RU2843610C1 - Polynucleotide vaccine and method of jet needle-free delivery thereof into body - Google Patents

Abstract

FIELD: medicine; biotechnology.

SUBSTANCE: invention relates to a polynucleotide vaccine, a method for jet needle-free delivery thereof and can be used in genetic engineering, biotechnology and medicine.

EFFECT: higher immunogenicity and reduced reactogenicity of the vaccine against the infection of Covid-19 due to the use of naked DNA-RBD or mRNA-C1-RBD molecules and optimization of parameters of jet needle-free delivery into an individual’s body.

3 cl, 10 dwg, 1 tbl, 7 ex

Description

The invention relates to a polynucleotide vaccine, a method for its jet needle-free delivery into the body and can be used in immunology, genetic engineering, biotechnology and medicine.

The spread of highly pathogenic and pandemic dangerous viruses has stimulated the development of DNA and mRNA vaccines. Since such a platform allows for a very quick transition to mass production of a new vaccine taking into account current virus strains. Despite all the advantages of DNA and mRNA vaccines, they have low immunogenicity when administered as a “naked” molecule. At the time of the pandemic, lipid nanoparticles became a temporary solution. However, liposome components are expensive and can cause side effects, and the technology of production, storage and transportation presents certain difficulties. In an attempt to find a promising alternative, a wide range of strategies has been studied, in particular, needle-free injection is of particular interest.

The invention relates to a physical method for delivering DNA and mRNA vaccines, which greatly enhances their immunogenic properties, and can be used in the field of medicine and immunology.

Over the past few years, impressive advances have been made in the field of nucleic acid-based vaccines. DNA and mRNA vaccines are becoming a promising alternative to other types of vaccines based on live or inactivated viruses, vector vaccines, subunit proteins, virus-like particles, etc. These vaccines have demonstrated high efficacy (ability to induce both humoral and cellular immunity) and very acceptable safety. Construction of a plasmid, obtaining a producer strain, fermentation and purification of the plasmid can be accomplished within 2-4 weeks. The technology for producing such vaccines does not require complex manipulations or work with dangerous pathogens, which significantly simplifies the process of their creation and reduces its overall cost.

Needle-free vaccine delivery systems are known: patent RU2733424, RU2474437 and US2007/0293447. In particular, patent RU2474437 uses means to prevent cross-infection by blowing off with a gas stream, which cannot fully ensure the safety of the device, but at the same time complicates and increases the cost of the procedure. Patents RU2733424, RU2008932 and US2007/0293447 use the classic jet injection procedure, which has no protection against cross-infection.

Electroporation (EP) is a physical method that results in improved delivery of large molecules across the cell membrane (https://translated.turbopages.org/proxy_u/en-ru.ru.d76332f5-66139600-8f53ba5e-74722d776562/https/pubmed.ncbi.nlm.nih.gov/27987151/?_уа_mt_enable_static_t_ranslations=1). In the case of plasmid DNA and mRNA, electroporation enhances both the uptake and expression of the delivered nucleic acids. Muscle is an attractive tissue for nucleic acid vaccination in clinical settings due to the availability and abundance of target tissue.

The use of lipid nanoparticles (US Patent No. 10703789, IPC A61 K 39/395, published 07.07.2020) containing mRNA vaccines against COVID-19, which have vaccinated more than a billion people over the past 3 years, is known. Despite the fact that the delivery of mRNA using liposomes has proven to be very effective, certain disadvantages have been revealed, associated with both the complexity of storage and logistics of such vaccines (at -80 ° C), and with adverse post-vaccination effects. It is believed that these phenomena are associated with the nature of lipids. In particular, cationic liposomes can attach to the cell surface, thereby contributing to irreversible destabilization of the plasma membrane, causing side effects in the vaccinated person. They can cause an allergic reaction.

In addition, cases of reactions characterized by pericarditis, myocarditis, facial paralysis and other symptoms have been described.

mRNA vaccines are considered a promising platform, but DNA vaccines are an alternative. It is important to recognize that both approaches have certain advantages and disadvantages that may be important for solving various problems in medicine.

One of the disadvantages of nucleic acid-based vaccines is their low immunogenicity. Intramuscular administration of "naked" mRNA and DNA vaccines using a syringe with a needle is ineffective, since it provides a low level of immune response. To increase immunogenicity, various delivery methods are used, including liposomes, cationic polymers, cytokines, proteins, etc. Delivery using jet injection allows you to get rid of many of the disadvantages of chemical methods, in particular, those related to the toxicity of the compounds used for delivery and the occurrence of proapoptotic reactions in cells. Due to the use of the jet injection method, on the one hand, the immunogenicity of "naked" mRNA and DNA vaccines increases, and on the other hand, their reactogenicity decreases, since they do not contain additional components, which in turn affects the reduction in the cost of using the method.

The principle of jet injection is associated with a change in shear stress, which induces the formation of pores. Through these pores, increased absorption of nucleic acid by the cell occurs, which leads to a significant increase in the immunogenicity of mRNA vaccines.

In summary, new-generation needle-free jet injectors offer several important advantages over traditional injection delivery of DNA and mRNA vaccines. Jet injection typically produces immunogenicity comparable to EP and an enhanced response compared to conventional injection, which may sometimes fail to induce effective immunogenicity. This is not surprising given that jet injection can more efficiently deliver DNA and mRNA into cells to produce the desired antigens.

The closest analogue (prototype) is a method of genetic vaccination developed by the German company Kurevak AG and comprising administering to an individual in need thereof a modified RNA which comprises one open reading frame and a modification that increases the expression of the encoded peptide or protein, wherein said modification comprises a modification of the GC content in its coding region compared to the coding region of its wild type, and replaces at least 5% of the codons suitable for replacement in the coding region encoding the peptide or protein, thereby increasing the G/C content in said coding region, but the translated amino acid sequence of the wild type coding region is preserved, using a needle-free injection (RU Patent No. 2733424, C12N15/68, published 01.10.2020).

However, in the prototype patent, the technical result (increased expression of a protein encoded by an mRNA molecule used for genetic vaccination of guinea pigs using a needle-free jet injection) is achieved in a model experiment on a vaccine containing RSV-F mRNA for the prevention of respiratory syncytial virus infection, and the jet injection parameters have a wide, non-optimized range:

- liquid jet output speed from 60-150 m/sec;

- the diameter of the nozzle hole is from 20 to 600 microns;

- injection duration is from 0.1 to 1 sec.

The technical result of the claimed invention is to increase the immunogenicity and reduce the reactogenicity of the vaccine against COVID-19 infection by using naked DNA-RBD or mRNA-C1-RBD molecules and optimizing the parameters of its jet needle-free delivery to the individual's body.

The specified technical result is achieved by the proposed use of naked pVAX-RBD plasmid DNA molecules or naked mRNA-C1-RBD molecules as a polynucleotide vaccine for jet needle-free delivery into the body.

