaths are in USA (275,744 and 7,086, respectively), Italy (119,827 and 14,681, respectively), Spain (119,199 and 11,198, respectively), Germany (91,159 and 1,275, respectively), in China (81,620 and 3,322, respectively). Cases of coronavirus and deaths due to it have been recorded already in 205 countries of the world [https://www.worldometers.info/coronavirus/]. Obviously, the infection rate is extremely high, despite the measures taken.

[0006] Among the conditions observed in sick people - ARDS, acute kidney injury, cardiac injury, liver dysfunction, arrhythmia, lethal outcome is not uncommon. For most patients external support for respiratory function is required [Arabi, Y.M., Murthy, S.

& Webb, S. COVID-19: a novel coronavirus and a novel challenge for critical care. Intensive Care Med (2020)].

[0007] 2019-nCoV is a virus that has a serious impact on public health, the economics, and socio-political issues. Containment of COVID-19 should be the top priority for all countries

[https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-th e-mission-briefing-on- covid-19 — 4-march-2020].

[0008] Since this is a new infectious disease, there is currently no registered vaccine against the new coronavirus

[https://www.rospotrebnadzor.ru/about/info/news_time/news_details.php7ELEMENT_

ID=13566J.

[0009] There is also an acute unmet need for optimal treatment, including specific antiviral therapy, studying the role of the immune system modulation and how to provide support for failed organ systems the best way. At the time of writing the mentioned above article by the Arabi Y.M. and co-authors, over 160 randomized and non-random studies were recorded (http://www.chictr.org.cn/searchprojen.aspx and http://apps.who.int/trialsearch/default.aspx). Several drugs are used to treat COVID-19, including corticosteroids, various combinations of ribavirin, lopinavir/ritonavir, chloroquine, hydroxychloroquine, interferons and other agents. Randomised controlled trial (RCT) is performed using remdesivir in severe course of COVID-19 (NCT04257656). [Arabi, Y. M., Murthy, S. & Webb, S. COVID-19: a novel coronavirus and a novel challenge for critical care. Intensive Care Med (2020)]. Treatment is also carried out using traditional medicine.

Summary of Invention

[0010] The purpose of the present invention is to create a vaccine against coronavirus which is safe, quick and easy to produce and use, which would be effective and low-cost, and which can be used both for the prevention and treatment of coronavirus, especially the new coronavirus.

[0011] From all the directions of the development of new vaccines, the authors of the present group of inventions consider the use of vaccines obtained using recombinant DNA technology being the most promising. However, their effectiveness varies and is caused by a lot of factors, one of the most important of which is the selection of components and their shape (structure).

[0012] To create the present invention, fragments of coronavirus antigens — of M, S, N, E proteins of the new coronavirus — have been selected. A fusion protein has been developed on their basis. The components of the fusion protein are connected by flexible bridges, which allows to achieve the full functioning of each component of the protein. The developed protein is also effective against SARS due to cross-activity.

[0013] It is important that, due to its structure, the developed fusion protein doesn’t have homology with the known proteins of other organisms except coronavirus, which ensures the specificity of their action.

[0014] The vaccine is important to be specifically immunogenic and protective against coronavirus, and also safe from the point of view that it will not induce complications caused by coronavirus, as well as other side effects.

[0015] An important feature of the COVID-19 virus is that the SI subunit (aaOl - aa550) of its S glycoprotein has an RBD (receptor-binding domain) which interacts with the host cell receptor, angiotensin-converting enzyme 2 (ACE2). Penetration of the coronavirus into the host cell and its uptake occurs mainly due to the N-terminal domain of SI. It would seem that the use of this fragment as a target for generating an immune response could be important for blocking the penetration of the virus. However, during the formation of antibodies to this fragment, there is a chance that they will capture molecules, and as a result of the lack of penetration of such organisms molecules into the necessary tissues, hence, the effect of such vaccination can be completely unsafe, due to which serious complications mentioned above may develop. As a consequence, the use of vaccines containing such a fragment, i.e. vaccines based on attenuated or inactivated new coronavirus, of recombinant vaccines containing such a fragment of S protein is potentially unsafe.

[0016] That is why a different fragment of the S 1 subunit of S glycoprotein is used in our developed fusion protein, which will allow the formation of an immune response, including to S glycoprotein, and the prevention of the penetration of the virus, without causing a disruption in the functioning of body systems, inter alia cardiovascular and respiratory systems.

[0017] A vaccine is proposed by us for coronavirus infection prevention or treatment in a human or animal containing at least one genetic construct as an active agent, in an effective amount, and also a physiologically acceptable carrier and a buffer solution. The genetic construct encodes the fusion protein described above.

[0018] No analogues of the vaccine proposed by us have been identified. Vaccines based on inactivated or attenuated strains of coronavirus are not considered as analogues by us because of the significantly lower safety, including due to the presence of full-size S protein. All types of vaccines that do not contain live microorganisms are safer not only because of the absence of infection risks, but also because of the lack of risks of side effects in people with mutations in the genes contributing to the structure of the molecules, which are crucial for the proper functioning of the immune system. However, it is important not to cause the formation of side effects with them, including those described above, including due to the presence of the S protein or the N-terminal fragment of its SI subunit.

[0019] The technical result from the use of the invention is expressed, first of all, in a significant acceleration of vaccine production due to the simple, with the least re- quirements, process of obtaining an active vaccine agent. With regard to the rapidly spreading infection, the rate of vaccine production plays a key role in defeating the infection, and also has an economic impact - under quarantine measures the economics will fall into a severe decline in several months, and only quick vaccination in shortest period will allow quarantine and self-isolation to be removed, and will help the economics not to fall into a severe decline.

[0020] The technical result of using the vaccine is also to increase the safety of the vaccine.

[0021] The mentioned technical result is achieved mainly by the fact that the synthesized fusion protein according to the invention does not contain fragments homologous to fragments of the molecules of the body.

[0022] The mentioned technical result is achieved by using DNA as the active substance, which exists in the form of an episome and does not integrate into the genome, from which the protein is synthesized and then, in one of the variants, is secreted from the cell.

[0023] The mentioned technical result is also achieved by the fact that the used DNA is not replicated after the introduction into the body of a mammal, which allows control over the amount of synthesized protein, and, accordingly, over the effect.

[0024] The mentioned technical result is achieved also due to the presence of regulatory sequences such as silencer and/or insulator in the used DNA, in one embodiment of the invention, due to which protein synthesis and, accordingly, the effect are also monitored: if necessary, there is a possibility to stop or decrease the gene expression in a short time.

[0025] The mentioned technical result is also achieved by the fact that the infectious agent as such is not used in production.

[0026] The mentioned technical result is achieved, including in one of the variants, by the fact that the used polynucleotide contains exact fragments of genes encoding proteins of the coronavirus; accordingly, the effect of such a genetic construct will not be unexpected.

[0027] The technical result is also expressed in the increased immunogenicity of the vaccine due to the nature of the molecules used. Thus, it is shown that DNA itself along with the injection with a needle, acts as an adjuvant, which mediates the presentation of the synthesized target antigens, mainly with the help of MHC I, which, in turn, induces the formation of a cytotoxic response. It has also been shown that the synthesis of single intracellular protein in large quantities may contribute to the active presentation by myofibrils using MHC I [Hartikka J. et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle // Hum. Gene Ther. 1996. Vol. 7, No. 10. P. 1205-1217].

[0028] The technical result from the use of a vaccine based on a genetic construct according to the invention also consists in increased efficiency due to the fact that the fusion protein is synthesized from a genetic construct for a long time, and from several days to several weeks, depending on the structure of the used genetic construct. It is also achieved by the structure of the encoded fusion protein.

[0029] The technical result also consists in simplifying and cheapening the production of vaccines by avoiding the production and purification processes of protein preparations in vitro due to the fact that protein synthesis occurs in vivo. The production, purification and storage of DNA preparations are economically more profitable than of protein ones, since the former are more stable, they can be produced in large quantities at a lower cost.

[0030] The technical result also consists in expanding the spectrum of active substances and vaccines based on them against coronavirus. When reluctance to use analogues due to their disadvantages mentioned above, or precautions for the use of analogues, this active substance or vaccine will allow protection against coronavirus. The mentioned technical result is achieved by using the active substance or the vaccine of the present invention.

[0031] In the context of a pandemic, it is extremely important to implement vaccination quickly and on a large-scale, as this is the only way to protect against the spread and severe course of the disease, and deaths. The proposed vaccine like no other currently satisfies this requirement, in addition to the high safety.

[0032] A method is proposed by us for producing the vaccine according to the invention, namely that the genetic construct — a linear DNA fragment — is amplified using PCR, purified and mixed with a physiologically acceptable carrier and a buffer solution.

[0033] The technical result of using the method of obtaining the developed vaccine is to obtain the vaccine as soon as possible. So, the process of obtaining a purified preparation of genetic construct - a linear DNA fragment - the active agent of the vaccine - takes only a few hours (from 2-3 hours)! No other type of coronavirus vaccine can be obtained in such a short time.

