WO2021206581A1 - Vaccin basé sur une construction génétique contre une infection à coronavirus - Google Patents

Vaccin basé sur une construction génétique contre une infection à coronavirus Download PDF

Info

Publication number
WO2021206581A1
WO2021206581A1 PCT/RU2020/000257 RU2020000257W WO2021206581A1 WO 2021206581 A1 WO2021206581 A1 WO 2021206581A1 RU 2020000257 W RU2020000257 W RU 2020000257W WO 2021206581 A1 WO2021206581 A1 WO 2021206581A1
Authority
WO
WIPO (PCT)
Prior art keywords
genetic construct
polynucleotide
vaccine
sequence
dna
Prior art date
Application number
PCT/RU2020/000257
Other languages
English (en)
Inventor
Ilya Vladimirovich DUKHOVLINOV
Ekaterina Alekseevna FEDOROVA
Nikolai Nikolaevich KOLMAKOV
Evgenii Leonidovich CHIRAK
Aleksei Victorovich ALEKSEEV
Original Assignee
Dukhovlinov Ilya Vladimirovich
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dukhovlinov Ilya Vladimirovich filed Critical Dukhovlinov Ilya Vladimirovich
Publication of WO2021206581A1 publication Critical patent/WO2021206581A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to molecular biology, biotechnology, medicine and can be used to prevent or treat coronavirus infection.
  • Coronaviruses are a large family of viruses that cause illness from a common cold to more serious illnesses, such as the Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV).
  • MERS-CoV Middle East respiratory syndrome
  • SARS-CoV severe acute respiratory syndrome
  • the new coronavirus (nCoV, COVID-19, SARS-CoV-2) is a new strain that has never been detected in humans before
  • 2019-nCoV is a virus that has a serious impact on public health, the economics, and socio-political issues. Containment of COVID-19 should be the top priority for all countries
  • Randomised controlled trial is performed using remdesivir in severe course of COVID-19 (NCT04257656).
  • NCT04257656 remdesivir in severe course of COVID-19
  • Treatment is also carried out using traditional medicine.
  • the purpose of the present invention is to create a vaccine against coronavirus which is safe, quick and easy to produce and use, which would be effective and low-cost, and which can be used both for the prevention and treatment of coronavirus, especially the new coronavirus.
  • fragments of coronavirus antigens — of M, S, N, E proteins of the new coronavirus — have been selected.
  • a fusion protein has been developed on their basis. The components of the fusion protein are connected by flexible bridges, which allows to achieve the full functioning of each component of the protein.
  • the developed protein is also effective against SARS due to cross-activity.
  • the developed fusion protein doesn’t have homology with the known proteins of other organisms except coronavirus, which ensures the specificity of their action.
  • the vaccine is important to be specifically immunogenic and protective against coronavirus, and also safe from the point of view that it will not induce complications caused by coronavirus, as well as other side effects.
  • SI subunit aaOl - aa550
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting enzyme 2
  • a vaccine is proposed by us for coronavirus infection prevention or treatment in a human or animal containing at least one genetic construct as an active agent, in an effective amount, and also a physiologically acceptable carrier and a buffer solution.
  • the genetic construct encodes the fusion protein described above.
  • DNA as the active substance, which exists in the form of an episome and does not integrate into the genome, from which the protein is synthesized and then, in one of the variants, is secreted from the cell.
  • the technical result from the use of a vaccine based on a genetic construct according to the invention also consists in increased efficiency due to the fact that the fusion protein is synthesized from a genetic construct for a long time, and from several days to several weeks, depending on the structure of the used genetic construct. It is also achieved by the structure of the encoded fusion protein.
  • the technical result also consists in simplifying and cheapening the production of vaccines by avoiding the production and purification processes of protein preparations in vitro due to the fact that protein synthesis occurs in vivo.
  • the production, purification and storage of DNA preparations are economically more profitable than of protein ones, since the former are more stable, they can be produced in large quantities at a lower cost.
  • the technical result also consists in expanding the spectrum of active substances and vaccines based on them against coronavirus.
  • this active substance or vaccine will allow protection against coronavirus.
  • the mentioned technical result is achieved by using the active substance or the vaccine of the present invention.
  • a method is proposed by us for producing the vaccine according to the invention, namely that the genetic construct — a linear DNA fragment — is amplified using PCR, purified and mixed with a physiologically acceptable carrier and a buffer solution.
  • the technical result of using the method of obtaining the developed vaccine is to obtain the vaccine as soon as possible. So, the process of obtaining a purified preparation of genetic construct - a linear DNA fragment - the active agent of the vaccine - takes only a few hours (from 2-3 hours)! No other type of coronavirus vaccine can be obtained in such a short time.
  • the technical result also consists in the simplicity of the vaccine production. It is achieved by the fact that the production of such a vaccine does not require the development of a multi-stage production technology, and a long adjustment of the process steps, it requires a minimal set of reagents that are readily available, approvals for working with pathogenic organisms are not required, as well as significant investments in the construction and refurbishment of premises and equipment. Such a method can even be carried out in a standard DNA laboratory.
