RU2733831C1 - Artificial gene coding a bicistronic structure formed by receptor-binding domain sequences of the glycoprotein s of the sars-cov-2 coronavirus, p2a-peptide and glycoprotein g vsv, recombinant plasmid pstem-rvsv-stbl_rbd_sc2, providing expression of artificial gene, and a recombinant strain of vesicular stomatitis virus rvsv-stbl_rbd_sc2, used to create a vaccine against sars-cov-2 coronavirus - Google Patents

Abstract

FIELD: biotechnology; molecular biology; genetic engineering; medicine.

SUBSTANCE: described is an artificial gene used to create a vaccine against SARS-CoV-2 coronavirus, coding an artificial protein-immunogen, which is a bicistronic structure consisting of receptor-binding domain (RBD) sequences of SARS-CoV-2 coronavirus glycoprotein S, heterological signal peptide haemagglutinin (HA) of influenza A virus, a linker, P2A-peptide for splitting polyprotein during translation and glycoprotein G with mutation M(1)>P(1), preventing alternative translation initiation, presented in SEQ ID NO: 1, length of 2355 bp. Disclosed is a recombinant plasmid pStem-rVSV-Stbl_RBD_SC2, used to create a vaccine against SARS-CoV-2 coronavirus, having molecular weight 8.85⋅10⁶ Da, size 14309 bp and comprising, in accordance with physical and genetic map presented in Fig. 2, target gene according to item 1, coding artificial protein-immunogen RBD of SARS-CoV-2 coronavirus glycoprotein S, having amino acid sequence SEQ ID NO: 2 and under control of viral promoter providing its expression in mammalian cells. Strain rVSV-Stbl_RBD_SC2 of a recombinant virus of vesicular stomatitis obtained using a recombinant plasmid pStem-rVSV-Stbl_RBD_SC2, providing independent synthesis of coronavirus antigen (receptor-binding domain of SARS-CoV-2 glycoprotein S) and protein G of virus of vesicular stomatitis used to develop a vaccine against SARS-CoV-2 coronavirus and deposited in the State collection of viral infection causative agents, rickettsioses of State Research Center of Virology and Biotechnology VECTOR of Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, No. V-984.

EFFECT: improved folding and higher infectious titre of the recombinant virus, stability of the target transgene and immunogenic / antigenic properties of the declared recombinant virus.

3 cl, 5 dwg, 3 tbl, 5 ex

Description

The invention relates to an artificial gene encoding a bicistronic structure formed by the sequences of the receptor-binding domain (RBD) of the glycoprotein S of the coronavirus SARS-CoV-2, P2A peptide and the glycoprotein G of the vesicular stomatitis virus, a recombinant plasmid providing expression of the said artificial gene and a recombinant strain vesicular stomatitis, expressing the antigens of the SARS-CoV-2 coronavirus, inducing a specific immune response to SARS-CoV-2 and used to create a vaccine against the SARS-CoV-2 coronavirus and can be used in biotechnology, molecular biology, genetic engineering and medicine.

Known patent solution (US, 20190062785 A1, IPC A61K 39/215, A61K 39/205; C12N15 / 86, publ. 02/28/2019), which describes a vaccine based on a recombinant rabies virus, which provides protection against rabies and severe acute respiratory syndrome (SARS-CoV). The transcriptional cassette is located in the intergenic region N and P, which guarantees a high level of transgene expression due to the peculiarities of the organization of the rhabdovirus genome. The patent discloses the main existing approaches for the design of target coronavirus immunogens / antigens, such as the full-length S glycoprotein, the variant with the truncated cytoplasmic domain (Δ19), and the RBD receptor-binding domain with the transmembrane region and the cytoplasmic domain of the rabies virus.

However, the use of this vector is complicated by residual neurovirulence and possible side effects. At the same time, the use of functional glycoprotein S of coronaviruses as an immunogen can lead to the creation of a recombinant rabies virus, which has double tropism due to exposed viral glycoproteins. The disadvantage of this solution is also the use of antigens of the SARS-CoV virus, which probably will not allow the formation of antiviral immunity against the new SARS-CoV-2 coronavirus.

Known immunobiological agent for the prevention of diseases caused by the virus of severe respiratory syndrome SARS-CoV-2 based on recombinant human adenovirus 5th serotype or recombinant human adenovirus 26th serotype, containing optimized for expression in mammalian cells sequence of protective antigen S of SARS-CoV virus -2 with a deletion of 18 amino acids at the C'-end of the gene (RU, 2720614, IPC A61K39 / 215; A61P31 / 12, publ. 05/12/2020), or the sequence of the complete protective antigen S of the SARS-CoV-2 virus and the Fc sequence -fragment from human IgG1, or the sequence of the receptor-binding domain of the protein S of the SARS-CoV-2 virus with the sequence of the leader peptide of the virus, or the sequence of the receptor-binding domain of the protein S of the SARS-CoV-2 virus with the transmembrane domain of the glycoprotein of the vesicular stomatitis virus, or the sequence the receptor-binding domain of the SARS-CoV-2 protein S with the leader sequence a peptide and an Fc sequence from human IgG1, or a combination thereof.