[0034] The technical result also consists in the simplicity of the vaccine production. It is achieved by the fact that the production of such a vaccine does not require the development of a multi-stage production technology, and a long adjustment of the process steps, it requires a minimal set of reagents that are readily available, approvals for working with pathogenic organisms are not required, as well as significant investments in the construction and refurbishment of premises and equipment. Such a method can even be carried out in a standard DNA laboratory.

[0035] A genetic construct is proposed for the expression of the polynucleotide according to the invention in the cells of a target organism, including such a polynucleotide and other elements allowing to realize the indicated purpose — to express the polynu- cleotide in the cells of the target organism. The target organism is a human or animal. The genetic construct can be represented by a plasmid DNA for transient expression in mammalian cells, a virus-based vector, a linear DNA fragment containing the polynucleotide according to the invention.

[0036] The technical result consists mainly in the induction of an immune response which does not cause side effects associated with the use of other fragments of S protein of the new coronavirus, or of such full-sized protein in genetic constructs or strains of coronavirus.

[0037] The technical result consists in a significant acceleration and simplification of obtaining the active substance of the vaccine against coronavirus - a genetic construct. This is achieved, first of all, by the safety of the genetic constructs types being used and, accordingly, by the absence of strict requirements for working with them.

[0038] The technical result also consists in the fact that the genetic construct serves as a matrix for PCR to produce a vaccine according to the invention.

[0039] In addition, the technical result consists in increasing the duration of the encoded protein synthesis and is achieved by the fact that the nucleotide sequence encoding the target protein contains elements that determine mRNA stability and, accordingly, increase the half-life of the mRNA, resulting in the synthesis of the protein from one mRNA molecule carried out more times, and also in the synthesized protein amount increase; and also by the fact that that the nucleotide sequence of the fusion protein is optimized in codon composition for expression in the cells of the target organism — of a human and animal, mammals, resulting in more intensive protein synthesis.

[0040] When put into practice, this will allow to use an extremely small amount of the plasmid DNA for injection.

[0041] The technical result also consists in expanding the spectrum of genetic constructs for the synthesis of immunogenic coronavirus antigens in an organism.

[0042] A polynucleotide is proposed for expression in the cells of a target organism, encoding a fusion protein set forth as SEQ ID NO.:l or 2 amino acid sequence comprising coronavirus M, S, N, E proteins fragments connected by flexible bridges. The polynucleotide may contain a fragment encoding a heterologous secretory sequence.

[0043] The technical result of using the polynucleotide proposed by us is mainly in obtaining a genetic construct, due to the use of which in the vaccine a specific protective immune response to the coronavirus, mainly to the new coronavirus, is formed in a human or animal.

[0044] The technical result of using the polynucleotide proposed by us is also to expand the spectrum of polynucleotides for the synthesis of immunogenic coronavirus antigens in an organism. [0045] A prokaryotic recombinant cell - producer - is proposed for producing a genetic construct according to the present invention.

[0046] The technical result of using the producer of the developed genetic construct is in the stable storage and reproduction of the genetic construct, as well as in obtaining the developed genetic construct.

[0047] A polynucleotide, a genetic construct, a producer of a genetic construct, a method for producing a vaccine — all these objects are used to produce a vaccine according to the present invention.

[0048] Due to the fact that the problem of the new coronavirus is very acute, but a few manage to bring the drug on the market, and the effectiveness of analogues in humans is also unknown yet, this group of inventions will allow to increase the chances in fighting this infection.

Description of Embodiments

[0049] A group of inventions is proposed: a polynucleotide, a genetic construct, a recombinant cell, a vaccine based on at least 1 genetic construct for the prevention or treatment of coronavirus, and a method of its production.

[0050] A fusion protein has been developed which includes immunogenic fragments of the coronavirus M, S, N, E proteins connected by flexible bridges, the protein is characterized by SEQ ID NO.: 1 or 2 amino acid sequence. The peculiarity of the protein is that it does not have homology with proteins of other organisms than coronavirus, and also does not contain a binding site for ACE2. This provides an important advantage of a vaccine based on it - an additional aspect of the vaccine safety. Such protein will be synthesized in the cells of a target organism — a human or an animal — from the proposed genetic construct containing the proposed polynucleotide.

[0051] A polynucleotide for synthesis in the cells of a target organism encoding a developed fusion protein is proposed. When the amino acid sequence of a protein is known, one skilled in the art will be able to obtain the nucleotide sequence. This can be a sequence either based on fragments of the native nucleotide sequence of viral proteins, or artificially optimized by codon composition. Codon optimization can be carried out independently, using information on the frequency of codons occurrence in a producer, for example, in a database [for example, Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res . 2000 Jan 1; 28 (1): 292], or using specialized programs, for example, available at http://www.encorbio.com/protocols/Codon.htm or molbiol.ru or other resources.

[0052] A polynucleotide may not contain (e.g., SEQ ID NO.: 1 or 4) or contain (e.g., SEQ ID NO.:5 or 6) a heterologous secretory sequence optimized in codon composition for the target organism - for example, such of TPA (tissue-type plasminogen activator), hGH (human growth hormone), IGF (insulin-like growth factor), EPO (erythropoietin), but not limited to such. In the case without a secretory sequence, the immune response will be the most shifted towards a cytotoxic one, in the second case, in addition to the cytotoxic one, a humoral - antibody-based immune response will also be significantly induced. Since, especially in the context of the complexity of testing for coronavirus, it is difficult to assess whether a person is infected, the obvious advantage of the proposed vaccine is the induction of an immune response profile, where the cytotoxic response is manifested significantly, in contrast to the immune response that forms on the introduced protein immunogen or a virus particle. This means, both polynucleotide variants are suitable.

[0053] A genetic construct for expression of the polynucleotide described above in producer cells is proposed; each construct includes, in addition to the polynucleotide, elements allowing to realize the indicated purpose — to express the polynucleotide. Due to the fact that the genetic construct from which the antigen is synthesized is the active substance of the vaccine, it is possible to obtain an immune response profile in which the cytotoxic immune response will play a significant role, however, an antibody response will also be formed. Also, due to the duration of the antigen synthesis in a body and the absence of infectivity, the expectation of causing the formation of an immune response which is adequate, with the formation of immunological memory, increases. The molecule itself also has an adjuvant effect.

[0054] By genetic construct primarily a linear DNA fragment or a recombinant vector is meant, which can be represented by a viral or plasmid vector.

[0055] The recombinant vector must comprise elements essential for the organisms of its maintenance and use, together with the corresponding regulatory sequences.

Regulatory sequences are nucleotide sequences which can affect gene expression at the level of transcription and/or translation, as well as mechanisms that ensure the existence and maintenance of the recombinant vector functioning.

[0056] An origin of replication, for maintaining in a cell with medium and preferably high copy number, and a marker gene for a producer strain selection are essential to the prokaryotic system. Bacterial elements of a plasmid DNA should not adversely affect expression in mammalian cells and cause a side effect of using the plasmid DNA. A suitable origin of replication is represented by pMl (der.), ColEl (der.), and FI, pUC, and FI, but is not limited to such. A suitable marker gene is represented by a reporter gene or antibiotic resistance gene, for example, ampicillin, mainly kanamycin, but is not limited to such. There is evidence in the literature that the use of ampicillin resistance gene as a marker gene may be undesirable due to the development of the patient's response to ampicillin, however, the authors consider such consequences be associated with a poor quality of DNA purification, but not with the element itself.

[0057] Elements for efficient functioning, for expression of the encoded gene, - a promoter, including transcription initiation signals, mRNA leader sequence, a termination sequence, regulatory sequences, - are essential elements of a recombinant vector for use in mammals.

[0058] A promoter is an important component of a vector that triggers the expression of a gene of interest. Human CMV / immediate early or CMV-chicken-b actin (CAGG) promoter are classic promoters for recombinant vectors - drug components. CMV promoters are used for most DNA vaccines, as they mediate high levels of constitutive expression in a wide range of mammalian tissues [Manthorpe M, Comefert-Jensen F, Hartikka J, et al. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum. Gene Ther. 1993; 4 (4): 419-431] and do not inhibit downstream expression. An increase in the expression level is observed when the CMV promoter is changed, for example, by incorporating HTLV-1R-U5 downstream the cytomegalovirus promoter or when using the chimeric SV40-CMV promoter [Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol. Adv. 2009; 27 (4): 353-370]. Tissue-specific host promoters which allow avoiding constitutive expression of antigens in inappropriate tissues are an alternative to CMV promoters [Cazeaux N, Bennasser Y, Vidal PL, Li Z, Paulin D, Bahraoui E. Comparative study of immune responses induced after immunization with plasmids encoding the HIV- 1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter. Vaccine 2002; 20 (27-28): 3322-3331].