  • a genetic construct is proposed for the expression of the polynucleotide according to the invention in the cells of a target organism, including such a polynucleotide and other elements allowing to realize the indicated purpose — to express the polynu- cleotide in the cells of the target organism.
  • the target organism is a human or animal.
  • the genetic construct can be represented by a plasmid DNA for transient expression in mammalian cells, a virus-based vector, a linear DNA fragment containing the polynucleotide according to the invention.
  • the technical result consists mainly in the induction of an immune response which does not cause side effects associated with the use of other fragments of S protein of the new coronavirus, or of such full-sized protein in genetic constructs or strains of coronavirus.
  • the technical result consists in a significant acceleration and simplification of obtaining the active substance of the vaccine against coronavirus - a genetic construct. This is achieved, first of all, by the safety of the genetic constructs types being used and, accordingly, by the absence of strict requirements for working with them.
  • the technical result also consists in the fact that the genetic construct serves as a matrix for PCR to produce a vaccine according to the invention.
  • the technical result consists in increasing the duration of the encoded protein synthesis and is achieved by the fact that the nucleotide sequence encoding the target protein contains elements that determine mRNA stability and, accordingly, increase the half-life of the mRNA, resulting in the synthesis of the protein from one mRNA molecule carried out more times, and also in the synthesized protein amount increase; and also by the fact that that the nucleotide sequence of the fusion protein is optimized in codon composition for expression in the cells of the target organism — of a human and animal, mammals, resulting in more intensive protein synthesis.
  • the technical result also consists in expanding the spectrum of genetic constructs for the synthesis of immunogenic coronavirus antigens in an organism.
  • a polynucleotide is proposed for expression in the cells of a target organism, encoding a fusion protein set forth as SEQ ID NO.:l or 2 amino acid sequence comprising coronavirus M, S, N, E proteins fragments connected by flexible bridges.
  • the polynucleotide may contain a fragment encoding a heterologous secretory sequence.
  • the technical result of using the polynucleotide proposed by us is mainly in obtaining a genetic construct, due to the use of which in the vaccine a specific protective immune response to the coronavirus, mainly to the new coronavirus, is formed in a human or animal.
  • a prokaryotic recombinant cell - producer - is proposed for producing a genetic construct according to the present invention.
  • the technical result of using the producer of the developed genetic construct is in the stable storage and reproduction of the genetic construct, as well as in obtaining the developed genetic construct.
  • a polynucleotide, a genetic construct, a producer of a genetic construct, a method for producing a vaccine all these objects are used to produce a vaccine according to the present invention.
  • a group of inventions is proposed: a polynucleotide, a genetic construct, a recombinant cell, a vaccine based on at least 1 genetic construct for the prevention or treatment of coronavirus, and a method of its production.
  • a fusion protein has been developed which includes immunogenic fragments of the coronavirus M, S, N, E proteins connected by flexible bridges, the protein is characterized by SEQ ID NO.: 1 or 2 amino acid sequence.
  • the peculiarity of the protein is that it does not have homology with proteins of other organisms than coronavirus, and also does not contain a binding site for ACE2. This provides an important advantage of a vaccine based on it - an additional aspect of the vaccine safety.
  • Such protein will be synthesized in the cells of a target organism — a human or an animal — from the proposed genetic construct containing the proposed polynucleotide.
  • a polynucleotide for synthesis in the cells of a target organism encoding a developed fusion protein is proposed.
  • the amino acid sequence of a protein is known, one skilled in the art will be able to obtain the nucleotide sequence.
  • This can be a sequence either based on fragments of the native nucleotide sequence of viral proteins, or artificially optimized by codon composition. Codon optimization can be carried out independently, using information on the frequency of codons occurrence in a producer, for example, in a database [for example, Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res . 2000 Jan 1; 28 (1): 292], or using specialized programs, for example, available at http://www.encorbio.com/protocols/Codon.htm or molbiol.ru or other resources.
  • a polynucleotide may not contain (e.g., SEQ ID NO.: 1 or 4) or contain (e.g., SEQ ID NO.:5 or 6) a heterologous secretory sequence optimized in codon composition for the target organism - for example, such of TPA (tissue-type plasminogen activator), hGH (human growth hormone), IGF (insulin-like growth factor), EPO (erythropoietin), but not limited to such.
  • TPA tissue-type plasminogen activator
  • hGH human growth hormone
  • IGF insulin-like growth factor
  • EPO erythropoietin
  • the obvious advantage of the proposed vaccine is the induction of an immune response profile, where the cytotoxic response is manifested significantly, in contrast to the immune response that forms on the introduced protein immunogen or a virus particle.
  • both polynucleotide variants are suitable.
  • each construct includes, in addition to the polynucleotide, elements allowing to realize the indicated purpose — to express the polynucleotide. Due to the fact that the genetic construct from which the antigen is synthesized is the active substance of the vaccine, it is possible to obtain an immune response profile in which the cytotoxic immune response will play a significant role, however, an antibody response will also be formed. Also, due to the duration of the antigen synthesis in a body and the absence of infectivity, the expectation of causing the formation of an immune response which is adequate, with the formation of immunological memory, increases. The molecule itself also has an adjuvant effect.
