WO2016064063A1 - 대상포진 예방 및 치료용 dna 백신 조성물 및 이를 이용한 vzv 항원에 대한 t세포 활성화 방법 - Google Patents
대상포진 예방 및 치료용 dna 백신 조성물 및 이를 이용한 vzv 항원에 대한 t세포 활성화 방법 Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
- A61K39/25—Varicella-zoster virus
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- A61K2039/70—Multivalent vaccine
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16741—Use of virus, viral particle or viral elements as a vector
Definitions
- the present technology is a technique in the vaccine field, and more particularly, a technique for preventing and treating shingles, which is excellent in productivity, stability, and immune response to T cells, and which can be applied to various disease forms.
- Herpes Zoster is a skin rash disease in which Varicella-Zoster Virus (VZV) is latent in the ganglion, and when immunity is weakened, reactivation of VZV triggers. to be.
- VZV Varicella-Zoster Virus
- a shingles develops, a bullous lesion appears and, even when it is recovered, there is a sequelae of postherpetic neuralgia, and once the pain occurs, it is difficult to cure and patients suffer extreme pain.
- Reactivation of VZV and the development of shingles are associated with attenuated cellular immune responses centered on T cells, especially in older people and those receiving immunosuppressive treatment.
- shingles develops, they are treated with antiviral agents, but the goal is to administer within 72 hours after the onset of the rash so that the rash no longer spreads to other sites and to reduce or alleviate complications after shingles.
- the incidence of shingles is increasing rapidly in Korea, but since there is no underlying treatment, the importance of preventive vaccines is considered to be a very serious disease.
- An object of the present invention is to solve the problems of the foregoing and the prior art, and to consider the practical needs, and to provide a DNA vaccine composition for the treatment and treatment of shingles, which is applicable to patients with various forms of diseases and has greatly improved stability. will be.
- Still another object of the present invention is to provide a method for activating T cells against VZV antigens by effectively administering the DNA vaccine composition for preventing and treating shingles in the body.
- Plasmid DNA for preventing and treating shingles includes an insertion site of a VZV-derived gene encoding a shingles virus (VZV) protein, and is directly administered to the body by an electroporation method to a VZV antigen. Induces T cell immune activity.
- VZV shingles virus
- Inovio inovio
- CELLECTRA electrical perforator
- the gene is any one of a first gene encoding the IE-62 protein (SEQ ID NO: 1), a second gene encoding the IE-63 protein (SEQ ID NO: 2), and a third gene encoding the gE protein (SEQ ID NO: 3). It can be one.
- the plasmid may be any one of plasmid DNA of SEQ ID NO: 4, plasmid DNA of SEQ ID NO: 5, and plasmid DNA of SEQ ID NO: 6.
- the DNA vaccine composition for preventing and treating shingles includes at least one plasmid containing an insertion site of a VZV-derived gene encoding shingles virus (VZV) protein, and other pharmaceutically acceptable components. It may include. As said other component, water, such as a saline solution, is mentioned, for example.
- the plasmid constituting the DNA vaccine may include a plurality of plasmids containing different heterologous gene insertion sites.
- a plurality of plasmids containing different gene regions encoding proteins of VZV can be used as the plasmid.
- the first plasmid included in the DNA vaccine composition including the insertion site of the first gene encoding the IE-62 protein (SEQ ID NO: 1) and the second gene encoding the IE-63 protein (SEQ ID NO: 2)
- Three plasmids can be used, including a second plasmid comprising the insertion site and a third plasmid containing the insertion site of the third gene encoding the gE protein (SEQ ID NO: 3).
- the content in the composition of each plasmid may be adjusted in various ways in consideration of the disease form, the age of the subject to be administered.
- T cell activation method for a VZV antigen comprises the steps of preparing a plasmid containing the insertion site of the VZV-derived gene encoding shingles virus (VZV) protein, and the plasmid in an electroporation method Thereby administering to the body.