However, it has been shown that the use of vectors based on agents circulating in the human population (adenoviruses, herpes viruses, respiratory syncytial virus, etc.) can be complicated by the presence of specific immunity against the "wild-type" virus, as a result of which there will not be sufficient productive infection cells with a recombinant virus, and as a result, the expression of the target transgene will not be efficiently provided.

The closest analogue (prototype) is the solution under the patent (CN, 111088283 A, IPC A61K 39/215; A61P 31/14; C12N 15/86; publ. 05/01/2020), where viral vectors are proposed based on an attenuated vesicular virus stomatitis, in which the chimeric gene of the heterologous antigen is localized between the G and L genes. The patent also discloses classical approaches to the design of target coronavirus immunogens / antigens (codon optimization, full-length S (Spike), RBD). The patent discloses an approach to obtain chimeric surface glycoproteins of the vesicular stomatitis virus in order to induce immunity to the coronavirus component.

The disadvantage of this solution is the design of immunogens in the form of N- and C-terminal fusion proteins, which can affect the folding of coronavirus antigenic components and the infectious titer of the recombinant virus. The proposed strict localization of the transgene in the genome of the virus (between genes G and L, in fact, the 5th transcriptional cassette, starting from the 3'-end of the genome) does not allow full control of the transcriptional level of the transgene. Also, the issue of stability of target transgenes in the composition of RNA-containing viruses remains unresolved, which may result in a decrease in immunogenic / antigenic properties and / or a change in the tropism and virulent properties of recombinant viruses.

Thus, in the prior art, there is an urgent need for the development of new recombinant vaccines against coronavirus infection COVID-19 (SARS-CoV-2).

Disclosure of invention

The aim of the claimed invention is to provide a recombinant vesicular stomatitis virus that provides expression of SARS-CoV-2 coronavirus antigens and induces a specific immune response to SARS-CoV-2.

The technical result is to improve folding and increase the infectious titer of the recombinant virus, the stability of the target transgene, and the immunogenic / antigenic properties of the claimed recombinant virus.

The specified technical result is achieved by creating an artificial gene used to create a vaccine against the SARS-CoV-2 coronavirus, which encodes an artificial immunogen protein, which is a bicistronic structure consisting of the receptor-binding domain (RBD) sequences of the S coronavirus SARS-CoV-2 glycoprotein, heterologous signal peptide hemagglutinin (HA) of influenza A virus, linker, P2A peptide for cleavage of polyprotein during translation, and glycoprotein G with mutation M (1)> P (1), preventing alternative translation initiation, shown in SEQ ID NO: 1 length 2355 bp

The specified technical result is also achieved by creating a recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 used to create a vaccine against the SARS-CoV-2 coronavirus, having a molecular weight of 8.85x10 ⁶ daltons, size 14309 bp. and containing, in accordance with the physical and genetic map shown in FIG. 2 the target gene according to claim 1, encoding an artificial immunogen RBD of the glycoprotein S of the coronavirus SARS-CoV-2, having the amino acid sequence of SEQ ID NO: 2 and under the control of a viral promoter that ensures its expression in mammalian cells and consists of the following fragments:

- ORI - (ORI, origin of replication) the origin of replication of the plasmid vector pUC (coordinates 13483-14071 bp);

- AmpR - β-lactamase gene providing resistance of E. coli transformants to the selective antibiotic ampicillin (coordinates 12452-13312 bp);

- T7 promoter - the nucleotide sequence of the T7 bacteriophage promoter required for transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 166-183 bp);

- T7 terminator - the nucleotide sequence of the T7 bacteriophage terminator, which is necessary for terminating the transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 12224-12330 bp);

- HDV-Rbz - the nucleotide sequence of the HDV ribozyme, which, after transcription, is cleaved with the formation of an authentic 3'-terminal non-coding sequence (coordinates 12137-12219 bp);

- N - open reading frame of the N gene (nucleoprotein) (coordinates 247-1515 bp);

- P - open reading frame of the P gene (phosphoprotein) (coordinates 1579-2376 bp);

- L - open reading frame of the L gene (RNA-dependent RNA polymerase) (coordinates 5708-12037 bp);

- VSV-G - open reading frame of glycoprotein G with mutation M (1)> P (1) (coordinates 3322-4032 bp);

- P2A - 2A-peptide of teshovirus-1, providing cleavage of the polyprotein at the translation stage (coordinates 4033-4089 bp);

- Signal peptide - hemagglutinin (HA) signal peptide of influenza A virus, which provides efficient intracellular transport of synthesized polyprotein (coordinates 3268-3321 bp);

- Spike protein - a fragment of the S (Spike) SARS-CoV-2 gene (coordinates 3322-4032 bp);

- Receptor-binding domain (RBD) - receptor-binding domain of glycoprotein S (Spike) SARS-CoV-2 (coordinates 3334-4002 bp).

The specified technical result is also achieved by creating a strain rVSV-Stbl_RBD_SC2 of the recombinant vesicular stomatitis virus, obtained using the recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 according to claim 2, providing an independent synthesis of the coronavirus antigen (receptor-binding domain S of SARS-CoV protein) G of the vesicular stomatitis virus used to create a vaccine against the SARS-CoV-2 coronavirus and deposited in the State Collection of pathogens of viral infections, rickettsioses of the FBSI SSC VB "Vector" of Rospotrebnadzor under No. V-984.