  • genetic construct primarily a linear DNA fragment or a recombinant vector is meant, which can be represented by a viral or plasmid vector.
  • the recombinant vector must comprise elements essential for the organisms of its maintenance and use, together with the corresponding regulatory sequences.
  • Regulatory sequences are nucleotide sequences which can affect gene expression at the level of transcription and/or translation, as well as mechanisms that ensure the existence and maintenance of the recombinant vector functioning.
  • An origin of replication, for maintaining in a cell with medium and preferably high copy number, and a marker gene for a producer strain selection are essential to the prokaryotic system.
  • Bacterial elements of a plasmid DNA should not adversely affect expression in mammalian cells and cause a side effect of using the plasmid DNA.
  • a suitable origin of replication is represented by pMl (der.), ColEl (der.), and FI, pUC, and FI, but is not limited to such.
  • a suitable marker gene is represented by a reporter gene or antibiotic resistance gene, for example, ampicillin, mainly kanamycin, but is not limited to such.
  • Elements for efficient functioning, for expression of the encoded gene, - a promoter, including transcription initiation signals, mRNA leader sequence, a termination sequence, regulatory sequences, - are essential elements of a recombinant vector for use in mammals.
  • a promoter is an important component of a vector that triggers the expression of a gene of interest.
  • Human CMV / immediate early or CMV-chicken-b actin (CAGG) promoter are classic promoters for recombinant vectors - drug components.
  • CMV promoters are used for most DNA vaccines, as they mediate high levels of constitutive expression in a wide range of mammalian tissues [Manthorpe M, Comefert-Jensen F, Hartikka J, et al. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum. Gene Ther. 1993; 4 (4): 419-431] and do not inhibit downstream expression.
  • CMV promoter is changed, for example, by incorporating HTLV-1R-U5 downstream the cytomegalovirus promoter or when using the chimeric SV40-CMV promoter [Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol. Adv. 2009; 27 (4): 353-370].
  • Tissue-specific host promoters which allow avoiding constitutive expression of antigens in inappropriate tissues are an alternative to CMV promoters [Cazeaux N, Bennasser Y, Vidal PL, Li Z, Paulin D, Bahraoui E.
  • the promoter can be with corresponding regulatory sequences from natural promoters with its own regulatory elements (CaM kinase P , CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of the kainate receptor, and subunit B of glutamate receptor, and others), or from synthetic promoters with regulatory sequences to obtain the desired expression rate (ratio of duration and expression level) of a target gene at the transcription level.
  • CaM kinase P CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of the kainate receptor, and subunit B of glutamate receptor, and others
  • Regulatory sequences are nucleotide sequences that may affect gene expression at the level of transcription and/or translation, as well as mechanisms that ensure the existence and maintenance of the genetic construct functioning.
  • the recombinant vector, the plasmid or viral DNA of the present invention contains at least one of the abovementioned regulatory sequences, depending on the DNA variant, based on the selection of promoter and the desired expression parameters of the target gene. Based on the existing level of technology and known and obvious variants of such elements and their use, die recombinant vector according to the present invention may contain any combinations meeting the above-mentioned criteria, using which synthesis of the developed fusion gene is performed from it, in cells of a target organism - a human or an animal. When using a silencer or an insulator in the construct, it is possible to regulate the expression of the target gene.
  • the recombinant vector of the present invention in one embodiment additionally comprises such a regulatory element.
  • the recombinant vector of the present invention also contains such an important element as mRNA leader sequence containing translation initiation signals, a start codon.
  • Translation initiation signals is Kozak sequence in eukaryotes [Kozak M.
  • the recombinant vector also contains a site, mainly different sites, for cloning of the target gene, for die correct target gene orientation in the recombinant vector, and a site, mainly sites, for primers annealing for its sequencing.
  • the recombinant vector also contains the polynucleotide described above.
  • the recombinant vector also contains a termination sequence containing sequentially a stop codon, a 3' untranslated region with a polyadenylation signal and site, a stop codon; due to which mRNA stability is maintained, and transcription is terminated appropriately, and mRNA is exported from the nucleus.
  • Gene expression can be influenced by changing the termination sequence, which is necessary for mRNA stability maintaining, for proper transcription termination and mRNA export from the nucleus, including by its shortening.
  • the bovine growth hormone transcriptional terminator sequence is used [Montgomery DL, Shiver JW, Leander KR, et al. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors.
  • the recombinant vector according to the present invention may contain any termination sequence meeting the above-mentioned criteria, using which synthesis of a target protein is performed in cells of a human or animal.
  • An example of a termination sequence for mammalian cells is that of bovine growth hormone (BGH).
  • Vectors tested on humans containing the elements described above with appropriate regulatory sequences, possibly modified to meet the above-mentioned criteria are preferred recombinant vectors for use in a human, thereby reducing the number of studies required to register a drug.