- the plasmid can be mass produced by a mass production system.
- the DNA vaccine for shingles prevention and treatment according to the present invention is a vaccine by direct administration of plasmid, it does not cause the safety problem of live attenuated vaccines. In addition, mass production of the plasmid is easy, and stability of the distribution process of the vaccine can be dramatically improved. Since the DNA vaccine is administered in the body by the electroporation method, it can exhibit an innovative in vivo delivery efficiency compared to a general injection. Meanwhile, the DNA vaccine for preventing and treating shingles can be used for prevention as well as for therapeutic purposes, and can be appropriately used for patients with various diseases and patients of various ages. The DNA vaccine for preventing and treating shingles has the advantage of being able to design a customized vaccine through various combinations of a plurality of plasmids having different gene insertion sites of plasmids.
- FIG. 1 is a view showing an inoculation schedule according to an embodiment of the present invention.
- FIG. 2 is a graph showing immunogenicity results for experimental groups according to an embodiment of the present invention.
- 3 and 4 are graphs showing the results of comparing the protein overlap peptide immune response of each experimental group.
- 5 to 7 are electrophoretic photographs confirming the product expressed by each plasmid gene.
- the DNA vaccine for preventing and treating shingles according to the present invention is not a virus-based live vaccine but a DNA-based vaccine which administers DNA itself to the body. DNA itself functions as a vaccine.
- the DNA vaccine encodes an antigenic protein derived from VZV, and ultimately forms an antigenic protein in the body.
- Proteins derived from VZV are preferably selected in consideration of the characteristics of the required DNA vaccine, and the proteins may be selected alone or in combination of multiple species.
- the protein is a protein capable of inducing a cellular immune response IE-62 (SEQ ID NO: 1), IE-63 (SEQ ID NO: 2), the glycoprotein gE (SEQ ID NO: 3) gene
- the plasmid is designed to insert a gene encoding such a protein.
- the plasmid DNA is mixed and administered to the liquid phase in the body, the plasmid DNA of the present invention is designed to be injected into the body with high efficiency by the electroporation method, it is possible to maximize the delivery efficiency in the body only by such an electroporation method Ultimately, the therapeutic efficacy can be maximized.
- the site of administration in the body by the electroporation method is not particularly limited, such as muscle areas, it may be determined in consideration of various factors such as the age, disease type, parallel disease of the subject.
- an electroporation device for implementing the electroporation method for example, Inobio's electroporator (CELLECTRA ® ) may be used.
- the first gene, the second gene, and the third gene which are genes capable of encoding and expressing the IE-62, IE-63, and gE proteins, are mounted on the plasmid in a conventional manner, but maximize the effect and administration efficiency.
- the plasmids of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively it is possible to maximize the efficiency of the DNA vaccine.
- Each of the plasmids may be used alone, but is preferably included in the DNA vaccine in the form of a combination of two or more different plasmids.
- a DNA vaccine containing all three plasmids can be considered.
- other kinds of plasmids not specified in this embodiment can be introduced newly.
- the plasmid may be administered to the body via an injection or the like, and the plasmid should be prepared in high concentration and high purity for delivery efficiency and immune response efficiency.
- the ease of mass production must be ensured. Plasmids according to an embodiment of the present invention is currently secured the basis for such high concentration and high purity mass production.
- the DNA vaccine for herpes prophylaxis and treatment is a vaccine containing at least one plasmid DNA as described above, that is, a vaccine of injection, and includes a plasmid DNA and a liquid component.
- the liquid component may be pure water.
- the DNA vaccine composition according to one embodiment of the present invention may further include pharmaceutically acceptable functional additives, and such additives may be introduced at the level of tolerance.
- the DNA vaccine composition may include, for example, all three plasmids described above.
- the proteins IE-62, IE-63 and glycoprotein gE genes capable of inducing cellular immune responses were selected as candidates.
- the type of the VZV genome is divided into a total of five clades, and there is no significant difference between the clade IE-62, IE-63, and gE amino acid sequences.
