Expression vectors against severe acute respiratory syndrome virus sars-cov-2

Abstract

This invention relates to biotechnology, immunology, and virology. An expression vector containing the genome of recombinant human adenovirus serotype 26 was generated, wherein the E1 and E3 regions were deleted, and the ORF6-Ad26 region was replaced by ORF6-Ad5. The integrated expression cassette was selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (variant 1). Sequence SEQ ID NO: 5 was used as the parental sequence for human adenovirus serotype 26. Furthermore, an expression vector containing the genome of recombinant simian adenovirus serotype 25 was generated, wherein the E1 and E3 regions were deleted. The integrated expression cassette was selected from SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3 (variant 2). Sequence SEQ ID NO: 6 was used as the parental sequence for simian adenovirus serotype 25. Additionally, an expression vector containing the genome of recombinant human adenovirus serotype 5 was generated, wherein the E1 and E3 regions were deleted. The integrated expression cassette was selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (variant 3). The sequence SEQ ID NO: 7 is used as the parental sequence for human adenovirus serotype 5. A method has also been developed that utilizes the developed expression vector to produce an immunobiological agent for inducing specific immunity against SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to biotechnology, immunology, and virology. It covers recombinant vectors that can be used in the pharmaceutical industry to develop immunobiological agents for inducing specific immunity against SARS-CoV-2, the severe acute respiratory syndrome virus. Background Technology

[0002] The disease caused by the novel coronavirus (SARS-CoV-2) has presented public health experts and physicians with complex tasks, including rapid diagnostic methods and clinical management of patients. The SARS-CoV-2 virus has spread rapidly worldwide, developing into an unprecedented pandemic. As of August 19, 2020, the number of cases exceeded 22 million, and the death toll reached 791,000.

[0003] To date, only limited data are available regarding the epidemiology, clinical symptoms, prevention, and treatment of this disease. Pneumonia is known to be the most common clinical manifestation of novel coronavirus infection, and a significant number of patients have reportedly developed acute respiratory distress syndrome (ARDS). This virus, like other viruses in the same family (SARS-CoV and MERS-CoV), belongs to Group II of dangerous pathogens. Currently, there are no specific preventative or therapeutic agents for novel coronavirus disease.

[0004] The high mortality rate, the rapid geographical spread of SARS-CoV-2, and the fact that the cause of the disease is not yet fully determined have led to an urgent need to develop effective products to prevent and treat the disease caused by this virus.

[0005] One promising area in vaccinology is the focus on developing viral vector-based agents to prevent disease. In this context, systems based on human adenovirus serotype 5 are the most widely used tools in the pharmaceutical industry.

[0006] This type of vector has advantages such as high safety, ability to enter different cell types, high packaging capacity, and the possibility of obtaining products with high potency.

[0007] One proposal (CN1276777C) suggests using a vaccine against severe acute respiratory syndrome based on recombinant human adenovirus serotype 5 containing the SARS-CoV virus S protein sequence.

[0008] One embodiment according to claim US20080267992A1 describes a vaccine against severe acute respiratory syndrome based on recombinant human adenovirus serotype 5, containing a full-length SARS-CoV protective S antigen sequence, or a sequence including a SARS-CoV S antigen S1 domain, a SARS-CoV S antigen S2 domain, or both domains. Furthermore, the recombinant virus within the expression cassette contains a human cytomegalovirus promoter (CMV-promoter) and a bovine growth hormone polyadenylation (bgh-PolyA) signal.

[0009] One protocol, based on CN111218459, describes the development of an expression vector for human adenovirus serotype 5 with deleted E1 and E3 regions, containing the S protein gene. This vector is intended for designing vaccines against COVID-19.

[0010] Meanwhile, the widespread use of vectors based on human adenovirus serotype 5 is limited because some individuals already have an immune response. Therefore, the focus has shifted to the development of multiple vectors with genetic variations, such as those based on other adenovirus serotypes. Detailed Implementation

[0011] The technical objective of the invention group protected by the claims is to induce a sustained immune response against the SARS-CoV-2 glycoprotein and to ensure the presence of a bioeffective protective antibody titer against the SARS-CoV-2 glycoprotein. It will be able to produce an immunobiological agent for inducing specific immunity against the severe acute respiratory syndrome virus SARS-CoV-2.