  • FDA guideline (2007) states that studies of the biodistribution of a substance after its introduction into an organism can be canceled for DNA vaccines produced by cloning of a new gene into a plasmid vector, for which acceptable biodistribution and integration profiles have been previously documented.
  • a WHO guideline (2007) states that studies of biological distribution and conservation are required if significant experience with an almost identical or similar product is not yet available.
  • EMEA guideline (2006) states that experience with a vector system will allow to optimize and focus on preclinical studies.
  • pcDNA3.1 (+) vector elements that provide expression of the gene causing antibiotic resistance to neomycin, as well as plasmid replication in the form of episome in mammalian cells, do not function in the target organism, since such cells - cells of the organism of such plasmid DNA introduction - do not contain the large SV40 T-antigen.
  • the pcDNA3.1 (+) vector does not contain transposons, as well as elements responsible for the transmissibility. Such DNA does not integrate into the genome and does not replicate in mammalian cells. All this certainly indicates the safety of drugs based on it.
  • a virus-based vector in which the polynucleotide is cloned a safe vector is meant, such as, i.e. a recombinant adeno-associated virus, but not limited to such.
  • a DNA fragment is meant containing a promoter, an mRNA leader sequence, the polynucleotide described above, a termination sequence, including 1 or 2 stop codons, regulatoiy sequences. Specification described above is applicable to the elements. Such elements are crucial, but other elements may also be contained. A fusion protein is synthesized from such a fragment in the cells of a target organism. The arrangement sequence of the described elements is clear to the average person skilled in the art.
  • a method for producing a vaccine against coronavirus based on a linear DNA fragment which is in amplification of a linear DNA fragment using a polymerase chain reaction - PCR, purification from impurities, and mixing with a physiologically acceptable carrier and buffer solution.
  • This method of obtaining the vaccine is the fastest, and not a single vaccine registered in Russia is produced in this way.
  • only such a way of producing the vaccine meets the challenge of nature.
  • a vaccine based on other genetic constructs can be obtained by cultivating microorganisms - a prokaryotic recombinant producer cell, followed by isolation and purification of the genetic construct. This method will take more time (about 4-5 days), however, it is also fast and applicable in the realities of the new coronavirus.
  • Vaccines based on proteins or mRNA, or attenuated or inactivated viruses - are much more complicated and longer to be produced, especially the first period - the development of a technology for producing an active agent - a protein in a monomeric, active form that does not cause side effects; the production of mRNA itself - a molecule that is less stable than DNA; work with a dangerous virus.
  • a prokaryotic recombinant cell is proposed for obtaining the genetic construct.
  • a prokaryotic producer is represented, for example, by Escherichia coli, Bacillus subtilis. In one embodiment, this is Escherichia coli DH10B/R bacterial strain, containing the pcDNA3.1 (+) vector, which contains the nucleotide sequence of SEQ ID NO.:3-6.
  • a vaccine is proposed for the prevention or treatment of coronavirus, containing the above-described genetic construct as an active agent in an effective amount, as well as a physiologically acceptable carrier and a buffer solution.
  • Pharmaceutically acceptable carriers and buffers are known from the prior art and include those described in various texts, such as, for example, Remington's Pharmaceutical Sciences. Both a human and an animal, inter alia of domestic or farm animals, can be a consumer of the vaccine.
  • the authors propose parenteral administration of the vaccine, and in one of the options it contains a genetic construct with and without secretory sequence. Also, the vaccine can be administered using electroporation to increase the efficiency of delivery to cells for polynucleotide expression.
  • Example 1 Modeling of a fusion protein
  • the modelled fusion protein in one embodiment consists of 422 aa, with methionine at the N-terminus - 423 aa, is represented by the amino acid sequence SEQ ID NO.:l.
  • SEQ ID NO.:l amino acid sequence SEQ ID NO.:l.
  • Analysis of the amino acid sequence of this protein using the ProtParam program http.V/au.expasy.org/tools/protparam.html has shown that the fusion protein has a molecular mass of 46.4 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 100 hours. With methionine at the N-terminus - 46.5 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 30 hours.
  • the fusion protein When adding the secretory sequence of IGF (GKISSLPTQLFKCCFCDFLK) , with methionine at the N-terminus, the fusion protein consists of 443 aa, has a molecular weight of 48.8 kDa, pi 9.55, the protein is stable.
  • the hGH secretory sequence When adding the hGH secretory sequence
  • the fusion protein consists of 448 aa, molecular mass is 49.1 kDa, pi 9.57, the protein is stable.
  • the fusion protein consists of 445 aa, molecular mass is 48.8 kDa, pi 9.53, the protein is stable.
  • the modelled fusion protein in one embodiment consists of 424 aa, with methionine at the N-terminus - 425 aa, is represented by SEQ ID NO.:2 amino acid sequence.
  • SEQ ID NO.:2 amino acid sequence Analysis of the amino acid sequence of this protein using the ProtParam program (http://au.expasy.org/tools/protparam.html) has shown that the fusion protein has a molecular mass of 46.5 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 100 hours. With methionine at the N-terminus - 46.6 kDa, pi 9.61, the protein is stable, the half-life in mammals is about 30 hours.
  • the fusion protein When adding the secretory sequence of IGF, with methionine at the N-terminus, the fusion protein consists of 445 aa, molecular mass is 48.9 kDa, pi 9.55, the protein is stable.
