RECOMBINANT PLASMID DNA TEMPLATE pVAX-C3-H1-PolyA AND POLYNUCLEOTIDE mRNA VACCINE SYNTHESIZED USING DNA TEMPLATE pVAX-C3-H1-PolyA, ENCODING HEMAGGLUTININ OF INFLUENZA A / H1N1 VIRUS AND ENSURING ITS EXPRESSION AND INDUCTION OF SPECIFIC IMMUNE RESPONSE WHEN ADMINISTERED TO BODY OF MAMMALS

Abstract

FIELD: biotechnology.SUBSTANCE: plasmid DNA matrix pVAX-C3-H1-PolyA and the polynucleotide mRNA vaccine synthesized using the said DNA matrix pVAX-C3-H1-PolyA, encoding the hemagglutinin of the influenza A / H1N1 virus and ensuring its expression and induction of a specific immune response when introduced into the body of mammals, used in immunology, genetic engineering, biotechnology and medicine. A recombinant plasmid DNA matrix pVAX-C3-H1-PolyA was obtained, providing expression of mRNA in mammalian cells, having the nucleotide sequence SEQ ID NO: 1 with a size of 4849 bp, a molecular weight of 3.1 MDa and including a fragment of 1680 bp, having coordinates from 699 to 2378 bp and containing an artificial gene encoding the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus without the transmembrane and cytoplasmic domain with the addition of the trimerizing domain of phage T4 with the initiation codon ATG. An mRNA vaccine was obtained having the nucleotide sequence SEQ ID NO: 2, obtained using a DNA matrix and containing an open reading frame encoding the amino acid sequence SEQ ID NO:3 of the influenza virus hemagglutinin immunogen protein, and ensuring the synthesis and secretion of the said protein in the body of mammals.EFFECT: expansion of the range of more relevant mRNA vaccines against the influenza virus.2 cl, 6 dwg, 3 ex

Description

[0001] The invention relates to a recombinant plasmid DNA matrix pVAX-C3-H1-PolyA and a polynucleotide mRNA vaccine synthesized using the said DNA matrix pVAX-C3-H1-PolyA, encoding the hemagglutinin of the influenza A / H1N1 virus and ensuring its expression and induction of a specific immune response when introduced into the body of mammals, and can be used in immunology, genetic engineering, biotechnology and medicine.

[0002] Seasonal influenza is an acute respiratory disease caused by influenza A and B viruses, which circulate worldwide. The development of seasonal influenza vaccines is complicated by the high variability of these viruses. Influenza A and B viruses have the ability to rapidly change their genetic and antigenic structure. Antigenic drift occurs due to the accumulation of point mutations in the genes encoding viral surface proteins, such as hemagglutinin and neuraminidase. These changes allow the virus to evade the immune response generated by a previous vaccination or infection. The constant antigenic drift of circulating influenza viruses can render vaccines against them ineffective the following year, necessitating vaccine redevelopment. Traditional inactivated or attenuated influenza vaccines require a multi-step and lengthy production process, making their adaptation to new strains slow and expensive.Every year, the WHO predicts which strains will circulate in the following season, and mass production of vaccines begins based on these predictions. However, forecasting errors and the limited ability to quickly adapt vaccines reduce their effectiveness.

[0003] So, starting in 2023, in the northern hemisphere, the WHO has recommended using the influenza virus strain A / Wisconsin / 67 / 2022 (H1N1pdm09) as a vaccine strain, and as of the current year 2025, it has not yet changed.

[0004] Incorrect prediction of the predominant strains can significantly reduce vaccine effectiveness. Furthermore, traditional vaccine production methods, such as culturing viruses in chicken embryos, have their limitations and cannot flexibly respond to new mutations. The advent of mRNA vaccine technology has made it possible to overcome these challenges. Advantages of mRNA vaccines include the speed of production and the ability to quickly update the vaccine when new virus strains emerge. Developers can quickly modify the antigen sequence embedded in the mRNA vaccine, adapting it to current virus strains without changing the core production process. Compared to traditional vaccines, which can require months of development, mRNA vaccine production can be adapted in a matter of weeks.