Naked plasmid DNA molecules pVAX-RBD have the nucleotide sequence SEQ ID NO: 1, 3710 bp in size, molecular weight 2.3 MDa, containing the target gene encoding the chimeric protein 176-RBD, having the amino acid sequence SEQ ID NO: 2:

- vector fragment 1484-754 of DNA plasmid pVAX with a size of 2981 bp;

- the neomycin/kanamycin resistance gene (NeoR/KanR) (1944-2738, 795 bp) and the ColE1 ori replication origin (3064-3652, 589 bp), which ensure selection and amplification of the target plasmid in Escherichia coli bacterial cells;

- fragment 755-1483, 729 bp in size, containing the artificial gene 176-RBD with the initiation codon ATG.

The molecules of the plasmid DNA template for the synthesis of mRNA pVAX-C1-RBD have the nucleotide sequence SEQ ID NO: 3, 3864 bp in size, molecular weight 2.3 MDa, containing the target gene encoding the chimeric protein 176-RBD, having the amino acid sequence SEQ ID NO: 5, before which there is a promoter of the phage T7 RNA polymerase, providing transcription of the gene, and consisting of the following fragments:

- vector fragment 1668-641 of DNA plasmid pVAX, 2844 bp in size, containing the promoter of RNA polymerase of phage T7 (625-641, 17 bp in size);

- the neomycin/kanamycin resistance gene (NeoR/KanR) (2091-2885, 795 bp) and the ColE1 ori replication origin (3211-3799, 589 bp), which ensure selection and amplification of the target plasmid in Escherichia coli bacterial cells;

- fragment 642-1661, 1020 bp in size, containing 5'UTR, artificial gene 176-RBD with initiation codon ATG, 3'UTR, poly(A) tail 100 nucleotides long. Naked C1-RBD mRNA molecules that induce SARS-CoV-2-specific antibodies, having the nucleotide sequence of SEQ ID NO: 4, obtained using the pVAX-C1-RBD DNA template having the nucleotide sequence of SEQ ID NO: 3, containing an open reading frame encoding the amino acid sequence of SEQ ID NO: 5, which ensures the synthesis and secretion of the SARS-CoV 2 RBD immunogen protein in a mammalian organism, and a 176 hybrid signal sequence, a Kozak consensus sequence, a 7-methylguanosine cap at the 5' end of the mRNA, a polyadenosine (poly A) tail at the 3' end of the mRNA, 100 nucleotides in length, and 5'- and 3'-untranslated regions (UTRs) located upstream and downstream of the open reading frame.

The said technical result is also achieved by the fact that in the method of jet needleless injection for delivering a polynucleotide vaccine that enhances the activation of a specific humoral and T-cell immune response, including intramuscular administration of a polynucleotide vaccine in liquid form using a needleless syringe, using the elastic force of the elastic element to drive the piston, according to the invention, a DNA vaccine is used as a polynucleotide vaccine, made in the form of naked plasmid DNA molecules pVAX-RBD, which are used to immunize animals at a dose of 50-200 μg, administered twice with an interval of 3 weeks between vaccinations, or an mRNA vaccine according to claim 1, made in the form of naked mRNA-C1-RBD molecules that induce SARS-CoV-2-specific antibodies, administered twice with an interval of 3 weeks between vaccinations at a dose of 30 µg mRNA-C1-RBD in 50 µl PBS using a jet-jet needle-free injector with individual nozzles positioned at a right angle to the injection surface area and providing the following characteristics: jet velocity of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 s.

The invention is illustrated by the following graphic materials.

Fig. 1, A, B show: (A) the nucleotide sequence SEQ ID NO: 1 of the plasmid DNA molecules pVAX-RBD and (B) the amino acid sequence SEQ ID NO: 2 of the chimeric protein 176-RBD. Fig. 2, A, B, C show: (A) the nucleotide sequence SEQ ID NO: 3 of the DNA template molecules pVAX-C1-RBD for mRNA synthesis; (B) the nucleotide sequence SEQ ID NO: 4 of the mRNA-C1-RBD molecules encoding the sequence of the immunogen protein 176-RBD SARS-COV-2 against SARS-COV-2; (C) the amino acid sequence SEQ ID NO: 5, ensuring the synthesis and secretion of the immunogen protein 176-RBD SARS-CoV 2 in the body of mammals. Figure 3 shows light micrographs showing changes in muscle tissue of mice injected with DNA encoding GFP using different jet injection protocols (see Table 1). Hematoxylin and eosin stained sections: (A) control mice, (B) Protocol #1; (C) Protocol #2; (D) Protocol #3. Tissue from mice injected with the plasmid encoding GFP was cut into 1 mm thick sections. Key: 1, necrosis. Figure 4 shows fluorescence micrographs of muscle tissue obtained 3 days after jet injection of the GFP plasmid. Injected muscles were collected and then cut into 1 mm thick sections to visualize GFP expression: (A) control mice (no jet injection); (B) Protocol #1; (C) Protocol #2; (D) Protocol #3. Figure 5 shows a graphical representation of fluorescence intensity using computer signal processing. Significance was assessed using the nonparametric Kruskal-Wallis one-way ANOVA with Dunn's multiple comparison correction (** p < 0.01, **** p < 0.0001). Figure 6 shows the analysis of the immune response in mice immunized with different doses of pVAX-RBD plasmid (50 mg, 100 mg, and 200 mg) using a jet injector. (A) Reciprocal titers of SARS-CoV-2 RBD-specific IgG antibodies were determined by ELISA. (B) Virus neutralizing activity was determined using the hCoV-19/Australia/VICO 1/2020 strain and Vero cells. (C) The number of IFN-γ-releasing splenocytes was quantified by ELISpot assay. (D) Threshold cycle viral load in BALB/c mouse lung tissues on day 4 after SARS-CoV-2 gamma infection. Data are presented as median and range in all panels. Significance was calculated using the nonparametric Mann-Whitney test (* p<0.01, ** p<0.05). Fig. 7 shows the analysis of the immune response in mice immunized with different doses of pVAX-RBD plasmid (50 mg, 100 mg, and 200 mg) using a jet injector. (A) Reciprocal titers of SARS-CoV-2 RBD-specific IgG antibodies were determined by ELISA. (B) Virus-neutralizing activity was determined using the hCoV-19/Australia/VICO 1/2020 strain and Vero cells. (C) Threshold PCR cycle of viral load in lung tissues of BALB/c mice on day 4 after infection with the gamma variant of SARS-CoV-2. Data are presented as median with range in all panels. Significance was calculated using the nonparametric Kruskal-Wallis one-way analysis of variance with correction for multiple comparisons and Dunn's statistical hypothesis test. (* p < 0.01, ** p < 0.05). Figure 8 shows the humoral immune response in BALB/c mice immunized with mRNA-C1-RBD. A. Specific IgG titers

antibodies to RBD in the sera of immunized animals (ELISA data). B. Results of virus neutralization using the SARS-CoV-2 nCoV/Victoria/1/2020 strain (100 TCID50). C. Results of virus neutralization using pseudotyped viruses carrying the SARS-CoV nCoV/Victoria/1/2020 S glycoprotein on their surface.