  • the fusion protein When adding the secretion sequence of hGH, with methionine at the N-terminus, the fusion protein consists of 450 aa, molecular mass is 49.2 kDa, pi 9.57, the protein is stable.
  • the fusion protein When adding the secretory sequence of TPA (MDAMKRGLCCVLLLCGAVFVSPS), the fusion protein consists of 447 aa, has a molecular mass of 48.9 kDa, pi 9.53, the protein is stable.
  • a protein is synthesized with methionine at the N-terminus, since translation always starts from the start codon. Further, methionine can be cleaved naturally, for example, if the protein is secreted, as part of the secretory peptide.
  • Example 2 Obtaining highly purified genetic constructs according to the invention
  • a nucleotide sequence of a gene collinear to the amino acid sequence of the fusion protein encoded by it was calculated, set forth as SEQ ID NO.:l or 2, with the target gene flanking by restriction sites, as well as with the addition of the Kozak sequence before the start codon to initiate translation, and after the start codon - in a part of embodiments - a signal sequence, for example, of TPA, hGH, IGF, EPO, or represented by aa MLLLLLLLLALALA, for secretion of the synthesized protein from an eukaryotic cell, with simultaneous optimization by codon composition for expression in human cells, the tool on the site molbiol.ru was used. For example, sequences set forth as SEQ ID NO.:3,4 and set forth as SEQ ID NO.:6, respectively were obtained.
  • nucleotide sequences of the new coronavirus were taken to obtain a genetic construct, since these sequences are expressed in mammals, the remaining fragments were optimized as described above. For example, the nucleotide sequence set forth as SEQ ID NO.:5 was obtained.
  • the mixture was heated at +95°C for 10 min and purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 mpi (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.
  • E. coli cells of strain DH10B / R (F-mcrA, D (mrr-hsdRMS-mcrBC), cp80dlacZAM 15, AlacX74, deoR, recAl, endAl, araD139, D (ara, leu) 769, galU, galKX-, rpsL, nupG) were transformed with the obtained plasmid DNA by electroporation using a MicroPulser electroporator (BioRad).
  • This strain does not contain methylase, which allows to minimize the possibility of mutations occurrence in the DNA, including the gene cloned in the plasmid maintained in this strain.
  • 1 m ⁇ of dialyzed ligase mixture was added to 12 m ⁇ of competent cells, placed between the electrodes of an electroporation cuvette and processed by a current pulse.
  • the cells were placed in 1 ml of SOC broth (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgC12, 10 mM MgS04, 20 mM glucose) and incubated for 40 min at + 37°C.
  • SOC broth 2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgC12, 10 mM MgS04, 20 mM glucose
  • Clones of E. coli cells containing the obtained plasmid DNA were detected on a selective medium containing LB agar, 50 pg/ml of kanamycin, or ampicillin (for plasmid DNA based on pcDNA3.1+).
  • Plasmid DNA was isolated from the grown clones. Plasmid DNA was isolated using the Wizard Minipreps DNA Purification System kit (Promega, USA). Purified recombinant plasmid DNA was checked by sequencing.
  • a PCR reaction was performed, then the reaction mixture was purified from free labeled ddNTP according to the instructions for the BigDye X-Terminator Purification Kit (Applied Biosystems, USA) and the products of the sequencing reaction were separated using an Applied Biosystems 3500 / 3500xL Genetic Analyzer capillary sequencer (Applied Biosystems, USA) and 3500/3500xL Genetic Analyzer Polymer “POP-6 TM” reagent (Applied Biosystems, USA).
  • E. coli cell clones were selected containing the full-length sequences of the target genes in the plasmids - DNA sequences encoding the developed fusion proteins. Such clones were used as the producer strain of the plasmids according to the present invention. In one embodiment, this is Escherichia coli DH10B/R bacterial strain, containing the pcDNA3.1(+) vector, which contains the nucleotide sequence of SEQ ID NO.:3-6.
  • Protein impurities in the resulting plasmid DNA preparations were also quantified using the microBCA assay [Smith, P.K., et all, Measurement of protein using bicin- choninic acid. Analyt. Biochem. 150, 76-85 (1985)], measuring the optical density of the resulting colored protein complexes with copper and bicinchoninic acid with a wavelength of 562 nm.
  • the sensitivity of the microBCA assay method is 0.5-20 mg/ml of protein. The concentration of total protein in none of the studied plasmid DNA preparations exceeded the norm.
  • the content of bacterial lipopoly saccharide in plasmid DNA preparations was also determined using a gel-thrombus version of the LAL test with a sensitivity of >0.25 EU/ml (ToxinSensor, GenScript, USA).
  • a Limulus polyphemus horseshoe crab amebocytes lysate was used as the LAL reagent.
  • the LAL reagent specifically reacts with bacterial endotoxins; as a result of the enzymatic reaction, the reaction mixture changes in proportion to the concentration of endotoxin.
  • the results were evaluated by the presence or absence of a dense clot at the bottom of the tube by inverting the tube.
  • a gel clot did not form when examining a sample diluted 10 times, for all obtained plasmid DNA preparations, i.e. when the sensitivity of the method is 2.5 EU/ml, which, given the concentration of plasmid DNA in the sample, indicates an acceptable rate of endotoxin removal.
  • the yield of plasmid DNA ranged from 3.1 mg to 4.7 mg per liter of culture medium.
  • the synthesized genes were cloned into a vector based on the pAAVK-EFla-MCS (System Biosciences (SBI)) adeno-associated virus, on the basis of which a producer strain of this vector was created using E. coli cells (RecA-). Bacillus subtilis bacteria cells were also successfully used as producer.
  • SBI System Biosciences
  • a vector was isolated for use in mammals, all according to the instructions for the vector.
  • the yield of the vector ranged from 2 mg to 3.2 mg per liter of culture medium.
  • the plasmid DNA obtained according to 2.1.2., or the viral vector of item 2.2., or a fragment amplified from them was used.
  • the DNA fragment was amplified containing a promoter, an mRNA leader sequence, and also regulatory sequences for these elements, a polynucleotide - a fusion gene, a termination sequence.
  • the solution may contain also other elements, and the elements indicated in the previous sentence are the key ones.
  • Amplification of this sequence was carried out in a volume of 50 m ⁇ , in 650 m ⁇ thin- walled polypropylene tubes containing 5 m ⁇ of lOx Taq buffer (700 mM Tris-HCl, pH 8.6/25°C, 166 mM (NH4) 2S04), 5 m ⁇ of MgC12 (1.25 mM), 1 m ⁇ of dNTP, 31.5 m ⁇ of water, 1 m ⁇ of forward and 1 m ⁇ of reverse primers, 5 m ⁇ of plasmid DNA and 0.5 m ⁇ Taq polymerase (Fermentas, Lithuania).
  • lOx Taq buffer 700 mM Tris-HCl, pH 8.6/25°C, 166 mM (NH4) 2S04
  • 1 m ⁇ of dNTP 31.5 m ⁇ of water
  • 1 m ⁇ of forward and 1 m ⁇ of reverse primers 5 m ⁇ of
  • the reaction mixture was warmed for 5 minutes at 95°C for DNA denaturation.
  • 30 m ⁇ of Bayol F mineral oil (Sigma, USA) were layered onto a reaction mixture of 50 m ⁇ volume.
  • the amplification reaction was carried out in a C1000 Thermal Cycler (Bio-Rad, USA) thermal cycler. 35 cycles were carried out: 95°C - 20 sec, 50-62°C (depending on the primers) - 20 sec, 72°C - 1 min.
  • an additional cycle was carried out: 5 min at 72°C.
  • Amplified DNA fragments were concentrated and purified using preparative electrophoresis in 0.8-1.2% agarose gel (Gibko BRL, USA). A sample of the mixture after PCR was mixed with 6x buffer (0.25% bromophenol blue, 30% glycerin) (ThermoScientific, USA) and loaded into the gel wells, 18 m ⁇ per well. Electrophoresis was carried out in a horizontal apparatus in TAE buffer (40 mM Tris-acetate, 2 mM EDTA pH 8.0, 0.5 mg/ml ethidium bromide) at a voltage of 5-10 V/cm.
  • TAE buffer 40 mM Tris-acetate, 2 mM EDTA pH 8.0, 0.5 mg/ml ethidium bromide
  • the result of DNA separation was recorded in transmitted UV light (302 nm) of the Macrovue transilluminator (LKB, Sweden).
  • the length of the amplified fragment was determined by the logarithmic dependence of DNA mobility on the length of the fragments in the marker.
  • markers a proprietary mixture of DNA fragments “GeneRuler 1000 bp DNA Ladder” (Fermentas, Lithuania) was used.
  • a plot of agarose containing DNA strip of the required size was excised and the DNA fragment was purified using the DNA & Gel Band Purification Kit (GE Healthcare, UK) according to the instructions.
  • the isolated genetic construct was used in mammals.
  • mice were injected intramuscularly with pcDNA3.1 (+)seqidno5 plasmid DNA in the amount of 50 mg in PBS, animals not injected with any substance were used as a negative control. Animals were sacrificed on days 2, 5, and 7, blood was taken to prepare the serum. Some animals of all groups were not withdrawn from the experiment to assess the safety of the vaccine.
  • the obtained serums were analyzed by electrophoresis in PAGE followed by transfer of proteins to the nitrocellulose membrane and visualization of the target protein using chemiluminescence.
  • a ‘sandwich’ was assembled on a Semi-phor TE70 Semi-dry transfer unit apparatus for horizontal transfer: 6 Whatman papers fragments soaked in Solution I, 3 Whatman papers fragments soaked in Solution II, BioRad nitrocellulose membrane soaked in Solution P, PAAG after electrophoresis, which was located on the membrane and immobilized, 9 fragments of Whatman paper soaked in Solution PI.
  • the lid of the device was closed, the power was connected, 100 V for an hour,
  • Constant current PS unit model PS50 was the current source.
  • a fusion protein a specific antibody — a secondary antibody — horseradish peroxidase.
  • the membrane was treated with 1% dry skim milk in phosphate-buffered saline with the addition of 0.5% Tween-20 (125 m ⁇ of a 20% aqueous solution in 50 ml of milk), incubation was carried out for 15 minutes at room temperature. Then the mixture was poured, antibodies were added to the coronavirus proteins - Anti- SARS-CoV-2 spike glycoprotein monoclonal antibody (CABT-RM321) rabbit antibodies to S protein of the coronavirus in 1% skimmed milk at a dilution of 1: 3000, then incubated for 16 hours at +4°C, after which it was heated to room temperature.
  • CABT-RM321 Anti- SARS-CoV-2 spike glycoprotein monoclonal antibody
  • the formed membrane-immobilized complexes were developed using the Amersham ECL Western Blotting Detection Reagent (GE Healthcare) chemiluminescence kit. A mixture of equal volumes of 1 and 2 reagents from this kit was applied to a wet membrane with immobilized complexes of a fusion protein with specific antibody with a secondary antibody with horseradish peroxidase, all was packed between layers of a transparent plastic paper folder (slide). This construct was placed in an X-ray cassette (Kodak). Amersham Hyperfilm ECL X-ray film fragment was glued on top and fixed. The cassette was closed and kept for 12 hours. The film was developed using commercial solutions of developer and fixer (Krok-rentgen) according to the instruction and dried. These actions were carried out in the dark in the light of a red lamp.
  • the ability of the developed genetic construct to express the encoded target gene after introduction into the muscles, as well as the functioning of the domains of the developed fusion protein, in particular, represented by the new coronavirus S and N proteins, were evaluated.
  • the synthesis of the fusion protein was demonstrated already on the second day after the introduction of the developed genetic construct carrying the gene encoding it, and within a week, with the maximum level of synthesis being detected 4-5 days after immunization. This suggests that the used genetic construct according to the invention allows the expression of the target gene in mammalian cells.
  • N domain from 216 to 360 amino acid residues of N protein of the new coronavirus [0175] ⁇ 220>
  • Val Thr Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg lie Asn Trp [0182] 1 5 10 15
  • N domain from 216 to 360 amino acid residue of N protein of the new coronavirus [0252] ⁇ 220>
  • He Thr Gly Gly lie Ala He Ala Met Ala Cys Leu Val Gly Leu Met [0261] 2025 30

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Pulmonology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne la biologie moléculaire, la biotechnologie, la médecine et peut être utilisée pour prévenir et traiter une infection à coronavirus, principalement provoquée par le 2019-nCoV. Un vaccin est proposé basé sur une construction génétique codant pour une protéine de fusion, comprenant des fragments de protéines de nouveau coronavirus M, S, N, E. La vitesse et la simplicité de préparation (de 2-3 heures à 4-5 jours), la sécurité sont les avantages du vaccin développé, du fait de la nature de la molécule de la substance active, ainsi que l'absence d'un site de liaison ACE2 dans la protéine de fusion synthétisée à partir de la construction génétique, et l'absence de son homologie avec des protéines de l'organisme, l'induction du profil de réponse immunitaire, représenté dans une large mesure par la réponse immunitaire cytotoxique, en plus de la réponse humorale. Le vaccin peut être administré de manière standard ou par électroporation.
PCT/RU2020/000257 2020-04-05 2020-06-02 Vaccin basé sur une construction génétique contre une infection à coronavirus WO2021206581A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2020112937A RU2747762C1 (ru) 2020-04-05 2020-04-05 Вакцина для профилактики или лечения коронавирусной инфекции на основе генетической конструкции
RU2020112937 2020-04-05

Publications (1)

Publication Number Publication Date
WO2021206581A1 true WO2021206581A1 (fr) 2021-10-14

Family

ID=75919813

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2020/000257 WO2021206581A1 (fr) 2020-04-05 2020-06-02 Vaccin basé sur une construction génétique contre une infection à coronavirus

Country Status (2)