[0012] The technological achievement is the generation of an expression vector containing the genome of recombinant human adenovirus serotype 26, wherein the E1 and E3 regions are deleted and the ORF6-Ad26 region is replaced by ORF6-Ad5, and the expression cassette is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (variant 1). Thus, sequence SEQ ID NO:5 is used as the parental sequence of human adenovirus serotype 26.

[0013] Furthermore, the technological achievement is the generation of an expression vector containing the genome of recombinant simian adenovirus serotype 25, wherein the E1 and E3 regions are deleted, and the expression cassette is selected from SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:3 (variant 2). Thus, sequence SEQ ID NO:6 is used as the parental sequence of simian adenovirus serotype 25.

[0014] Furthermore, the technological achievement is the generation of an expression vector containing the genome of recombinant human adenovirus serotype 5, wherein the E1 and E3 regions are deleted, and the expression cassette is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (variant 3). Thus, the sequence SEQ ID NO:7 is used as the parental sequence of human adenovirus serotype 5.

[0015] The achievement of this technology also lies in the development of a method that utilizes the developed expression vector to produce an immunobiological agent for inducing specific immunity against SARS-CoV-2, the severe acute respiratory syndrome virus.

[0016] Embodiments of the present invention

[0017] The method for obtaining an expression vector containing the genome of recombinant human adenovirus serotype 26 (HAV 26) involves constructing a plasmid containing two homologous regions of the HAV 26 genome in the first stage, linearizing it using restriction endonucleases, mixing it with DNA isolated from viral particles of HAV 26, and performing homologous recombination in *E. coli* cells. This yielded a plasmid carrying the recombinant HAV 26 genome with the E1 region deleted. Next, using genetic engineering methods, open reading frame 6 (ORF6) was replaced with the ORF6 of human adenovirus serotype 5 (HAV 5). Then, the E3 region was deleted to increase the packaging capacity. Finally, the expression cassette was inserted into the vector.

[0018] The method for obtaining an expression vector containing the genome of recombinant simian adenovirus serotype 25 is as follows: In the first stage, a plasmid containing two homologous regions of the genome of simian adenovirus serotype 25 was constructed, then linearized using restriction endonucleases and mixed with DNA isolated from viral particles of simian adenovirus serotype 25, and homologous recombination was performed in E. coli cells. As a result, a plasmid carrying the genome of simian adenovirus serotype 25 with the E1 region deleted was obtained. Then, the E3 region was deleted to increase the packaging capacity. Finally, the expression cassette was inserted into the vector.

[0019] The method for obtaining an expression vector containing the recombinant human adenovirus serotype 5 genome is as follows: In the first stage, a plasmid containing two homologous regions of the human adenovirus serotype 5 genome was constructed. This plasmid was then linearized using restriction endonucleases and mixed with DNA isolated from viral particles of human adenovirus serotype 5, followed by homologous recombination in *E. coli* cells. The resulting plasmid carried the human adenovirus serotype 5 genome with the E1 region deleted. Next, using genetic engineering methods, the E3 region was deleted to increase the packaging capacity. Finally, the expression cassette was inserted into the vector.

[0020] To maximize the effectiveness of inducing an immune response, the authors requested protection for multiple variants of the expression cassette.

[0021] The spike (S) protein of the SARS-CoV-2 virus, optimized for expression in mammalian cells, was used as the antigen in all boxes. The S protein is a structural protein of coronaviruses. It is exposed on the surface of the viral particle and is responsible for binding to the ACE2 (angiotensin-converting enzyme 2) receptor. The results of the completed studies indicate the production of virus-neutralizing antibodies against the S protein, thus it is considered a promising antigen for drug formulation development.