Figure 9 shows the results of the ELISpot assay for SARS-CoV-2-specific T cell-mediated response in immunized BALB/c mice. M, control (non-immunized) mice; mRNA-C1-RBD, mice immunized IM, mRNA-C1-RBD inj, mice immunized with the plasmid using jet injection. (A) Number of cells expressing IFN-γ in response to stimulation with a pool of RBD-specific peptides per 1× ¹⁰⁶ splenocytes. (B) Representative images of ELISpot wells (top row: splenocytes not stimulated with peptides; bottom row: splenocytes stimulated with a pool of peptides or mitogen). Significance was assessed using the nonparametric Kruskal-Wallis one-way ANOVA with correction for multiple comparisons and Dunn's hypothesis testing (* p < 0.05). Figure 10 shows the viral load in the lung tissues of BALB/c mice on the 4th day after infection with the gamma variant of SARS-CoV-2. Individual Ct values in real-time RT-PCR are shown as dots; medians in groups are shown as histogram peaks, with numerical Ct values indicated at the base of the histograms; the boundaries of 95% confidence intervals are marked with vertical lines. Statistical analysis was performed using the Kruskal-Wallis test (** p < 0.01, **** p < 0.0001).

Below are examples of specific implementation of the invention.

Example 1. Design of plasmid pVAX-RBD and production and purification of recombinant plasmid pVAX-RBD.

Design of the pVAX-RBD plasmid. The sequence of the gene encoding the full-length SARS-CoV-2 S protein (GenBank Ace. No. MN908947) was used to construct immunogens. The GeneOptimizer program (https://www.thermofisher.com/ru/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html) was used to optimize the codon composition and the secondary structure of RNA for efficient expression in mammalian cells. The resulting nucleotide sequence was synthesized at DNA Synthesis LLC (Russia) and cloned into the pGH vector. To amplify the fragment encoding the RBD of protein S (320V-542N) and the signal sequence 176 (MMRTLILAVLLVYFCATVHC) at the 5' end, the following primers were used: 5'-TAATACGACTCACTATAGGCTAGCCT-3' (forward) and 5'-AAAAAAGCGGCCGCTCATTAGTTGAAGTTCACGCATTTGTTCTTC-3' (reverse), - and a template containing the specified fragment. The amplification product was inserted into the pVAX vector (Thermo Fisher Scientific, USA) at the NheI and NotI sites under the immediate early promoter of human cytomegalovirus (CMV). The structure of the resulting construct was confirmed by Sanger sequencing at the Genomics Collective Use Center (Russia). As a result, the DNA construct pVAX-RBD was obtained, encoding RBD with signal sequence 176, which ensures the secretion of the produced protein from the cell.

Production and purification of the recombinant plasmid pVAX-RBD. Escherichia coli Stb13 cells were transformed with the plasmids pVAX and pVAX-RBD using calcium chloride. Plasmid DNA for immunization was produced in 2.7 l of LB nutrient medium supplemented with kanamycin (25 μg/ml). Plasmid DNA was isolated and purified using the EndoFree Plasmid Giga Kit (QIAGEN, Germany) according to the manufacturer's recommendations. This kit allows for very effective purification of the plasmid DNA preparation from bacterial endotoxins. After purification, the plasmid structure was checked by restriction analysis and spectrophotometry. As a result, naked molecules of plasmid DNA pVAX-RBD were obtained, having the nucleotide sequence SEQ ID NO: 1 (Fig. 1, A), containing the target gene encoding the chimeric protein 176-RBD, having the amino acid sequence SEQ ID NO: 2 (Fig. 1, B). More detailed information on the recombinant plasmid pVAX-RBD is presented in the Russian Federation patent No. 2781294.

Example 2. Design of a template for the synthesis of pVAX-C1-RBD mRNA and production and purification of the recombinant plasmid pVAX-C1-RBD.

Design of the pVAX-C1-RBD plasmid. The pVAX vector (Thermo Fisher Scientific, USA) was used to obtain the DNA matrix. A sequence containing 3'UTR and a 100-nucleotide-long poly(A) tail, necessary for obtaining mRNA, was inserted into it. The oligonucleotide containing these elements was synthesized at BIOSSET LLC (Novosibirsk, Russia). Insertion into the pVAX plasmid was performed at the Psp124BI and PspOMI sites, resulting in the pVAX-C1 vector.

To construct immunogens, the sequence of the gene encoding the full-length SARS-CoV-2 S protein (GenBank Ace. No. MN908947) was used. To optimize the codon composition, as well as the secondary structure of RNA for efficient expression in mammalian cells, the GeneOptimizer program was used (https://www.thermofisher.com/ru/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html). The resulting nucleotide sequence was synthesized at DNA Synthesis LLC (Russia) and cloned into a vector. To amplify the fragment encoding the RBD of the S protein (320V-542N) and the signal sequence 176 (MMRTLILAVLLVYFCATVHC) at the 5' end, the following primers were used: 5'- (reverse), - and a matrix containing the indicated fragment. The forward primer contained the T7 promoter and 5'UTR sequences.

The amplification product was inserted into the pVAX-C1 vector at the Psp124BI and BamHI sites. The structure of the resulting construct was confirmed by sequencing using the Sanger method at the Genomics Collective Use Center (Russia). As a result, the DNA construct pVAX-C1-RBD was obtained, encoding RBD with the signal sequence 176, which ensures the secretion of the produced protein from the cell. This construct is a DNA template for mRNA synthesis.

Production and purification of the recombinant plasmid pVAX-C1-RBD. Escherichia coli StbB cells were transformed with the plasmids pVAX and pVAX-C1-RBD using calcium chloride. Plasmid DNA for immunization was produced in 2.7 l of LB nutrient medium supplemented with kanamycin (25 μg/ml). Plasmid DNA was isolated and purified using the EndoFree Plasmid Giga Kit (QIAGEN, Germany) according to the manufacturer's recommendations. This kit allows for very effective purification of the plasmid DNA preparation from bacterial endotoxins. After purification, the plasmid structure was checked by restriction analysis and spectrophotometry.

As a result, naked molecules of plasmid DNA pVAX-C1-RBD were obtained, having the nucleotide sequence SEQ ID NO: 3 (Fig. 2, A), containing the target gene encoding the chimeric protein 176-RBD, having the amino acid sequence SEQ ID NO: 5 (Fig. 2, B). Example 3. Obtaining mRNA-CI-RBD

The mRNA encoding the SARS-COV-2 RBD was synthesized from the linearized and purified DNA template pVAX-C1-RBD, which has the nucleotide sequence SEQ ID NO: 3 (Fig. 2, A) and contains the bacteriophage T7 RNA polymerase promoter, 5'UTR, the target RBD gene, 3'UTR and a 100 nucleotide poly(A) tail.

A fragment of the S gene corresponding to the RBD (320-542 aa) (GenBank MN908947) was used to construct the plasmid. The codon composition of the sequence was optimized using the GeneOptimizer program. science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html). To ensure efficient transport of the synthesized protein from the cell, a fragment encoding the signal sequence MMRTLILAVLLVYFCATVHC was added to the 5'-end of the gene. The transcription reaction was performed using bacteriophage T7 RNA polymerase (BioLabMix, Novosibirsk, Russia). The reaction mixture included 1 μg of linearized template, T7 polymerase with buffer, ribonucleotide mixture in which uridine was replaced by pseudouridine or methylpseudouridine, cap structure analog m7GmAmG (BioLabMix, Novosibirsk, Russia), RNase inhibitor and nuclease-free water. The mixture was incubated for 2 hours at 37°C. After incubation, 1 μl of DNase was added to the mixture and incubated for 30 minutes at 37°C to destroy the DNA template. The resulting product was analyzed using electrophoresis in 2% agarose gel.