[0005] The production of an mRNA vaccine begins with a DNA template, which contains the necessary structural elements of mRNA and the target antigen sequence. A key step in constructing mRNA vaccines is optimizing its nucleotide sequence, both the coding and non-coding regions, which can influence the rate of translation initiation and mRNA degradation.

[0006] We previously developed the pVAX-C3-PolyA DNA cassette, which carries effective regulatory UTR sequences and other elements necessary for mRNA function, such as the cap and poly(A) tail (RU Patent No. 2839841, published May 13, 2025). This design allows for rapid insertion of the target protein sequence and the production of a DNA template for the synthesis of the desired mRNA.

[0007] Lipid nanoparticles for delivering mRNA vaccines against the influenza virus, methods for their production and use are known (patent US 11771653, published 03.10.2023).

[0008] The closest analogue (prototype) is an mRNA multivalent vaccine against influenza virus and a method for producing it, containing mRNA of recombinant H1N1 protein (strain A / Beijing / 262 / 1995 (H1N1), mRNA of recombinant H3N2 protein (strain A / Perth / 16 / 2009 (H3N2), mRNA of recombinant H5N1 protein (strain A / Anhui / 1 / 2005 (H5N1), mRNA of recombinant H6N1 protein (A / quail / Hong Kong / 1721-30 / 99 (H6N1), mRNA of recombinant H7N9 protein (strain A / Anhui / 01 / 2013 (H7N9), mRNA of recombinant H9N2 protein (A / Hong Kong / 1073 / 1999 (H9N2), mRNA of recombinant protein H11N2 (A / duck / Yangzhou / 906 / 2002 (H11N2), mRNA of recombinant protein B-Victoria and mRNA of recombinant protein B-Yamagata (Chinese Patent No. CN 116785424 B, IPC A61K39 / 145, C12N15 / 62, published on September 22, 2023). The mass concentration of each mRNA in the vaccine is 100 μg / ml.According to the invention, the antigen sequence is optimized, and the prepared mRNA multivalent influenza vaccine can stimulate the body to produce relatively strong immunogenicity.

[0009] All of the above analogs and the prototype involve the use of lipid nanoparticles to deliver mRNA vaccines to mammals. Furthermore, these vaccines use less relevant influenza virus strains, including the A / Beijing / 262 / 1995 (H1N)1 strain.

[0010] Thus, the construction of new, more relevant DNA templates for the synthesis of mRNA vaccines against influenza will make it possible to obtain a more effective mRNA vaccine.

[0011] The technical result of the claimed invention is to expand the range of more relevant mRNA vaccines against influenza virus by creating the pVAX-C3-H1-PolyA DNA matrix and a polynucleotide mRNA vaccine using it, which provides the formation of an immune response in mammals. The mRNA-C3-H1 polynucleotide mRNA vaccine can be used in combination with other mRNA vaccines against seasonal influenza virus, depending on the circulating strains of this virus.

[0012] The said technical result is achieved by obtaining a recombinant plasmid DNA matrix pVAX-C3-H1-PolyA, which provides expression of mRNA in mammalian cells, has a nucleotide sequence SEQ ID NO: 1 with a size of 4849 bp, a molecular weight of 3.1 MDa and contains, in accordance with the physical and genetic map (Fig. 1), the following elements:

[0013] - NeoR / KanR gene for neomycin / kanamycin resistance, 795 bp in size, with coordinates from 3076 to 3870 bp, and the ColEl ori replication origin, 589 bp in size, with coordinates from 4196 to 4784, which ensure selection and amplification of the target plasmid in Escherichia coli bacterial cells;

[0014] - a 20 bp fragment - a modified promoter of phage T7 RNA polymerase with initiating nucleotides AGG, necessary for the use of the AG-Cap analogue and having coordinates from 625 to 644 bp;

[0015] - a 54 bp fragment with coordinates from 645 to 698 bp and is a 5'UTR-modified chimeric sequence for the COVID-19 mRNA vaccine that includes the Kozak sequence;