[0059] The promoter can be with corresponding regulatory sequences from natural promoters with its own regulatory elements (CaM kinase P , CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of the kainate receptor, and subunit B of glutamate receptor, and others), or from synthetic promoters with regulatory sequences to obtain the desired expression rate (ratio of duration and expression level) of a target gene at the transcription level.

[0060] Possible regulatory sequences in relation to the promoter:

[0061] - enhancer, to increase the level of expression through improve of RNA polymerase and DNA interaction.

[0062] - insulator, to modulate the functions of the enhancer,

[0063] - silencers, or fragments thereof, to reduce the level of transcription, for example, for tissue-specific expression,

[0064] - 5' untranslated region upstream promoter, including an intron.

[0065] Regulatory sequences are nucleotide sequences that may affect gene expression at the level of transcription and/or translation, as well as mechanisms that ensure the existence and maintenance of the genetic construct functioning. The recombinant vector, the plasmid or viral DNA of the present invention, contains at least one of the abovementioned regulatory sequences, depending on the DNA variant, based on the selection of promoter and the desired expression parameters of the target gene. Based on the existing level of technology and known and obvious variants of such elements and their use, die recombinant vector according to the present invention may contain any combinations meeting the above-mentioned criteria, using which synthesis of the developed fusion gene is performed from it, in cells of a target organism - a human or an animal. When using a silencer or an insulator in the construct, it is possible to regulate the expression of the target gene.

[0066] Other regulatory sequences:

[0067] - untranslated region downstream the promoter, including an intron, to increase mRNA stability and the target gene expression.

[0068] The recombinant vector of the present invention in one embodiment additionally comprises such a regulatory element.

[0069] The recombinant vector of the present invention also contains such an important element as mRNA leader sequence containing translation initiation signals, a start codon. Translation initiation signals is Kozak sequence in eukaryotes [Kozak M.

(1986) "Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes", Cell 44, 283-292].

[0070] The recombinant vector also contains a site, mainly different sites, for cloning of the target gene, for die correct target gene orientation in the recombinant vector, and a site, mainly sites, for primers annealing for its sequencing.

[0071] The recombinant vector also contains the polynucleotide described above.

[0072] The recombinant vector also contains a termination sequence containing sequentially a stop codon, a 3' untranslated region with a polyadenylation signal and site, a stop codon; due to which mRNA stability is maintained, and transcription is terminated appropriately, and mRNA is exported from the nucleus. Gene expression can be influenced by changing the termination sequence, which is necessary for mRNA stability maintaining, for proper transcription termination and mRNA export from the nucleus, including by its shortening. In many modem DNA vaccines the bovine growth hormone transcriptional terminator sequence is used [Montgomery DL, Shiver JW, Leander KR, et al. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors. DNA Cell Biol. 1993; 12 (9): 777-783]. Polyadenylation (poly A) is necessary to stabilize the transcript. Alterations to the polyA sequence can lead to the increase of gene expression [Norman JA, Hobart P, Manthorpe M, Feigner P, Wheeler C. Development of improved vectors for DNA- based immunization and other gene therapy applications. Vaccine 1997; 15 (8): 801-803]. In pVAXl plasmid (Invitrogen, Carlsbad, Calif.), the bovine growth hormone terminator region contains a homopurin region which is sensitive to nuclease. It is shown that an alternative polyA sequence may significantly improve plasmid stability to nuclease [Azzoni AR, Ribeiro SC, Monteiro GA, Prazeres DMF. The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J Gene Med. 2007; 9: 392-402]. The introduction of two stop codons preceding the 3' untranslated region allows to increase the efficiency of the transcription terminator. Based on the existing art and known and obvious variants of such an element, the recombinant vector according to the present invention may contain any termination sequence meeting the above-mentioned criteria, using which synthesis of a target protein is performed in cells of a human or animal. An example of a termination sequence for mammalian cells is that of bovine growth hormone (BGH).

[0073] Other elements may also be contained for the functioning of the expression system.

[0074] Vectors tested on humans, containing the elements described above with appropriate regulatory sequences, possibly modified to meet the above-mentioned criteria are preferred recombinant vectors for use in a human, thereby reducing the number of studies required to register a drug. However, it is possible to use other recombinant vectors containing the required described elements.

[0075] Numerous clinical studies of DNA vaccines have been performed which have demonstrated their safety.

[0076] For 2019, the number of clinical trials conducted in the field of gene therapy is 3001 [http://www.abedia.com/wiley/countries.php], including 184 studies conducted on infectious diseases. At the same time, 15% of all clinical trials in the field of gene therapy have been carried out for agents based on plasmid DNA, 8% - based on adeno- associated virus. Thus, this direction is real and implementable.

[0077] FDA guideline (2007) states that studies of the biodistribution of a substance after its introduction into an organism can be canceled for DNA vaccines produced by cloning of a new gene into a plasmid vector, for which acceptable biodistribution and integration profiles have been previously documented. A WHO guideline (2007) states that studies of biological distribution and conservation are required if significant experience with an almost identical or similar product is not yet available. EMEA guideline (2006) states that experience with a vector system will allow to optimize and focus on preclinical studies. Safety studies using DNA vectors with different cloned genes have demonstrated similar biodistribution [Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ, Gomez PL. Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ, Gomez PL.Toxicol Sci. 2006 Jun; 91 (2): 610-9. Epub 2006 Mar 28.] and toxicology [Sheets RL, Stein J, Manetz TS, Andrews C, Bailer R, Rathmann J, Gomez PL. Toxicological safety evaluation of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar despite differing plasmid backbones or gene-inserts. Toxicol Sci. 2006 Jun; 91 (2): 620-30. Epub 2006 Mar 28]. For plasmid DNA for use in mammals except humans, the requirements are less stringent, and therefore it is possible to use a wider range of plasmids.

[0078] The sequence of arrangement of the described elements in a recombinant vector is clear to the average person skilled in the art.

[0079] Regarding the pcDNA3.1 (+) vector, elements that provide expression of the gene causing antibiotic resistance to neomycin, as well as plasmid replication in the form of episome in mammalian cells, do not function in the target organism, since such cells - cells of the organism of such plasmid DNA introduction - do not contain the large SV40 T-antigen. The pcDNA3.1 (+) vector does not contain transposons, as well as elements responsible for the transmissibility. Such DNA does not integrate into the genome and does not replicate in mammalian cells. All this certainly indicates the safety of drugs based on it.

[0080] By a virus-based vector in which the polynucleotide is cloned, a safe vector is meant, such as, i.e. a recombinant adeno-associated virus, but not limited to such.

[0081] By a linear DNA fragment a DNA fragment is meant containing a promoter, an mRNA leader sequence, the polynucleotide described above, a termination sequence, including 1 or 2 stop codons, regulatoiy sequences. Specification described above is applicable to the elements. Such elements are crucial, but other elements may also be contained. A fusion protein is synthesized from such a fragment in the cells of a target organism. The arrangement sequence of the described elements is clear to the average person skilled in the art.

[0082] Synthesis from a linear DNA fragment is less prolonged than from a plasmid or viral vector, but its production is much faster. Thus, each genetic construct has its own advantages and can be applied in a particular situation. For example, if vaccine doses are needed at the earliest possible time - within a few hours (from 2-3 hours), then a linear DNA fragment will be used, if there is more time, then other variants are possible.

[0083] Accordingly, a method is proposed for producing a vaccine against coronavirus based on a linear DNA fragment, which is in amplification of a linear DNA fragment using a polymerase chain reaction - PCR, purification from impurities, and mixing with a physiologically acceptable carrier and buffer solution. This method of obtaining the vaccine is the fastest, and not a single vaccine registered in Russia is produced in this way. However, in conditions of such a rapidly spreading infection as the coronavirus infection of 2020 affecting the world, only such a way of producing the vaccine meets the challenge of nature.

[0084] A vaccine based on other genetic constructs can be obtained by cultivating microorganisms - a prokaryotic recombinant producer cell, followed by isolation and purification of the genetic construct. This method will take more time (about 4-5 days), however, it is also fast and applicable in the realities of the new coronavirus.

[0085] These two methods are also safe as both the production and the use of the vaccine, since the drug cannot cause an infection even hypothetically.

[0086] Vaccines based on proteins or mRNA, or attenuated or inactivated viruses - are much more complicated and longer to be produced, especially the first period - the development of a technology for producing an active agent - a protein in a monomeric, active form that does not cause side effects; the production of mRNA itself - a molecule that is less stable than DNA; work with a dangerous virus.

[0087] A prokaryotic recombinant cell is proposed for obtaining the genetic construct. A prokaryotic producer is represented, for example, by Escherichia coli, Bacillus subtilis. In one embodiment, this is Escherichia coli DH10B/R bacterial strain, containing the pcDNA3.1 (+) vector, which contains the nucleotide sequence of SEQ ID NO.:3-6.

