CN116947982B - Three dominant epitope peptide sequences and application thereof in influenza virus vaccine - Google Patents
Three dominant epitope peptide sequences and application thereof in influenza virus vaccine Download PDFInfo
- Publication number
- CN116947982B CN116947982B CN202310850666.5A CN202310850666A CN116947982B CN 116947982 B CN116947982 B CN 116947982B CN 202310850666 A CN202310850666 A CN 202310850666A CN 116947982 B CN116947982 B CN 116947982B
- Authority
- CN
- China
- Prior art keywords
- influenza
- vaccine
- dominant epitope
- influenza virus
- epitope
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 229940124873 Influenza virus vaccine Drugs 0.000 title claims description 6
- 229960005486 vaccine Drugs 0.000 claims abstract description 43
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 230000002163 immunogen Effects 0.000 claims abstract 2
- 206010022000 influenza Diseases 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 238000008416 Ferritin Methods 0.000 claims description 10
- 102000008857 Ferritin Human genes 0.000 claims description 9
- 108050000784 Ferritin Proteins 0.000 claims description 9
- 239000002671 adjuvant Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 241001263478 Norovirus Species 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000010255 intramuscular injection Methods 0.000 claims description 2
- 239000007927 intramuscular injection Substances 0.000 claims description 2
- 238000010254 subcutaneous injection Methods 0.000 claims description 2
- 239000007929 subcutaneous injection Substances 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims 1
- 239000007922 nasal spray Substances 0.000 claims 1
- 229940097496 nasal spray Drugs 0.000 claims 1
- 229960003971 influenza vaccine Drugs 0.000 abstract description 19
- 230000036039 immunity Effects 0.000 abstract description 8
- 229920001184 polypeptide Polymers 0.000 abstract description 7
- 241001465754 Metazoa Species 0.000 abstract description 6
- 239000000427 antigen Substances 0.000 abstract description 6
- 102000036639 antigens Human genes 0.000 abstract description 6
- 108091007433 antigens Proteins 0.000 abstract description 6
- 230000007969 cellular immunity Effects 0.000 abstract description 6
- 230000004727 humoral immunity Effects 0.000 abstract description 5
- 230000001939 inductive effect Effects 0.000 abstract description 5
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 241001591005 Siga Species 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 230000001404 mediated effect Effects 0.000 abstract 1
- 230000003389 potentiating effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 18
- 241000699670 Mus sp. Species 0.000 description 17
- 241000700605 Viruses Species 0.000 description 15
- 238000001514 detection method Methods 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 11
- 238000002649 immunization Methods 0.000 description 10
- 230000003053 immunization Effects 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- 230000027455 binding Effects 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 230000001932 seasonal effect Effects 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 4
- 101710154606 Hemagglutinin Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 4
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 4
- 101710176177 Protein A56 Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000000185 hemagglutinin Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000702620 H-1 parvovirus Species 0.000 description 2
- 241000702437 Parvovirus H3 Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 229940126580 vector vaccine Drugs 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 1
- 229940124718 AIDS vaccine Drugs 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 241000713196 Influenza B virus Species 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000010485 coping Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- 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
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Pulmonology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of immunology, relates to a composition containing peptide immunogens for preventing and treating influenza virus-mediated diseases, and discloses dominant epitope peptide sequences of three influenza viruses HA2 and application of the dominant epitope peptide sequences in research and development of influenza virus vaccines. The amino acid sequences of the three dominant epitope peptides are shown in SEQ ID NO. 1-3. According to the invention, three dominant epitope peptides with immunogenicity are screened out by combining immunoinformatics prediction and animal experiment verification of the influenza virus HA2, and the three dominant epitope peptides have T/B cell epitopes and can induce cellular immunity and humoral immunity; immunogens as intranasal vaccines are capable of inducing potent mucosal immunity, inducing high titers of sIgA against influenza virus. Compared with other polypeptides aiming at the antigen region, the dominant epitope peptide has high potency and high conservation, and can be used as a candidate immunogen of influenza vaccine.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to three dominant epitope peptide sequences and application thereof in influenza virus vaccines.