As a result, mRNA with the nucleotide sequence SEQ ID NO: 4 (Fig. 2, B) was obtained, which was capable of providing the synthesis of the target RBD protein with the amino acid sequence SEQ ID N0:5 (Fig. 2, C) in a eukaryotic cell.

More detailed information on obtaining mRNA-C1-RBD molecules is contained in the Dissertation of the author of this application for invention Sharabrin S.V. "Development of experimental mRNA vaccines against influenza and COVID-19", for the degree of candidate of biological sciences in specialty 1.5.3 - molecular biology, biological sciences. Published on the website of the applicant FBSI SRC VB "Vector" of Rospotrebnadzor on 01.03.2024.

Example 4. Jet needle-free injection method for delivering a polynucleotide vaccine

A jet needle-free injection method for delivering a polynucleotide vaccine comprises intramuscularly administering a polynucleotide vaccine in liquid form using a needle-free syringe, using the elastic force of an elastic element to drive a piston.

4.1. A DNA vaccine is used as a polynucleotide vaccine. The vaccine is naked molecules of plasmid DNA pVAX-RBD, which are used to immunize animals in a dose of 50-200 μg, administered twice with an interval of 3 weeks between vaccinations. The vaccine is administered using a jet needleless injector with individual nozzles, located at a right angle to the injection surface area and providing the following characteristics: jet speed of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 s.

4.2. An mRNA vaccine is used as a polynucleotide vaccine. The vaccine is naked mRNA-C1-RBD molecules that induce SARS-CoV-2-specific antibodies, administered twice with an interval of 3 weeks between vaccinations at a dose of 30 μg mRNA-C1-RBD in 50 μl PBS. The vaccine is also administered using a jet needle-free injector with individual nozzles located at a right angle to the injection surface area and providing the following characteristics: jet speed of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 s.

4.3. Development of a jet injection protocol

Optimal parameters for DNA or mRNA vaccine delivery were determined using the model plasmid phMGFP as an example using a needleless jet injector device. To select the optimal protocol for phMGFP plasmid delivery, 4 groups of mice were selected, consisting of 10 animals: 3 experimental groups and one control group (Table 1).

The plasmid was injected into the left hind paw muscle of mice (30 μg in 50 μl PBS/dose/mouse) using a prototype of a needleless jet injector. Jet injection is proposed to be performed using a prototype injector (reusable, compact, with a spring mechanism, an injection button, measuring 13*2*7.17 cm and weighing 110 g ±5%) with optimal characteristics: jet speed of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 sec using individual nozzles (disposable packaging, gamma-sterilization, single use, diameter 0.95 cm, length 4.7 cm, weight 3.8 g). In this example, the COMFORT-IN jet injector (Mika Medical, China) was used.

To assess the extent of damage, histological analysis of skin preparations with adjacent muscles was performed; samples were taken from the injection site on the 2nd day after jet injection.

The extent of histopathological changes depended on the protocol used, as shown in Fig. 3A-D (Light micrographs of mouse tissue samples showing histological changes with different jet injection protocols). Hematoxylin and eosin stained sections: (a) control mice, (b) protocol #1; (c) protocol #2; (d) protocol #3. Mice injected with GFP plasmid and tissue cut into 1 mm thick sections. Key: 1 - moderate infiltration.

In the group of animals immunized according to protocols No. 1 and No. 2, as in the control, pathohistological changes were absent. In the group of animals vaccinated according to protocol No. 3, moderate infiltration of the subcutaneous fat layer was observed after jet injection.

GFP fluorescence was assessed on day 3 after jet injection by UV microscopy of thin sections of animal muscle tissue, since this is the period when intense GFP fluorescence begins. Representative micrographs are shown in Fig. 4a-d (Fluorescence micrographs of muscle tissue obtained 3 days after immunization by jet injection with GFP plasmid). Injected muscles were collected and then cut into 1 mm thick sections to visualize GFP expression: (a) Control mice (no jet injection); (b) Protocol #1; (c) Protocol #2; (d) Protocol #3.

To quantify GFP fluorescence, computer processing was performed to determine the fluorescence intensity of green fluorescent protein in muscle sections of BALB/c mice using the imputed value of GFP+ fibers as described previously (Fig. 3 (Graphical representation of fluorescence intensity using computer signal processing). Significance was assessed using the nonparametric one-way Kruskal-Wallis ANOVA with correction for multiple comparisons and Dunn's statistical hypothesis tests (** p < 0.01, **** p < 0.0001).

As shown in Fig. 4 and Fig. 5, the highest number of fibers with GFP fluorescence was recorded in mice immunized according to protocol #3. The intensity of the GFP signal in this group of mice was statistically significantly different from the group of control animals and protocol #1. However, considering that protocol #3 caused minor histological damage, it was decided to choose protocol #2.

As a result of the experiments, the following conditions of jet injection were determined, namely, the pressure in the injector was 6.5 bar, which provided a jet velocity of 220 meters per second and an injection time of 50 μl of solution of 0.33 ms. Injection of RNA under such conditions did not lead to the occurrence of serious tissue damage and subsequent inflammatory reaction and provided a high level of GFP expression.

Example 5. Immunization of mice with pVAX-RBD DNA vaccine using jet injection significantly enhances the immune response against SARS-COV-2.

The work with animals was carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals". The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the State Research Center of Virology and Biotechnology "Vector" (permit number: NRC VB "Vector" / 02-03.2023, access mode: 03.04.2023). To assess the immunogenicity of the created constructs, female BALB / c mice weighing 16-18 g were used.

For the experiment, mice were divided into three groups of 16 animals each and immunized as follows: pVAX-RBD group, 100 μg pVAX-RBD administered intramuscularly; pVAX-RBD injection group: 100 μg pVAX-RBD administered using a jet injector; naive group, no immunization. Mice were immunized twice with an interval of 3 weeks between immunizations. Fourteen days after the second immunization, blood was collected from six mice from each group for analysis of the humoral immune response and the spleen was collected for analysis of the cellular response. Ten mice from each group were infected with live virus.

Serum ELISA. Eukaryotic RBD protein kindly provided by the immunochemistry laboratory of the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor (Russia) were used as antigens for ELISA. RBD protein (1 μg/ml) was adsorbed in the wells of a 96-well plate in PBS (Greiner_bio one, Germany) at 4 ⁰ C for 12 hours, then washed with PBST and blocked with 1% casein solution in wash buffer for 60 minutes at room temperature. After that, serum samples were added in three-fold serial dilutions starting from 1:50 and incubated for 60 minutes at 37°C. After washing, rabbit anti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma, USA) were added and incubated for 60 minutes at 37°C. The plate was washed and a TMB substrate solution (Amresco, USA) was added to it. After stopping the reaction with a stop solution, the optical density was measured at a wavelength of 450 nm using an ELISA reader (ChroMate Awareness Technology inc., USA).