[0088] A vaccine is proposed for the prevention or treatment of coronavirus, containing the above-described genetic construct as an active agent in an effective amount, as well as a physiologically acceptable carrier and a buffer solution. Pharmaceutically acceptable carriers and buffers are known from the prior art and include those described in various texts, such as, for example, Remington's Pharmaceutical Sciences. Both a human and an animal, inter alia of domestic or farm animals, can be a consumer of the vaccine.

[0089] Injection of the DNA constructs encoding a pathogen antigen, as a rule, is carried out in muscle tissue, and this construct enters somatic cells, where the target gene is expressed. The synthesized protein is exposed on the surface of the somatic cell using MHC I molecules, which causes the formation of cytotoxic T-lymphocytes. However, when a secretory peptide is introduced into a protein, it is mainly secreted into the extracellular space, and a mechanism of a humoral immune response is realized.

[0090] The authors propose parenteral administration of the vaccine, and in one of the options it contains a genetic construct with and without secretory sequence. Also, the vaccine can be administered using electroporation to increase the efficiency of delivery to cells for polynucleotide expression.

Examples

[0091] The authors of the present invention have conducted laboratory studies confirming the feasibility of the characterized invention. The obtained research results are illustrated by the following examples (1-3).

[0092] Example 1. Modeling of a fusion protein [0093] To model proteins, the following actions have been performed:

[0094] 1. Search for components of a fusion protein

[0095] 2. Building a model of the whole protein to determine the orientation of the domains

[0096] 3. Building models for each domain (using samples of 3D structures and ab initio)

[0097] 4. Docking of models using the whole protein model.

[0098] To obtain the most realistic results in automatic mode, I-Tasser algorithm was used to model proteins.

[0099] The modelled fusion protein in one embodiment consists of 422 aa, with methionine at the N-terminus - 423 aa, is represented by the amino acid sequence SEQ ID NO.:l. Analysis of the amino acid sequence of this protein using the ProtParam program (http.V/au.expasy.org/tools/protparam.html) has shown that the fusion protein has a molecular mass of 46.4 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 100 hours. With methionine at the N-terminus - 46.5 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 30 hours. When adding the secretory sequence of IGF (GKISSLPTQLFKCCFCDFLK) , with methionine at the N-terminus, the fusion protein consists of 443 aa, has a molecular weight of 48.8 kDa, pi 9.55, the protein is stable. When adding the hGH secretory sequence

(ATGSRTSLLLAFGLLCLPWLQEGSA), with methionine at the N-terminus, the fusion protein consists of 448 aa, molecular mass is 49.1 kDa, pi 9.57, the protein is stable. By adding the TPA secretory sequence

(MDAMKRGLCCVLLLCGAVFVSPS), the fusion protein consists of 445 aa, molecular mass is 48.8 kDa, pi 9.53, the protein is stable.

[0100] The modelled fusion protein in one embodiment consists of 424 aa, with methionine at the N-terminus - 425 aa, is represented by SEQ ID NO.:2 amino acid sequence. Analysis of the amino acid sequence of this protein using the ProtParam program (http://au.expasy.org/tools/protparam.html) has shown that the fusion protein has a molecular mass of 46.5 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 100 hours. With methionine at the N-terminus - 46.6 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 30 hours. When adding the secretory sequence of IGF, with methionine at the N-terminus, the fusion protein consists of 445 aa, molecular mass is 48.9 kDa, pi 9.55, the protein is stable. When adding the secretion sequence of hGH, with methionine at the N-terminus, the fusion protein consists of 450 aa, molecular mass is 49.2 kDa, pi 9.57, the protein is stable. When adding the secretory sequence of TPA (MDAMKRGLCCVLLLCGAVFVSPS), the fusion protein consists of 447 aa, has a molecular mass of 48.9 kDa, pi 9.53, the protein is stable.

[0101] When using other secretory signals, the indicators change.

[0102] When a fusion polynucleotide is expressed in any cell, a protein is synthesized with methionine at the N-terminus, since translation always starts from the start codon. Further, methionine can be cleaved naturally, for example, if the protein is secreted, as part of the secretory peptide.

[0103] Example 2. Obtaining highly purified genetic constructs according to the invention

[0104] A nucleotide sequence of a gene collinear to the amino acid sequence of the fusion protein encoded by it was calculated, set forth as SEQ ID NO.:l or 2, with the target gene flanking by restriction sites, as well as with the addition of the Kozak sequence before the start codon to initiate translation, and after the start codon - in a part of embodiments - a signal sequence, for example, of TPA, hGH, IGF, EPO, or represented by aa MLLLLLLLLLLALALA, for secretion of the synthesized protein from an eukaryotic cell, with simultaneous optimization by codon composition for expression in human cells, the tool on the site molbiol.ru was used. For example, sequences set forth as SEQ ID NO.:3,4 and set forth as SEQ ID NO.:6, respectively were obtained.

[0105] In one embodiment, instead of artificial optimization, the corresponding nucleotide sequences of the new coronavirus were taken to obtain a genetic construct, since these sequences are expressed in mammals, the remaining fragments were optimized as described above. For example, the nucleotide sequence set forth as SEQ ID NO.:5 was obtained.

[0106] The calculated nucleotide sequences were synthesized by a chemical method using ASM-800 DNA synthesizer (BIOSSET, Russia).

[0107] 2.1. Obtaining plasmid DNA

[0108] 2.1.1. The creation of a producer strain of plasmid DNA

[0109] The synthesized genes were cloned in pVAXl (Invitrogen), pcDNA3.1+ (Invitrogen) eukaryotic expression vectors at restriction sites flanking the target genes, according to the vector manual. A pcDNA3.1+ vector, incapable of expressing neomycin, was also obtained by exposing this vector to Nsil restriction enzyme in the SV40 promoter region (-71 bp). The obtained fragments were also cloned into the resulting vector.

[0110] 3 mΐ of synthesized DNA solution, 1 mΐ of obtained vector solution, 5 mΐ of buffer for ligation x2 and 1 mΐ of T4 ligase were taken for the ligation reaction. The reaction was carried out at +20° C for 2 hours.

[0111] After this, the mixture was heated at +95°C for 10 min and purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 mpi (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.

[0112] Then E. coli cells of strain DH10B / R (F-mcrA, D (mrr-hsdRMS-mcrBC), cp80dlacZAM 15, AlacX74, deoR, recAl, endAl, araD139, D (ara, leu) 769, galU, galKX-, rpsL, nupG) were transformed with the obtained plasmid DNA by electroporation using a MicroPulser electroporator (BioRad). This strain does not contain methylase, which allows to minimize the possibility of mutations occurrence in the DNA, including the gene cloned in the plasmid maintained in this strain. 1 mΐ of dialyzed ligase mixture was added to 12 mΐ of competent cells, placed between the electrodes of an electroporation cuvette and processed by a current pulse.

[0113] After transformation, the cells were placed in 1 ml of SOC broth (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgC12, 10 mM MgS04, 20 mM glucose) and incubated for 40 min at + 37°C.

[0114] Clones of E. coli cells containing the obtained plasmid DNA were detected on a selective medium containing LB agar, 50 pg/ml of kanamycin, or ampicillin (for plasmid DNA based on pcDNA3.1+).

[0115] Plasmid DNA was isolated from the grown clones. Plasmid DNA was isolated using the Wizard Minipreps DNA Purification System kit (Promega, USA). Purified recombinant plasmid DNA was checked by sequencing.

[0116] Sequencing of the cloned fragments was performed according to the Sanger method using the Applied Biosystems BigDye® Terminator (BDT) v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the instructions attached to it. To label the reaction products, ddNTP labeled with a fluorescent dye were used, with each ddNTP corresponding to its own dye. For sequencing, unlabeled plasmid specific primers were used. A PCR reaction was performed, then the reaction mixture was purified from free labeled ddNTP according to the instructions for the BigDye X-Terminator Purification Kit (Applied Biosystems, USA) and the products of the sequencing reaction were separated using an Applied Biosystems 3500 / 3500xL Genetic Analyzer capillary sequencer (Applied Biosystems, USA) and 3500/3500xL Genetic Analyzer Polymer “POP-6 ™” reagent (Applied Biosystems, USA).

[0117] The results of the products separation of the sequencing reaction were recorded by laser scanning and detection of four fluorescent dyes included in all types of ddNTP.

[0118] Computer analysis of DNA sequences was performed on PC using the Chromas and BioEdit programs. The nucleotide sequences of the studied DNA fragments were aligned with the calculated ones, the identity of the synthesized fragments with the calculated ones was demonstrated. As a result, E. coli cell clones were selected containing the full-length sequences of the target genes in the plasmids - DNA sequences encoding the developed fusion proteins. Such clones were used as the producer strain of the plasmids according to the present invention. In one embodiment, this is Escherichia coli DH10B/R bacterial strain, containing the pcDNA3.1(+) vector, which contains the nucleotide sequence of SEQ ID NO.:3-6.

[0119] Cells of the bacterium Bacillus subtilis have also been successfully used as a producer of the genetic construct.