Background
Influenza viruses continue to threaten global public health safety, and vaccination with seasonal influenza vaccines is still the most effective means of preventing and controlling influenza epidemics today. Due to antigenic drift and antigenic shift of influenza viruses, seasonal influenza vaccines must be reconstituted and re-vaccinated annually to match epidemic strains, and thus the range of protection afforded by seasonal influenza vaccines is very limited, the effectiveness of which depends largely on the degree of matching of vaccine cover strains to epidemic strains. Conventional seasonal influenza vaccines are generally prepared by culturing viruses in chicken embryos or cells and processing the virus into the vaccine, and are complex in production, time-consuming and highly dependent. Therefore, there is a need to develop a "universal" influenza vaccine that is effective against most influenza viruses, independent of chicken embryo and cells, and that is produced at a fast rate. Development of "universal" influenza vaccines requires focusing of the immune response on conserved epitopes, thereby increasing the breadth of the immunity. The epitope vaccine is possible to induce strong immune response to non-dominant or recessive epitopes in the natural immune process, has simple manufacturing process, can be rapidly prepared in escherichia coli, has low cost, and is more suitable for mass production. Hemagglutinin (HA) protein is a key protein mediating viral invasion into host cells, its stem region (HA 2) is highly conserved among IAVs of the isotype, and antibodies targeting HA2 have broad cross-reactivity, which can neutralize viral infection by preventing membrane fusion between the virus and host cells. There is currently a lack of known HA2 epitopes that can induce protective immunity.
The disadvantages and problems with current influenza vaccine technology include mainly the following:
1. Limited protection range: the scope of protection of traditional seasonal influenza vaccines is limited by the degree of matching of vaccine cover strains with epidemic strains. The antigenic drift and antigenic shift of influenza virus cause the strain to change continuously, so that the protective effect of the vaccine is limited.
2. Production is complex and time-consuming: the conventional seasonal influenza vaccine production process requires the virus to be cultured in chicken embryos or cells and processed to produce the vaccine. The production method is complex, time-consuming and highly dependent, and is unfavorable for rapidly coping with influenza virus variation.
3. Need to be reconstituted and inoculated annually: seasonal influenza vaccines must be reconstituted and vaccinated annually due to variations in influenza virus, placing a significant burden on public health systems.
4. General influenza vaccine development faces challenges: a "universal" influenza vaccine requires focusing of the immune response on conserved epitopes to improve immune breadth. However, there is still a lack of known HA2 epitopes that can induce protective immunity, making the development of universal influenza vaccines challenging.
5. Uncertainty of epitope vaccine: although epitope vaccines have the advantages of simple manufacturing process, low cost, etc., the effect of inducing strong immune responses to non-dominant or recessive epitopes in the course of natural immunity remains uncertain, requiring further research and validation.
Disclosure of Invention
Aiming at the technical problems, one of the purposes of the invention is to screen out three dominant epitope peptide sequences aiming at an influenza virus HA2 conserved region, wherein the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 1-3, any one polypeptide sequence of the SEQ ID NO. 1-3 is substituted and/or deleted and/or inserted by one or more amino acid residues, and the polypeptide HAs the same function and is derived from any one polypeptide of the SEQ ID NO. 1-3.
It is a second object of the present invention to provide a gene encoding the dominant epitope peptide.
The invention further provides application of the dominant epitope peptide in preparation of anti-influenza virus vaccines, antibodies and medicaments.
The fourth object of the invention is to provide the application of the gene of the dominant epitope peptide in preparing anti-influenza virus vaccine, antibody and medicine.
It is a fifth object of the present invention to provide an influenza epitope vaccine whose immunogens are derived from a combination of one or more and different copy numbers of the dominant epitope peptide.