[0022] The expression cassette SEQ ID NO:1 contains the CMV promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0023] The expression cassette SEQ ID NO:2 contains the CAG promoter, the SARS-CoV-2 viral S protein gene, and a polyadenylation signal.

[0024] The expression cassette SEQ ID NO:3 contains the EF1 promoter, the SARS-CoV-2 viral S protein gene, and a polyadenylation signal.

[0025] The expression cassette SEQ ID NO:4 contains the CMV promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0026] To demonstrate the effectiveness of the present invention, the ability of the developed expression vector to induce an immune response against SARS-CoV-2 in animals was evaluated.

[0027] The embodiments of the present invention are demonstrated by the following examples.

[0028] Example 1

[0029] An expression vector containing the genome of recombinant human adenovirus serotype 26 was generated.

[0030] In the first phase, the plasmid construct pAd26-Ends was designed, carrying two regions (two homologous arms) homologous to the genome of human adenovirus serotype 26 and the ampicillin resistance gene. One homologous arm contains the start portion of the human adenovirus serotype 26 genome (repeated from the left inverted terminal to the E1 region) and the viral genome sequence including the pIX protein. The other homologous arm contains a nucleotide sequence located after the ORF3E4 region up to the end of the genome. The synthesis of the pAd26-Ends construct was performed by the Moscow-based company Eurogen ZAO.

[0031] Human adenovirus serotype 26 DNA isolated from viral particles was mixed with pAd26-Ends. The plasmid pAd26-dlE1, carrying the genome of human adenovirus serotype 26 without the E1 region, was obtained by homologous recombination of pAd26-Ends and viral DNA.

[0032] Then, in the obtained plasmid pAd26-dlE1, using conventional cloning techniques, the sequence containing open reading frame 6 (ORF6-Ad26) was replaced with a similar sequence from the genome of human adenovirus serotype 5 to ensure that human adenovirus serotype 26 could replicate efficiently in HEK293 cell cultures. The result was plasmid pAd26-dlE1-ORF6-Ad5.

[0033] Furthermore, using conventional genetic engineering techniques, the E3 region of the adenovirus genome (approximately 3321 base pairs between gene pVIII and U-exon) was deleted from the constructed plasmid pAd26-dlE1-ORF6-Ad5 to expand the vector's packaging capacity. Finally, a recombinant vector pAd26-only-null based on the genome of human adenovirus serotype 26 was obtained, which carries the open reading frame ORF6 of human adenovirus serotype 5 and lacks both the E1 and E3 regions. Sequence SEQ ID NO:5 was used as the parental sequence for human adenovirus serotype 26.

[0034] In addition, the author developed several designs for the expression box:

[0035] - Expression cassette SEQ ID NO:1 contains the CMV promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0036] - Expression cassette SEQ ID NO:2 contains the CAG promoter, the SARS-CoV-2 viral S protein gene, and a polyadenylation signal.

[0037] - Expression cassette SEQ ID NO:3 contains the EF1 promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0038] Based on the plasmid construct pAd26-Ends, using genetic engineering techniques, constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, and pArms-26-EF1-S-CoV2 were obtained, each containing an expression cassette (SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) and a carrier homologous arm of the adenovirus serotype 26 genome, respectively. Subsequently, constructs pArms-26-CMV-S-CoV2, pArms-26-CAG-S-CoV2, and pArms-26-EF1-S-CoV2 were linearized using unique hydrolysis sites between the homologous arms; each plasmid was then mixed with the recombinant vector pAd26-only-null. Homologous recombination allows the acquisition of plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, and pAd26-only-EF1-S-CoV2, which carry the genome of recombinant human adenovirus serotype 26 with the open reading frame ORF6 of human adenovirus serotype 5 and the E1 and E3 regions deleted, and have expression cassettes SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively.

[0039] In the fourth stage, plasmids pAd26-only-CMV-S-CoV2, pAd26-only-CAG-S-CoV2, and pAd26-only-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion. The resulting DNA products were used for transfection of НЕК293 cell cultures.