- Plasmids were prepared from the above IE-62, IE-63, and gE gene sequences.
- the amino acid sequence of the proteins is shown in SEQ ID NOS: 1-3
- the DNA sequence of the prepared plasmid is shown in SEQ ID NOS: 4-6.
- the DNA vaccine contained no components other than water as the plasmid and the liquid component.
- IE-62, IE-63, and gE genes were prepared as DNA vaccine candidates, administered to C57BL / 6 mice, and splenocytes were isolated to assess T cell immunogenicity against the VZV antigen.
- the test group consisted of 5 groups. Each group consisted of 5 animals.
- CELLECTRA ® manufactured by INNOBIOS was used to inoculate three times at intervals of two weeks.
- the amount of DNA at each inoculation was 30 ⁇ g per horse, and the mixed group was inoculated with a total of 90 ⁇ g at 30 ⁇ g per DNA.
- splenocytes were isolated and subjected to immunoassay.
- the inoculation schedule is as shown in FIG. 1. 1. 1 is a view showing an inoculation schedule according to an embodiment of the present invention.
- Each overlapping peptide consists of 15 amino acids and is designed to overlap a neighboring peptide with 10 amino acids.
- Duplicate peptides were designed to include IE-62, IE-63, gE protein full amino acid sequences. 261 IE-62 duplicate peptides, 54 IE-63 duplicate peptides, and 124 gE duplicate peptides were prepared. A total of seven peptide mixtures were used by mixing 37 or 38 consecutive IE-62 duplicate peptides. A total of two peptide mixtures were made by mixing 27 consecutive IE-63 duplicate peptides.
- a total of three peptide mixtures were made by mixing contiguous 40 or 41 gE duplicate peptides. 5% DMSO was used as a negative control, and PMA (Phorbol Myristate Acetate) ionomycin was used as a positive control. Peptide mixtures made from a mixture of duplicate peptides are shown in Table 1 below.
- a lysate prepared by infecting MZ-5 cells with VZV was used as an antigen and MRC-5 cell lysate was used as a corresponding negative antigen.
- the Neg group used as a negative control did not show an immune response to any duplicate peptide stimulation.
- the immune responses were shown only against overlapping peptides of the protein.
- the IE62 + IE63 + gE group which received all of the IE-62, IE-63, and gE DNA vaccines, all of the overlapping peptides of the three proteins showed immune responses.
- the immune response was shown only against the overlapping peptide specific for the candidate. 2 is a graph showing immunogenicity results for experimental groups according to an embodiment of the present invention.
- FIGS. 3 and 4 are graphs showing the results of comparing the protein-peptide immunoglobulin in each experimental group.
- the comparison of the protein overlap peptide immune response of the group administered with a single DNA vaccine and a mixture of three DNA vaccines, a combination of three DNA vaccines would be administered a single DNA vaccine.
- the immune response to IE-62 was reduced by 43.3%, the immune response to IE-63 was not different, and the immune response to gE was decreased by 9.6%.
- DNA vaccines prepared with genes encoding IE-62, IE-63, gE proteins were evaluated and analyzed in experimental animal models and showed sufficient T cell immunogenicity upon single or mixed administration.
- T cell immunogenicity analysis through VZV lysate antigen stimulation it was determined that IE-62, IE-63, and gE were candidate candidates for the vaccine.
- 5 to 7 are electrophoretic photographs confirming the product expressed by each plasmid gene.
- a plasmid encoding the IE-62 gene using Lipofectamine ® 2000 was introduced into RD cells derived from human rhabdomyosarcoma cells (rhabdomyosarcoma), and the cells were lysed to extract proteins and detect expression antigens. Western blot was performed using the antibody. Expression proteins of the plasmid encoding the IE-62 gene were detected using anti-VZV IE62 (SantaCruz, cat.no.SC-2020) and visualized using HRP Chromogenic Substrates (Invitrogen Cat. No. WP20004). .