It is a sixth object of the present invention to provide an influenza epitope vaccine, wherein the vector is KLH vector, norovirus P particle vector, ferritin nm vector, but not limited to these vectors.
It is a seventh object of the present invention to provide an influenza epitope vaccine whose immunization route is intramuscular injection, subcutaneous injection, nasal injection, inhalation, but not limited to these routes.
It is an eighth object of the present invention to provide an influenza epitope vaccine that may be devoid of or contain an adjuvant, such AS03, al (OH) 3, but is not limited to these adjuvants.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
first, the invention provides three HA2 epitope peptide sequences capable of inducing protective immunity, which improves the protection scope of the existing influenza vaccine. Because of the lack of known HA2 epitopes capable of inducing protective immunity, the invention is based on the highly conserved HA2 sequence of H3N2, firstly uses immunoinformatics to predict the epitopes, then fuses a plurality of predicted epitopes with protein carriers and expresses the fused epitopes in escherichia coli, then verifies the immunogenicity and the protectiveness of the fused proteins on an animal model, and finally obtains three B cell epitopes, th epitopes and CTL epitopes with high conservation and broad-spectrum neutralization activity. The dominant epitope peptide can be regarded as a virus subunit capable of effectively stimulating body humoral immunity and cellular immunity, can accurately target immune response to a conserved epitope, further improves the protection efficacy and range, and is obviously superior to the existing influenza vaccine. The dominant epitope peptide is prepared from escherichia coli, is simple and rapid to produce, is safe and effective to use, and has wide application prospects in anti-influenza virus vaccine, antibody and drug research and development. The research method of the invention is also suitable for screening dominant epitopes of other highly variable pathogenic pathogens.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention provides three dominant epitope peptide sequences, which can be prepared in escherichia coli by taking the epitope peptide as an antigen component, can excite effective adaptive immune response and improve the protective range of vaccine, and has the incomparable advantage of the traditional vaccine.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
(1) The expected benefits and commercial values after the technical scheme of the invention is converted are as follows:
The invention provides three dominant epitope peptide sequences, which can be prepared by depending on escherichia coli, and are obviously superior to the traditional influenza vaccine in the aspects of production cost, production speed, safety, protection range and the like.
(2) Whether the technical scheme of the invention solves the technical problems that people want to solve all the time but fail to obtain success all the time is solved:
The invention provides three dominant epitope peptide sequences, overcomes the defect of insufficient number of the existing HA2 epitopes, and expands the protection breadth of influenza vaccines. The invention screens out specific antigen epitope peptide based on antigen epitope predictive analysis technology, protein expression technology and animal experiment technology, and has important significance in rapid immunodiagnosis and prevention of virus infectious diseases and development of therapeutic vaccines.
(4) The technical scheme of the invention overcomes the technical bias: .
The technical scheme solves the problem that the production of the existing influenza vaccine can not be separated from chicken embryo or cells.