Significant differences from the prototype, ensuring the achievement of the technical result, are:

1. Optimization of an artificial gene by normalizing the codon composition and excluding elements from the sequence that have a negative effect on the level of expression and stability of mRNA;

2. The use of a heterologous signal peptide of hemagglutinin (HA) of the influenza A virus, which is necessary for efficient intracellular transport of the synthesized polyprotein;

3. Use of P2A-peptide to form a bicistronic structure for independent synthesis and folding of coronavirus antigen (receptor-binding domain of S SARS-CoV-2 glycoprotein S) and vesicular stomatitis virus glycoprotein G necessary for viral reproduction;

4. Introduction of the M (1)> P (1) mutation into the open reading frame of glycoprotein G as part of an artificial gene, which is necessary to prevent alternative translation initiation of the glycoprotein necessary for viral reproduction.

Implementation of the invention

The essence of the claimed invention is illustrated by drawings. FIG. 1 shows a scheme for cloning the nucleotide sequences of genes encoding constructive variants of glycoprotein S and RBD in a plasmid vector for reverse genetics of vesicular stomatitis virus pStem-rVSV_P2A_G. FIG. 2 shows a diagram of the construction of the recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 used to create a vaccine against the SARS-CoV-2 coronavirus.

FIG. 3 shows the nucleotide sequence (SEQ ID NO: 1) of an artificial gene encoding an artificial immunogen protein of the receptor binding domain (RBD) of the glycoprotein S of the SARS-CoV-2 coronavirus with a heterologous hemagglutinin (HA) signal sequence of the influenza A virus, a linker and P2A- a peptide for cleavage of a polyprotein during translation, a glycoprotein G with a mutation M (1)> P (1).

FIG. 4 shows the amino acid sequence of the secreted receptor-binding domain (RBD) of the S (Spike) glycoprotein SARS-CoV-2 with a heterologous signal sequence, a linker and a P2A peptide providing independent synthesis of coronavirus antigen and G protein of vesicular stomatitis virus (SEQ ID NO: 2 ).

FIG. 5 shows the immunohistochemistry of a monolayer of a Vero E6 cell culture: A - intact; B - SARS CoV-2 infected strain nCoV / Victoria / 1/2020.

Artificial gene design and recombinant plasmid production. The first step in creating a recombinant vesicular stomatitis virus to create a vaccine was the design of the target antigen / immunogen. According to the literature, the most promising viral components for the induction of protective immunity are surface transmembrane glycoprotein S (Spike), as well as nucleoprotein N.

The bioinformatic analysis of the published whole genome nucleotide sequences of the SARS-CoV-2 virus (reference sequence GenBank: NC_045512.2), as well as fragmentary nucleotide sequences encoding glycoprotein S, confirmed the relative stability of the amino acid sequences of the target antigens.

It is important to note that the native viral glycoprotein S is represented by a transmembrane trimmer, with retention signals in the ER / Golgi at the C-terminal fragment of the protein. Using the online tools SignalIP-5.0 and TMHMM, the ectodomain of glycoprotein S was localized, the receptor-binding domain (RBD) was isolated by homology with the glycoprotein S SARS-CoV, with flanking regions necessary for correct folding of the domain. According to the literature, immunization of animals with the recombinant RBD protein of related coronaviruses (SARS-CoV, MERS-CoV, etc.), or constructs that ensure its expression, leads to the induction of specific virus-neutralizing antibodies.

Therefore, the expression of the ectodomain of glycoprotein S, or the receptor binding domain (RBD) by means of the recombinant virus in the vaccine will induce the synthesis of virus-specific antibodies to SARS-CoV-2. The invention provides an optimized nucleotide sequence encoding the receptor binding domain (RBD) of the S (Spike) SARS-CoV-2 glycoprotein. Optimization of synthetic constructs encoding the full-length SARS-CoV glycoprotein S, as well as the receptor-binding domain (RBD) of glycoprotein S, was carried out by normalizing the codon composition and excluding from the sequences elements that negatively affect the level of expression or stability of mRNA. Nucleotide sequences of artificial genes were chemically synthesized and used to obtain recombinant constructs for reverse genetics of the replication-competent vesicular stomatitis virus.

Vesicular stomatitis virus has been used as a vector because of its safety, wide cellular tropism, and the lack of preexisting immunity in most people, which is a prerequisite for effective immunization. It has been shown that the use of vectors based on other agents circulating in the human population (adenoviruses, herpes viruses, respiratory syncytial virus, etc.) can be complicated by the presence of specific immunity against the wild-type virus, as a result of which there will be no productive cell infection recombinant virus and, as a consequence, the expression of the target transgene will not be provided. An important property that allows rhabdoviruses to be recommended as vaccine viral vectors is a high accumulation titer in transplanted cell cultures (up to 10 ¹⁰ TCD ₅₀ / ml), as well as relatively low immunizing doses (10 ⁵ - 10 ⁷ TCD ₅₀ ). It has also been shown that recombinant vesicular stomatitis viruses expressing glycoproteins of heterologous viruses (Ebola, Marburg, Lassa, etc.) provide the formation of protective immunity against a wide range of infectious diseases.