[0120] 2.1.2. The production of plasmid DNA encoding a fusion protein

[0121] A separate colony of E. coli producer cells grown on LB agar in a Petri plate with the addition of kanamycin or ampicillin, depending on the contained plasmid DNA, was placed in 10 ml of selective medium. Cells were grown for 12 hours at + 37 °C under constant stirring (250 rpm). The resulting cells were harvested by centrifugation at 4000g. Further isolation and purification of plasmid DNA was carried out using the EndoFree Plasmid Mega Kit (Qiagen), allowing to obtain pyrogen-free DNA. The isolated plasmid DNA was analyzed by electrophoresis in 0.8% agarose gel, and its concentration was measured using fluorimetry. Other methods for plasmid purification, in particular, chromatography, were also used.

[0122] The values determined in the experiment corresponded to the values of the ratios A260/A280 and A260/A230 for pure preparations, for all obtained preparations of plasmid DNA.

[0123] Protein impurities in the resulting plasmid DNA preparations were also quantified using the microBCA assay [Smith, P.K., et all, Measurement of protein using bicin- choninic acid. Analyt. Biochem. 150, 76-85 (1985)], measuring the optical density of the resulting colored protein complexes with copper and bicinchoninic acid with a wavelength of 562 nm. The sensitivity of the microBCA assay method is 0.5-20 mg/ml of protein. The concentration of total protein in none of the studied plasmid DNA preparations exceeded the norm.

[0124] The content of bacterial lipopoly saccharide in plasmid DNA preparations was also determined using a gel-thrombus version of the LAL test with a sensitivity of >0.25 EU/ml (ToxinSensor, GenScript, USA). A Limulus polyphemus horseshoe crab amebocytes lysate was used as the LAL reagent. The LAL reagent specifically reacts with bacterial endotoxins; as a result of the enzymatic reaction, the reaction mixture changes in proportion to the concentration of endotoxin. The results were evaluated by the presence or absence of a dense clot at the bottom of the tube by inverting the tube.

A gel clot did not form when examining a sample diluted 10 times, for all obtained plasmid DNA preparations, i.e. when the sensitivity of the method is 2.5 EU/ml, which, given the concentration of plasmid DNA in the sample, indicates an acceptable rate of endotoxin removal.

[0125] The yield of plasmid DNA ranged from 3.1 mg to 4.7 mg per liter of culture medium.

The process took about 4 days.

[0126] 2.2. Obtaining a vector based on adeno-associated virus encoding a fusion protein

[0127] The synthesized genes were cloned into a vector based on the pAAVK-EFla-MCS (System Biosciences (SBI)) adeno-associated virus, on the basis of which a producer strain of this vector was created using E. coli cells (RecA-). Bacillus subtilis bacteria cells were also successfully used as producer.

[0128] A vector, further, was isolated for use in mammals, all according to the instructions for the vector. The yield of the vector ranged from 2 mg to 3.2 mg per liter of culture medium.

[0129] 2.3. Obtaining a short linear construct encoding a fusion protein of the invention

[0130] To produce a short linear construct, the plasmid DNA obtained according to 2.1.2., or the viral vector of item 2.2., or a fragment amplified from them was used. Using specific primers and PCR, and the DNA specified in the previous sentence as a matrix, the DNA fragment was amplified containing a promoter, an mRNA leader sequence, and also regulatory sequences for these elements, a polynucleotide - a fusion gene, a termination sequence. After amplification the solution may contain also other elements, and the elements indicated in the previous sentence are the key ones.

[0131] Amplification of this sequence was carried out in a volume of 50 mΐ, in 650 mΐ thin- walled polypropylene tubes containing 5 mΐ of lOx Taq buffer (700 mM Tris-HCl, pH 8.6/25°C, 166 mM (NH4) 2S04), 5 mΐ of MgC12 (1.25 mM), 1 mΐ of dNTP, 31.5 mΐ of water, 1 mΐ of forward and 1 mΐ of reverse primers, 5 mΐ of plasmid DNA and 0.5 mΐ Taq polymerase (Fermentas, Lithuania).

[0132] The reaction mixture was warmed for 5 minutes at 95°C for DNA denaturation. To prevent evaporation, 30 mΐ of Bayol F mineral oil (Sigma, USA) were layered onto a reaction mixture of 50 mΐ volume. The amplification reaction was carried out in a C1000 Thermal Cycler (Bio-Rad, USA) thermal cycler. 35 cycles were carried out: 95°C - 20 sec, 50-62°C (depending on the primers) - 20 sec, 72°C - 1 min. To complete the formed DNA chains, an additional cycle was carried out: 5 min at 72°C.

[0133] The result of PCR was analyzed by electrophoresis in agarose gel. Upon a positive result, preparative electrophoresis was performed.

[0134] Amplified DNA fragments were concentrated and purified using preparative electrophoresis in 0.8-1.2% agarose gel (Gibko BRL, USA). A sample of the mixture after PCR was mixed with 6x buffer (0.25% bromophenol blue, 30% glycerin) (ThermoScientific, USA) and loaded into the gel wells, 18 mΐ per well. Electrophoresis was carried out in a horizontal apparatus in TAE buffer (40 mM Tris-acetate, 2 mM EDTA pH 8.0, 0.5 mg/ml ethidium bromide) at a voltage of 5-10 V/cm. The result of DNA separation was recorded in transmitted UV light (302 nm) of the Macrovue transilluminator (LKB, Sweden). The length of the amplified fragment was determined by the logarithmic dependence of DNA mobility on the length of the fragments in the marker. As markers, a proprietary mixture of DNA fragments “GeneRuler 1000 bp DNA Ladder” (Fermentas, Lithuania) was used. A plot of agarose containing DNA strip of the required size was excised and the DNA fragment was purified using the DNA & Gel Band Purification Kit (GE Healthcare, UK) according to the instructions.

[0135] Modem methods of DNA purification, in particular using silicon dioxide, make it possible to get rid of all impurities and to obtain DNA suitable for use in animals and a human. It is also possible to purify the amplified DNA without the use of preparative electrophoresis, using other methods, for example, with chromatography, on a column. Thus, it is possible to obtain a preparation ready for use in 2-3 hours.

[0136] Other genetic constructs according to the invention were also obtained, including those containing other components, in addition to the above key ones, as well as other variants of the polynucleotide according to the invention than set forth as SEQ ID NO.:3-6, polynucleotide according to the invention is expressed from such constructs in mammalian cells.

[0137] The isolated genetic construct was used in mammals.

[0138] Example 3. Demonstration of efficacy of the developed vaccine

[0139] The experiment was conducted on white mice of inbred lines (“Rappolovo”

Laboratory animal kennels). Animals weighing 19-22 g were kept under standard conditions, at an ambient temperature of +27+2°C with 55% constant humidity, with a 12-hour daylight. They received dry standardized food and water without restriction.

[0140] The animals were injected intramuscularly with pcDNA3.1 (+)seqidno5 plasmid DNA in the amount of 50 mg in PBS, animals not injected with any substance were used as a negative control. Animals were sacrificed on days 2, 5, and 7, blood was taken to prepare the serum. Some animals of all groups were not withdrawn from the experiment to assess the safety of the vaccine.

[0141] The obtained serums were analyzed by electrophoresis in PAGE followed by transfer of proteins to the nitrocellulose membrane and visualization of the target protein using chemiluminescence.

[0142] After electrophoresis in PAGE, proteins were transferred to the membrane. The following solutions were used: Solution I - 10 ml of ethanol, 17 ml Tris-HCl 1M pH10.4, up to 50 ml dH20, Solution II - 10 ml ethanol, 1.25 ml Tris-HCl 1M pH10.4, up to 50 ml dH20, Solution PI - 10 ml of ethanol, 1.25 ml of Tris-HCl 1M pH9.4, up to 50 ml dH20. A ‘sandwich’ was assembled on a Semi-phor TE70 Semi-dry transfer unit apparatus for horizontal transfer: 6 Whatman papers fragments soaked in Solution I, 3 Whatman papers fragments soaked in Solution II, BioRad nitrocellulose membrane soaked in Solution P, PAAG after electrophoresis, which was located on the membrane and immobilized, 9 fragments of Whatman paper soaked in Solution PI.

The lid of the device was closed, the power was connected, 100 V for an hour,

Constant current PS unit model PS50, Hoefler Scentific Instruments (HSI) was the current source.

[0143] Further, the complex immobilized on the membrane was formed: a fusion protein — a specific antibody — a secondary antibody — horseradish peroxidase.