Drawings
FIG. 1 is an HA2 sequence analysis of H3N 2; wherein, the amino acid conservation analysis of each position of HA2 of 9532H 3N2 viruses in A.1968-2020; B. the highly conserved HA2 sequence, namely the HA2 sequence of A/Nicaragua/8774_10/2016; C. sequence alignment of influenza a virus HA2 of each subtype;
FIG. 2 is a dominant epitope peptide sequence; wherein, a three-dimensional structure diagram of the A.H3N2 (PDB: 6 BKP); B. a dominant epitope peptide sequence obtained by combining immunoinformatics prediction and animal experiment verification;
FIG. 3 is a transmission electron microscope view of a fusion protein of a dominant epitope peptide and Ferritin carriers;
FIG. 4 is a graph showing the detection of antibody binding activity of serum after the immunization of fusion proteins, ferritin is a negative control group, and H3 Vaccine is an H3 monovalent split Vaccine group; wherein a. Epitope specifically binds to the antibody; b and c.h3n2 binding antibodies specifically; h1n1 specifically binds to the antibody; E. influenza b virus specifically binds to antibodies; F. typing and detecting antibodies;
FIG. 5 is a graph showing the detection of antibody neutralization activity of serum after the immunization of a fusion protein;
FIG. 6 is a graph showing cellular immunity evaluation after the fusion protein is avoided; wherein, A and B. CD3 +CD4+、CD3+CD8+ T cell proliferation detection of post-immune mice; C. peptide-specific in vivo CTL detection;
FIG. 7 is a graph showing the change in body weight and survival of mice after challenge;
FIG. 8 is a graph of immunogenicity testing of a multi-epitope vaccine after nasal drip inoculation; wherein, A. The epitope in serum specifically binds to the antibody; B. detecting proliferation of CD3 +CD4+、CD3+CD8+ T cells of the post-immune mice; C. detecting the long-acting performance of the epitope specific binding antibody in serum; D. sIgA titers in nasal wash (NLF) and alveolar lavage (BALF).
FIG. 9 is a graph of protective evaluation of a multi-epitope vaccine and H3 split vaccine for nasal drip vaccination; wherein, A, weight change of mice after toxin attack; B. survival curve of mice after challenge.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1: antigen conservation analysis
HA2 sequences of 1968-2020 9532H 3N2 subtype viruses are downloaded from NCBI influenza database (http:// www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), amino acid conservation analysis and virus circularity analysis of each site are carried out by BioEdit software, and highly conserved HA2 sequences (A/Nicaragua/8774_10/2016) are obtained, wherein the amino acid conservation of the rest sites is above 95% except the 18 th, 32 th, 46 th, 57 th, 77 th, 121 th, 123 th, 149 th, 155 th and 160 th sites. The amino acids at the remaining sites of the heterosubtype of HA2 were more diverse except for the fusion peptide region (positions 1-24 of HA 2) (FIG. 1).
Example 2: epitope prediction and validation
Based on a conserved HA2 sequence of H3N2, firstly, using an IEDB database to predict B cell epitopes (http:// tools.iedb.org/main/bcell /), and using Kolaskar & Tongaonkar method to predict antigen epitopes, wherein the default threshold is 1; bepipred Linear Epitope Prediction 2.0.0 predicts linear B cell epitope, default threshold is 0.5; b cell epitopes were then predicted using ABCpred (https:// webs. Iiitd. Edu. In/cgibin/abcpred /), default threshold of 0.5, 16 peptides; then, the IEDB database is used for carrying out the prediction of the binding sites of the MHC class II and MHC class I molecules (http:// tools. IEDB. Org), and the comprehensive prediction method recommended by the IEDB and the default threshold value are used; two direct CTL epitope prediction methods were used simultaneously, one being CTL PRED (https:// webs. Iiitd. Edu. In/cgibin/ctlpred/ctlpred. Pl) based on consensus and combined ANN/SVM algorithm and the other being NETCTLPAN (https:// services. Health. Dtu. Dk/service. PhpNetCTLpan-1.1) based on ANN. Animal experiments prove that three dominant epitope peptides which can effectively stimulate body humoral immunity and cellular immunity are finally obtained and named as E1-E3 (figure 2).
Example 3: preparation of Single epitope vaccine
The present example uses Ferritin vector obtained by construction and expression in the national engineering laboratory for AIDS vaccine at Jilin university, but is not limited to this vector. Inserting Ferritin (NCBI Reference Sequence:WP_ 000949190.1) encoding helicobacter pylori and a gene of dominant epitope peptide of the invention into a prokaryotic expression vector pET-20b (+), converting into expression competent BL21 (DE 3) with His-tag at N end, adding 0.5mM IPTG to induce the bacterium at 18 ℃ overnight when the bacterium is in logarithmic growth phase, centrifuging to collect the bacterium, ultrasonically crushing, and sequentially subjecting supernatant to steps of affinity chromatography purification, dialysis and the like to obtain fusion proteins of the epitope and Ferritin vectors, which are named E1-Ferritin-E3-Ferritin (figure 3).