Neutralizing activity of immune sera. The study used the 2019-nCoV coronavirus strain nCoV / Victoria / 1/2020 (State Collection of Pathogens of Viral Infections and Rickettsioses, Federal Budgetary Scientific Institution, State Research Center of Viral Virology and Virology "Vector" of Rospotrebnadzor, Russian Federation). The working dose of the virus was 100 CPE _50. To assess the effectiveness of the neutralizing activity of the sera, their serial two-fold dilutions were prepared, starting with a dilution from 1:10 to 1: 1000. To dilute the sera, MEM medium with glutamine and the addition of antibiotics (penicillin 100 U / ml, streptomycin 100 μg / ml) was used. A mixture of serum dilutions and a working dilution of the virus was prepared in equal volumes. The mixture was incubated for 1 h at room temperature and then added to the wells of a 96-well plate with a monolayer of Vero E6 cell culture. The dishes were incubated for 4 days at 37°C, 5% CO2. To stain the cells, 150 μl of a 0.2% gentian violet solution were added to each well of the plate (1 g of gentian violet was added to 20 ml of 96% ethanol, 120 ml of 40% formalin and 350 ml of Hank's solution). After 30 minutes, the liquid was removed from the wells and washed with water. The results were recorded visually. Any specific damage to the cell culture in the well was considered a cytopathic effect of the virus. The neutralizing activity of the sera of immunized animals was assessed by the titer (dilution) of the sera, at which protection in 50% of the wells with the cell culture from the cytopathic effect of the virus was recorded. The titer of neutralizing antibodies was calculated using the Reed-Munch formula. The cellular response was examined by ELISpot analysis using the Mouse IFN-gamma ELISpot kit (BD, USA). Splenocytes isolated from immunized animals were stimulated with a pool of 9 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide), restricted by MHC class I and MHC class II. The number of IFN-γ-producing cells was counted using an ELISpot reader (Carl Zeiss, Germany).

IFN-γ ELISpot assay. The assay was performed using Mabtech kits (Cincinnati, USA) according to the manufacturer's recommendations. Splenocytes isolated from immunized animals were stimulated with a pool of 13 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide) recognized by the major histocompatibility complex (MHC) class I (H-2-Dd, H-2-Kd, H-2-Ld) and class II molecules (H2-IAd, H2-IEd) for BALB/c mice. The peptides were synthesized by AtaGenix Laboratories (Wuhan, China), the peptide purity was > 80%. The number of IFN-γ-producing cells was counted using an ELISpot analyzer from Carl Zeiss (Oberkochen, Germany).

Analysis of protective properties. The vaccine's protective properties were assessed against the Gamma variant of the SARS-CoV-2 virus. The hCoV-19/Russia/SA-17620-080521/2021 strain was used in the work.

On day 35 after the first immunization, animals were infected intranasally with a dose of 50 ID ₅₀ (3.8 log ₁₀ CPI ₅₀ ) of the SARS-CoV-2 virus.

Statistics. Statistical processing of the obtained data was performed using the nonparametric Mann-Whitney analysis in the GraphPad Prism 9.0 program; at p<0.05, the differences were considered statistically significant.

The DNA vaccine pVAX-RBD, previously developed at the State Research Center of Virology and Biotechnology Vector, was described in the work by Borgoyakova, M.V et al [26]. It was shown that pVAX-RBD induces both humoral and cellular immune responses after traditional intramuscular administration with syringes. Since jet injection has a number of advantages, we decided to study the specific immune response and protective capacity of the DNA vaccine pVAX-RBD in this study after immunization of BABL/c mice using jet injection.

The serum was tested by ELISA for the presence of RBD-specific antibodies and for the ability to neutralize live virus. According to the results of RBD-specific ELISA, at the end of the experiment, an 80-fold increase in the median titers of specific IgG was observed in animals immunized with the DNA vaccine by jet method compared with the group immunized by traditional administration (3633 versus 291600, p-value = 0.0022, Fig. 6A).

The neutralizing properties of the sera were analyzed using the hCoV-19/Australia/VICO 1/2020 strain (Wuhan lineage) in vitro cell culture. The results correlated with the ELISA results: sera from animals immunized by jet injection showed significantly higher activity in inhibiting the cytopathic effect of the virus. Median neutralizing titers increased more than 35-fold compared to the group immunized by traditional injection (12 vs. 453, p value = 0.0022, Fig. 6B).

To assess the T cell response in the IFN-γ-ELISpot assay, splenocytes isolated from immunized and control mice were stimulated with a mixture of RBD protein peptides. The difference between the groups receiving pVAX-RBD intramuscularly or by bolus injection was about 3.5 (238 SFU vs. 875 SFU, p-value=0.0022, Figure 6C).

To assess the protective efficacy of the pVAX-RBD vaccine, animals were administered 50 ID ₅₀ (hCoV-19/Poccra/SA-17620-080521/2021 strain) intranasally. The endpoint was day 4 after the trial. Analysis of the protective efficacy by real-time PCR in lung homogenates showed a reduction in viral load in the experimental groups (median Ct 31.53 and 32.17, respectively, for the pVAX-RBD intramuscular and bolus injection groups, Figure 6D).

Thus, the immunogenicity study of the pVAX-RBD DNA vaccine administered by different routes confirms that jet injection can significantly enhance both the humoral and cellular immune response induced by the DNA vaccine. At the same time, significant differences in Ct values were found in both experimental groups (p value = 0.0084).

Example 6. Study of the dose-response effect of pVAX-RBD using a jet injector

The work with animals was carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals". The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the State Research Center of Virology and Biotechnology "Vector" (permit number: NRC VB "Vector" / 02-03.2023, access mode: 03.04.2023). To assess the immunogenicity of the created constructs, female BALB / c mice weighing 16-18 g were used.

For the pVAX-RBD dose-response experiment using a jet injector to evaluate the immunogenicity, female BALB/c mice weighing 16-18 g were used. The mice were divided into four groups of 10 animals each and immunized by jet injection as follows: Group 1: 50 μg pVAX-RBD; Group 2: 100 μg pVAX-RBD; Group 3: 200 μg pVAX-RBD; naive group, no immunization. Mice were immunized twice with an interval of 3 weeks between immunizations. Fourteen days after the second immunization, mice from each group were bled for humoral immune response analysis and challenged with hCoV-19/Australia/VICO 1/2020.

Serum ELISA. Eukaryotic RBD proteins kindly provided by the immunochemistry laboratory of the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor (Russia) were used as antigens for ELISA. RBD protein (1 μg/ml) was adsorbed in the wells of a 96-well plate in PBS (Greiner_bio one, Germany) at 4°C for 12 hours, then washed with PBST and blocked with 1% casein solution in wash buffer for 60 minutes at room temperature. After that, serum samples were added in three-fold serial dilutions starting from 1:50 and incubated for 60 minutes at 37°C. After washing, rabbit anti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma, USA) were added and incubated for 60 minutes at 37°C. The plate was washed and a TMB substrate solution (Amresco, USA) was added to it. After stopping the reaction with a stop solution, the optical density was measured at a wavelength of 450 nm using an ELISA reader (ChroMate Awareness Technology inc., USA).