[0144] For this, the membrane was treated with 1% dry skim milk in phosphate-buffered saline with the addition of 0.5% Tween-20 (125 mΐ of a 20% aqueous solution in 50 ml of milk), incubation was carried out for 15 minutes at room temperature. Then the mixture was poured, antibodies were added to the coronavirus proteins - Anti- SARS-CoV-2 spike glycoprotein monoclonal antibody (CABT-RM321) rabbit antibodies to S protein of the coronavirus in 1% skimmed milk at a dilution of 1: 3000, then incubated for 16 hours at +4°C, after which it was heated to room temperature. After that, 3 washes were performed with 1% skimmed milk powder with the addition of 0.5% Tween-20, each for 10 minutes. After the last wash, secondary antibodies conjugated with horseradish peroxidase, - Goat Anti-Rabbit IgG H&L (HRP) (Abeam), - were added in 1% skimmed milk powder, diluted 1: 6000, then incubated for 1 h, after which they were washed 3 times (each for 10 minutes) with phosphate-buffered saline.

[0145] The formed membrane-immobilized complexes were developed using the Amersham ECL Western Blotting Detection Reagent (GE Healthcare) chemiluminescence kit. A mixture of equal volumes of 1 and 2 reagents from this kit was applied to a wet membrane with immobilized complexes of a fusion protein with specific antibody with a secondary antibody with horseradish peroxidase, all was packed between layers of a transparent plastic paper folder (slide). This construct was placed in an X-ray cassette (Kodak). Amersham Hyperfilm ECL X-ray film fragment was glued on top and fixed. The cassette was closed and kept for 12 hours. The film was developed using commercial solutions of developer and fixer (Krok-rentgen) according to the instruction and dried. These actions were carried out in the dark in the light of a red lamp.

[0146] Then, under normal lighting, a picture with the best quality was selected and scanned in parallel with the original membrane, on which molecular weight markers and track markings are located. The resulting images were combined using PhotoFiltre 7.

[0147] A similar experiment was performed using antibodies to the coronavirus N protein - Rabbit anti-SARS-CoV-2 NP monoclonal antibody, clone 120 (CABT-RM320).

[0148] In both experiments, it was demonstrated that the target gene encoded in pcDNA3.1(+)seqidno5 plasmid DNA is expressed, the maximum expression level being observed on the fifth day after the plasmid DNA injection, and the protein is present after a week, and this protein can bind to antibodies to coronavirus S, N proteins.

[0149] Similar results were also demonstrated using other declared genetic constructs, including a linear construct, a viral vector, and using other polynucleotides of the present invention, including with and without a fragment encoding a secretory sequence, and in both variants of the fusion protein , and when using more than one genetic construct according to the present invention, and with the standard introduction in the body or using electroporation. When genetic constructs were introduced into the body by electroporation, a higher level of polynucleotide expression and a stronger immune response were demonstrated.

[0150] Thus, the ability of the developed genetic construct to express the encoded target gene after introduction into the muscles, as well as the functioning of the domains of the developed fusion protein, in particular, represented by the new coronavirus S and N proteins, were evaluated. The synthesis of the fusion protein was demonstrated already on the second day after the introduction of the developed genetic construct carrying the gene encoding it, and within a week, with the maximum level of synthesis being detected 4-5 days after immunization. This suggests that the used genetic construct according to the invention allows the expression of the target gene in mammalian cells.

[0151] Since rabbit antibodies to the S and N proteins of the new coronavirus were used in this analysis, and since it was shown that they bind to the fusion protein of the invention synthesized in mouse cells, it is possible to speak about the full functioning of the S and N domains in the composition of the fusion protein, as well as of the fusion protein synthesized from the developed genetic construct, due to the production in a form recognizable specifically by the immune system. Accordingly, it may be said, that both availability of the protein synthesized from the developed plasmid DNA to be recognized by the immune system and the formation of an adequate immune response have been demonstrated.

[0152] To induce a long-lasting response related to the reaction of germination centers, it is advisable for a human to make the interval between primary and secondary immunizations for at least 1-2 months, optimally for at least 4-6 months, for appropriate maturation of B-cell affinity. By shortening the interval, the booster response may be smaller.

[0153] Other studies including using different amounts of the genetic construct have also been conducted demonstrating the effectiveness of the developed vaccine, all the variants, for the prevention and treatment of the new coronavirus.

[0154] The safety of the proposed vaccine was also demonstrated: animals of the corresponding groups survived and were forcibly removed from the experiment, no side effects were observed during the tests.

Sequence Listing Free Text

[0155] <110> Dukhovlinov I.V.

[0156] <120> Gen. constr. -based vac. for the prev. or tr-t of coronav. inf-on

[0157] <160> 6

[0158]

[0159] <210> SEQ ID NO: 1

[0160] <211> 422

[0161] <212> PRT [0162] <213> Artificial sequence

[0163] <220>

[0164] <221> domain

[0165] <222> from 1 to 121 amino acid residue

[0166] <223> M domain, from 60 to 180 amino acid residues of M protein of the new coronavirus [0167] <220>

[0168] <221> domain

[0169] <222> from 128 to 202 amino acid residue

[0170] <223> S domain, from 306 to 380 amino acid residues of S protein of the new coronavirus [0171] <220>

[0172] <221> domain

[0173] <222> from 208 to 352 amino acid residue

[0174] <223> N domain, from 216 to 360 amino acid residues of N protein of the new coronavirus [0175] <220>

[0176] <221> domain

[0177] <222> from 358 to 422 amino acid residue

[0178] <223> E domain, from 6 to 70 amino acid residues of E protein of the new coronavirus [0179] <400> 1

[0180]

[0181] Val Thr Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg lie Asn Trp [0182] 1 5 10 15

[0183] He Thr Gly Gly He Ala He Ala Met Ala Cys Leu Val Gly Leu Met [0184] 202530

[0185] Trp Leu Ser Tyr Phe He Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg [0186] 354045

[0187] Ser Met Trp Ser Phe Asn Pro Glu Thr Asn He Leu Leu Asn Val Pro [0188] 5055 60

[0189] Leu His Gly Thr He Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val

[0190] 657075 80

[0191] He Gly Ala Val lie Leu Arg Gly His Leu Arg He Ala Gly His His [0192] 859095

[0193] Leu Gly Arg Cys Asp He Lys Asp Leu Pro Lys Glu He Thr Val Ala [0194] 100 105 110

[0195] Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Gly Gly Gly Gly Gly Gly Phe [0196] 115 120 125

[0197] Thr Val Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Val Gin Pro [0198] 130 135 140

[0199] Thr Glu Ser He Val Arg Phe Pro Asn He Thr Asn Leu Cys Pro Phe [0200] 145 150 155 160

[0201] Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn [0202] 165 170 175

[0203] Arg Lys Arg He Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn [0204] 180 185 190

[0205] Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Gly Gly Gly Gly Asp [0206] 195200205

[0207] Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gin Leu Glu Ser [0208] 210215 220

[0209] Lys Met Ser Gly Lys Gly Gin Gin Gin Gin Gly Gin Thr Val Thr Lys [0210] 225230235240

[0211] Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gin Lys Arg Thr Ala [0212] 245 250255

[0213] Thr Lys Ala Tyr Asn Val Thr Gin Ala Phe Gly Arg Arg Gly Pro Glu [0214] 260265 270

[0215] Gin Thr Gin Gly Asn Phe Gly Asp Gin Glu Leu lie Arg Gin Gly Tin^¬ gle] 275280285

[0217] Asp Tyr Lys His Trp Pro Gin He Ala Gin Phe Ala Pro Ser Ala Ser [0218] 290295 300

[0219] Ala Phe Phe Gly Met Ser Arg He Gly Met Glu Val Thr Pro Ser Gly [0220] 305310315320

[0221] Thr Trp Leu Thr Tyr Thr Gly Ala He Lys Leu Asp Asp Lys Asp Pro

[0222] 325330335

[0223] Asn Phe Lys Asp Gin Val He Leu Leu Asn Lys His He Asp Ala Tyr [0224] 340345 350

[0225] Gly Gly Gly Gly Gly Ser Glu Glu Thr Gly Thr Leu He Val Asn Ser [0226] 355 60365

[0227] Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala [0228] 370375 380

[0229] He Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn He Val Asn [0230] 385 390395400

[0231] Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

[0232] 405410415

[0233] Leu Asn Ser Ser Arg Val [0234] 420

[0235]

[0236] <210> SEQ ID NO: 2

[0237] <211> 424

[0238] <212> PRT

[0239] <213> Artificial sequence

[0240] <220>

[0241] <221> domain

[0242] <222> from 1 to 121 amino acid residue

[0243] <223> M domain, from 60 to 180 amino acid residue of M protein of the new coronavirus [0244] <220>

[0245] <221> domain

[0246] <222> from 128 to 202 amino acid residue

[0247] <223> S domain, from 306 to 380 amino acid residues of S protein of the new coronavirus [0248] <220>

[0249] <221> domain

[0250] <222> from 209 to 353 amino acid residue

[0251] <223> N domain, from 216 to 360 amino acid residue of N protein of the new coronavirus [0252] <220>

[0253] <221> domain

[0254] <222> from 360 to 424 amino acid residue

[0255] <223> E domain, from 6 to 70 amino acid residue of E protein of the new coronavirus [0256] <400> 2

[0257]

[0258] Val Thr Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg lie Asn Trp [0259] 1 5 10 15