Example 4: subcutaneous immunization and immunogenicity and protective evaluation of single epitope vaccines
Animal immunization: BALB/c female mice with 6-8 weeks of age were immunized three times at two week intervals, the subcutaneous immunization dose was 20 μg/mouse, and the immunization adjuvant was aluminum adjuvant (Thermo FISHER SCIENTIFIC) 3:1 volume mixed.
Binding activity assay: the results of detection of the titer of binding antibodies after immunization with the single epitope vaccine by indirect ELISA showed that the single epitope vaccine induced high levels of binding antibodies against polypeptides, against H3 virus and E1 polypeptides induced cross-binding antibodies against H1 virus (FIGS. 4. A-E).
Antibody typing detection: coating, blocking and detecting the binding activity of the primary antibody, adding a rabbit anti-parting antibody (Sigma) into the secondary antibody, adding an HRP-goat anti-rabbit antibody into the tertiary antibody, and performing color development and stopping detection. The results show that the E3 epitope-induced IgG1/IgG2a ratio tended to be 1, i.e. induced a relatively balanced Th1 and Th2 immune bias; the E1 and E2 epitopes tended to induce Th 2-type immune responses (fig. 4.F).
Neutralization activity detection: adding 50 mu L of virus culture medium into 8 EP pipes of 1.5mL, adding 50 mu L of serum into the first pipe, sequentially diluting to 8 th pipe in multiple ratio, finally discarding 50 mu L, adding 50 mu L of 100 xTCID 50 influenza virus into each pipe, slowly blowing and mixing uniformly, and incubating in an incubator at 37 ℃ for 1h; adding the serum-virus mixture into 96-well plate, adding MDCK cells (1.5X10 4/well) into each well, and culturing in 37 deg.C incubator; after 48h incubation, the ID 50 values were calculated using visualization of cytopathic effect (CPE). The 50% inhibitor dose (ID 50) was defined as the serum dilution that neutralized 50% of the virus. The results show that the single epitope vaccine can induce high levels of neutralizing antibodies against H3 virus and that the E1 epitope can induce cross-neutralizing antibodies against H1 virus (fig. 5).
Lymphocyte detection: spleen lymphocytes from immunized mice were isolated using erythrocyte lysate (Biolegend), stained with FITC-labeled anti-mouse-CD 3 antibody, PE-labeled anti-mouse-CD 4 antibody, and APC-labeled anti-mouse-CD 8a antibody; incubating for 20min in the dark on ice, centrifuging for 5min at 350 Xg, and discarding the supernatant; after washing twice with CELL STAINING buffer, 500. Mu. L CELL STAINING buffer was added to the cells to suspend the cells, followed by 5. Mu.L of 7-AAD, and incubated on ice for 5min in the absence of light, immediately followed by detection using a flow cytometer. The results showed that the single epitope vaccine stimulated proliferation of CD3 +CD4+ and CD3 +CD8+ T cells in immunized mice (fig. 6. A-B).
In vivo CTL detection: spleen lymphocytes of a blank mouse are separated by using erythrocyte lysate, half of the cells are incubated with polypeptide for 2 hours and then stained with high-concentration CFSE (5 mu M) as target cells, the other half of the cells are incubated with BSA for 2 hours and then stained with low-concentration CFSE (0.5 mu M) as control cells, two groups of cells with the same number are mixed and then tail vein is injected into a post-immune mouse, spleen cells are separated after 10 hours, and the proportion of the two groups of cells is detected by using a flow cytometer. The results show that the E2, E3 epitopes can induce peptide-specific CTLs in mice (fig. 6. C).