[0016] - a 1680 bp fragment with coordinates from 699 to 2378 bp and containing an artificial gene encoding the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus without the transmembrane and cytoplasmic domain with the addition of the trimerizing domain of the T4 phage with the ATG initiation codon;

[0017] - a 135 bp fragment with coordinates from 2385 to 2519 bp and being the 3'UTR sequence of human β-globin;

[0018] - a fragment of 110 bp in size - a poly(A) tail of 100 nucleotides in length with an internal linker 30(A)GCATATGACT70(A), having coordinates from 2526 to 2636 bp;

[0019] - 6 bp Bso31I restriction endonuclease site with coordinates from 2642 to 2647 bp.

[0020] The said technical result is also achieved by the fact that a polynucleotide mRNA vaccine is obtained that induces specific antibodies to the influenza virus, having the nucleotide sequence SEQ ID NO: 2, obtained using the DNA matrix pVAX-C3-H1-PolyA according to claim 1, and containing an open reading frame encoding the amino acid sequence SEQ ID NO: 3 of the hemagglutinin immunogen protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus and ensuring the synthesis and secretion of the said protein in the body of mammals, 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 with a length of 100 nucleotides, 5' and 3' - untranslated regions (UTR) before and after the open reading frame, respectively.

[0021] A polynucleotide mRNA vaccine synthesized in vitro using the pVAX-C3-H1-PolyA DNA template elicits a strong immune response upon immunization of animals. Animals are immunized using a method that activates specific humoral and T-cell immune responses upon administration of the mRNA vaccine, such as jet injection or lipid nanoparticles. In this example, jet injection was used to deliver the mRNA vaccine. Thus, the mRNA vaccine, made in the form of molecules inducing specific antibodies against the influenza virus, was administered twice with an interval of three weeks between vaccinations at a dose of 30 μg mRNA-C3-H1 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.

[0022] The invention is illustrated by the following graphic materials.

[0023] Figure 1 shows the physical and genetic map of the pVAX-C3-H1-PolyA plasmid DNA template molecules.

[0024] Fig. 2 shows the nucleotide sequence SEQ ID NO: 1 of the molecules of the plasmid DNA template pVAX-C3-H1-PolyA.

[0025] Fig. 3 shows the nucleotide sequence SEQ ID NO:2 of the mRNA vaccine molecules encoding the sequence of the hemagglutinin immunogen protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus.

[0026] Fig. 4 shows the amino acid sequence SEQ ID NO:3 of the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus.

[0027] Figure 5 shows the humoral immune response in BALB / c mice immunized with the mRNA vaccine. The mRNA vaccine, which contained molecules inducing specific antibodies against the influenza virus, was administered twice with a three-week interval between vaccinations. Significance was calculated using the nonparametric Mann-Whitney analysis. Statistical analysis was performed using GraphPad Prism 8.0 software (*** p < 0.001).

[0028] Figure 6 shows the results of ELISpot analysis of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus-specific T cell-mediated response in BALB / c mice immunized with mRNA vaccine. The number of cells expressing IFN-y in response to stimulation with a pool of hemagglutinin-specific peptides per 1×10 6splenocytes. Significance was assessed using the nonparametric one-way Kruskal-Wallis analysis of variance with correction for multiple comparisons and Dunn's statistical hypothesis testing (* p < 0.01).

[0029] Below are examples of specific implementation of the invention.

[0030] Example 1. Design of pVAX-C3-H1-PolyA Plasmid DNA Template for mRNA Synthesis

[0031] The pVAX-C3-PolyA vector was used to produce the pVAX-C3-H1-PolyA plasmid DNA template. This vector contains the elements necessary for efficient expression of the mRNA vaccine in eukaryotic cells. Co-transcriptional capping with a chemical cap analog, AG-cap analog (m7GmAmG), is used for one-step mRNA synthesis, and a 100-nucleotide poly(A) tail sequence is inserted directly into the DNA template with an internal linker of 10 random nucleotides, such as 30(A)GCATATGACT70(A). A modified chimeric sequence proposed by Moderna for the mRNA vaccine against COVID-19 was used as the 5'UTR, and the human β-globin sequence was used as the 3'UTR. The 5'UTR sequence ended with a CciNI restriction endonuclease site, and the 3'UTR sequence began with a BamHI restriction endonuclease site. These sites were used to insert the hemagglutinin gene into the DNA cassette.