- a plasmid encoding a gE gene into RD cells derived from human rhabdomyosarcoma cells (rhabdomyosarcoma) using Lipofectamine ® 2000, the cells were lysed to extract proteins and the antibody for detecting the expression antigen. Western Blot was performed using. Expression proteins of the plasmid encoding the gE gene were detected using HA-probe HRP (SantaCruz, cat.no. SC-805HRP) and visualized using HRP Chromogenic Substrates (Invitrogen Cat. no. WP20004).
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Abstract
Description
단백질 | 항원 이름 | 아미노산 번호 | 혼합된 중복펩타이드 개수 |
IE-62 | IE62-1 | 1-37 | 37 |
IE62-2 | 28-74 | 37 | |
IE62-3 | 75-111 | 37 | |
IE62-4 | 112-143 | 37 | |
IE62-5 | 149-185 | 37 | |
IE62-6 | 186-223 | 38 | |
IE62-7 | 224-261 | 38 | |
IE-63 | IE63-1 | 1-27 | 27 |
IE63-2 | 28-54 | 27 | |
gE | gE-1 | 1-27 | 41 |
gE-2 | 28-54 | 40 | |
gE-3 | 1-41 | 41 |
Claims (7)
- 대상포진 바이러스(VZV) 단백질을 부호화하는 VZV 유래 유전자의 삽입 부위를 포함하고,전기천공 방식에 의하여 체내에 직접 투여되어 VZV 항원에 대한 T세포 면역활성을 유도하는 대상포진 예방 및 치료용 플라스미드 DNA.
- 제1항에 있어서,상기 유전자는 IE-62 단백질(서열목록 1)을 코딩하는 제1 유전자, IE-63 단백질(서열목록 2)을 코딩하는 제2 유전자 및 gE 단백질(서열목록 3)을 코딩하는 제3 유전자 중 어느 하나인 것을 특징으로 하는 대상포진 예방 및 치료용 플라스미드 DNA.
- 제1항에 있어서,서열목록 4의 플라스미드 DNA, 서열목록 5의 플라스미드 DNA 및 서열목록 6의 플라스미드 DNA 중 어느 하나인 것을 특징으로 하는 플라스미드 DNA.
- 대상포진 바이러스(VZV) 단백질을 부호화하는 VZV 유래 유전자의 삽입 부위를 포함하는 적어도 일종의 플라스미드, 및 약제학적으로 허용 가능한 기타의 성분을 포함하는 대상포진 예방 및 치료용 DNA 백신 조성물.
- 제4항에 있어서,상기 플라스미드는 서로 다른 이종(異種)의 유전자 삽입부위를 포함하는 복수의 플라스미드를 포함하는 것을 특징으로 하는 대상포진 예방 및 치료용 DNA 백신 조성물.
- 제4항에 있어서,상기 플라스미드는 IE-62 단백질(서열목록 1)을 코딩하는 제1 유전자의 삽입부위를 포함하는 제1 플라스미드, IE-63 단백질(서열목록 2)을 코딩하는 제2 유전자의 삽입부위를 포함하는 제2 플라스미드 및 gE 단백질(서열목록 3)을 코딩하는 제3 유전자의 삽입부위를 포함하는 제3 플라스미드를 포함하는 것을 특징으로 하는 대상포진 예방 및 치료용 DNA 백신 조성물.