The invention also provides a recombinant vesicular stomatitis virus strain containing an optimized nucleotide sequence (SEQ ID NO: 1) of an artificial gene encoding an artificial immunogen protein receptor binding domain (RBD) of the SARS-CoV-2 coronavirus glycoprotein S with a heterologous hemagglutinin peptide signal sequence (HA) influenza A virus, a linker and a P2A peptide for cleavage of the polyprotein during translation, glycoprotein G with a mutation M (1)> P (1), which results in independent synthesis of the SARS-CoV-2 antigen and the surface fusion protein.

The method of inducing specific immunity to SARS-CoV-2 includes:

1) 1-, 2- or 3-fold introduction into the human or animal organism of one, two or more immunogenic compositions based on the inventive strain of the recombinant vesicular stomatitis virus.

The implementation of the invention is confirmed by the following examples.

Example 1. Obtaining a constructive variant of the glycoprotein S RBD of the SARS-CoV-2 virus.

The prototype for the design of a synthetic construct of glycoprotein S variants was the amino acid sequence encoded by the S (Spike) gene presented in the deposited genome-wide sequence of the SARS-CoV-2 virus (GenBank: NC_045512). A chimeric synthetic immunogen is a secreted receptor-binding domain (RBD) of the S (Spike) SARS-CoV-2 glycoprotein with a heterologous signal sequence, a linker, and a P2A peptide that provides independent synthesis of coronavirus antigen and vesicular stomatitis G virus protein (SEQ ID NO: 2). When this variant is expressed in eukaryotic cells, synthesis and the necessary post-translational modifications of RBD glycoprotein S occur, after which the coronavirus antigen is transported into the extracellular space, which ensures the induction of an immune response, including to conformational antigenic determinants. P2A peptide provides the cleavage of the coronavirus antigen at the stage of translation, followed by the independent synthesis and folding of the G protein, which is necessary for the reproduction of the virus. This strategy provides a higher stability of the target transgene due to the fact that the occurrence of any mutations leading to a shift in the reading frame (insertions, deletions, inversions, etc.) excludes the possibility of synthesizing the viral protein necessary for a full reproductive cycle, located after P2A- peptide.

Example 2. Obtaining a genetic construct.

To obtain the genetic construct for glycoprotein S and RBD, an optimized artificial gene with a nucleotide sequence (SEQ ID NO: 1, Fig. 3) was calculated. The optimization of the gene was aimed at normalizing the codon composition of the artificial gene and excluding from the sequences elements that have a negative effect on the level of expression or stability of mRNA.

Nucleotide sequences of genes were obtained by the method of chemical synthesis of ZAO Biocad and ZAO Evrogen.

To obtain a recombinant vesicular stomatitis virus, with the formation of a bicistronic structure of transgenes with glycoprotein G by means of a self-cleaving P2A peptide (to stabilize transgenes in the composition of recombinant viruses), the nucleotide sequences of genes encoding constructive variants of glycoprotein S and RBD were reverse vector cloned in plasmid vesicular stomatitis virus pStem-rVSV_P2A_G. The cloning scheme is shown in FIG. 1. Directed cloning was carried out at the recognition sites of restriction endonucleases NheI and SmaI located in the polylinker of the G gene transcriptional cassette in the genomic cDNA sequence. For this, 2 μg of plasmid DNA pStem-rVSV_P2A_G was digested with restriction endonuclease SmaI (NEB) in CutSmart buffer at 25 ° C for 1 hour, after which 10 ua was added. restriction enzymes NheI (NEB) and 2 ea. recombinant shrimp alkaline phosphatase (rSAP) (NEB), necessary to prevent the "self-ligation" of the vector, with incubation at 37 ° C for 1 hour. The hydrolysis products were separated by electrophoresis, DNA fragments corresponding to the hydrolyzed vector (~ 13500 bp) were isolated from the agarose gel using a commercial QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions. The nucleotide insert encoding the target transgene can be prepared by setting up the hydrolysis of the cloning plasmid, amplifying the nucleotide sequences in LAMP or PCR, or by any other method available to the researcher. Cloning can be performed by any of the existing methods (ligation, Gibson method, homologous recombination, etc.), while the target transgene must have a start codon (ATG) in the optimal flanking context (Kozak consensus sequence; GCCACC) and the frame must not be disturbed readout of glycoprotein G. FIG. 1 shows the scheme of "blunt-sticky" cloning of the transgene, with the formation of a single reading frame. For this, 1 μg of the hydrolyzed vector pStem-rVSV_P2A_G / SmaI + NheI was mixed with the NheI-hydrolyzed transgene DNA fragment in an equimolar ratio, reaction buffer and T4 DNA ligase were added. After incubation at 20 ° C for 1 hour, the ligase mixture was used to transform competent E. coli cells. Screening of transformant clones was performed using PCR methods and restriction analysis of recombinant plasmids.

The correctness of the nucleotide sequences of the plasmid constructs was confirmed by Sanger sequencing. A genetic construct was obtained that encodes the nucleotide sequence of the constructive variant of the SARS-CoV-2 antigen (SEQ ID NO: 1, Fig. 3).

The recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 has a molecular weight of 8.85⋅10 ⁶ daltons, a size of 14309 bp. and contains, in accordance with the physical and genetic map shown in FIG. 2 target artificial gene according to claim 1, encoding artificial protein-immunogen RBD glycoprotein S coronavirus SARS-CoV-2, having the amino acid sequence SEQ ID NO: 2 (Fig. 4) length 784 aa. and under the control of a viral promoter, ensuring its expression in mammalian cells and consisting of the following fragments:

- ORI - (ORI, origin of replication) the origin of replication of the plasmid vector pUC (coordinates 13483-14071 bp);

- AmpR - β-lactamase gene providing resistance of E. coli transformants to the selective antibiotic ampicillin (coordinates 12452-13312 bp);

- T7 promoter - the nucleotide sequence of the T7 bacteriophage promoter required for transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 166-183 bp);

- T7 terminator - the nucleotide sequence of the T7 bacteriophage terminator, which is necessary for terminating the transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 12224-12330 bp);

- HDV-Rbz - the nucleotide sequence of the HDV ribozyme, which, after transcription, is cleaved with the formation of an authentic 3'-terminal non-coding sequence (coordinates 12137-12219 bp);

- N - open reading frame of the N gene (nucleoprotein) (coordinates 247-1515 bp);

- P - open reading frame of the P gene (phosphoprotein) (coordinates 1579-2376 bp);

- L - open reading frame of the L gene (RNA-dependent RNA polymerase) (coordinates 5708-12037 bp);

- VSV-G - open reading frame of glycoprotein G with mutation M (1)> P (1) (coordinates 3322-4032 bp);

- P2A - 2A-peptide of teshovirus-1, providing cleavage of the polyprotein at the translation stage (coordinates 4033-4089 bp);

- Signal peptide - hemagglutinin (HA) signal peptide of influenza A virus, which provides efficient intracellular transport of synthesized polyprotein (coordinates 3268-3321 bp);

- Spike protein - a fragment of the S (Spike) SARS-CoV-2 gene (coordinates 3322-4032 bp);

- Receptor-binding domain (RBD) - receptor-binding domain of glycoprotein S (Spike) SARS-CoV-2 (coordinates 3334-4002 bp).

Example 3. Obtaining a replication-competent strain of rVSV-Stbl_RBD_SC2 vesicular stomatitis virus expressing the SARS-CoV-2 antigen.

Isolation of plasmid DNA of the genetic construct for reverse genetics of the vesicular stomatitis virus encoding SARS-CoV-2 antigens was performed using the commercial QIAGEN Plasmid Maxi Kit, according to the manufacturer's instructions.

Generation of replication-competent vesicular stomatitis virus was carried out using reverse genetics methods in a transplantable cell line Vero and / or 4647 (collection of cell cultures of the Federal State Budgetary Scientific Institution SSC VB Vector of Rospotrebnadzor). For this, a cell culture monolayer (90% confluence) was transfected using Lipofectamine 3000 (Thermo Scientific, USA) with the obtained genetic plasmid construct pStem-rVSV-Stbl_RBD_SC2 together with the helper plasmids pStem-vN, pStem-vP, pStem-vL, providing expression of the protein replication machine vesicular stomatitis virus (N, P and L), as well as DNA-dependent RNA polymerase of bacteriophage T7. The amount of the plasmid construct was pStem-rVSV-Stbl_RBD_SC2 (constructive variant) - 2 μg. A day later, the culture medium was changed to a supporting one with 2% fetal blood serum, followed by cultivation for 48-72 hours in a CO ₂ incubator (5% CO ₂ ; 37 ° C; humid atmosphere). Optical microscopy showed complete destruction of the cell monolayer at the end of the cultivation period. The virus-containing culture fluid of the recombinant virus was collected, clarified by centrifugation (10 min at 10 thousand rpm), aliquoted and stored at minus 80 ° C until use.

The strain was identified at the FBSI SSC VB Vector of Rospotrebnadzor in April 2020 and deposited in the State collection of causative agents of viral infections, rickettsioses of the FBSI SSC VB Vector of Rospotrebnadzor under number V-984.

The infectious activity of the recombinant vesicular stomatitis virus was assessed when performing classical virological titration methods in sensitive cell cultures Vero and / or 4647, expressed in 50% tissue cytopathic doses (TCID ₅₀ ), or in plaque-forming units (PFU). When studying the cultural properties, it was found that the resulting recombinant virus retained the basic properties of the original vesicular stomatitis virus strain (cellular sensitivity, the nature of cytopathic action, optimal cultivation parameters, etc.).

The titer of accumulation in the culture of Vero cells of the recombinant strain of the vesicular stomatitis virus encoding the nucleotide sequence of the SARS-CoV-2 antigen (SEQ ID NO: 15) was 7.80 ± 0.19 lg TCD ₅₀ / cm ³ . The titer of accumulation in 4647 cell culture of the recombinant vesicular stomatitis virus encoding the nucleotide sequence of the SARS-CoV-2 antigen (SEQ ID NO: 15) was 9.25 ± 0.2 lg TCD ₅₀ / cm ³ .