Country Link
RU (1) RU2747762C1 (fr)
WO (1) WO2021206581A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957410A (zh) * 2022-06-08 2022-08-30 华兰基因工程有限公司 一种κ株2019-nCoV的表面蛋白受体结合区制备方法
US11547673B1 (en) 2020-04-22 2023-01-10 BioNTech SE Coronavirus vaccine
WO2023123722A1 (fr) * 2021-12-31 2023-07-06 中国科学院微生物研究所 Polypeptide anti-coronavirus, ses dérivés et son utilisation
WO2023109979A3 (fr) * 2021-12-16 2023-08-03 浙江迪福润丝生物科技有限公司 Protéine de fusion affichant une protéine s du sars-cov-2, virion recombinant, et utilisation de la protéine de fusion et du virion recombinant
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006056103A1 (fr) * 2004-11-26 2006-06-01 Dna Shuttle Biopharm Co., Ltd. Vecteur d'expression codant des particules de type coronavirus
TW201729835A (zh) * 2015-10-22 2017-09-01 現代公司 呼吸道病毒疫苗

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2280288C2 (ru) * 2004-09-16 2006-07-20 Вирусологический центр НИИ Микробиологии Министерства обороны Российской Федерации Способ моделирования заболевания тяжелого острого респираторного синдрома у экспериментальных животных

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006056103A1 (fr) * 2004-11-26 2006-06-01 Dna Shuttle Biopharm Co., Ltd. Vecteur d'expression codant des particules de type coronavirus
TW201729835A (zh) * 2015-10-22 2017-09-01 現代公司 呼吸道病毒疫苗

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11547673B1 (en) 2020-04-22 2023-01-10 BioNTech SE Coronavirus vaccine
US11925694B2 (en) 2020-04-22 2024-03-12 BioNTech SE Coronavirus vaccine
WO2023109979A3 (fr) * 2021-12-16 2023-08-03 浙江迪福润丝生物科技有限公司 Protéine de fusion affichant une protéine s du sars-cov-2, virion recombinant, et utilisation de la protéine de fusion et du virion recombinant
WO2023123722A1 (fr) * 2021-12-31 2023-07-06 中国科学院微生物研究所 Polypeptide anti-coronavirus, ses dérivés et son utilisation
CN114957410A (zh) * 2022-06-08 2022-08-30 华兰基因工程有限公司 一种κ株2019-nCoV的表面蛋白受体结合区制备方法
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Also Published As

Publication number Publication date
RU2747762C1 (ru) 2021-05-13

Similar Documents

Publication Publication Date Title
RU2747762C1 (ru) Вакцина для профилактики или лечения коронавирусной инфекции на основе генетической конструкции
Sathaliyawala et al. Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines
KR101255870B1 (ko) 재조합 바이러스 벡터를 포함하는 다가 백신
CN110951756A (zh) 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN110269932A (zh) 非洲猪瘟病毒疫苗及其用途
US20240123053A1 (en) Coronavirus vaccine through nasal immunization
Sykes et al. Linear expression elements: a rapid, in vivo, method to screen for gene functions
US20230285535A1 (en) Recombinant Pseudorabies Virus and Vaccine Composition thereof
WO1995027069A1 (fr) Arn d'alphavirus utilise comme vecteur de vaccins
Cai et al. Production of pharmaceutical-grade plasmids at high concentration and high supercoiled percentage
CN111808176B (zh) 牛疱疹病毒抗原组合物及其应用
Xiong et al. Immunization against the transgene but not the TetON switch reduces expression from gutless adenoviral vectors in the brain
WO2017113050A1 (fr) Procédé de préparation d'une protéine de capside de circovirus porcin de type 2 et composition pharmaceutique la contenant
JPH02500327A (ja) マイコバクテリアに対する遺伝子操作ワクチン
WO2022049409A1 (fr) Diagnostic express pour sars-cov-2
WO2022119481A1 (fr) Vaccin pour le traitement et la prévention d'une infection par coronavirus
WO2019059817A1 (fr) Vaccin à base de protéine de fusion et d'adn plasmidique
CN116904489B (zh) 一种鸭坦布苏病毒核酸疫苗及应用
CN106636138A (zh) 一种针对羊种3型布鲁氏菌感染的dna疫苗及其制备方法
CN112592410B (zh) 犬腺病毒基因工程亚单位疫苗、其制备方法及应用
KR0178110B1 (ko) 대장균에서 발현된 인간유래 한탄바이러스의 뉴클레오캡시드 단백질을 이용한 백신
WO2023202711A1 (fr) Vaccin à arnm basé sur un nouveau coronavirus
CN107827986A (zh) 猪O/Mya98和O/PanAsia型口蹄疫基因工程灭活疫苗
TWI633114B (zh) 豬第二型環狀病毒之外鞘蛋白質的製備方法及含該外鞘蛋白質的醫藥組合物
TWI659966B (zh) 豬第二型環狀病毒之外鞘蛋白質的製備方法及含該外鞘蛋白質的醫藥組合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20929751

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20929751

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20929751

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 20929751

Country of ref document: EP

Kind code of ref document: A1