WO2022087856A1 - Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine rbd, et son utilisation - Google Patents

Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine rbd, et son utilisation Download PDF

Info

Publication number
WO2022087856A1
WO2022087856A1 PCT/CN2020/124173 CN2020124173W WO2022087856A1 WO 2022087856 A1 WO2022087856 A1 WO 2022087856A1 CN 2020124173 W CN2020124173 W CN 2020124173W WO 2022087856 A1 WO2022087856 A1 WO 2022087856A1
Authority
WO
WIPO (PCT)
Prior art keywords
rbd
salmonella
novel coronavirus
attenuated salmonella
protein
Prior art date
Application number
PCT/CN2020/124173
Other languages
English (en)
Chinese (zh)
Inventor
华子春
吴乐阳
李霖
Original Assignee
南京吉芮康生物科技研究院有限公司
江苏靶标生物医药研究所有限公司
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 南京吉芮康生物科技研究院有限公司, 江苏靶标生物医药研究所有限公司 filed Critical 南京吉芮康生物科技研究院有限公司
Publication of WO2022087856A1 publication Critical patent/WO2022087856A1/fr

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella

Definitions

  • the invention relates to the field of biotechnology, in particular to a novel coronavirus vaccine antigen presentation system for secreting and expressing RBD domain proteins by attenuated Salmonella and its application.
  • coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most serious challenge civilization has faced in a century, as of September 1, 2020, More than 27 million people have been infected and more than 800,000 people have died.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • 2019-nCoV spreads more widely, more rapidly, and more lethally than SARS virus (Zhu N et al., 2020, N Engl J Med, 382:727-733.). Therefore, in order to effectively prevent the spread of the new coronavirus, it is urgent to develop a safe, efficient and inexpensive SARS-CoV-2 vaccine.
  • Attenuated Salmonella is a widely used bacterial oral vaccine carrier, as well as a natural mucosal immune adjuvant, antigen expression and delivery tool.
  • Genetically engineered recombinant vaccine strains enter the body through M cells in the gut after oral administration (Jensen VB et al., 1998, Infect Immun 66:3758-3766.), and the strains entering the in vivo environment can be processed by antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • the antigenic protein can be effectively secreted into the interior of APC cells, and further effectively decomposed into polypeptide fragments and passed through the histocompatibility complex MHC-I or MHC-II.
  • the pathway is displayed to T-help cells to stimulate the body to generate cellular, humoral and mucosal immune responses against antigenic molecules (Mei Y et al., 2017, Cancer Immunol Res 5:503-514.).
  • a safe, efficient and stable Salmonella secretion expression vector must be constructed to achieve the expression of antigen molecules in Salmonella and It can be effectively secreted into antigen-presenting cells to efficiently induce immune response of the body, which is the problem to be solved by the present invention.
  • the main purpose of the present invention is to construct an efficient attenuated Salmonella expression and secretion vector, and to screen out the combination of novel coronavirus antigenic epitopes that can induce the best immune response, so as to establish a stable expression and high protective efficiency for the attenuated Salmonella.
  • novel coronavirus vaccine antigen is a combination of novel coronavirus antigenic epitopes that have been screened and can induce the best immune response.
  • the combination of novel coronavirus antigenic epitopes that can induce the best immune response is SARS-CoV.
  • S protein Spike protein
  • the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6, which is located at amino acids 319-541 of the overall amino acid sequence of S protein.
  • the intracellular inducible promoter of the antigen-presenting cell is the Salmonella sifB promoter; the gene sequence of the Salmonella sifB promoter is the nucleotide sequence shown in SEQ ID No.4.
  • the intracellular inducible promoter of the antigen presenting cell is the Escherichia coli NirB promoter, or the Salmonella SseA promoter, or the Salmonella SseJ promoter; the gene sequence of the Escherichia coli sifB promoter is shown in SEQ ID No.1.
  • the nucleotide sequence shown, the gene sequence of the Salmonella SseA promoter is the nucleotide sequence shown in SEQ ID No.2, and the gene sequence of the Salmonella SseJ promoter is the nucleotide sequence shown in SEQ ID No.3 acid sequence.
  • the bacterial secretion signal secretion expression system is a Salmonella type III secretion expression system
  • the type III secretion signal is the Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide
  • the Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide is the Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide.