[0032] The immunogen was constructed using the gene sequence encoding the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus without the transmembrane and cytoplasmic domains (1-528 aa). GeneOptimizer (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 RNA secondary structure for efficient expression in mammalian cells.

[0033] The resulting nucleotide sequence was synthesized at DNA Synthesis LLC (Russia) and cloned into a vector. The following primers were used to amplify the hemagglutinin fragment: 5'-TTTGCGGCCGCCACCATG-3' (forward) and 5'-GAGCACCTTCCTGTAATAAGGATCC-3' (reverse), along with a template containing the fragment.

[0034] The amplification product was inserted into the pVAX-C3-PolyA vector at the CciNI and BamHI sites. The structure of the resulting construct was confirmed by Sanger sequencing at the Genomics Research Center (Russia). This resulted in the pVAX-C3-H1-PolyA DNA construct, encoding influenza virus hemagglutinin. The plasmid DNA molecules have the nucleotide sequence SEQ ID NO: 1 (Fig. 2), containing the elements necessary for mRNA and the influenza virus hemagglutinin gene. This construct serves as a DNA template for the synthesis of an mRNA vaccine.

[0035] Example 2. Obtaining an mRNA vaccine

[0036] The synthesis of mRNA encoding the influenza virus hemagglutinin was carried out from the linearized and purified DNA template pVAX-C3-H1-PolyA, which has the nucleotide sequence SEQ ID NO: 1 (Fig. 2) and contains the bacteriophage T7 RNA polymerase promoter, 5'UTR, the HI target gene, 3'UTR, and a 100 nucleotide poly(A) tail.

[0037] 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, a ribonucleotide mixture in which uridine was replaced by pseudouridine or methylpseudouridine, an analog of the m7GmAmG cap structure (BioLabMix, Novosibirsk, Russia), an 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 the mixture was incubated for 30 minutes at 37°C to degrade the DNA template. The resulting product was analyzed using electrophoresis in a 2% agarose gel.

[0038] As a result, mRNA with the nucleotide sequence SEQ ID NO: 2 (Fig. 3) was obtained, which was capable of providing the synthesis of the target protein hemagglutinin of the influenza virus with the amino acid sequence SEQ ID NO: 3 (Fig. 4) in a eukaryotic cell.

[0039] Example 3. Delivery of an experimental mRNA vaccine encoding influenza virus hemagglutinin by jet injection and study of its immunogenicity

[0040] Balb / c mice (female) weighing 16-18 g were used to study the immunogenicity of the mRNA vaccine. Animal experiments were carried out in compliance with the principles of humaneness in accordance with the protocols approved by the Bioethics Committee of the State Research Center of Virology and Biotechnology Vector (02-03.2023, Protocol BEC No. 2 dated 03.04.2023). Two groups of 6 animals 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, using jet injection. The first group of animals was injected with 30 μg of mRNA vaccine in 50 μl PBS; the second group was injected intramuscularly with 50 μl of saline. Jet injection was carried out 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 day 10 after the second injection, mice were bled, sacrificed, and their spleens were collected for further immunogenicity analysis.

[0041] Serum ELISA. Eukaryotic hemagglutinin proteins obtained at the Bioengineering Department of the State Research Center of Virology and Biotechnology Vector, Rospotrebnadzor (Russia) were used as antigens for the ELISA. Hemagglutinin protein (1 μg / ml) was adsorbed in the wells of a 96-well plate in PBS (Greiner_bio one, Germany) at 4°C overnight, then washed with PBST and blocked with 1% casein solution in wash buffer for 90 minutes at room temperature. Serum samples were then added in three-fold serial dilutions, starting with 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 (Imtek, Russia) was added. After stopping the reaction with stop solution, the optical density was measured at 450 nm using an ELISA reader (Feyond A300, China).