Neutralizing activity of immune sera. The study used the 2019-nCoV coronavirus strain nCoV / Victoria / 1/2020 (State Collection of Pathogens of Viral Infections and Rickettsioses, Federal Budgetary Scientific Institution, State Research Center of Viral Virology and Virology Vector, Rospotrebnadzor, Russian Federation). The working dose of the virus was 100 CPD _50. To assess the effectiveness of the neutralizing activity of the sera, their serial two-fold dilutions were prepared, starting with a dilution from 1:10 to 1: 1000. To dilute the sera, MEM medium with glutamine and the addition of antibiotics (penicillin 100 U / ml, streptomycin 100 μg / ml) was used. A mixture of serum dilutions and a working dilution of the virus was prepared in equal volumes. The mixture was incubated for 1 h at room temperature and then added to the wells of a 96-well plate with a monolayer of Vero E6 cell culture. The dishes were incubated for 4 days at 37 ⁰ C, 5% CO ₂ . To stain the cells, 150 μl of a 0.2% gentian violet solution were added to each well of the plate (1 g of gentian violet was added to 20 ml of 96% ethanol, 120 ml of 40% formalin and 350 ml of Hanks' solution). After 30 minutes, the liquid was removed from the wells and washed with water. The results were recorded visually. Any specific damage to the cell culture in the well was considered a cytopathic effect of the virus. The neutralizing activity of the sera of immunized animals was assessed by the titer (dilution) of the sera, at which protection in 50% of the wells with the cell culture from the cytopathic effect of the virus was recorded. The titer of neutralizing antibodies was calculated using the Reed-Munch formula. The cellular response was examined by ELISpot analysis using the Mouse IFN-gamma ELISpot kit (BD, USA). Splenocytes isolated from immunized animals were stimulated with a pool of 9 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide), restricted by MHC class I and MHC class II. The number of IFN-γ-producing cells was counted using an ELISpot reader (Carl Zeiss, Germany).

IFN-γ ELISpot assay. The assay was performed using Mabtech kits (Cincinnati, USA) according to the manufacturer's recommendations. Splenocytes isolated from immunized animals were stimulated with a pool of 13 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide) recognized by the major histocompatibility complex (MHC) class I (H-2-Dd, H-2-Kd, H-2-Ld) and class II molecules (H2-IAd, H2-IEd) for BALB/c mice. The peptides were synthesized by AtaGenix Laboratories (Wuhan, China), the peptide purity was> 80%. The number of IFN-γ-producing cells was counted using an ELISpot analyzer from Carl Zeiss (Oberkochen, Germany).

Analysis of protective properties. The vaccine's protective properties were assessed against the Gamma variant of the SARS-CoV-2 virus. The hCoV-19/Russia/SA-17620-080521/2021 strain was used in the work.

On day 35 after the first immunization, animals were infected intranasally with a dose of 50 ID ₅₀ (3.8 log ₁₀ CPI ₅₀ ) of the SARS-CoV-2 virus.

Statistics. Statistical processing of the obtained data was performed using the nonparametric Mann-Whitney analysis in the GraphPad Prism 9.0 program; at p<0.05, the differences were considered statistically significant.

The vaccine was administered twice at a three-week interval in three different doses: 50, 100 and 200 μg using a jet injector. Ten days after the second immunization, serum was collected from the animals, followed by a challenge.

IgG binding to the recombinant RBD protein increased in a dose-dependent manner, reaching median titers of 1:83,700, 1:539,040, and 1:876,450 for the 50, 100, and 200 μg groups, respectively (Fig. 7A). Similarly, a dose-dependent increase in neutralizing activity measured with live SARS-CoV-2 virus was observed. Animals vaccinated with 50 μg pVAX-RBD had a reciprocal median ID50 titer of 43, whereas sera from the 100 and 200 μg groups had 11- and 16-fold higher neutralizing titers (495 and 704, respectively, Fig. 7B). It should be noted that the difference between titers in the groups receiving 100 and 200 μg was not significant (p value>0.9999).

The median Cts were 31.27, 32.17, and 32.02 for the groups that received 50, 100, and 200 μg of DNA vaccine, respectively (no significant difference between them, Fig. 7C). This is a significant (5-10-fold) decrease compared to the intact control, which had a Ct of 28.81 (p-value 0.0125; 0.0004; 0.0018 for doses of 50, 100, and 200, respectively).

Thus, decreasing the dose to 50 mcg leads to a significant decrease in the neutralizing activity of the serums, but does not lead to a strong decrease in the effectiveness of protection. At the same time, increasing the plasmid dose to 200 mcg leads to an insignificant increase in protective properties.

Example 7. Delivery of an experimental mRNA vaccine encoding the SARS-CoV-2 RBD via jet injection

To study the immunogenicity of the mRNA vaccine, Balb/c mice (female) weighing 16-18 g were used. Experiments with animals were carried out in compliance with the principles of humanity in accordance with the protocols approved by the Bioethics Committee of the State Research Center of Virology and Biotechnology Vector/02-03.2023, Protocol of the Bioethics Committee No. 2 dated 03.04.2023). Three groups of 8 animals each were formed. Hair was removed from the hairy part of the paw using depilatory gel. Mice were immunized in the quadriceps muscle of the left hind paw twice, on days 0 and 21. The first group of animals was administered 30 μg mRNA-C1-RBD intramuscularly (i.m.) in 50 μl PBS; the second group - 30 μg mRNA-C1-RBD in 50 μl PBS, i.m., using jet injection; The third group was injected intramuscularly with 50 μl of physiological solution using jet injection. Jet injection was performed using our own prototype injector with the following characteristics: jet speed of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 s. using individual nozzles. On the 10th day after the second injection, blood was taken from the mice, they were sacrificed and their blood and spleens were collected for further immunogenicity analysis.

Serum ELISA. Eukaryotic RBD proteins kindly provided by the immunochemistry laboratory of the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor (Russia) were used as antigens for ELISA. RBD protein (1 μg/ml) was adsorbed in the wells of a 96-well plate in PBS (Greiner_bio one, Germany) at 4°C for 12 hours, then washed with PBST and blocked with 1% casein solution in wash buffer for 60 minutes at room temperature. After that, serum samples were added in three-fold serial dilutions starting from 1:50 and incubated for 60 minutes at 37°C. After washing, rabbit anti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma, USA) were added and incubated for 60 minutes at 37°C. The plate was washed and a TMB substrate solution (Amresco, USA) was added to it. After stopping the reaction with a stop solution, the optical density was measured at a wavelength of 450 nm using an ELISA reader (ChroMate Awareness Technology inc., USA).