[0260] He Thr Gly Gly lie Ala He Ala Met Ala Cys Leu Val Gly Leu Met [0261] 2025 30

[0262] Trp Leu Ser Tyr Phe He Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg

[0263] 354045

[0264] Ser Met Trp Ser Phe Asn Pro Glu Thr Asn He Leu Leu Asn Val Pro [0265] 5055 60

[0266] Leu His Gly Thr He Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val [0267] 657075 80 [0268] He Gly Ala Val lie Leu Arg Gly His Leu Arg lie Ala Gly His His [0269] 859095

[0270] Leu Gly Arg Cys Asp lie Lys Asp Leu Pro Lys Glu He Thr Val Ala [0271] 100 105 110

[0272] Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Gly Gly Gly Gly Gly Gly Phe [0273] 115 120 125

[0274] Thr Val Glu Lys Gly He Tyr Gin Thr Ser Asn Phe Arg Val Gin Pro [0275] 130 135 140

[0276] Thr Glu Ser He Val Arg Phe Pro Asn He Thr Asn Leu Cys Pro Phe [0277] 145 150 155 160

[0278] Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn [0279] 165 170 175

[0280] Arg Lys Arg He Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn [0281] 180 185 190

[0282] Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Gly Gly Gly Gly Gly [0283] 195 200205

[0284] Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gin Leu Glu [0285] 210215 220

[0286] Ser Lys Met Ser Gly Lys Gly Gin Gin Gin Gin Gly Gin Thr Val Thr [0287] 225230235240

[0288] Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gin Lys Arg Thr [0289] 245250255

[0290] Ala Thr Lys Ala Tyr Asn Val Thr Gin Ala Phe Gly Arg Arg Gly Pro [0291] 260265 270

[0292] Glu Gin Thr Gin Gly Asn Phe Gly Asp Gin Glu Leu He Arg Gin Gly [0293] 275280285

[0294] Thr Asp Tyr Lys His Trp Pro Gin He Ala Gin Phe Ala Pro Ser Ala [0295] 290295 300

[0296] Ser Ala Phe Phe Gly Met Ser Arg He Gly Met Glu Val Thr Pro Ser [0297] 305 310315 320

[0298] Gly Thr Trp Leu Thr Tyr Thr Gly Ala He Lys Leu Asp Asp Lys Asp [0299] 325 330335

[0300] Pro Asn Phe Lys Asp Gin Val He Leu Leu Asn Lys His He Asp Ala [0301] 340345 350

[0302] Tyr Gly Gly Gly Gly Gly Gly Ser Glu Glu Thr Gly Thr Leu He Val [0303] 355360365

[0304] Asn Ser Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr [0305] 370375 380 [0306] Leu Ala He Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn He [0307] 385 390395400

[0308] Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val

[0309] 405410415

[0310] Lys Asn Leu Asn Ser Ser Arg Val

[0311] 420

[0312] <210> SEQ ID NO: 3

[0313] <211> 1281

[0314] <212> DNA

[0315] <213> Artificial sequence

[0316] <220>

[0317] <223> polynucleotide codon-optimized for mammals encoding a fusion protein of

422 aa

[0318] <400> 3

[0319]

[0320] gccgccacca tggtgaccct ggcctgcttc gtgctggccg ccgtgtaccg gatcaactgg 60

[0321] atcaccggcg gcatcgccat cgccatggcc tgcctggtgg gcctgatgtg gctgtcctac 120

[0322] ttcatcgcct ccttccggct gttcgcccgg acccggtcca tgtggtcctt caaccccgag 180 [0323] accaacatcc tgctgaacgt gcccctgcac ggcaccatcc tgacccggcc cctgctggag 240 [0324] tccgagctgg tgateggcgc cgtgatectg cggggccacc tgcggatcgc cggccaccac 300 [0325] ctgggccggt gcgacatcaa ggacctgccc aaggagatca ccgtggccac ctcccggacc 360 [0326] ctgtcctact acaagggcgg cggcggcggc ggcttcaccg tggagaaggg catctaccag 420 [0327] acctccaact tccgggtgca gcccaccgag tecatcgtgc ggttccccaa catcaccaac 480

[0328] ctgtgcccct tcggcgaggt gttcaacgcc acccggttcg cctccgtgta cgcctggaac 540

[0329] cggaagcgga tctccaactg cgtggccgac tactccgtgc tgtacaactc cgcctccttc 600

[0330] tccaccttca agtgctacgg cggcggcggc ggcgacgccg ccctggccct gctgctgctg 660 [0331 ] gaccggctga accagctgga gtccaagatg tccggcaagg gccagcagca gcagggccag 720

[0332] accgtgacca agaagtccgc cgccgaggcc tccaagaagc cccggcagaa gcggaccgcc 780

[0333] accaaggcct acaacgtgac ccaggccttc ggccggcggg gccccgagca gacccagggc 840

[0334] aacttcggcg accaggagct gatccggcag ggcaccgact acaagcactg gccccagatc 900

[0335] gcccagttcg ccccctccgc ctccgccttc ttcggcatgt cccggatcgg catggaggtg 960 [0336] accccctccg gcacctggct gacctacacc ggcgccatca agctggacga caaggacccc 1020 [0337] aacttcaagg accaggtgat cctgctgaac aagcacatcg acgcctacgg cggcggcggc 1080 [0338] ggctccgagg agaccggcac cctgatcgtg aactccgtgc tgctgttcct ggccttcgtg 1140 [0339] gtgttcctgc tggtgaccct ggccatcctg accgccctgc ggctgtgcgc ctactgctgc 1200

[0340] aacatcgtga acgtgtccct ggtgaagc cc tccttctacg tgtactcccg ggtgaagaac 1260

[0341 ] ctgaactcct cccgggtgtg a 1281

[0342] [0343] <210> SEQ ID NO: 4

[0344] <211> 1287

[0345] <212> DNA

[0346] <213> Artificial sequence

[0347] <220>

[0348] <223> polynucleotide codon-optimized for mammals encoding a fusion protein of

424 aa

[0349] <400> 4

[0350]

[0351] gccgccacca tggtgaccct ggcctgcttc gtgctggccg ccgtgtaccg gatcaactgg 60

[0352] atcaccggcg gcatcgccat cgccatggcc tgcctggtgg gcctgatgtg gctgtcctac 120

[0353] ttcatcgcct ccttccggct gttcgcccgg acccggtcca tgtggtcctt caaccccgag 180 [0354] accaacatcc tgctgaacgt gcccctgcac ggcaccatcc tgacccggcc cctgctggag 240 [0355] tccgagctgg tgatcggcgc cgtgatcctg cggggccacc tgcggatcgc cggccaccac 300 [0356] ctgggccggt gcgacatcaa ggacctgccc aaggagatca ccgtggccac ctcccggacc 360 [0357] ctgtcctact acaagggcgg cggcggcggc ggcttcaccg tggagaaggg catctaccag 420 [0358] acctccaact tccgggtgca gcccaccgag tecatcgtgc ggttccccaa catcaccaac 480

[0359] ctgtgcccct tcggcgaggt gttcaacgcc acccggttcg cctccgtgta cgcctggaac 540

[0360] cggaagcgga tctccaactg cgtggccgac tactccgtgc tgtacaactc cgcctccttc 600

[0361] tccaccttca agtgctacgg cggcggcggc ggcggcgacg ccgccctggc cctgctgctg 660 [0362] ctggaccggc tgaaccagct ggagtccaag atgtccggca agggccagca gcagcagggc 720 [0363] cagaccgtga ccaagaagtc cgccgccgag gcctccaaga agccccggca gaagcggacc 780

[0364] gccaccaagg cctacaacgt gacccaggcc ttcggccggc ggggccccga gcagacccag 840

[0365] ggcaacttcg gcgaccagga gctgatccgg cagggcaccg actacaagca ctggccccag 900 [0366] atcgcccagt tcgccccctc cgcctccgcc ttcttcggca tgtcccggat cggcatggag 960 [0367] gtgaccccct ccggcacctg gctgacctac accggcgcca tcaagctgga cgacaaggac 1020 [0368] cccaacttca aggaccaggt gatcctgctg aacaagcaca tcgacgccta cggcggcggc 1080 [0369] ggcggcggct ccgaggagac cggcaccctg atcgtgaact ccgtgctgct gttcctggcc 1140 [0370] ttcgtggtgt tcctgctggt gaccctggcc atcctgaccg ccctgcggct gtgcgcctac 1200 [0371] tgctgcaaca tcgtgaacgt gtccctggtg aagccctcct tctacgtgta ctcccgggtg 1260 [0372] aagaacctga actcctcccg ggtgtga 1287 [0373] <210> SEQ ID NO: 5

[0374] <211> 1350

[0375] <212> DNA

[0376] <213> Artificial sequence

[0377] <220>

[0378] <223> polynucleotide based on viral seq-s encoding a fusion protein of 424 aa with the addition of IGF secretory seq. [0379] <400> 5

[0380]