- 대상포진 바이러스(VZV) 단백질을 부호화하는 VZV 유래 유전자의 삽입 부위를 포함하는 플라스미드를 준비하는 단계; 및상기 플라스미드를 전기천공 방식에 의하여 체내에 투여하는 단계를 포함하는 VZV 항원에 대한 T세포 활성화 방법.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP15852539.4A EP3210631A4 (en) | 2014-10-21 | 2015-06-16 | Dna vaccine composition for preventing and treating herpes zoster, and method for activating t cells for vzv antigen by using same |
JP2017540952A JP2017532983A (ja) | 2014-10-21 | 2015-06-16 | 帯状疱疹予防及び治療用dnaワクチン組成物並びにこれを利用したvzv抗原に対するt細胞活性化方法 |
CN201580070052.9A CN107106705A (zh) | 2014-10-21 | 2015-06-16 | 用于预防和治疗带状疱疹的dna疫苗组合物,以及使用其激活针对vzv抗原的t细胞的方法 |
US15/520,282 US20180117141A1 (en) | 2014-10-21 | 2015-06-16 | Dna vaccine composition for preventing and treating herpes zoster, and method for activating t cells for vzv antigen by using same |
Applications Claiming Priority (2)
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US20220001007A1 (en) * | 2018-11-06 | 2022-01-06 | Oxford University Innovation Limited | Compositions and methods |
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WO2019035605A2 (ko) | 2017-08-16 | 2019-02-21 | 주식회사 차백신연구소 | 리포펩티드가 삽입된 리포좀을 유효성분으로 포함하는 백신 아쥬반트 및 이의 용도 |
KR102098097B1 (ko) | 2017-08-16 | 2020-05-26 | 주식회사 차백신연구소 | 리포펩티드가 삽입된 리포좀을 유효성분으로 포함하는 백신 아쥬반트 및 이의 용도 |
AU2019272184B2 (en) * | 2018-05-23 | 2022-01-27 | Mogam Institute For Biomedical Research | Antigen variant of varicella zoster virus and use thereof |
CA3114658A1 (en) * | 2018-09-27 | 2020-04-02 | Bravovax Co., Ltd | Immune composition, preparation method therefor and use thereof |
CN112142829B (zh) * | 2019-06-28 | 2022-02-22 | 怡道生物科技(苏州)有限公司 | 水痘-带状疱疹病毒gE蛋白突变体及其表达方法 |
CN112941031B (zh) * | 2021-02-20 | 2023-06-27 | 安徽智飞龙科马生物制药有限公司 | 一种gE-HEK293细胞的构建方法及其应用 |
CN118401544A (zh) * | 2021-11-24 | 2024-07-26 | 旗舰创业创新六公司 | 水痘-带状疱疹病毒免疫原组合物及其用途 |
CN114891830B (zh) * | 2022-04-02 | 2024-03-01 | 武汉博沃生物科技有限公司 | 基于水痘-带状疱疹病毒的重组表达载体、重组病毒及用途 |
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KR19990082360A (ko) * | 1996-02-09 | 1999-11-25 | 장 스테판느 | 수두 대상포진 바이러스 유전자 63 생성물에 대한 백신 |
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EP2091558B1 (en) * | 2006-11-17 | 2018-04-04 | Genetronics, Inc. | Methods of enhancing immune response using electroporation-assisted vaccination and boosting |
AU2008331673B2 (en) * | 2007-11-12 | 2014-12-11 | The Trustees Of The University Of Pennsylvania | Novel vaccines against multiple subtypes of influenza virus |
US9243041B2 (en) * | 2011-01-31 | 2016-01-26 | The Trustees Of The University Of Pennsylvania | Nucleic acid molecules encoding novel herpes antigens, vaccine comprising the same, and methods of use thereof |
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DATABASE GenBank 14 August 2009 (2009-08-14), Database accession no. BAC44882.1 * |
DATABASE GenBank 16 December 2013 (2013-12-16), Database accession no. AGU41816.1 * |
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US20220001007A1 (en) * | 2018-11-06 | 2022-01-06 | Oxford University Innovation Limited | Compositions and methods |
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EP3210631A4 (en) | 2018-04-25 |
CN107106705A (zh) | 2017-08-29 |
KR20160047033A (ko) | 2016-05-02 |
JP2017532983A (ja) | 2017-11-09 |
US20180117141A1 (en) | 2018-05-03 |
EP3210631A1 (en) | 2017-08-30 |
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