Neutralizing activity of immune sera. The study used the 2019-nCoV coronavirus strain nCoV / Victoria / 1/2020 (State Collection of Pathogens of Viral Infections and Rickettsioses, Federal Budgetary Scientific Institution, State Research Center of Viral Virology and Virology "Vector" of Rospotrebnadzor, Russian Federation). The working dose of the virus was 100 CPE _50. To assess the effectiveness of the neutralizing activity of the sera, their serial two-fold dilutions were prepared, starting with a dilution from 1:10 to 1: 1000. To dilute the sera, MEM medium with glutamine and the addition of antibiotics (penicillin 100 U / ml, streptomycin 100 μg / ml) was used. A mixture of serum dilutions and a working dilution of the virus was prepared in equal volumes. The mixture was incubated for 1 h at room temperature and then added to the wells of a 96-well plate with a monolayer of Vero E6 cell culture. The dishes were incubated for 4 days at 37°C, 5% CO ₂ . To stain the cells, 150 μl of a 0.2% gentian violet solution were added to each well of the plate (1 g of gentian violet was added to 20 ml of 96% ethanol, 120 ml of 40% formalin and 350 ml of Hank's solution). After 30 minutes, the liquid was removed from the wells and washed with water. The results were recorded visually. Any specific damage to the cell culture in the well was considered a cytopathic effect of the virus. The neutralizing activity of the sera of immunized animals was assessed by the titer (dilution) of the sera, at which protection in 50% of the wells with the cell culture from the cytopathic effect of the virus was recorded. The titer of neutralizing antibodies was calculated using the Reed-Munch formula. The cellular response was examined by ELISpot analysis using the Mouse IFN-gamma ELISpot kit (BD, USA). Splenocytes isolated from immunized animals were stimulated with a pool of 9 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide), restricted by MHC class I and MHC class II. The number of IFN-γ-producing cells was counted using an ELISpot reader (Carl Zeiss, Germany).

IFN-γ ELISpot assay. The assay was performed using Mabtech kits (Cincinnati, USA) according to the manufacturer's recommendations. Splenocytes isolated from immunized animals were stimulated with a pool of 13 SARS-CoV-2 RBD peptides (20 μg/ml of each peptide) recognized by the major histocompatibility complex (MHC) class I (H-2-Dd, H-2-Kd, H-2-Ld) and class II molecules (H2-IAd, H2-IEd) for BALB/c mice. The peptides were synthesized by AtaGenix Laboratories (Wuhan, China), the peptide purity was > 80%. The number of IFN-γ-producing cells was counted using an ELISpot analyzer from Carl Zeiss (Oberkochen, Germany).

Analysis of protective properties. The vaccine's protective properties were assessed against the Gamma variant of the SARS-CoV-2 virus. The hCoV-19/Russia/SA-17620-080521/2021 strain was used in the work.

On day 35 after the first immunization, animals were infected intranasally with a dose of 50 ID ₅₀ (3.8 log ₁₀ CPI ₅₀ ) of the SARS-CoV-2 virus.

Statistics. Statistical processing of the obtained data was performed using the nonparametric Mann-Whitney analysis in the GraphPad Prism 9.0 program; at p 0.05, the differences were considered statistically significant.

To analyze the immunogenicity, laboratory animals were immunized with 30 μg mRNA-C1-RBD by jet injection, and animals immunized with mRNA-C1-RBD i/m were used as a comparison group. ELISA analysis of animal sera showed that the titer of RBD-specific antibodies in mice immunized by injection significantly exceeded the titer of antibodies in mice of the control group (Fig. 8A).

An important criterion for the efficacy of a vaccine is its ability to induce antibodies capable of neutralizing the virus. In our study, the serum of mice immunized with mRNA-C1-RBD demonstrated the ability to neutralize both the SARS-CoV-2 nCoV/Victoria/1/2020 virus strain on Vero cell culture in vitro in the virus neutralization reaction (Fig. 8B) and pseudotyped viruses carrying SARS-CoV-2 S glycoproteins on their surface (Fig. 8C).

The results of the virus neutralization assay correlated with the ELISA results: sera from animals immunized with mRNA-C1-RBD by jet injection showed higher activity in inhibiting the cytopathic effect of the virus.

The cellular response was analyzed by ELISpot assay. It was shown that the number of splenocytes producing IFN-γ in response to stimulation with RBD-specific peptides in the mRNA-C1-RBD group administered by jet injection was two-fold higher than that in the mRNA-C1-RBD group administered by i.m. A lower response was recorded in the group of mice immunized with mRNA-C1-RBD without jet injection (p>0.05, Fig. 9).

In control groups of animals immunized with saline, the cellular response was at the background level. These data indicate an increase in the specific post-vaccination T-cell response when using jet injection, which correlates with an increase in the humoral immune response.

The vaccine protectivity was assessed against the Gamma variant of the SARS-CoV-2 virus. The hCoV-19/Russia/SA-17620-080521/2021 strain was used in the study. On the 35th day after the first immunization, the animals were infected intranasally with a dose of 50 ID ₅₀ (3.8 log ₁₀ CPE ₅₀ ) of the SARS-CoV-2 virus. On the 4th day after infection, the animals were sacrificed from the experiment. Quantitative determination of the virus in the lung tissues was performed by real-time RT-GPCR in 10% homogenate. The threshold cycle (Ct) values are presented graphically in Fig. 10 (Viral load in the lung tissues of BALB/c mice on the 4th day after infection with the Gamma variant of SARS-CoV-2). Individual Ct values in real-time RT-GPCR are shown as dots; medians in groups are shown as peaks of histograms, with numerical values of Ct indicated at the base of the histograms; the boundaries of 95% confidence intervals are marked with vertical lines. Statistical analysis was performed using the Kruskal-Wallis test (**p<0.01,****p<0.0001).

As a result of the study of protective activity against the Gamma variant of SARS-CoV-2 in the BALB/c mouse model, it was shown that the mRNA-C1-RBD vaccine has a protective effect when administered at a dose of 30 μg, significantly reducing the viral load in lung tissue compared to intramuscular administration (p<0.01) and control (p<0.0001).

--->

<?xml version="1.0" encoding="UTF-8"?>

<!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing

1.3//EN" "ST26SequenceListing_V1_3.dtd">

<ST26SequenceListing dtdVersion="V1_3" fileName="App_Vaccine

and the method of its delivery_09.04.2024.xml" softwareName="WIPO Sequence"

softwareVersion="2.3.0" productionDate="2024-04-09">

<ApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

<ApplicationNumberText>123456</ApplicationNumberText>

<FilingDate>2024-03-16</FilingDate>

</ApplicationIdentification>

<ApplicantFileReference>1234</ApplicantFileReference>

<EarliestPriorityApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

<ApplicationNumberText>1234567</ApplicationNumberText>

<FilingDate>2024-03-06</FilingDate>

</EarliestPriorityApplicationIdentification>

<ApplicantName languageCode="ru">Federal budgetary institution

science "State Research Center of Virology and Biotechnology

"Vector" of the Federal Service for Supervision of Human Rights Protection

consumers and human well-being (Federal Budgetary Institution of Science State Research Center of Virology and Biotechnology "Vector"