[0381] gccgccacca tgggcaagat cagcagcctg cccacccagc tgttcaagtg ctgcttctgc 60 [0382] gacttcctga aggtaacttt agcttgtttt gtgcttgctg ctgtttacag aataaattgg 120 [0383] atcaccggtg gaattgctat cgcaatggct tgtcttgtag gctgatgtg gctcagctac 180 [0384] ttcattgctt ctttcagact gtttgcgcgt acgcgttcca tgtggtcat caatccagaa 240 [0385] actaacattc ttctcaacgt gccactccat ggcactattc tgaccagacc gcttctagaa 300 [0386] agtgaactcg taatcggagc tgtgatcctt cgtggacatc ttcgtattgc tggacaccat 360 [0387] ctaggacgct gtgacatcaa ggacctgcct aaagaaatca ctgttgctac atcacgaacg 420 [0388] ctttcttatt acaaaggagg aggaggagga ggattcactg tagaaaaagg aatctatcaa 480 [0389] acttctaact ttagagtcca accaacagaa tctattgtta gatttcctaa tattacaaac 540 [0390] ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta tgcttggaac 600 [0391] aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc cgcatcattt 660 [0392] tccactttta agtgttatgg aggaggagga ggaggagatg ctgctettgc tttgctgctg 720 [0393] cttgacagat tgaaccagct tgagagcaaa atgtctggta aaggccaaca acaacaaggc 780 [0394] caaactgtca ctaagaaatc tgctgctgag gcttctaaga agcctcggca aaaacgtact 840 [0395] gccactaaag catacaatgt aacacaagct ttcggcagac gtggtccaga acaaacccaa 900 [0396] ggaaattttg gggaccagga actaatcaga caaggaactg attacaaaca ttggccgcaa 960 [0397] attgcacaat ttgcccccag cgcttcagcg ttcttcggaa tgtcgcgcat tggcatggaa 1020 [0398] gtcacacctt cgggaacgtg gttgacctac acaggtgcca tcaaattgga tgacaaagat 1080 [0399] ccaaattca aagatcaagt cattttgctg aataagcata ttgacgcata cggaggagga 1140 [0400] ggaggaggat cggaagagac aggtacgtta atagttaata gcgtacttct ttttcttgct 1200 [0401] ttcgtggtat tcttgctagt tacactagcc atccttactg cgctcgatt gtgtgcgtac 1260 [0402] tgctgcaata tgtaacgt gagtcttgta aaaccttctt tttacgttta ctctcgtgtt 1320 [0403] aaaaatctga attcttctag agtttaatga 1350 [0404]

[0405] <210> SEQ ID NO: 6

[0406] <211> 1356

[0407] <212> DNA

[0408] <213> Artificial sequence

[0409] <220>

[0410] <223> polynucleotide codon-optimized for mammals encoding a fusion protein of

422 aa with the addition of HGH secretory seq.

[0411] <400> 6

[0412]

[0413] gccgccacca tggccaccgg ctcccggacc tccctgctgc tggccttcgg cctgctgtgc 60 [0414] ctgccctggc tgcaggaggg ctccgccgtg accctggcct gcttegtgct ggccgccgtg 120 [0415] taccggatca actggatcac cggcggcatc gccatcgcca tggcctgcct ggtgggcctg 180 [0416] atgtggctgt cctacttcat cgcctccttc cggctgttcg cccggacccg gtccatgtgg 240 [0417] tccttcaacc ccgagaccaa catcctgctg aacgtgcccc tgcacggcac catcctgacc 300 [0418] cggcccctgc tggagtccga gctggtgatc ggcgccgtga tcctgcgggg ccacctgcgg 360 [0419] atcgccggcc accacctggg ccggtgcgac atcaaggacc tgcccaagga gatcaccgtg 420 [0420] gccacctccc ggaccctgtc ctactacaag ggcggcggcg gcggcggctt caccgtggag 480 [0421] aagggcatct accagacctc caacttccgg gtgcagccca ccgagtccat cgtgcggttc 540

[0422] cccaacatca ccaacctgtg ccccttcggc gaggtgttca acgccacccg gttcgcctcc 600

[0423] gtgtacgcct ggaaccggaa gcggatctcc aactgcgtgg ccgactactc cgtgctgtac 660 [0424] aactccgcct ccttctccac cttcaagtgc tacggcggcg gcggcggcga cgccgccctg 720 [0425] gccctgctgc tgctggaccg gctgaaccag ctggagtcca agatgtccgg caagggccag 780 [0426] cagcagcagg gccagaccgt gaccaagaag tccgccgccg aggcctccaa gaagccccgg 840 [0427] cagaagcgga ccgccaccaa ggcctacaac gtgacccagg ccttcggccg gcggggcccc 900

[0428] gagcagaccc agggcaactt cggcgaccag gagctgatcc ggcagggcac cgactacaag 960

[0429] cactggcccc agatcgccca gttcgccccc tccgcctccg ccttcttcgg catgtcccgg 1020 [0430] atcggcatgg aggtgacccc ctccggcacc tggctgacct acaccggcgc catcaagctg 1080 [0431] gacgacaagg accccaactt caaggaccag gtgatcctgc tgaacaagca catcgacgcc 1140 [0432] tacggcggcg gcggcggctc cgaggagacc ggcaccctga tcgtgaactc cgtgctgctg 1200 [0433] ttcctggcct tcgtggtgtt cctgctggtg accctggcca tcctgaccgc cctgcggctg 1260 [0434] tgcgcctact gctgcaacat cgtgaacgtg tecctggtga agccctcctt ctacgtgtac 1320 [0435] tcccgggtga agaacctgaa ctcctcccgg gtgtga 1356

Claims

Claims

[Claim 1] A polynucleotide for expression in the cells of a target organism, encoding a fusion protein set forth as SEQ ID NO.:l or SEQ ID NO.:2 amino acid sequence comprising fragments of coronavirus M, S, N, E proteins connected by flexible bridges.

[Claim 2] The polynucleotide of claim 1, set forth as SEQ ID NO.:3 or SEQ ID NO.:4 nucleotide sequence.

[Claim 3] The polynucleotide of claim 1, wherein it also contains a fragment encoding a heterologous secretory sequence.

[Claim 4] The polynucleotide of claim 3, wherein the heterologous secretory sequence is from that of TPA, EPO, hGH or IGF.

[Claim 5] The polynucleotide of claim 4, set forth as SEQ ID NO.:5 or SEQ ID NO.:6 nucleotide sequence.

[Claim 6] A genetic construct for the expression in the cells of a target organism of the polynucleotide of any one of claims 1 to 5, comprising the polynucleotide of any one of claims 1 to 5 and other elements allowing to realize the indicated aim.

[Claim 7] The genetic construct of claim 6, wherein it is a recombinant vector for transient expression in mammalian cells, comprising prokaryotic elements, an origin of replication and a marker gene, and eukaryotic elements, a strong promoter, mRNA leader sequence, and also regulatory sequences for these elements, at least one site for cloning of a gene of interest and at least one site for annealing of at least one primer for analysis of the recombinant vector composition, the polynu cleotide of any one of claims 1 to 5 codon-optimized for expression in mammalian cells, and a termination sequence.

[Claim 8] The genetic construct of claim 7, wherein it is a plasmid DNA of any one of pcDNA3.1(+), pcDNA3.1(+) without a fragment encoding the neomycin resistance gene, pVAXl, the polynucleotide of any one of the claims 1 to 5 being cloned in the plasmid DNA.

[Claim 9] The genetic construct of claim 8, wherein it is the pcDNA3.1(+) vector comprising insertion of the polynucleotide of claim 2 or 5.

[Claim 10] The genetic construct of claim 6, wherein it is a linear DNA fragment comprising a promoter, mRNA leader sequence, as well as regulatory sequences for these elements, the polynucleotide of any one of claims 1 to 5, a termination sequence.

[Claim 11] The genetic construct of claim 6 or 7, wherein it is a virus-based vector, the polynucleotide of any one of claims 1 to 5 being cloned in the vector.

[Claim 12] The genetic construct of claim 11, wherein it is an adeno-associated virus, in which the polynucleotide of any one of claims 1 to 5 is cloned.

[Claim 13] A prokaryotic recombinant cell for obtaining the genetic construct of any one of claims 6 to 12.

[Claim 14] The prokaryotic recombinant cell of claim 13, wherein it is Escherichia coli DH10B/R bacterial strain, containing pcDNA3.1(+) vector which comprises a nucleotide sequence of any one of SEQ ID NO.: 3 to 6.

[Claim 15] A vaccine for coronavirus infection prevention or treatment in a human or animal containing as an active agent at least one genetic construct of any one of claims 6 to 12, in an effective amount, and also a physiologically acceptable carrier and a buffer solution.

[Claim 16] A method of producing a vaccine for coronavirus infection prevention or treatment, wherein the genetic construct of claim 10 is amplified using PCR, purified and mixed with a physiologically acceptable carrier and a buffer solution.