[0040] Therefore, an expression vector containing the genome of recombinant human adenovirus serotype 26 was obtained, wherein the E1 and E3 regions were deleted and the RF6-Ad26 region was replaced by ORF6-Ad5. The integrated expression cassette was selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0041] Example 2

[0042] Production of expression vectors containing the genome of recombinant simian adenovirus serotype 25.

[0043] In the first phase, the plasmid construct pSim25-Ends was designed, carrying two regions (two homologous arms) homologous to the genome of simian adenovirus serotype 25. One homologous arm contains the start portion of the simian adenovirus serotype 25 genome (from the left inverted terminal repeat sequence to the E1 region) and the sequence from the end of the E1 region to the pIVa2 protein. The other homologous arm contains the sequence of the terminal portion of the adenovirus genome, including the right inverted terminal repeat sequence. The synthesis of the pSim25-Ends construct was performed by the Moscow-based company “Eurogen” ZAO.

[0044] Simian adenovirus serotype 25 DNA isolated from viral particles was mixed with pSim25-Ends. The plasmid pSim25-dlE1, carrying the genome of simian adenovirus serotype 25 with the E1 region deleted, was obtained by homologous recombination of pSim25-Ends and viral DNA.

[0045] Furthermore, using conventional genetic engineering techniques, the E3 region of the adenovirus genome (approximately 3921 base pairs from the beginning of gene 12.5K to gene 14.7K) was deleted from the constructed plasmid pSim25-dlE1 to increase the vector's packaging capacity. Finally, the plasmid construct pSim25-null was obtained, encoding the full-length genome of simian adenovirus serotype 25 with both E1 and E3 regions deleted. Sequence SEQ ID NO:6 was used as the parental sequence for simian adenovirus serotype 25.

[0046] In addition, the author developed several designs for the expression box:

[0047] - Expression cassette SEQ ID NO:4 contains the CMV promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0048] - Expression cassette SEQ ID NO:2 contains the CAG promoter, the SARS-CoV-2 viral S protein gene, and a polyadenylation signal.

[0049] - Expression cassette SEQ ID NO:3 contains the EF1 promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0050] Then, based on the plasmid construct pSim25-Ends, using genetic engineering techniques, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, and pArms-Sim25-EF1-S-CoV2 were obtained, each containing an expression cassette SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:3, and a carrier homologous arm from the genome of simian adenovirus serotype 25, respectively. Next, constructs pArms-Sim25-CMV-S-CoV2, pArms-Sim25-CAG-S-CoV2, and pArms-Sim25-EF1-S-CoV2 were linearized using unique hydrolysis sites between the homologous arms; each plasmid was then mixed with the recombinant vector pSim25-null. As a result of homologous recombination, recombinant plasmid vectors pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, and pSim25-EF1-S-CoV2 were obtained. These recombinant plasmid vectors contain the full-length genome of simian adenovirus serotype 25 with the E1 and E3 regions deleted, and expression cassettes SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:3, respectively.

[0051] In the third stage, plasmids pSim25-CMV-S-CoV2, pSim25-CAG-S-CoV2, and pSim25-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion. The resulting DNA products were used for transfection of НЕК293 cell cultures. The generated material was used to produce a prepared quantity of recombinant adenovirus.

[0052] As a result, recombinant human adenovirus serotype 25 was obtained, which contains the SARS-CoV-2 virus S protein gene: simAd25-CMV-S-CoV2 (containing expression cassette SEQ ID NO:4); simAd25-CAG-S-CoV2 (containing expression cassette SEQ ID NO:2); simAd25-EF1-S-CoV2 (containing expression cassette SEQ ID NO:3).

[0053] Therefore, an expression vector containing the genome of recombinant simian adenovirus 25 was obtained, with the E1 and E3 regions deleted, and the integrated expression cassette selected from SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:3.

[0054] Example 3

[0055] An expression vector containing the genome of recombinant human adenovirus serotype 5 was generated.