And (3) toxicity attack protective detection: two weeks after the third immunization, the mice were challenged nasally at a dose of 20. Mu.L 10 8EID50 A/Wisconsin/67/2005 (H3N 2). The change in body weight and survival rate of mice were recorded daily after challenge, and mice with 25% weight loss were considered dead for a total of 14 days. The results showed that the E3 mice lost more than 20% of their body weight after challenge with a final survival of 67%. The weight of the mice in the E1 and E2 groups was reduced by about 15% after challenge, the survival rate of the mice in the E2 group was 83%, and all the mice in the E1 group survived (FIG. 7).
The above-described embodiments are merely illustrative of the preferred modes of the present invention, and do not limit the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
The preparation method of the Ferritin vector vaccine is the same as that of the norovirus P particle vector vaccine.
The sequence table
2. Application example. In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
Example 5: mucosal immune assessment of multiple epitope vaccine
Mucosal immunity is the first barrier of the body against invasion of respiratory viruses, and can simulate the natural infection path. Three dominant epitope peptides are fused and expressed with Ferritin in a serial form, the plasmid construction and the protein expression purification modes are the same as those described above, and the prepared multi-epitope vaccine (E1-E2-E3-F) is subjected to animal immunization in a nose-drop form, and the humoral immunity, cellular immunity and toxicity attack protective detection methods are the same as those described above.
Mucosal immunity detects secretory IgA (sIgA) titers in mouse nasal washes (NLF) and alveolar lavages (BALF). The results show that the multi-epitope vaccine can directly stimulate respiratory tract mucous membrane to generate a large amount of sIgA through nasal drip inoculation, can effectively prevent virus from adhering and colonizing respiratory tract epithelium, simultaneously induces extensive and durable humoral immunity and cellular immunity, and can protect mice from being attacked by H3N2 virus (figure 8). The invention provides three dominant epitope peptide sequences, and the multi-epitope vaccine produced and prepared by using escherichia coli based on the sequences can produce the protection effect consistent with that of the intramuscular monovalent split vaccine (H3 vaccine) prepared by using chick embryos through nasal drip inoculation (figure 9). The implementation of the invention is obviously superior to the prior art in production cost and inoculation mode, and the invention has important significance in the development of rapid immunodiagnosis, prevention and treatment of virus infectious diseases. The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Sequence table information:
DTD version v1_3
File name three dominant epitope peptide sequences and application thereof in influenza virus vaccine
Software name WIPO Sequence
Software version 2.3.0
Date of formation 2023-07-12
Basic information:
current application/intellectual property office CN
Current application/applicant archive name Jilin University
Applicant name or name Jilin university
Applicant name or name/language zh
Applicant name or name/latin name Jilin University
The names Shan Yaming, dan Yuhua, nie Jiaojiao and Wang Qingyu of the inventors
Inventor name/language zh
Inventor name/latin name SHAN YAMING, shi Yuhua, nie Jiaojiao, wang Qingyu
Three dominant epitope peptide sequences and their use in influenza virus vaccines (zh)
Total sequence 3
Sequence:
serial number (ID) 1
Serial number (ID) 2
Serial number (ID) 3
Claims (8)
1. The dominant epitope peptide of the influenza virus HA2 is characterized in that the amino acid sequence of the dominant epitope peptide is shown as SEQ ID NO. 2 or SEQ ID NO. 3.
2. A gene encoding the dominant epitope peptide of claim 1.
3. Use of a dominant epitope peptide for the preparation of an anti-H3N 2 influenza virus vaccine, characterized in that the dominant epitope peptide of influenza virus HA2 according to claim 1 is used.
4. Use of a gene according to claim 2 for the preparation of a vaccine against H3N2 influenza virus.
5. An influenza epitope vaccine, characterized in that its immunogen is derived from one or more of the dominant epitope peptides of claim 1 and a combination of different copy numbers.