Example 4. Confirmation of the presence of the Stbl_RBD gene encoding the SARS-CoV-2 antigens in the genome of the inventive rVSV-Stbl_RBD_SC2 strain of the recombinant vesicular stomatitis virus.

Confirmation of the presence and expression of the artificial gene Stbl_RBD encoding the antigen (receptor-binding domain of the S glycoprotein SARS-CoV-2 coronavirus) was carried out in polymerase chain reaction combined with reverse transcription (RT-PCR). Isolation of RNA from culture media samples obtained after infection of a monolayer of Vero cells with a recombinant vesicular stomatitis virus was performed using the Ribo-prep reagent kit (Interlabservice, Russia) according to the manufacturer's instructions. To assess the expression and presence of transgenes, specific oligonucleotide primers flanking the open reading frames of chimeric genes were used:

F-Sig_NheI (5'-ACAAAGCTAGCCACCATGAAGGCAATACTAGTAGTTC TG-3 ') and R-vG_SbfI (5'- TTTTGCCCTGCAGGAGTGCGGCCGCTTAC-3'), and F-NCR (5'-ACGAAGACAAACAAACCATTATTATCATTAAAAGGC-3 ') and R-VSV_1_seq (5'-TTGTGTTCTGCCCACTCTGT -3 ').

The first strand of cDNA was synthesized using the RNAscribe RT reverse transcriptase reagent kit (Biolabmix, Russia) in accordance with the manufacturer's instructions. Amplification of vesicular stomatitis virus cDNA fragments was performed with Phusion polymerase (NEB, USA) in accordance with the manufacturer's instructions. Amplification products were analyzed by electrophoretic separation in an agarose gel and subsequent staining of the gels with a working solution of ethidium bromide (0.5 μg / ml). The correctness of the open reading frames of the transgenes was confirmed by Sanger sequencing of the target amplicons eluted from the agarose gel.

The presence and functional activity of the G gene encoding the surface glycoprotein of the vesicular stomatitis virus is confirmed by sequencing of the transgene amplicons according to Sanger, as well as the reproduction of the recombinant virus in permissive cell lines (Vero, 4647, BHK-21/13, etc.) with cultural properties similar to To the "wild type" of the Air Force.

Example 5. Research and use of the rVSV-Stbl_RBD_SC2 strain of the recombinant vesicular stomatitis virus expressing the SARS-CoV-2 coronavirus antigen (RBD SARS_CoV-2) and inducing a specific immune response to SARS-CoV-2.

The compositions may include suitable adjuvants, stabilizers, excipients and the claimed strain of recombinant vesicular stomatitis expressing SARS-CoV-2 coronavirus antigens. Induction of a specific immune response to SARS-CoV-2 antigens is provided by introducing into the human or animal body immunogenic compositions based on the inventive rVSV-Stbl_RBD_SC2 strain of the recombinant vesicular stomatitis virus expressing the SARS-CoV-2 coronavirus antigens, by any of the known methods (intramuscularly, intravenously , subcutaneously, intranasally, etc.).

The study of the immunogenic properties of the inventive strain rVSV-Stbl_RBD_SC2 was carried out by means of a single intramuscular immunization of mice and ferrets with a recombinant vesicular stomatitis virus expressing the antigens of the SARS-CoV-2 coronavirus at a dose of 2x10 ⁷ PFU. A single immunization of this dose is justified by the results of previous studies at the Center, in the development of vaccine preparations against OVI, as well as by current publications in the development of candidate vaccine preparations on this platform.

Immunogenic properties were assessed by the titer of virus-specific antibodies in a solid-phase enzyme-linked immunosorbent assay using inactivated natural culture antigen SARS-CoV-2. To identify the antigen-antibody complex, we used anti-species conjugates, or protein G.

Table 1.

Group of animals Virus-specific antibody titer
28 days after immunization rVSV-Stbl_RBD_SC2 1: 200 - 4 pieces
1: 400 - 2 pieces
1: 800 - 1 piece
1: 1600 - 2 pieces rVSV control <1: 100 - 9 pcs

Table 1 shows the titers of virus-specific antibodies in the blood sera of laboratory mice after immunization with the recombinant vesicular stomatitis virus encoding the receptor-binding domain (RBD) of the glycoprotein S of the SARS-CoV-2 coronavirus with a heterologous signal sequence, P2A peptide and glycoprotein G (SEQ ID NO : 1).

It was shown that on the 28th day after immunization, the titer of virus-specific antibodies in the blood serum of laboratory mice in the rVSV-Stbl_RBD_SC2 group was at least 1: 200.

Table 2 shows the titers of virus-specific antibodies in the blood sera of ferrets after immunization with a recombinant strain of vesicular stomatitis virus encoding the receptor-binding domain (RBD) of the glycoprotein S of SARS-CoV-2 coronavirus with a heterologous signal sequence, P2A peptide and glycoprotein ID NO G (SE : 1).

Table 2.

Group of animals Virus-specific antibody titer on day 28 after immunization rVSV-Stbl_RBD_SC2 1: 400 - 1 piece
1: 800 - 1 piece rVSV control <1: 100 - 2 pieces

It was shown that on the 28th day after immunization the titer of virus-specific antibodies in the blood serum of ferrets in the group "strain rVSV-Stbl_RBD_SC2" was at least 1: 400.