[0056] In the first phase, the plasmid construct pAd5-Ends was designed, carrying two regions (two homologous arms) homologous to the genome of human adenovirus serotype 5. One homologous arm contains the start portion of the human adenovirus serotype 5 genome (from the left-hand inverted terminal repeat to the E1 region) and the sequence of the viral genome including the pIX protein. The other homologous arm contains the nucleotide sequence following ORF3 in the E4 region up to the end of the genome. The synthesis of the pAd5-Ends construct was performed by the Moscow-based company "Eurogen" ZAO.

[0057] Human adenovirus serotype 5 DNA isolated from viral particles was mixed with pAd5-Ends. Plasmid pAd5-dlE1, carrying the genome of human adenovirus serotype 5 without the E1 region, was obtained by homologous recombination between pAd5-Ends and viral DNA.

[0058] Furthermore, using conventional genetic engineering techniques, the E3 region of the adenovirus genome (2685 base pairs from the 12,5K terminus to the beginning of the U-exon sequence) was deleted from the constructed plasmid pAd5-dlE1 to expand the vector's packaging capacity. Finally, based on the genome of human adenovirus serotype 5 with the E1 and E3 regions deleted, the recombinant plasmid vector pAd5-too-null was obtained. The sequence SEQ ID NO:7 was used as the parental sequence for human adenovirus serotype 5.

[0059] In addition, the author developed several designs for the expression box:

[0060] - Expression cassette SEQ ID NO:1 contains the CMV promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0061] - Expression cassette SEQ ID NO:2 contains the CAG promoter, the SARS-CoV-2 viral S protein gene, and a polyadenylation signal.

[0062] - Expression cassette SEQ ID NO:3 contains the EF1 promoter, the SARS-CoV-2 virus S protein gene, and a polyadenylation signal.

[0063] Then, based on the plasmid construct pAd5-Ends, genetic engineering techniques were used to obtain constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, and pArms-Ad5-EF1-S-CoV2. These constructs contain expression cassettes SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively, as well as a carrier homologous arm from the genome of human adenovirus serotype 5.

[0064] Next, the constructs pArms-Ad5-CMV-S-CoV2, pArms-Ad5-CAG-S-CoV2, and pArms-Ad5-EF1-S-CoV2 were linearized using unique hydrolysis sites between homologous arms; each plasmid was then mixed with the recombinant vector pAd5-too-null. As a result of homologous recombination, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, and pAd5-too-EF1-S-CoV2 were obtained, carrying the genome and expression cassettes SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 of recombinant human adenovirus serotype 5 with the E1 and E3 regions deleted, respectively.

[0065] In the fourth stage, plasmids pAd5-too-CMV-S-CoV2, pAd5-too-GAC-S-CoV2, and pAd5-too-EF1-S-CoV2 were hydrolyzed with specific restriction endonucleases to remove the vector portion. The resulting DNA products were used for transfection of НЕК293 cell cultures. The generated material was used to produce prepared quantities of recombinant adenovirus.

[0066] As a result, recombinant human adenovirus serotype 5 was obtained, which contains the SARS-CoV-2 virus S protein gene: Ad5-CMV-S-CoV2 (containing expression cassette SEQ ID NO:1); Ad5-CAG-S-CoV2 (containing expression cassette SEQ ID NO:2); and Ad5-EF1-S-CoV2 (containing expression cassette SEQ ID NO:3).

[0067] Therefore, an expression vector containing the genome of recombinant human adenovirus serotype 5 was obtained, with the E1 and E3 regions deleted, and the integrated expression cassette selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0068] Example 4

[0069] The expression of the SARS-CoV-2 virus S protein gene was verified in HEK293 cells using the developed expression vector.

[0070] The purpose of this experiment is to verify the ability of the constructed recombinant adenovirus to express the SARS-CoV-2 virus S protein gene in mammalian cells.