[0042] IFN-γ ELISpot assay. The assay was performed using Mabtech kits (Cincinnati, USA) according to the manufacturer's recommendations. Splenocytes isolated from the spleens of immunized animals were stimulated with a pool of 6 influenza virus hemagglutinin 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 (H2-IAd, H2-IEd) for BALB / c mice. The peptides were synthesized by AtaGenix Laboratories (Wuhan, China), the peptide purity is > 80%. The number of cells producing IFN-γ was counted using an ELISpot analyzer from Carl Zeiss (Germany, Oberkochen).

[0043] Statistics. Statistical processing of the obtained data was performed using the nonparametric Mann-Whitney analysis in GraphPad Prism 9.0; differences were considered statistically significant at p <0.05.

[0044] To analyze the immunogenicity, laboratory animals were immunized with 30 μg of mRNA-C3-H1 via jet injection. ELISA analysis of animal sera showed that the titer of hemagglutinin-specific antibodies in mice immunized with mRNA obtained using the developed pVAX-C3-H1-PolyA cassette significantly exceeded the antibody titer in mice in the control group (Fig. 5).

[0045] The cellular immune response was analyzed using the ELISpot method. It was shown that the number of splenocytes producing IFN-γ in response to stimulation with hemagglutinin-specific peptides in the mRNA-C3-H1 group exceeded that in the control group of mice (Fig. 6). In the 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 using jet injection, which correlates with an enhanced humoral immune response.

[0046] Thus, examples 1-3 confirm the achievement of the claimed technical result: the creation of the DNA matrix pVAX-C3-H1-PolyA and a polynucleotide mRNA vaccine using it, ensuring the formation of an immune response in the body of mammals.

Claims

1. Recombinant plasmid DNA matrix pVAX-C3-H1-PolyA, providing expression of mRNA in mammalian cells, having the nucleotide sequence SEQ ID NO: 1, 4849 bp in size, a molecular weight of 3.1 MDa and containing, in accordance with the physical and genetic map, the following structural elements: - the NeoR / KanR gene for resistance to neomycin / kanamycin, 795 bp in size, with coordinates from 3076 to 3870 bp, and the ColE1 ori replication origin, 589 bp in size, with coordinates from 4196 to 4784, which ensure selection and amplification of the target plasmid in Escherichia coli bacterial cells; - a 20 bp fragment - a modified promoter of the T7 phage RNA polymerase with the initiating nucleotides AGG, necessary for the use of the AG-Cap analogue and having coordinates from 625 to 644 bp; - a 54 bp fragment with coordinates from 645 to 698 bp and being a '5' UTR - a modified chimeric sequence for the mRNA vaccine against COVID-19, which includes the Kozak sequence; - a fragment of 1680 bp in size, having coordinates from 699 to 2378 bp and containing an artificial gene encoding the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus without the transmembrane and cytoplasmic domain with the addition of the trimerizing domain of the T4 phage with the ATG initiation codon; - a fragment of 135 bp in size, having coordinates from 2385 to 2519 bp and being the 3'UTR sequence of human β-globin; - a fragment of 110 bp in size; - a poly(A) tail of 100 nucleotides in length with an internal linker 30(A)GCATATGACT70(A), having coordinates from 2526 to 2636 bp; - a 6 bp Bso31I restriction endonuclease site with coordinates from 2642 to 2647 bp.

2. A polynucleotide mRNA vaccine that induces specific antibodies to the influenza virus, having the nucleotide sequence of SEQ ID NO: 2, obtained using the pVAX-C3-H1-PolyA DNA template according to claim 1, and containing an open reading frame encoding the amino acid sequence of SEQ ID NO: 3 of the hemagglutinin immunogen protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus and ensuring the synthesis and secretion of the said protein in the body of mammals, 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 with a length of 100 nucleotides, 5' and 3' untranslated regions (UTR) before and after the open reading frame, respectively.

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