For a detailed study of the immunogenic properties of the rVSV-Stbl_RBD_SC2 strain of the recombinant vesicular stomatitis virus encoding the receptor-binding domain (RBD) of the S glycoprotein of SARS-CoV-2 coronavirus with a heterologous signal sequence, P2A peptide and glycoprotein G: 1 (carried out by SEQ ID immunization) primates (3 pcs), hamsters (4 pcs) and ferrets (4 pcs).

The assessment of specific antiviral humoral immunity was carried out in the neutralization reaction against the SARS CoV-2 coronavirus strain nCoV / Victoria / 1/2020 (CPE ₅₀ ), as well as in the reaction of suppression (inhibition) of focus formation. The blood serum of a person who had recovered from COVID-19 was used as a positive control.

The reaction of suppression (inhibition) of focusing was carried out according to the developed protocol in a Vero E6 cell culture against 100 focusing units (FRU) SARS CoV-2 strain nCoV / Victoria / 1/2020, each dilution was applied to the wells of a 96-well culture plate.

The growth medium is removed from the wells of the plate, the monolayer of Vero E6 cells is washed twice with the supporting medium. Prepare 2-fold dilutions of immune and intact blood sera, then add a working dose of the virus (100 FRU), followed by incubation for 1 hour. The entire volume is transferred into the appropriate wells of the plate with a monolayer of cells, incubated for 60 min in a CO ₂ incubator at 37 ° C, after which the sample is removed from the wells, and the plate is washed once with a supporting medium. In all wells of the plate, add 100 μl of the supporting medium. After 16-18 hours of incubation, the culture medium is removed from the wells and 200 μl of acetone cooled to -20 ° C (80% acetone and 20% water) are added. Fixation takes 25-30 minutes, after which the acetone is removed and the plate is dried.

Prepare a dilution of a human monoclonal antibody to nucleoprotein N SARS-CoV-2 (AHA006 / Anti-N protein SARS-CoV-2 mAb (IgG) / Sanyou Biopharmaceuticals / Human) in PBS (phosphate buffered saline) in a working dilution of 1: 8000 , a solution of 50 μl is added to each well. The plate is incubated at 37 ° C for 1 hour, after which the wells are washed with PBS 3 times and affinity purified rabbit antibodies against human IgG conjugated with horseradish peroxidase are applied at a dilution of 1: 1000. After 60 min of incubation, the wells are washed 3 times with PBS, after which a solution of the chromogenic substrate AEC (3-amino-9-ethylcarbazole, Sigma) is added. After 30 min of incubation at room temperature in a dark place, the substrate solution is removed, the plate is washed once with PBS. Infected cells are counted by light microscopy by red-brown staining (FIG. 5). Virus neutralizing titer is the highest dilution of blood serum, at which the amount of FRU is less by 50% compared to the average FRU in control wells with a working dilution of the virus.

Table 3 - Titers of neutralizing antibodies in the blood serum of immunized animals

Animal species No., p / p RN (FOE) Primates 1 > 2560 2 > 2560 3 2560 Ferrets 4 5260 five 2560 6 5260 7 > 2560 Hamsters 8 > 2560 nine 5260 ten > 640 eleven > 640

It was found that the titer of neutralizing antibodies in the reaction of suppressing FRU in all immunized primates and ferrets was at least 1: 2560, in hamsters - at least 1: 640 (Table 3). Thus, the introduction of an immunogenic composition based on the rVSV-Stbl_RBD_SC2 strain of the recombinant vesicular stomatitis virus expressing the antigens of the SARS-CoV-2 coronavirus induces the synthesis of virus-specific and virus-neutralizing antibodies.

An immunogenic composition based on the inventive strain rVSV-Stbl_RBD_SC2 of the recombinant vesicular stomatitis virus expressing the antigens of the SARS-CoV-2 coronavirus can be considered as a candidate vaccine preparation for protecting humans and animals from COVID-19 coronavirus infection (SARS-CoV-2).

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Sequence listing

<110> Federal Budgetary Institution of Science "State Scientific Center of Virology and Biotechnology" Vector "of the Federal Service for Surveillance on Consumer Rights Protection and Human Welfare (FBSI SSC VB" Vector "of Rospotrebnadzor)

<120> Artificial gene encoding the bicistronic structure formed by the sequences of the receptor-binding domain of the SARS-CoV-2 coronavirus S glycoprotein, P2A-peptide and VSV glycoprotein G, the recombinant plasmid pStem-rVSV-Stbl_RBD_SC2, which provides the expression of the artificial gene virus and recombinant vascular gene stomatitis rVSV-Stbl_RBD_SC2, used to create a vaccine against the SARS-CoV-2 coronavirus

<160> SEQ ID NO 2

<210> SEQ ID NO: 1

<211> 2355

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of an artificial gene encoding an artificial immunogen protein, which is a bicistronic structure consisting of the receptor binding domain (RBD) sequences of the SARS-CoV-2 coronavirus S glycoprotein, a heterologous influenza A hemagglutinin (HA) signal peptide, a linker , A P2A peptide for cleavage of a polyprotein during translation, and a glycoprotein G with a mutation M (1)> P (1), which prevents alternative translation initiation.