Rospotrebnadzor)</ApplicantName>

<ApplicantNameLatin>Federal Scientific Research Institute

&quot;State Scientific Center of Virology and Biotechnology

"Vector" Federal Service for Supervision in the Sphere of Protection

consumer rights and the goodness of man (FBUN GNTS VB

&quot;Vektor&quot; Rospotrebnadzora) (RU)</ApplicantNameLatin>

<InventionTitle languageCode="en">Polynucleotide vaccine and method

jet needle-free delivery of it into the body</InventionTitle>

<SequenceTotalQuantity>5</SequenceTotalQuantity>

<SequenceData sequenceIDNumber="1">

<INSDSeq>

<INSDSeq_length>3710</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..3710</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gctgcttcgcgatgtacgggccagatatacgcgttgacattgattattg

actagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacat

aacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgta

tgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcc

cacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc

ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagt

catcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg

ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttc

caaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatat

aagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactat

agggagacccaagctggctagcctcgagaattcgtcctgctgcgcacgtgggaagccctggccccggccg

cccacatgatgcggacactgatcctggctgtgctgctggtgtacttctgtgccaccgtgcactgtgtgca

gcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaatgcc

accagatcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgc

tgtacaactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgtg

cttcacaaacgtgtacgccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggacag

acaggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaaca

gcaacaacctggactccaaagtcggcggcaactacaattacctgtaccggctgttccggaagtccaatct

gaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgtggaa

ggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccct

acagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcac

caatctcgtgaagaacaaatgcgtgaacttcaactaatgagcggccgctcgagtctagagggcccgttta

aacccgctgatcagcctcgactgtgccttctagttgccagccatctctgttgtttgcccctcccccgtgcct

tccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtc

tgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaa

tagcaggcatgctggggatgcggtgggctctatggcttctactgggcggttttatggacagcaagcgaac

cggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttctc

gccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatg

attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactggg

cacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttt

tgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggcc

acgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgg

gcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctga

tgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatc

gagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggc

tcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgaccca

tggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccgg

ctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcg

aatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcg

ccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcat

ctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgttt

atttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatag

cacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca

aaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttg

agatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgt

ttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaata

ctgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgc

tctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaaga

cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagc

gaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag

aaaggcggacaggtatccggtaagcggcagggtcggaacaggagcgcacgagggagcttccaggggga

aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgct

cgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctg

gccttttgctcacatgttctt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>244</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..244</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRK

RISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLP

DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG

FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFHHHHHHHHHH</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>3874</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..3874</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gactcttcgcgatgtacgggccagatatacgcgttgacattgattattg

actagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacat

aacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgta

tgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcc

cacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc

ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagt

catcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg

ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttc

caaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatat

aagcagagctcaaattaatacgactcactataagagaataaactagtattcttctggtccccacagactc

agagagaacccgccaccatgatgcggacactgatcctggctgtgctgctggtgtacttctgtgccaccgt

gcactgtgtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcggcgag

gtgttcaatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccg

actactccgtgctgtacaactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagct

gaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggagatgaagtgcggcagatt

gcccctggacagacaggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtga

ttgcctggaacagcaacaacctggactccaaagtcggcggcaactacaattacctgtaccggctgttccg

gaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgt

aacggcgtggaaggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgg

gctatcagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccc

taagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaactaaggatccgctggagcctcggtg

gcctagcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtc

tttgaataaagtctgagtgggcggcgaattcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatga

ctaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaaagagaccgggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatct

gttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa

atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacag

caagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctactggg

cggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctg

caaagtaaactggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagag

acaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtgg

agaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtc

agcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgag

gcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaag

cgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgc

cgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattc

gaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatg

atctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccga

cggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttt

tctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtg

atattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccga

ttcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctg

atgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaa

tgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataa

ccctgataaatgcttcaataatagcacgtgctaaaacttcatttttaatttaaaaggatctaggtgaaga

tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgt

agaaaagatcaaaggatcttcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaa

ccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct

tcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactc

tgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg

tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt

cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgaga

aagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag

cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgac

ttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctt

tttacggttcctgggcttttgctggccttttgctcacatgttctt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>909</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..909</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q12">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>agagaataaactagtattcttctggtccccacagactcagagagaaccc

gccaccatgatgcggacactgatcctggctgtgctgctggtgtacttctgtgccaccgtgcactgtgtgc

agcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcggcgaggtgttcaatgc

caccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtg

ctgtacaactccgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgacctgt

gcttcacaaacgtgtacgccgacagcttcgtgatccggggagatgaagtgcggcagattgcccctggaca

gacaggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaac

agcaacaacctggactccaaagtcggcggcaactacaattacctgtaccggctgttccggaagtccaatc

tgaagcccttcgagcgggacatctccaccgagatctatcaggccggcagcaccccttgtaacggcgtgga

aggcttcaactgctacttcccactgcagtcctacggctttcagcccacaaatggcgtgggctatcagccc

tacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagca

ccaatctcgtgaagaacaaatgcgtgaacttcaactaaggatccgctggagcctcggtggcctagcttct

tgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaag

tctgagtgggcggcgaattc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>240</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..240</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q14">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MRTLILAVLLVYFCATVHCVQPTESIVRFPNITNLCPFGEVFNATRFAS

VYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA

DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY

FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (6)

1. A method of jet needle-free injection for delivering a polynucleotide vaccine that enhances the activation of a specific humoral and T-cell immune response, comprising intramuscular administration of a polynucleotide vaccine in liquid form using a needle-free syringe, using the elastic force of an elastic element to drive a piston, characterized in that the polynucleotide vaccine used is a DNA vaccine in the form of naked pVAX-RBD plasmid DNA molecules, which are used to immunize animals at a dose of 50-200 μg, administered twice with an interval of 3 weeks between vaccinations, or an mRNA vaccine in the form of naked mRNA-C1-RBD molecules that induce SARS-CoV-2-specific antibodies, administered twice with an interval of 3 weeks between vaccinations at a dose of 30 μg mRNA-C1-RBD in 50 µl PBS using a jet needle-free injector with individual nozzles, located at a right angle to the injection surface area and providing the following characteristics: jet speed of 220 meters per second, pressure of 6.5 bar, injection time of 0.33 s. 2. The method according to claim 1, characterized in that the naked molecules of the plasmid DNA pVAX-RBD have a nucleotide sequence of SEQ ID NO: 1, 3710 bp in size, a molecular weight of 2.3 MDa, containing a target gene encoding a chimeric protein 176-RBD, having an amino acid sequence of SEQ ID NO: 2, in front of which there is a promoter of the phage T7 RNA polymerase, ensuring transcription of the gene, and consisting of the following fragments: vector fragment 1484-754 of the pVAX DNA plasmid, 2981 bp in size, containing the 19 bp T7 RNA polymerase promoter, having coordinates 664-682; the neomycin/kanamycin resistance gene (NeoR/KanR) of 795 bp in size, with coordinates 1944-2738, and the ColE1 ori replication origin of 589 bp in size, with coordinates 3064-3652, which ensure selection and amplification of the target plasmid in Escherichia coli bacterial cells; fragment 755-1483, 729 bp in size, containing the artificial gene 176-RBD with the initiation codon ATG. 3. The method according to claim 1, characterized in that the naked mRNA-C1-RBD molecules according to claim 1 are obtained using the DNA template pVAX-C1-RBD containing the nucleotide sequence of SEQ ID NO: 3, and have the nucleotide sequence of SEQ ID NO: 4, containing an open reading frame encoding the amino acid sequence of SEQ ID NO: 5, ensuring the synthesis and secretion of the immunogen protein 176-RBD SARS-CoV 2 in the body of mammals, a hybrid signal sequence 176, a Kozak consensus sequence located in front of the reading frame, a 7-methylguanosine cap at the 5' end of the mRNA, a polyadenosine (poly A) tail at the 3' end of the mRNA with a length of 100 nucleotides, and 5'- and 3'-untranslated regions (UTR) located in front of and after the open reading frames.