  • SPI-2 Salmonella virulence island 2
  • SPI-2) effector protein SseJ signal peptide The gene sequence of the effector protein SseJ signal peptide is the nucleotide sequence shown in SEQ ID No.5.
  • the plasmid loss prevention element is an AT element sequence
  • the gene sequence of the AT element sequence is the nucleotide sequence shown in SEQ ID No.8.
  • novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreting and expressing the RBD domain protein of the present invention through oral administration of the attenuated virus vaccine antigen presentation system of the novel coronavirus containing the attenuated Salmonella secreting and expressing the RBD domain protein Salmonella induces the production of high titer antibodies in a mouse model.
  • Beneficial effects a novel, efficient, safe, intracellular expression of antigen-presenting cells, low-cost, convenient, novel coronavirus vaccine secretion expression vector that can be used for humans and animals and its application.
  • the present invention can present the antigen to the antigen through the oral route and the unique secretion system after administration.
  • Cells efficiently deliver antigenic proteins.
  • the delivered antigenic proteins can be efficiently processed and presented by antigen-presenting cells, and finally activate/regulate the immune system, produce antibodies, and play the role of vaccines.
  • the present invention provides a novel coronavirus vaccine antigen expression vector using Salmonella type III secretion system signal to guide the novel coronavirus vaccine antigen expression vector, realizes the secretory expression of the novel coronavirus antigen, and induces the body to produce high-titer antibodies in a mouse model.
  • the present invention provides a novel oral bacteria-made novel coronavirus vaccine, which uses a novel attenuated Salmonella as a carrier, and the novel attenuated Salmonella itself can be used as an efficient adjuvant due to its weak pathogenicity. agent, eliminating the need for additional adjuvant addition.
  • the present invention provides a low-cost oral bacteria-made novel coronavirus vaccine.
  • the vaccine uses a novel attenuated Salmonella as a carrier. Due to the self-propagating property of the attenuated strain, the production cost is low and the preparation of the vaccine is greatly reduced. Time, simplifies the operation process, and makes the final product have a stronger price advantage.
  • the present invention provides a novel coronavirus vaccine expressed intracellularly in antigen-presenting cells.
  • the vaccine is only expressed intracellularly in antigen-presenting cells, which can not only effectively produce the immune effect of the vaccine, but also effectively prevent the antigen from The presence of normal tissue and possible side effects, thus enabling greater safety.
  • the present invention provides a safe and controllable oral vaccine system, which uses a novel attenuated Salmonella as a carrier, and can achieve effective and rapid regression by taking conventional Salmonella-sensitive antibiotics.
  • Figure 1 is a schematic diagram of the novel coronavirus RBD domain of the present invention and its binding to receptors;
  • 1A is a schematic structural diagram of the position of the RBD protein domain of the novel coronavirus of the present invention in the S protein;
  • Figure 1B is a schematic diagram of the binding of the novel coronavirus RBD protein domain to its receptor ACE2 protein of the present invention, 1. ACE2 protein; 2. RBD protein domain; 3. Enlarged schematic diagram of the binding region between ACE2 and RBD protein domain.
  • FIG. 2 is a schematic diagram of the construction of different new coronavirus RBD protein domain expression systems of the present invention
  • FIG. 3 is a graph showing the expression and secretion of the new coronavirus RBD protein of the recombinant attenuated Salmonella of different expression systems detected by WB of the present invention
  • 3A is a graph showing the expression and secretion of the new coronavirus RBD protein of the Ah-JP-RBD recombinant attenuated Salmonella of the present invention. 1. Collect the protein expression detection in the bacteria, 2. Collect the culture supernatant, and detect the secretion after protein expression. Ah-JP-RBD recombinant attenuated Salmonella can effectively express and secrete SARS-CoV-2 RBD protein;
  • Figure 3B is a graph showing the expression and secretion of the new coronavirus RBD proteins of four recombinant attenuated Salmonella species of the present invention, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD.