<400> 1

ATGAAGGCAATACTAGTAGTTCTGCTATATACATTTGCAACCGCAAACGCA 51

GATACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCC 102

AATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTC 153

GCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGAC 204

TACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGC 255

GTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGAC 306

AGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACA 357

GGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGT 408

GTGATTGCCTGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAACTAC 459

AATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGG 510

GACATCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTG 561

GAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTTCAGCCCACA 612

AATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTG 663

CTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTG 714

AAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACAGGCGGG 765

GCCACCAACTTCAGCCTCCTTAAACAGGCCGGCGACGTGGAGGAGAACCCT 816

GGCCCCAAGTGCCTTTTGTACTTAACCTTTTTATTCATTGGGGTGAATTGC 867

AAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTT 918

CCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAAT 969

GACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCT 1020

ATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGT 1071

GATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTC 1122

ACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGA 1173

ACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTG 1224

ACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTT 1275

GATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGC 1326

AGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGAC 1377

TATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACC 1428

TTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGG 1479

TTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATG 1530

CAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAG 1581

ATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAA 1632

GGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATT 1683

CAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGC 1734

AAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCT 1785

CCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTA 1836

AAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTC 1887

TCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGG 1938

GATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTG 1989

AGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATG 2040

TTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCT 2091

CACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTT 2142

GGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTC 2193

AGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATC 2244

ATTGGATATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAC 2295

AAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTT 2346

GGAAAGTAA 2355

<210> SEQ ID NO: 2

<211> 784

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of the secreted receptor-binding domain (RBD) of the S (Spike) glycoprotein SARS-CoV-2 2 with a heterologous signal sequence, linker, and P2A-peptide providing independent synthesis of coronavirus antigen and G protein of vesicular stomatitis virus

<400> 2

MKAILVVLLYTFATANADTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRK 60

RISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG 120

KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG 180

STPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN 240

KCVNFNFNGLTGTGGATNFSLLKQAGDVEENPGPKCLLYLTFLFIGVNCKFTIVFPHNQK 300

GNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFR 360

WYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTP 420

HHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSE 480

DGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFP 540

ECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPG 600

TGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN 660

GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLS 720

KNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMN 780

RLGK * 784

<---

Claims (16)

1. Gene for creating a vaccine against the coronavirus SARS-CoV-2, having the sequence SEQ ID NO: 1. 2. Recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 for obtaining a vaccine against the coronavirus SARS-CoV-2, having a molecular weight of 8.85x10 ⁶ daltons, size 14309 bp. and containing, in accordance with the physical and genetic map shown in FIG. 2, the target gene according to claim 1, encoding an artificial protein-immunogen RBD of the glycoprotein S of the coronavirus SARS-CoV-2, having the amino acid sequence of SEQ ID NO: 2 in length of 784 a.c. and is under the control of a viral promoter, ensuring its expression in mammalian cells, and consisting of the following fragments: - ORI - (ORI, origin of replication) the origin of replication of the plasmid vector pUC (coordinates 13483-14071 bp); - AmpR - β-lactamase gene providing resistance of E. coli transformants to the selective antibiotic ampicillin (coordinates 12452-13312 bp); - T7 promoter - the nucleotide sequence of the T7 bacteriophage promoter required for transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 166-183 bp); - T7 terminator - the nucleotide sequence of the T7 bacteriophage terminator, which is necessary for the termination of transcription of the antigenomic RNA sequence of the recombinant vesicular stomatitis virus (coordinates 12224-12330 bp); - HDV-Rbz - nucleotide sequence of the HDV ribozyme, which, after transcription, is cleaved to form an authentic 3'-terminal non-coding sequence (coordinates 12137-122219 bp); - N - open reading frame of the N gene (nucleoprotein) (coordinates 247-1515 bp); - P - open reading frame of the P gene (phosphoprotein) (coordinates 1579-2376 bp); - L - open reading frame of the L gene (RNA-dependent RNA polymerase) (coordinates 5708-12037 bp); - VSV-G - open reading frame of glycoprotein G with mutation M (1)> P (1) (coordinates 3322-4032 bp); - P2A - 2A-peptide of teshovirus-1, providing cleavage of polyprotein at the translation stage (coordinates 4033-4089 bp); - Signal peptide - hemagglutinin (HA) signal peptide of influenza A virus, which provides efficient intracellular transport of synthesized polyprotein (coordinates 3268-3321 bp); - Spike protein - a fragment of the S (Spike) SARS-CoV-2 gene (coordinates 3322-4032 bp); - Receptor-binding domain (RBD) - receptor-binding domain of glycoprotein S (Spike) SARS-CoV-2 (coordinates 3334-4002 bp). 3. Recombinant strain of vesicular stomatitis virus rVSV-Stbl_RBD_SC2, containing the recombinant plasmid pStem-rVSV-Stbl_RBD_SC2 according to claim 2, providing independent synthesis of the SARS-CoV-2 coronavirus antigen and protein G of the vesicular vaccine against SARS-CoV used to create a coronavirus vaccine against SARS -2 and deposited in the State collection of pathogens of viral infections, rickettsioses of the FBSI SSC VB "Vector" of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare under the number V-984.

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