[0071] HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 in an incubator. Cells were placed in 35 mm... 2They were placed in petri dishes and incubated for 24 hours until 70% confluence was achieved. Then, one expression vector formulation under study was added each time. Thus, the following groups were formed:

[0072] 1) Ad26-CMV-S-CoV2;

[0073] 2) Ad26-CAG-S-CoV2;

[0074] 3) Ad26-EF1-S-CoV2;

[0075] 4)Ad26-null;

[0076] 5) simAd25-CMV-S-CoV2;

[0077] 6) simAd25-CAG-S-CoV2;

[0078] 7) simAd25-EF1-S-CoV2;

[0079] 8)simAd25-null;

[0080] 9) Ad5-CMV-S-CoV2;

[0081] 10) Ad5-CAG-S-CoV2;

[0082] 11) Ad5-EF1-S-CoV2;

[0083] 12)Ad5-null;

[0084] 13) Phosphate buffered saline.

[0085] Two days after transduction, cells were collected and lysed in 0.5 ml of normal concentration buffer CCLR (Promega). The lysates were diluted with carbonate-bicarbonate buffer and placed in the wells of an ELISA plate. The plate was incubated overnight at +4°C.

[0086] Next, the plate wells were washed three times with 200 μl of wash buffer at normal concentration per well, and then 100 μl of blocking buffer was added to each well. The plate was capped and incubated at 37°C for 1 hour at 400 rpm on a shaker. Then, the plate wells were washed three times with 200 μl of wash buffer at normal concentration per well, and 100 μl of convalescent serum was added to each well. The plate was capped and incubated at room temperature for 2 hours at 400 rpm on a shaker. Then, the plate wells were washed three times with 200 μl of wash buffer at normal concentration per well, and 100 μl of biotin-conjugated secondary antibody was added. The plate was capped and incubated at room temperature for 2 hours at 400 rpm on a shaker. Next, a solution of streptavidin conjugated with horseradish peroxidase was prepared. For this purpose, 60 μl of the conjugate was diluted in 5.94 mL of analytical buffer. The wells were washed twice with 200 μl of normal strength wash buffer per well, and 100 μl of horseradish peroxidase-conjugated streptavidin solution was added to each well. The plates were incubated on a shaker at 400 rpm at room temperature for 1 hour. Then, the wells were washed twice with 200 μl of normal strength wash buffer per well, and 100 μl of TMB substrate was added to each well, and the plates were incubated at room temperature in the dark for 10 minutes. Finally, 100 μl of stop solution was added to each well. Optical density values ​​were measured at 450 nm using a multiskan FC (Thermo) plate spectrophotometer. The experimental results are presented in Table 1.

[0087] Table 1 - Experimental results verifying the expression of the SARS-CoV-2 virus S protein gene in HEK293 cells after adding the developed expression vector.

[0088] Average optical density at 450nm wavelength

[0089]

[0090]

[0091] As the data received indicate, expression of the SARS-CoV-2 target S protein was observed in all cells transduced using the developed expression vector.

[0092] Example 5

[0093] The effectiveness of animal immunization was evaluated using the developed expression vector.

[0094] One of the key characteristics of immunogenicity is antibody titer. Examples present data relating to changes in antibody titers against SARS-CoV-2 glycoproteins on day 21 post-immunization.

[0095] The experiment used BALB / c mice, specifically female mice weighing 18g. All animals were divided into 13 groups of 5 animals each, and were injected intramuscularly with 100 μl of 10... 8 The developed expression vector was used to dose viral particles. Therefore, the following animal groups were formed:

[0096] 1) Ad26-CMV-S-CoV2;

[0097] 2) Ad26-CAG-S-CoV2;

[0098] 3) Ad26-EF1-S-CoV2;

[0099] 4)Ad26-null;

[0100] 5) simAd25-CMV-S-CoV2;

[0101] 6) simAd25-CAG-S-CoV2;

[0102] 7) simAd25-EF1-S-CoV2;

[0103] 8)simAd25-null;

[0104] 9) Ad5-CMV-S-CoV2;

[0105] 10) Ad5-CAG-S-CoV2;

[0106] 11) Ad5-EF1-S-CoV2;

[0107] 12)Ad5-null;

[0108] 13) Phosphate buffered saline.