6. The influenza epitope vaccine of claim 5, wherein said influenza epitope vaccine carrier is a hemocyanin KLH carrier, a norovirus P particle carrier, a Ferritin nm carrier.
7. The influenza epitope vaccine of claim 5, wherein said influenza epitope vaccine is administered by intramuscular injection, subcutaneous injection, nasal spray, inhalation.
8. The influenza epitope vaccine of claim 5, wherein said adjuvant is AS03, al (OH) 3, is absent or comprised of an adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310850666.5A CN116947982B (en) | 2023-07-12 | 2023-07-12 | Three dominant epitope peptide sequences and application thereof in influenza virus vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310850666.5A CN116947982B (en) | 2023-07-12 | 2023-07-12 | Three dominant epitope peptide sequences and application thereof in influenza virus vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116947982A CN116947982A (en) | 2023-10-27 |
| CN116947982B true CN116947982B (en) | 2024-05-14 |
Family
ID=88442139
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310850666.5A Active CN116947982B (en) | 2023-07-12 | 2023-07-12 | Three dominant epitope peptide sequences and application thereof in influenza virus vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116947982B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101605558A (en) * | 2005-08-16 | 2009-12-16 | 夏威夷生物技术公司 | Influenza recombinant subunit vaccine |
| CN104066446A (en) * | 2011-11-28 | 2014-09-24 | 克鲁塞尔荷兰公司 | Influenza virus vaccines and uses thereof |
| CN105198971A (en) * | 2015-10-10 | 2015-12-30 | 长春百克生物科技股份公司 | Synthetic polypeptide and application thereof, and anti-influenza-virus vaccine |
| CN107427571A (en) * | 2014-12-31 | 2017-12-01 | ***合众国,由健康及人类服务部部长代表 | Novel multivalent vaccine based on nano particle |
| CN111315407A (en) * | 2018-09-11 | 2020-06-19 | 上海市公共卫生临床中心 | Broad-spectrum anti-influenza vaccine immunogen and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009543557A (en) * | 2006-07-11 | 2009-12-10 | アラーガン、インコーポレイテッド | Modified clostridial toxin with enhanced translocation ability and altered targeting activity against clostridial toxin target cells |
| EP4019044A3 (en) * | 2015-09-02 | 2022-08-24 | Janssen Vaccines & Prevention B.V. | Stabilized viral class i fusion proteins |
| EP4070110A4 (en) * | 2019-12-08 | 2024-02-28 | National Institute for Biotechnology in the Negev Ltd. | Influenza arrays and use thereof |
-
2023
- 2023-07-12 CN CN202310850666.5A patent/CN116947982B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101605558A (en) * | 2005-08-16 | 2009-12-16 | 夏威夷生物技术公司 | Influenza recombinant subunit vaccine |
| CN104066446A (en) * | 2011-11-28 | 2014-09-24 | 克鲁塞尔荷兰公司 | Influenza virus vaccines and uses thereof |
| CN107427571A (en) * | 2014-12-31 | 2017-12-01 | ***合众国,由健康及人类服务部部长代表 | Novel multivalent vaccine based on nano particle |
| CN105198971A (en) * | 2015-10-10 | 2015-12-30 | 长春百克生物科技股份公司 | Synthetic polypeptide and application thereof, and anti-influenza-virus vaccine |
| CN111315407A (en) * | 2018-09-11 | 2020-06-19 | 上海市公共卫生临床中心 | Broad-spectrum anti-influenza vaccine immunogen and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| "Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco";Mohamed Boukharta 等;《BMC Res Notes》;20140712;第7卷;第3-4页 * |
| "Self-adjuvant multiepitope nanovaccine based on ferritin induced long-lasting and effective mucosal immunity against H3N2 and H1N1 viruses in mice";Jiaojiao Nie 等;《Int J Biol Macromol》;20240106;第259卷(第Pt 1期);doi: 10.1016/j.ijbiomac.2024.129259 * |