  • Ah-NS-RBD and Ah-SS-RBD recombinant attenuated Salmonella could not induce the expression of 2019-nCoV RBD protein in macrophage RAW264.7 cells, while Ah-JJ-RBD and Ah-BJ-RBD reduced Salmonella virulence can effectively induce the expression and secretion of SARS-CoV-2 RBD protein in macrophage RAW264.7 cells.
  • FIG. 4 is a graph showing the intracellular expression and secretion of the novel coronavirus RBD protein of the recombinant attenuated Salmonella engineered bacteria detected by immunofluorescence of the present invention
  • Recombinant attenuated Salmonella was loaded with plasmids without RBD-HA sequence but with the same other elements; 2. Ah-JJ-RBD recombinant attenuated Salmonella; 3. Ah-BJ-RBD recombinant attenuated Salmonella. DAPI stained nuclei, Ah-1 was stained with Salmonella fluorescent antibody (arrow), and RBD-HA was stained with corresponding fluorescent antibody (arrow).
  • Fig. 5 is the experimental schematic diagram of the recombinant bacteria immunized mice of the present invention.
  • Figure 6 is an evaluation diagram of the antibody production of the new coronavirus RBD protein induced by three recombinant attenuated Salmonella species of the present invention, Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD;
  • the blank control group was Ah-NC recombinant attenuated Salmonella; 2.Ah-JP-RBD recombinant attenuated Salmonella; 3.Ah-JJ-RBD recombinant attenuated Salmonella; 4.Ah-BJ-RBD recombinant attenuated Salmonella.
  • the three recombinant attenuated Salmonella species can effectively induce the production of corresponding antibodies against the new coronavirus RBD protein in mice.
  • Ah-BJ-RBD recombinant attenuated Salmonella in inducing antibodies is significantly better than that of the other three recombinant bacteria, Ah-BJ - Compared with Ah-NC, Ah-JP-RBD and Ah-JJ-RBD recombinant attenuated Salmonella, the ELISA RBD protein titers of RBD attenuated Salmonella increased by 6.03, 3.55 and 2.19 times respectively, indicating that this system is more efficient .
  • the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
  • the following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.
  • the attenuated Salmonella used in the examples is the htrA-deficient VNP20009 attenuated strain (referred to as Ah-1). If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer.
  • RBD is a partial structural region of the new coronavirus S protein ( Figure 1). Structural analysis shows that RBD protein plays a key role in the binding of S protein to ACE2 (angiotensinase 2). Considering the large structural region of S, after extensive and multi-angle bioinformatics analysis and mutual comparison, it is predicted that the RBD located at positions 319-541 of the overall amino acid sequence of S protein contains more epitope sites. After a lot of experimental research, we tried various reported commonly used bacterial secretion systems, secretion signal peptides and matching promoters.
  • the present invention selects and uses strong constitutive promoter J23100, pelB signal peptide.
  • constitutive promoter J23100, pelB signal peptide For inducible expression type III secretion expression plasmids Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, Ah-BJ-RBD plasmids, the present invention attempts to use hypoxic promoter NirB, type III secretion
  • the system-related promoters SseA, SseJ, SifB, and the four plasmids all use the type III secretion system-related signal peptide SseJ.
  • N ends of the above-mentioned 5 plasmids are coupled with the antigen molecule NTD protein sequence by means of Linker, and the gene sequence of the Linker sequence is the nucleotide sequence shown in SEQ ID No.7.
  • the RBD was conjugated to the HA tag ( Figure 2).
  • NirB, sseA, sseJ and sifB promoters, sseJ, PelB signal peptide, and RBD in the S protein region of SARS-COV-2 were all obtained by PCR.
  • the relevant primer sequence is: PNirB P1, and the gene sequence of the PNirB P1 primer is the nucleotide sequence shown in SEQ ID No.9.
  • PNirB P2 the gene sequence of the PNirB P2 primer is the nucleotide sequence shown in SEQ ID No.10.
  • PsseA P1 the gene sequence of the PsseA P1 primer is the nucleotide sequence shown in SEQ ID No.11.
  • PsseA P2 the gene sequence of the PsseA P2 primer is the nucleotide sequence shown in SEQ ID No.12.
  • PsifB P1 5'-caaaatcccttataagaattctgccctaccgctaacatc-3'; the gene sequence of the PsifB P1 primer is the nucleotide sequence shown in SEQ ID No.13.
  • PsifB P2 5'-tgtccaacactcaatggcatccacaagtgattatatgata-3'; the gene sequence of the PsifB P2 primer is the nucleotide sequence shown in SEQ ID No.14.
  • SseJ P1 5'-tatcatataatcacttgtggatgccatttgagtgttggaca-3'; the gene sequence of the SseJ P1 primer is the nucleotide sequence shown in SEQ ID No.15.
  • SseJ P2 5'-gccttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the SseJ P2 primer is the nucleotide sequence shown in SEQ ID No.16.
  • PsseJ-sseJ P1 5'-caaaatcccttataagaatttcacataaaacactagcact-3'; the gene sequence of the PsseJ-sseJ P1 primer is the nucleotide sequence shown in SEQ ID No.17.
  • PsseJ-sseJ P2 5'-GCCttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the PsseJ-sseJ P2 primer is the nucleotide sequence shown in SEQ ID No.18.
  • RBD P2 5'-tctggaacatcgtatgggtacggcgcgtgcagcagttc-3'; the gene sequence of the RBD P2 primer is the nucleotide sequence shown in SEQ ID No.20.
  • Vec P1 5'-tacccatacgatgttccagattacg-3'; the gene sequence of the Vec P1 primer is the nucleotide sequence shown in SEQ ID No.21.
  • Vec P2 5'-gctgcctccacctccgctgc-3'; the gene sequence of the Vec P2 primer is the nucleotide sequence shown in SEQ ID No.22.
  • the plasmid pQE30 with AT element in our laboratory was used as the template.
  • the Linker sequence between the signal peptide and the target protein was obtained by thermal annealing self-linking method. After each fragment was obtained by PCR, the corresponding fragments were assembled by homologous recombination method, and finally various protein expression and secretion vectors of type III secretion system were obtained, including Ah-JP-RBD, Ah-NS-RBD, Ah-SS- RBD, Ah-JJ-RBD, Ah-BJ-RBD.
  • the recombinant vaccine DNA vector was transformed into attenuated Salmonella by electroporation: under sterile conditions, 0.5-5 ⁇ g of the constructed recombinant vector was added to the electroporation competent, and after mixing, it was transferred to a 2 mm electroporation cup. For electric shock, the electric transfer conditions are 1.8kV, 25 ⁇ F, 500 ⁇ . After electroporation, it was coated on a kanamycin plate for screening, and the grown colonies were the constructed recombinant bacteria, and the monoclonal strains were selected for sequencing verification.
  • the obtained recombinant attenuated Salmonella was cultured in kanamycin-resistant liquid LB medium to an OD600 of 0.8-1.0, the cells were collected and the OD600 value was adjusted to about 1.0 with PBS. Place 4 degrees for spare.
  • the macrophage cell line RAW264.7 was induced with 100 ng/mL LPS to obtain M1-type macrophages for 24 hours (referred to as RAW264.7(M1) below).
  • the obtained RAW264.7 (M1) was co-cultured at 1:10 with four recombinant attenuated Salmonella species, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD and Ah-BJ-RBD obtained above at 1:10 for 90 minutes, the supernatant was discarded, washed 2-3 times with PBS, and cultured in cell culture medium (10% serum, without double antibody) supplemented with 100 ng/mL gentamicin for 6 hours. Collect cells and collect total cell protein by thermal lysis method, that is, resuspend cells in 100 ⁇ L PBS, add 25 ⁇ L 5X Loading Buffer, and lyse at 100 degrees for 10-15 minutes. After centrifugation at 9,000 rpm for 5 minutes to collect the four recombinant bacteria in LB, the same method was used to collect the total protein in the bacteria.
  • the inoculated cells were expanded and cultured in 50 mL of kana-resistant liquid LB to an OD600 of about 1.0.
  • the total protein in the supernatant was collected by TCA (trichloroacetic acid)-acetone precipitation method. Briefly, the supernatant was transferred to a 50 ml centrifuge tube, and centrifuged at 15,000 g, 4 degrees, for 10 min using an ultracentrifuge. Take the supernatant and transfer it to a new 50ml centrifuge tube, add 10% TCA, vortex to mix well, and let stand on ice for 30min.
  • the collected total protein was detected by Western blotting (WB) method to detect the production and secretion of the target protein.
  • WB Western blotting
  • Rabbit monoclonal HA-tagged antibody was used as the primary antibody, and HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody.
  • Ah-JJ-RBD and Ah-BJ-RBD recombinant bacteria were analyzed by immunofluorescence. After the Ah-BJ-RBD recombinant RAW264.7 (M1) cells climbed the slides, they were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed with PBS three times. Wells were punched with 0.5% TritonX-100 in PBS for 30 minutes at room temperature. Washed 3 times with PBS, blocked with 3% BSA for 30 minutes at room temperature, washed 3 times with PBS, and incubated the slides overnight at 4 degrees using rabbit monoclonal HA-tagged antibody as the primary antibody.
  • M1 The expression and secretion of antigenic proteins in Ah-JJ-RBD and Ah-BJ-RBD recombinant bacteria were analyzed by immunofluorescence. After the Ah-BJ-RBD recombinant RAW264.7 (M1) cells climbed the slides, they were washed three
  • mice Female C57BL/6 mice aged 6-8 weeks were divided into groups of 4-5 mice per group, and each mouse was inoculated with empty bacteria or Ah-JP-RBD, Ah-JJ-RBD and Ah according to the oral dose of 1 ⁇ 109 CFU bacteria.

Landscapes

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

Abstract

Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine RBD, et son utilisation. Le système à présentation d'antigène est utilisé pour la prévention du nouveau coronavirus (SARS-CoV-2). Grâce à la construction d'un plasmide d'expression contrôlable et stable sécrétant et exprimant la protéine du domaine RBD et à l'ingénierie de salmonelles atténuées, une protéine antigénique peut être efficacement délivrée à une cellule présentant l'antigène par voie orale et au moyen de son système de sécrétion spécifique après administration. La protéine antigénique délivrée peut être efficacement traitée et présentée par des cellules présentatrices d'antigène, ce qui permet de réaliser finalement l'activation/la régulation du système immunitaire, de produire des anticorps et de présenter la fonction d'un vaccin.
PCT/CN2020/124173 2020-10-26 2020-10-27 Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine rbd, et son utilisation WO2022087856A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011155288 2020-10-26
CN202011155288.1 2020-10-26

Publications (1)

Publication Number Publication Date
WO2022087856A1 true WO2022087856A1 (fr) 2022-05-05

Family

ID=81381681

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/124173 WO2022087856A1 (fr) 2020-10-26 2020-10-27 Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine rbd, et son utilisation

Country Status (2)

Country Link
CN (1) CN114480463A (fr)
WO (1) WO2022087856A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703214A (zh) * 2022-03-03 2022-07-05 南京吉芮康生物科技研究院有限公司 一种抗丢失时空可控表达的新型质粒及其应用
CN116715737B (zh) * 2022-11-24 2024-02-23 扬州大学 新型冠状病毒受体结合域B细胞表位的靶标抗原及其在Peg菌毛展呈表达及应用
CN115716867B (zh) * 2022-11-24 2023-08-08 扬州大学 一种V型分泌系统MisL展呈表达新型冠状病毒受体结合域B细胞表位抗原和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1531549A (zh) * 2001-05-24 2004-09-22 健康保护代理公司 分泌性细菌效应蛋白的药物用途
CN1852921A (zh) * 2003-07-21 2006-10-25 美国政府卫生及公众服务部国家卫生协会 Sars冠状病毒刺突糖蛋白的可溶性片段
CN107001431A (zh) * 2014-05-21 2017-08-01 巴塞尔大学 基于细菌的蛋白质递送
CN108884466A (zh) * 2015-11-19 2018-11-23 巴塞尔大学 基于细菌的蛋白递送
CN111529701A (zh) * 2020-05-14 2020-08-14 内蒙古自治区国际蒙医医院(内蒙古自治区蒙医药研究所) 一种口服后产生新型冠状病毒抗体的制剂及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102335421B (zh) * 2011-08-03 2017-05-10 南京大学 一种减毒沙门氏菌诱导型分泌表达口服疫苗递呈系统及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1531549A (zh) * 2001-05-24 2004-09-22 健康保护代理公司 分泌性细菌效应蛋白的药物用途
CN1852921A (zh) * 2003-07-21 2006-10-25 美国政府卫生及公众服务部国家卫生协会 Sars冠状病毒刺突糖蛋白的可溶性片段
CN107001431A (zh) * 2014-05-21 2017-08-01 巴塞尔大学 基于细菌的蛋白质递送
CN108884466A (zh) * 2015-11-19 2018-11-23 巴塞尔大学 基于细菌的蛋白递送
CN111529701A (zh) * 2020-05-14 2020-08-14 内蒙古自治区国际蒙医医院(内蒙古自治区蒙医药研究所) 一种口服后产生新型冠状病毒抗体的制剂及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAZDANI ZAHRA, RAFIEI ALIREZA, YAZDANI MOHAMMADREZA, VALADAN REZA: "Design an Efficient Multi-Epitope Peptide Vaccine Candidate Against SARS-CoV-2: An in silico Analysis", INFECTION AND DRUG RESISTANCE, vol. Volume 13, 25 August 2020 (2020-08-25), pages 3007 - 3022, XP055817652, DOI: 10.2147/IDR.S264573 *

Also Published As

Publication number Publication date
CN114480463A (zh) 2022-05-13

Similar Documents

Publication Publication Date Title
WO2022087856A1 (fr) Système présentant l'antigène d'un nouveau vaccin contre le coronavirus utilisant des salmonelles atténuées pour sécréter et exprimer la protéine du domaine rbd, et son utilisation
WO2022087854A1 (fr) Système de présentation de l'antigène du vaccin contre le sars-cov-2 de la protéine à domaine ntd sécrétée et exprimée par salmonella typhimurium atténuée, et son utilisation
Wang et al. Engineered outer membrane vesicle is potent to elicit HPV16E7-specific cellular immunity in a mouse model of TC-1 graft tumor
US9187765B2 (en) In vitro and In vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine
Ameiss et al. Delivery of woodchuck hepatitis virus-like particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype
CN114478715B (zh) 一种减毒沙门氏菌分泌表达s2结构域蛋白的新型冠状病毒疫苗递呈系统及其应用
JP6917303B2 (ja) 人工多抗原融合タンパク質およびその製造と使用
Liu et al. Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus
EP1370284B1 (fr) Immunisation obtenue au moyen de bacteriophages
CN109303916A (zh) 焦亡相关蛋白gsdmd在制备菌蜕疫苗中的应用
WO2022087855A1 (fr) Nouveau vaccin contre le coronavirus basé sur l'expression sécrétoire contrôlable de salmonella atténuée, son procédé de préparation et son application
Lorenzen et al. Multi-component prime-boost Chlamydia trachomatis vaccination regimes induce antibody and T cell responses and accelerate clearance of infection in a non-human primate model
US20230256072A1 (en) Compositions and methods for vaccination against neisseria gonorrhoeae
CN112107682B (zh) M细胞靶向性重组乳酸菌疫苗及制备方法与应用
US20230137174A1 (en) Novel salmonella-based coronavirus vaccine
Sankar et al. Salivary gland delivery of pDNA‐cationic lipoplexes elicits systemic immune responses
Su et al. Intranasally inoculated bacterium-like particles displaying porcine epidemic diarrhea virus S1 protein induced intestinal mucosal immune response in mice
CN109207492B (zh) 一种包含IBV多表位EpiC和NDV F基因的重组基因、重组表达质粒、重组乳酸杆菌及其应用
CN112143750A (zh) 靶向m细胞的重组乳酸菌表面展示系统、重组乳酸菌疫苗及制备方法与应用
WO2024055273A1 (fr) Vaccin contre l'arnm de la rage et sa préparation et son utilisation
JP4535874B2 (ja) 肝炎に対するバクテリオファージを介する免疫
KR20190011687A (ko) 면역원성시스템 및 이를 포함한 동물 백신
CN114703214A (zh) 一种抗丢失时空可控表达的新型质粒及其应用
Bousbaine et al. Discovery and engineering of the antibody response against a prominent skin commensal
Smith et al. Group B meningococcal outer membrane protein vaccine promote potent anti-viral effect

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: 20959020

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: 20959020

Country of ref document: EP

Kind code of ref document: A1