[0109] Three weeks later, blood samples were collected from the tail vein of the animals, and serum was separated. Antibody titers were measured using enzyme-linked immunosorbent assay (ELISA) according to the following protocol:

[0110] 1) Adsorb the protein (S) onto the wells of a 96-well ELISA plate at +4°C for 16 hours.

[0111] 2) Then, to prevent nonspecific binding, the plate was "blocked" with 100 μl of 5% milk dissolved in TPBS per well. It was then incubated in a shaker at 37°C for 1 hour.

[0112] 3) The serum samples from immunized mice were diluted using the 2-fold dilution method. Twelve dilutions were prepared for each sample.

[0113] 4) Add 50 μl of diluted serum sample to each well of the plate.

[0114] 5) Then, incubate at 37°C for 1 hour.

[0115] 6) After incubation, wash the wells three times with phosphate buffer.

[0116] 7) In addition, a second antibody against mouse immunoglobulin conjugated with horseradish peroxidase was added.

[0117] 8) Next, incubate at 37°C for 1 hour.

[0118] 9) After incubation, wash the wells three times with phosphate buffer.

[0119] 10) Then, a solution of tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase was added, and the mixture was converted into a colored compound through reaction. After 15 minutes, the reaction was stopped by adding sulfuric acid. Next, the optical density (OD) of the solution in each well was measured at a wavelength of 450 nm using a spectrophotometer.

[0120] Antibody titer was determined when the optical density of the solution was significantly higher than that of the negative control group at the final dilution. The results (geometric mean) are presented in Table 1.

[0121] Table 1 - Antibody titer of anti-S protein in mouse serum (geometric mean of antibody titer)

[0122] serial number Naming of the animal group Antibody titer 1 Ad26-CMV-S-CoV2 14,703 2 Ad26-CAG-S-CoV2 12,800 3 Ad26-EF1-S-CoV2 16,890 4 Ad26-null 0 5 simAd25-CMV-S-CoV2 12,800 6 simAd25-CAG-S-CoV2 10,159 7 simAd25-EF1-S-CoV2 12,800 8 simAd25-null 0 9 Ad5-CMV-S-CoV2 11,143 10 Ad5-CAG-S-CoV2 16,127 11 Ad5-EF1-S-CoV2 12,800 12 Ad5-null 0 13 Phosphate buffered saline 0

[0123] As the data presented show, all developed expression vectors induced a sustained immune response to the SARS-CoV-2 glycoprotein and exhibited bioeffective protective antibody titers against it. Therefore, they can be used to generate immunobiological agents that induce specific immunity against SARS-CoV-2.

[0124] Therefore, as demonstrated by the provided examples, the specified technical objectives have been achieved, particularly the induction of a sustained immune response to the SARS-CoV-2 glycoprotein and the presence of a bioeffective protective antibody titer against the SARS-CoV-2 glycoprotein.

[0125] Industrial applicability

[0126] All the examples provided demonstrate the effectiveness of the expression vector and its suitability and industrial applicability for generating immunobiological agents that induce specific immunity against SARS-CoV-2.

Claims

1. An expression vector containing the genome of recombinant human adenovirus serotype 26, wherein the E1 and E3 regions are deleted, and the ORF6-Ad26 region is replaced by ORF6-Ad5, wherein the integrated expression cassette is selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and SEQ ID NO: 5 was used as the parent sequence for human adenovirus serotype 26.

2. An expression vector containing the genome of recombinant simian adenovirus serotype 25, wherein the E1 and E3 regions are deleted, and the integrated expression cassette is selected from any one of SEQ ID NO: 4, SEQ ID NO: 2, and SEQ ID NO: 3; and SEQ ID NO: 6 was used as the parental sequence for simian adenovirus serotype 25.

3. An expression vector containing the genome of recombinant human adenovirus serotype 5, wherein the E1 and E3 regions are deleted, and the integrated expression cassette is selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and SEQ ID NO: 7 was used as the parental sequence for human adenovirus serotype 5.

4. Use of the expression vector according to any one of claims 1 to 3 in the preparation of an immunobiological agent for inducing specific immunity against SARS-CoV-2, a severe acute respiratory syndrome virus.

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