| "Self-Assembled Multiepitope Nanovaccine Provides Long-Lasting Cross-Protection against Influenza Virus";Jiaojiao Nie 等;《Adv Healthc Mater》;20231120;doi: 10.1002/adhm.202303531 * |
| "基于H3流感病毒血凝素表位肽的重组Ferritin的免疫原性评价";聂娇娇;《中国优秀硕士学位论文全文数据库 (医药卫生科技辑)》;20200815(第8期);E059-100 * |
| "基于流感病毒H3亚型保守序列重组Ferritin免疫原性分析及保护活性的初步研究";郐子昱;《中国优秀硕士学位论文全文数据库 (医药卫生科技辑)》;20190115(第1期);E059-425 * |
| "基于诺如病毒P颗粒的甲型流感病毒多表位免疫原的构建及免疫活性研究";金圣惠;《中国优秀硕士学位论文全文数据库 (基础科学辑)》;20230115(第1期);E059-167 * |
| "铁蛋白纳米颗粒为载体的流感候选疫苗设计及其生物学活性评价";乔永波;《中国博士学位论文全文数据库 (基础科学辑)》;20221015(第10期);E059-32 * |
| Vester,S.K.等."hemagglutinin H3/SpyTag fusion protein [synthetic construct]".《genbank》.2022,ACCESSION USU81766. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116947982A (en) | 2023-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Qi et al. | Intranasal nanovaccine confers homo‐and hetero‐subtypic influenza protection | |
| WO2022218272A1 (en) | Novel coronavirus mutant strain s protein and novel coronavirus mutant strain subunit vaccine | |
| Guo et al. | Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein | |
| Stepanova et al. | Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes | |
| Lee et al. | The highly conserved HA2 protein of the influenza A virus induces a cross protective immune response | |
| Kamlangdee et al. | Broad protection against avian influenza virus by using a modified vaccinia Ankara virus expressing a mosaic hemagglutinin gene | |
| EP2069503A1 (en) | Papaya mosaic virus-based vaccines for influenza | |
| CN113354717B (en) | Novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen and its specific neutralizing antibody and application | |
| Zhao et al. | Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant | |
| Ding et al. | RETRACTED ARTICLE: Nanovaccine Confers Dual Protection Against Influenza A Virus And Porcine Circovirus Type 2 | |
| EP2168987A1 (en) | Multifunctional linker protein containing an antibody against hemagglutinin, a conserved influenza antigen and an immunostimulating carrier binding domain | |
| CN114716570A (en) | Fusion protein and preparation method and application thereof | |
| CN116947982B (en) | Three dominant epitope peptide sequences and application thereof in influenza virus vaccine | |
| Li et al. | Identification of linear peptide immunogens with verified broad-spectrum immunogenicity from the conserved regions within the hemagglutinin stem domain of H1N1 influenza virus | |
| CN110003314B (en) | Epitope peptide capable of inducing broad-spectrum protective antibody by H1N1 influenza virus hemagglutinin and application thereof | |
| WO2022085648A1 (en) | Fusion protein and vaccine | |
| EP0714307B1 (en) | Vaccine compositions | |
| Maes et al. | Truncated recombinant Dobrava hantavirus nucleocapsid proteins induce strong, long-lasting immune responses in mice | |
| US20030044936A1 (en) | Peptide repeat immunogens | |
| CN113248575B (en) | Recombinant protein vaccine for SARS-CoV-2 and its preparing method | |
| KR20190039065A (en) | Modification of engineered influenza hemagglutinin polypeptide | |
| Subbbiah et al. | Thermostable H1 hemagglutinin stem with M2e epitopes provides broad cross-protection against group1 and 2 influenza A viruses | |
| Wang et al. | Bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine | |
| JP6902804B2 (en) | Vaccine composition containing hepatitis B virus-like particles as an adjuvant | |
| Van et al. | Salmonella as a vaccine vector for influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |