WO2022039438A1 - 식물 발현 재조합 지카바이러스 외피단백질을 포함하는 백신 조성물 및 이의 제조방법 - Google Patents
식물 발현 재조합 지카바이러스 외피단백질을 포함하는 백신 조성물 및 이의 제조방법 Download PDFInfo
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- WO2022039438A1 WO2022039438A1 PCT/KR2021/010650 KR2021010650W WO2022039438A1 WO 2022039438 A1 WO2022039438 A1 WO 2022039438A1 KR 2021010650 W KR2021010650 W KR 2021010650W WO 2022039438 A1 WO2022039438 A1 WO 2022039438A1
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- zika virus
- envelope protein
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- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
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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
- A61K2039/525—Virus
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- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a vaccine composition
- a vaccine composition comprising a recombinant Zika virus envelope protein and an adjuvant as active ingredients, a method for preparing the same, and a plant-expressed recombinant vector for production of the recombinant Zika virus envelope protein, and the like.
- Zika virus is a mosquito-borne flavivirus of the Flaviviridae family , and is an infectious disease that induces Guillain-Barre syndrome and congenital malformations in the fetus and newborn . Since Zika virus was first reported in Brazil in May 2015, it has rapidly spread in Latin America and has recently emerged as of June 1, 2016 (since 2015). In 53 countries and Brazil alone in 2015, it was estimated that 440,000-1.3 million people were infected, and as of May 26, there were 240 confirmed microcephaly and more than 5,000 cases under suspicion and diagnosis. Considering the possibility, the market size is expected to be very large, but so far, there is no special prevention method other than an aerosol insecticide for the control of disease vectors as a method for preventing Zika virus infection.
- the present invention was derived to solve the problems of the prior art and the necessity of developing a Zika virus vaccine as described above, and not only enables efficient production using plants, but also has high immunogenicity and virus neutralization ability (neutralization ability)
- the present invention was completed by developing a recombinant Zika virus envelope protein representing
- an object of the present invention is a recombinant Zika virus (Zika virus) envelope protein comprising the amino acid sequence of SEQ ID NO: 1; And to provide a vaccine composition comprising alum (alum), MPL (monophosphoryl lipid A) or a combination thereof as an active ingredient as an adjuvant (adjuvant).
- Zika virus Zika virus
- a vaccine composition comprising alum (alum), MPL (monophosphoryl lipid A) or a combination thereof as an active ingredient as an adjuvant (adjuvant).
- Another object of the present invention is to provide a recombinant vector for Zika virus envelope protein expression comprising the gene sequence of the Zika virus envelope protein consisting of the nucleotide sequence of SEQ ID NO: 2.
- Another object of the present invention is to provide a transformant, transformed with the recombinant vector according to the present invention.
- Another object of the present invention is to provide a recombinant Zika virus envelope protein prepared using the recombinant vector according to the present invention.
- Another object of the present invention is to provide a method for producing a recombinant Zika virus envelope protein.
- the present invention provides a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1; And as an adjuvant (adjuvant) alum (alum), MPL (monophosphoryl lipid A) or a combination thereof as an active ingredient comprising a combination thereof, it provides a vaccine composition.
- the present invention is a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1; And it provides a method for preventing or treating Zika virus infection, comprising administering to a subject in need thereof a vaccine composition comprising alum, MPL, or a combination thereof as an adjuvant as an active ingredient.
- the present invention provides a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1; And it provides a prophylactic or therapeutic use of a composition comprising alum, MPL, or a combination thereof as an adjuvant as an active ingredient, Zika virus infection.
- the present invention provides a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1; and alum, MPL or a combination thereof as an adjuvant for producing a vaccine for use in Zika virus infection.
- the recombinant Zika virus envelope protein may be a fusion of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4.
- the recombinant Zika virus envelope protein may be a fusion of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6.
- the recombinant Zika virus envelope protein may be a fusion of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8.
- the recombinant Zika virus envelope protein may include the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12.
- the alum may be included in a ratio of 1:1 to 50 (envelope protein: alum) relative to the weight of the recombinant Zika virus envelope protein, but is not limited thereto.
- the MPL may be included in a ratio of 1: 0.4 to 2 (envelope protein: MPL) relative to the weight of the recombinant Zika virus envelope protein, but is not limited thereto.
- the combination of alum and MPL may be included in a ratio of 1: 1 to 50: 0.4 to 2 (envelope protein: alum: MPL) relative to the weight of the recombinant Zika virus envelope protein, but is limited thereto. it is not
- the vaccine composition may be inoculated 1 to 3 times, but is not limited thereto.
- the inoculation may be made at intervals of 14 to 28 days, but is not limited thereto.
- the vaccine composition is Zika virus PRVABC59 strain, Zika virus MR766 strain and dengue virus (Dengue virus) type 2 It may have a protective ability against one or more selected from the group consisting of, However, the present invention is not limited thereto.
- the vaccine composition is any one or more selected from the group consisting of interferon-gamma (IFN- ⁇ ), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF- ⁇ ) It can stimulate secretion.
- IFN- ⁇ interferon-gamma
- IL-12 interleukin-12
- TNF- ⁇ tumor necrosis factor-alpha
- the vaccine composition is capable of inducing the formation of a maternal antibody.
- the present invention provides a recombinant vector for Zika virus envelope protein expression comprising the gene sequence of the Zika virus envelope protein consisting of the nucleotide sequence of SEQ ID NO: 2.
- the vector may further include the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.
- the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4; Or it may further include the nucleotide sequence of SEQ ID NO: 5, but is not limited thereto.
- the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:6; Or it may further include the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.
- the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 8; Or it may further include the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.
- the vector may include the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 13, but is not limited thereto.
- the recombinant vector may be expressed in a plant.
- the present invention provides a transformant, transformed with the recombinant vector according to the present invention.
- the transformant may be a plant.
- the present invention provides a recombinant Zika virus envelope protein prepared using the recombinant vector according to the present invention.
- the recombinant Zika virus envelope protein may consist of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, but is not limited thereto.
- the present invention comprises the steps of (a) culturing the transformant according to the present invention; and (b) isolating and purifying the recombinant Zika virus envelope protein from the transformant or the culture medium.
- the purification of step (b) may be purification using a water-soluble fraction, but is not limited thereto.
- the recombinant Zika virus envelope protein of the present invention is not only effectively expressed in plants, but also has high water solubility, so it is easy to isolate and purify. Therefore, it can be used as a novel Zika virus vaccine.
- immune cells of mice inoculated with the recombinant Zika virus envelope protein-containing vaccine composition according to the present invention are interferon-gamma (IFN- ⁇ ), interleukin-12 (IL- 12) and tumor necrosis factor-alpha (TNF- ⁇ ) production was specifically increased, and it was found that the immunological defense/preventive efficacy against Zika virus infection was increased. Therefore, the vaccine composition of the present invention can be usefully used for the prevention of Zika virus infection.
- IFN- ⁇ interferon-gamma
- IL-12 interleukin-12
- TNF- ⁇ tumor necrosis factor-alpha
- the present invention can verify an expression system with more excellent immune efficacy by referring to the results of directly applying a recombinant Zika virus vaccine composition developed using a plant cell-derived expression system to an animal model, which is a recombinant Zika virus that can be applied to humans. While promoting the development of a Zika virus vaccine, it can be used for the development of vaccines for other mosquito-borne infectious diseases similar to Zika virus.
- NB new chaperone binding protein
- Zika Env Zika envelope protein ectodomain
- H polyhistidine tag
- hFc Fc fragment of human immunoglobulin heavy chain
- HDEL histidine-aspartic acid-glutamic acid-leucine tag
- Zika:Env1 NB-zika envelope protein ectodomain-H-HDEL
- Zika:Env NB- zika envelope protein ectodomain-human Fc-HDEL
- T total fraction
- S water-soluble fraction
- P pellet fraction
- FIG. 3 is a diagram showing the results of western blotting to check whether or not binding to a resin (resin) in the process of separating and purifying recombinant Zika virus envelope protein from a plant.
- Figure 4 is a view confirmed through Coomassie staining after electrophoresis (SDS-PAGE) of the recombinant Zika virus envelope protein isolated and purified from the plant.
- FIG. 5 is a transmission electron micrograph (top, Zika:Env1; bottom, Zika:Env) of two recombinant Zika virus envelope proteins.
- FIGS. 6A to 6c are roads showing the results of measuring the antibody titer using Indirect ELISA of the serum separated through orbital blood sampling after 7 to 14 days after inoculating mice with the vaccine composition according to the present invention 1-3 times.
- 6A is a flowchart showing the inoculation and blood collection schedule
- FIGS. 6B and 6C are graphs comparing the levels of antibodies recognizing Zika:Env1 and Zika:Env in immunized mouse serum according to the inoculation cycle and group ( one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- FIG. 7A and 7B show that mice were inoculated 1-3 times with the vaccine composition according to the present invention, and the sera of 5 mice of the same group isolated after 7 to 14 days were pooled, and Th1 through indirect ELISA.
- FIG. 7A is a result using a plate coated with Zika: Env1
- FIG. 7B is a result using a plate coated with Zika-Env.
- Figure 8 is a vaccine candidate group each inoculated once, 7 days later, the spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours to measure IFN- ⁇ producing cells through ELISPOT analysis. A diagram showing the results.
- FIG. 9 shows that each vaccine candidate group was inoculated once, and after 7 days, spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours to measure IL-12 producing cells through ELISPOT analysis.
- a diagram showing the results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 10 is a vaccine candidate group, each inoculated twice, 3 days later, the spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours to measure IFN- ⁇ producing cells through ELISPOT analysis.
- a diagram showing the results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 11 shows that the vaccine candidate group was inoculated twice, and after 3 days, spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours to measure IL-12 producing cells through ELISPOT analysis.
- a diagram showing the results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 13 shows that each vaccine candidate group was inoculated 3 times, and 3 days later, spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours to measure IL-12 producing cells through ELISPOT analysis.
- a diagram showing the results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- FIG. 14 shows that each vaccine candidate group was inoculated once, and spleen immune cells derived from the same group were pooled 7 days later and stimulated with each Zika virus envelope protein antigen for 48 hours through ELISA analysis for IFN- ⁇ (A), IL -12(B), TNF- ⁇ (C), IL-4(D) is a diagram showing the measurement results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; *** , p ⁇ 0.001).
- FIG. 15 shows that each of the vaccine candidates was inoculated twice, and 3 days later, spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours through ELISA analysis for IFN- ⁇ (A), IL -12(B), TNF- ⁇ (C), IL-4(D) is a diagram showing the measurement results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; *** , p ⁇ 0.001).
- FIG. 16 shows that each vaccine candidate group was inoculated 3 times, and 3 days later, spleen immune cells derived from the same group were pooled and stimulated with each Zika virus envelope protein antigen for 48 hours.
- -12(B), TNF- ⁇ (C), IL-4(D) is a diagram showing the measurement results (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; *** , p ⁇ 0.001).
- 17A and 17B show that the vaccine candidate group was inoculated once and 7 days later (day 7), or 3 days after inoculation (day 45), spleen immune cells derived from the same group were pooled and CD4, A diagram showing the results of measuring the ratio of CD8 T cells (CD3 + , CD4 + , CD8 + ).
- 18A to 18D show that each of the vaccine candidate groups was inoculated once and 7 days later (day 7), or 3 days after inoculation (day 45), spleen immune cells derived from the same group were pooled and exhausted through flow cytometry. It is a diagram showing the result of measuring the ratio of T cells (CD4 + PD-1 + or CD8 + PD-1 + ).
- 19A and 19B show that the vaccine candidate group was inoculated once and 7 days later (day 7), or 3 days after inoculation (day 45), spleen immune cells derived from the same group were pooled to control T through flow cytometry. It is a diagram showing the result of measuring the ratio of cells (CD4 + , CD25 + , Foxp3 + ).
- Figures 20a and 20b are diagrams showing the results of indirect ELISA analysis by pooling sera so that the vaccine candidate group (Zika: Env1, Zika: Env) was inoculated three times and crossed 7 days later to the same group ( one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 21 is a vaccine candidate group (Zika:Env1, Zika:Env) three times inoculation, 7 days later, the spleen from the pup mice obtained by mating, spleen immune cells stimulated with each antigen for 48 hours, IFN through ELISPOT analysis The results of measuring - ⁇ -producing cells and quantifying the number of spots are shown (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 22 is a vaccine candidate group (Zika:Env1, Zika:Env) three times inoculated, 7 days later, the spleen from the pups obtained by mating, spleen immune cells are stimulated with each antigen for 48 hours, IL through ELISPOT analysis The results of measuring -12 producing cells and quantifying the number of spots are shown (one-way ANOVA; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- Figure 23 is a vaccine candidate group (Zika:Env1, Zika:Env) inoculated three times, and 7 days later, the spleen was extracted from the pup mice obtained by mating, spleen immune cells were stimulated with each antigen for 48 hours, and ELISA analysis was performed.
- 24A and 24B show the survival rate (24a) by inoculating the vaccine candidate group (Zika:Env1, Zika:Env) three times and challenged with two kinds of Zika virus or dengue virus type 2 to baby mice obtained by mating after 7 days (24a) and a diagram showing the result of observing the change in body weight (24b).
- the present inventors developed two recombinant Zika virus vaccines (named Zika:Env1 and Zika:Env, respectively) in which the Zika virus envelope protein was expressed in plants to develop a vaccine capable of minimizing side effects in vivo and reducing production costs. , it was confirmed whether the composition according to the present invention induces an immune response such as activating various immune molecules capable of directly killing pathogens together with an antibody response to the antigen, similar to the in vivo virus penetration.
- the present invention relates to a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1; And as an adjuvant (adjuvant) alum (alum), MPL (monophosphoryl lipid A) or a combination thereof may provide a vaccine composition comprising as an active ingredient.
- alum alum
- MPL monophosphoryl lipid A
- the present invention relates to a vaccine composition containing a recombinant Zika virus envelope protein comprising the amino acid sequence of SEQ ID NO: 1.
- the recombinant Zika virus envelope protein which is a major immunogenicity-inducing antigen site of Zika virus, can be expressed.
- After constructing and cloning a plant expression vector it was introduced into Agrobacterium strain LBA4404, and the vector-introduced Agrobacterium was infiltrated into Nicotiana benthamiana, and the Nicotiana benthamiana Recombinant antigen protein was produced from me. It provides a prevention strategy against Zika virus by inducing an immune response such as activating various immune molecules capable of directly destroying an antibody response to an antigen similar to the in vivo virus penetration through the recombinant antigen protein and a pathogen.
- the recombinant Zika virus envelope protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, that is, polyhistidine and histidine-aspartic acid-glutamic acid-leucine (hereinafter referred to as 'HDEL') tags are fused (H-HDEL).
- the recombinant Zika virus envelope protein may be a fusion of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, that is, an Fc fragment of a human immunoglobulin heavy chain (hereinafter referred to as 'hFc').
- the binding site of the hFc may be directly linked to the C-terminus of a peptide or protein intended for expression or synthesis in plant cells or added (or linked) indirectly, that is, with another peptide or protein(s) interposed therebetween. there is.
- the recombinant Zika virus envelope protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8, that is, a HDEL tag fused thereto.
- the binding site of the HDEL may be directly linked to the C-terminus of a peptide or protein intended for expression or synthesis in plant cells or added (or linked) indirectly, that is, with another peptide or protein(s) interposed therebetween. there is.
- the recombinant Zika virus envelope protein may include the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12.
- the recombinant Zika virus envelope protein is one in which a Zika virus envelope protein, a polyhistidine tag, and an HDEL tag are sequentially fused;
- the Zika virus envelope protein, hFc and HDEL tag may be sequentially fused.
- Zika virus is a virus having a genome with a single-stranded length of about 10-kilobase positive-sense RNA belonging to the Flaviviridae family.
- the RNA genome encodes 7 nonstructural proteins and 3 structural proteins.
- the three major viral structural proteins are capsid (C), pre-membrane (prM) and envelope (E), and seven nonstructural proteins are NS1, NS2A, NS2B, NS3, NS4A, Contains one long open reading frame (ORF) encoding NS4B and NS5.
- the "envelope protein (E)” included in the structural protein is responsible for the influx of the Zika virus and is a main target of the neutralizing antibody. So far, monoclonal antibodies that neutralize a number of potent flavivirus types have been shown to target domain-III of the envelope protein. Therefore, the Zika virus envelope protein comprising the domain-III exhibits immunogenicity when administered to a subject, and can greatly contribute to the formation of antibodies capable of fighting Zika virus infection.
- a functional equivalent of the amino acid sequence represented by SEQ ID NO: 1 is also included in the scope of the present invention, and the functional equivalent is a result of the addition, substitution, or deletion of amino acids, the amino acid sequence and at least 60 % or more, preferably 70% or more, more preferably 80% or more, most preferably 90% or more of sequence homology, and a polypeptide that exhibits substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 (polypeptide), and the amino acid sequence of the Zika virus envelope protein that can be stably produced using a plant is not limited thereto.
- the term “antigen” refers to all substances that cause an immune response in the body, preferably viruses, compounds, bacteria, pollen, cancer cells, etc. or some peptides or proteins thereof, or immune responses in the body. As long as it is a material capable of causing a reaction, it is not limited thereto.
- the term “vaccine” refers to a biological preparation containing an antigen that induces an immune response in a living body, and is an immunogen that induces immunity in a living body by injecting or oral administration to a person or animal for the prevention of infection.
- the animal is a human or non-human animal, and the non-human animal refers to a pig, a cow, a horse, a dog, a goat, a sheep, and the like, but is not limited thereto.
- the term “adjuvant” refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase or affect an immune response. Typically, it refers to a carrier or auxiliary material for an immunogen and/or other pharmaceutically active material or composition. Typically, the term “adjuvant” is to be interpreted in a broad sense and encompasses a broad range of substances or strategies capable of enhancing the immunogenicity of an antigen incorporated into or administered with the adjuvant. means In addition, the adjuvant, without being limited thereto, may be divided into an immune potentiator, an antigen delivery system, or a combination thereof.
- adjuvants examples include aluminum hydroxide, Freud's complete or incomplete adjuvant, DEAE dextran, levamisole, PCG and poly I:C or poly A:U.
- alum and MPL monophosphoryl lipid A, also called alum, were used as adjuvants.
- the alum may be included in a ratio of 1:1 to 50 (envelope protein: alum) based on the weight of the recombinant Zika virus envelope protein. More specifically, the ratio of the weight of the recombinant Zika virus envelope protein to the weight of alum is 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1 It may be :40, 1:45 or 1:50, but is not limited thereto.
- the MPL may be included in a ratio of 1:0.4 to 2 (envelope protein:MPL) relative to the weight of the recombinant Zika virus envelope protein. More specifically, the weight ratio of the recombinant Zika virus envelope protein to the weight of MPL is 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.2, 1 :1.4, 1:1.6, 1:1.8, or 1:2, but is not limited thereto.
- the combination of alum and MPL may be included in a ratio of 1:1 to 50:0.4 to 2 (envelope protein:alum:MPL) relative to the weight of the recombinant Zika virus envelope protein. More specifically, the ratio of the weight of the recombinant Zika virus envelope protein to the weight of alum to the weight of MPL is 1:1:0.4, 1:5:0.4, 1:10:0.4, 1:20:0.4, 1:30:0.4 , 1:40:0.4, 1:50:0.4, 1:1:0.6, 1:5:0.6, 1:10:0.6, 1:20:0.6, 1:30:0.6, 1:40:0.6, 1 :50:0.6, 1:1:1, 1:5:1, 1:10:1, 1:20:1, 1:30:1, 1:40:1, 1:50:1, 1:1 :1.4, 1:5:1.4, 1:10:1.4, 1:20:1.4, 1:30:1.4, 1:40:1.4, 1:50:1.4, 1:1:2, 1:5:2 , 1:100
- alum when the recombinant Zika virus envelope protein is included in the weight of 50 ⁇ g in the vaccine composition according to the present invention, alum may contain 50 to 2500 ⁇ g by weight, and MPL may include 20 to 100 ⁇ g by weight.
- MPL may include 20 to 100 ⁇ g by weight.
- alum when the recombinant Zika virus envelope protein is included in the weight of 10 ⁇ g in the vaccine composition according to the present invention, alum may include 10 to 500 ⁇ g by weight, and MPL may include 4 to 20 ⁇ g by weight.
- the vaccine composition exhibits the highest neutralizing antibody when inoculated 1 to 3 times at intervals of 14 to 28 days, cellular immune response, maternal transfer antibody formation ability and protective efficacy.
- the vaccine composition may have a protective ability against an Asian-lineage Zika virus strain or an African-lineage Zika virus strain.
- suitable Zika viruses of the present invention include, without limitation, viruses from African and/or Asian strains. Several strains within the African and Asian strains of Zika virus have already been identified.
- the vaccine composition may have a protective ability against dengue virus type-2.
- the vaccine composition is interferon-gamma (IFN- ⁇ ), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF- ⁇ ) any one or more cytokines selected from the group consisting of It can stimulate the secretion of Cain.
- IFN- ⁇ interferon-gamma
- IL-12 interleukin-12
- TNF- ⁇ tumor necrosis factor-alpha
- the vaccine composition may induce the formation of a maternal antibody and induce maternal transfer cellular immunity.
- the “vaccine composition” of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods, respectively.
- oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods, respectively.
- it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the lecithin-like emulsifier, for example, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose), gelatin, etc. can be mixed and prepared.
- excipients for example, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose), gelatin, etc.
- lubricants such as magnesium stearate talc can also be used.
- Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, are used. may be included.
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and freeze-dried preparations.
- non-aqueous preparation and suspension propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
- the route of administration of the vaccine composition according to the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.
- parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
- the vaccine composition of the present invention may also be administered in the form of a suppository for rectal administration.
- the dosage of the vaccine composition or pharmaceutical composition according to the present invention is selected in consideration of the individual's age, weight, sex, physical condition, and the like.
- the amount necessary to induce an immunoprotective response in a subject without any side effects may vary depending on the recombinant protein used as the immunogen and any presence of excipients.
- the present invention can provide a recombinant vector for Zika virus envelope protein expression comprising the gene sequence of the Zika virus envelope protein consisting of the nucleotide sequence of SEQ ID NO: 2.
- Vaccines for preventing viral diseases cannot use bacteria due to problems such as protein folding and glycosylation, and are mainly produced using animal cells.
- the vaccine production method using animal cells is not easy to manufacture because it requires a large cost to expand facilities for mass production, and the price of the vaccine is high in most cases.
- inactivated virus vaccines prepared using animal cells have disadvantages in that they are not easily stored and are highly likely to be contaminated with viruses that can infect animals.
- the present invention compensates for these disadvantages by using plants. In other words, plant cells, unlike animal cells, are highly unlikely to be contaminated with viruses that can infect animals, and can be mass-produced at any time as long as the cultivated area is secured. This is possible.
- the recombinant vector for Zika virus envelope protein expression according to the present invention is a recombinant vector for plant expression.
- the recombinant vector is a polynucleotide encoding a new chaperone binding protein (NB), a polynucleotide encoding a human immunoglobulin heavy chain (Fc fragment of human immunoglobulin heavy chain; hFc), a polynucleotide It may further include a polynucleotide encoding a histidine tag or an HDEL (His-Asp-Glu-Leu) tag.
- NB new chaperone binding protein
- hFc human immunoglobulin heavy chain
- HDEL His-Asp-Glu-Leu
- the polynucleotide encoding the NB is a gene comprising the nucleotide sequence of SEQ ID NO: 3, preferably a gene represented by SEQ ID NO: 3, 80% or more, more preferably 90% or more of the nucleotide sequence of SEQ ID NO: 3 , more preferably, it may include a nucleotide sequence having 95% or more sequence homology, and may be located at the 5' end of the polynucleotide sequence encoding the recombinant Zika virus envelope protein.
- all or part of the amino acid sequence is cut off when the recombinant protein is expressed, so that the entire amino acid sequence may be removed from the recombinant Zika virus envelope protein of the present invention, or only some amino acids may remain.
- the polynucleotide encoding the hFc is a gene comprising the nucleotide sequence of SEQ ID NO: 7, preferably the gene represented by SEQ ID NO: 7, 80% or more of the nucleotide sequence of SEQ ID NO: 7, more preferably 90 % or more, more preferably, it may contain a nucleotide sequence having sequence homology of 95% or more, and may be located at the 3' end of the polynucleotide sequence encoding the recombinant Zika virus envelope protein.
- the polynucleotide encoding the HDEL tag is a gene comprising the nucleotide sequence of SEQ ID NO: 9, preferably a gene represented by SEQ ID NO: 9, 75% or more of the nucleotide sequence of SEQ ID NO: 9, more preferably It may include a nucleotide sequence having sequence homology of 80% or more, more preferably 90% or more, and may be located at the 3' end of the polynucleotide sequence encoding the recombinant Zika virus envelope protein.
- the polyhistidine tag may be fused with the HDEL tag, and the polynucleotide encoding it is a gene including the nucleotide sequence of SEQ ID NO: 5, preferably the gene represented by SEQ ID NO: 5, but the base of SEQ ID NO: 5 It may include a nucleotide sequence having sequence homology of 80% or more, more preferably 90% or more, more preferably 95% or more, and the 3' terminal direction of the polynucleotide sequence encoding the recombinant Zika virus envelope protein can be located in
- the recombinant vector according to the present invention comprises the nucleotide sequence of SEQ ID NO: 3; the nucleotide sequence of SEQ ID NO: 2; And the polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 5 may be sequentially linked.
- the above sequence that is, when it includes an expression cassette of Zika:Env1 shown in the cleavage map (A) at the top of FIG.
- the recombinant vector according to the present invention has the nucleotide sequence of SEQ ID NO: 11 and most preferably consists of the nucleotide sequence of SEQ ID NO: 11, but has 80% or more, more preferably 90% or more, more preferably 95% or more sequence homology to the nucleotide sequence of SEQ ID NO: 11 may include
- the recombinant vector according to the present invention comprises the nucleotide sequence of SEQ ID NO: 3; the nucleotide sequence of SEQ ID NO: 2; a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 or the nucleotide sequence of SEQ ID NO: 7; And the polynucleotide encoding the amino acid sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 9 may be sequentially linked.
- the above sequence that is, when it includes an expression cassette of Zika:Env shown in the cleavage map (B) at the bottom of FIG.
- the recombinant vector according to the present invention has the nucleotide sequence of SEQ ID NO: 13 and most preferably consists of the nucleotide sequence of SEQ ID NO: 13, but has 80% or more, more preferably 90% or more, more preferably 95% or more sequence homology to the nucleotide sequence of SEQ ID NO: 13 may include
- the term “recombinant vector” refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, preferably a target antigen (in the present invention, Zika It refers to a vector prepared to express viral envelope protein).
- the "vector” refers to any medium for introduction and/or transfer of a base into a host cell in vitro, in vivo or in vivo, and a replication unit ( replicon), and "replication unit” means any genetic unit (eg, plasmid, phage, cosmid, chromosomes, viruses, etc.).
- the recombinant vector of the present invention preferably comprises a promoter, which is a transcription initiation factor to which RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may include regulatory sequences, terminators, etc., such as NB gene, polyhistidine-tag (amino acid motif consisting of at least 5 histidine residues), endoplasmic reticulum signal peptide (ER targeting sequence) Meaning) may further include a gene, a cloning site, and the like. In addition, it may further include a selection marker gene, such as an antibiotic resistance gene for selecting an Fc fragment of human immunoglobulin and transformants.
- a promoter which is a transcription initiation factor to which RNA polymerase binds
- an optional operator sequence for regulating transcription a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may include regulatory sequence
- the term “Fc fragment of human immunoglobulin heavy chain (hFc)” refers to when immunoglobulin is digested by papain, only the heavy chain (H chain) portion.
- a portion linked by SS bond and not having an antigen-binding site is called an Fc fragment
- the Fc fragment of the present invention is preferably a human Fc fragment, more preferably a human Fc fragment consisting of the amino acid sequence of SEQ ID NO: 6 or A human Fc fragment encoded by the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.
- a variant of the nucleotide sequence represented by SEQ ID NO: 7 is included in the scope of the present invention.
- the gene may include a nucleotide sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the nucleotide sequence of SEQ ID NO: 7.
- the “cloning site” refers to a thing inserted for the purpose of linking/distinguishing each gene in a vector.
- ER signal peptide (ER signal sequence) is not limited in its type and amino acid sequence as long as it is a plant ER signal peptide known to those skilled in the art.
- references such as US 20130295065 and WO2009158716 may be referred to.
- the endoplasmic reticulum signal peptide may be encoded by the nucleotide sequence of SEQ ID NO: 3 (NB; new chaperone binding protein), and after being translated into protein, the sequence may be partially or completely removed. there is.
- the selection marker gene includes, for example, herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, chloram.
- herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, chloram.
- Antibiotic resistance genes such as phenylchol (chloramphenicol), aadA gene, etc.
- the promoters include, for example, pEMU promoter, MAS promoter, histone promoter, Clp promoter, 35S promoter derived from cauliflower mosaic virus, flower 19S RNA promoter derived from cauliflower mosaic virus, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, EF-1 ⁇ (Elongation) factor-1 alpha) promoter, pEMU promoter, MAS promoter, histone promoter, Clp promoter, etc.
- the terminator is, for example, nopaline synthase (NOS), rice amylase RAmy1 A terminator, phaseolin terminator, agro Bacterium tumafaciens octopine (Octopine) gene terminator, E. coli rrnB1/B2 terminator, etc. may be included, but those listed above are merely examples and are not limited thereto.
- NOS nopaline synthase
- rice amylase RAmy1 A terminator phaseolin terminator
- Octopine agro Bacterium tumafaciens octopine gene terminator
- E. coli rrnB1/B2 terminator etc.
- the present invention provides a transformant, transformed with the above recombinant vector.
- the transformant is preferably a microorganism such as Escherichia coli, Bacillus, Salmonella, yeast, etc., insect cells, animal cells including humans, mice, rats, dogs, monkeys, pigs, horses, cattle, etc.
- a microorganism such as Escherichia coli, Bacillus, Salmonella, yeast, etc., insect cells, animal cells including humans, mice, rats, dogs, monkeys, pigs, horses, cattle, etc.
- transformation refers to a change in the genetic properties of an organism due to the injected DNA
- transgenic organism refers to an external gene by injecting a molecular genetic method.
- the produced organism it is preferably an organism transformed by the recombinant expression vector of the present invention, and the organism is not limited as long as it is a living organism such as a microorganism, eukaryotic cell, insect, animal, plant, etc., preferably E. coli, Salmonella, bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cattle, Acrobacterium tumafaciens, plants, and the like, but not limited thereto.
- plant may be used without limitation as long as it is a plant capable of mass-producing the protein containing the recombinant Zika virus envelope protein of the present invention, but more specifically, tobacco, Arabidopsis, corn, rice, soybean, canola , alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper may be selected from the group consisting of, preferably tobacco.
- Tobacco in the present invention is not limited in type as long as it is a plant of the genus Tobacco (Nicotiana genus) that can overexpress protein, and the present invention may be carried out by selecting an appropriate variety according to the transformation method and the purpose of mass production of protein. can For example, Nicotiana benthamiana L. or Nicotiana tabacum cv. xanthi and other varieties are available.
- the transformant may be prepared by transformation, transfection, Agrobacterium-mediated transformation method, particle gun bombardment, sonication, and electroporation. ) and PEG (Polyethylen glycol)-mediated transformation method, etc., but there is no limitation as long as it is a method capable of injecting the vector of the present invention.
- the present invention may provide a recombinant Zika virus envelope protein prepared using the recombinant vector according to the present invention.
- the recombinant Zika virus envelope protein may consist of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, but is not limited thereto.
- the present invention comprises the steps of (a) culturing the transformant of claim 16; and (b) isolating and purifying the recombinant Zika virus envelope protein from the transformant or the culture medium.
- the purification of step (b) may be purified using an aqueous fraction, but is not limited thereto.
- Alum and TLR4-type monophosphoryl-lipid A (MPL) approved for use in human vaccines were selected as adjuvant candidates, and tests were conducted to select the most effective antigen and adjuvant combination and concentration.
- the vaccine candidates (Zika virus recombinant antigen) are as follows: Zika virus envelope protein fused with polyhistidine and HDEL (hereinafter 'Zika:Env1') and Zika virus envelope protein fused with hFc and HDEL (hereinafter 'Zika: Env').
- Antibodies recognizing the vaccine candidates Zika:Env1 and Zika:Env were measured by using the Indirect ELISA method constructed in-house to evaluate the production of the target level of antibody during vaccine administration. After intramuscular injection of PBS or vaccine candidates into mice (C57BL/6, female, 8 weeks old), blood was collected from mice 7 to 14 days later (Day-1, 7, 28, 49) and serum was separated. . Each of the vaccine candidates (Zika:Env1, Zika:Env) was dispensed into plates (Nunc-Immuno Plates; Thermo Scientific, UK) at 0.1 ⁇ g/well, and reacted at 4°C for at least 16 hours, and then wash buffer (wash buffer; 0.05%).
- mice C57BL/6, female, 8 weeks old
- the vaccine candidate group was administered 1-3 times (Day 0, 14, 42) intramuscularly, and after 7 to 14 days (Day-1, 7, 28, 49), blood from mice was collected and serum was separated.
- Each of the candidate vaccine groups (Zika:Env1, Zika:Env) was dispensed into plates (Nunc-Immuno Plates; Thermo Scientific) at 0.1 ⁇ g/well, reacted at 4°C for 16 hours or longer, and then washed with a wash buffer (0.05% Tween20 in PBS).
- Neutralizing antibodies produced during vaccine administration were measured using the Plaque reduction neutralization test (PRNT).
- Mice C57BL/6, female, 8 weeks old) were inoculated with a vaccine candidate group or PBS 3 times (Day 0, 14, 42) intramuscularly, and after 7 days (Day 49), blood was collected from the mice to separate the serum. .
- the serum of 5 mice of the same group was pooled and used, and after being immobilized at 56°C for 30 minutes, the control serum and the experimental group serum were diluted at the same dilution ratio, respectively.
- the spleen was removed on the 7th day (Day 7), or after inoculation twice or 3 times (Day 14, 42) On the third day (Day 17, 45), the spleen was removed.
- the extracted spleen was placed in cold RPMI 1640 medium, the spleen was disrupted using a 5 ml syringe, and then flowed into a 40 ⁇ m nylon cell strainer (BD Bioscience, USA) to separate splenocytes. After centrifuging the obtained cells at 2,000 rpm for 10 minutes, RBC lysis buffer was treated for 2 minutes to remove red blood cells. After removing the RBC lysis buffer by centrifugation, the cells were suspended in RPMI1640 (10% FBS, 1% penicillin-streptomycin) and used for ELISPOT, ELISA and flow cytometry experiments.
- ELISPOT was used to evaluate whether the increase in immune cells producing IFN- ⁇ and IL-12 was performed. After inoculating a mouse (C57BL/6, female, 8 weeks old) with PBS or a vaccine candidate once (Day 0), the spleen was removed on the 7th day (Day 7), or after inoculation twice or 3 times (Day 14, 42) On the third day (Day 17, 45), the spleen was removed. Splenocytes were isolated from the spleen, stimulated with an antigen (Zika:Env1 or Zika:Env) at 1 ⁇ g/ml, and cultured on ELISPOT plates for 48 hours.
- an antigen Zika:Env1 or Zika:Env
- IFN- ⁇ and IL-12 ELISPOT were performed using BD TM ELISPOT mouse IFN- ⁇ ELISPOT set (BD Life Sciences, USA) and mouse IL-12(p70) ELISpotBASIC (MABTECH, USA), respectively, according to the manufacturer's instructions.
- IFN- ⁇ capture antibody was diluted 1:200 and 100 ⁇ l/well was added, followed by reaction at 4° C. for 16 hours. After washing 3 times with a wash buffer, 200 ⁇ l/well of a blocking reagent (RPMI1640, 10% FBS) was added and reacted at room temperature for 2 hours. After washing again with wash buffer 3 times, splenocytes were aliquoted at 5 ⁇ 10 5 cells/well, and antigens (vaccine candidates Zika:Env1, Zika:Env) were treated at a concentration of 1 ⁇ g/ml at 37°C, 5 % CO 2 Incubated in an incubator.
- a blocking reagent RPMI1640, 10% FBS
- the IL-12 capture antibody was diluted to 15 ⁇ g/ml and added at 100 ⁇ l/well, followed by reaction at 4° C. for 16 hours. After washing 4 times with PBS, 200 ⁇ l/well of a blocking reagent (RPMI1640, 10% FBS) was added and reacted at room temperature for 1 hour. After washing again with PBS 4 times, splenocytes were aliquoted at 5 ⁇ 10 5 cells/well, and antigens (vaccine candidates Zika:Env1, Zika:Env) were treated at a concentration of 1 ⁇ g/ml at 37°C, 5% It was cultured in a CO 2 incubator.
- a blocking reagent RPMI1640, 10% FBS
- the detection antibody was diluted to 1 ⁇ g/ml and added at 100 ⁇ l/well, followed by reaction at room temperature for 2 hours.
- streptavidin-HRP was diluted 1:100 and added at a time of 100 ⁇ l/well, followed by reaction at room temperature for 1 hour.
- the color development reaction was observed using the BD AEC substrate set (BD Life Sciences), the wells were washed with DDW to stop the reaction, and then the spots in each well were counted after drying in the air. and analyzed.
- Cytokine production was evaluated using an ELISA kit. After inoculating a mouse (C57BL/6, female, 8 weeks old) with PBS or a vaccine candidate once (Day 0), the spleen was removed on the 7th day (Day 7), or after inoculation twice or 3 times (Day 14, 42) On the third day (Day 17, 45), the spleen was removed. Splenocytes were isolated from the spleen, aliquoted at 5 ⁇ 10 5 cells/well in a 96-well cell culture plate, and restimulated with an antigen (vaccine candidate Zika:Env1 or Zika:Env) corresponding to a concentration of 1 ⁇ g/ml.
- an antigen vaccine candidate Zika:Env1 or Zika:Env
- cytokines IFN- ⁇ , IL-12, IL-4, TNF- ⁇
- the measurement of cytokines (IFN- ⁇ , IL-12, IL-4, TNF- ⁇ ) secreted into the cell culture medium is mouse IFN- ⁇ ELISA MAX TM standard set, mouse IL-12 ELISA MAX TM standard set, mouse IL-4 ELISA MAX TM standard set, mouse TNF- ⁇ ELISA MAX TM standard set (Bio Legend, USA) was used according to the manufacturer's instructions.
- the capture antibody corresponding to each cytokine was diluted 1:200 and put into plates (Nunc-Immuno Plates; Thermo Scientific) at 100 ⁇ l/well, and then reacted at 4°C for 16 hours. After washing 4 times with wash buffer, it was reacted with 10% FBS (in PBS), a blocking reagent, for 1 hour. After washing 4 times with wash buffer, 100 ⁇ l of each cell culture solution was added, and the reaction was performed at room temperature for 2 hours. After washing 4 times with wash buffer, 100 ⁇ l/well of the detection antibody diluted 1:200 was added, and the reaction was performed at room temperature for 1 hour.
- Analysis of immune cells activated at the time of vaccination includes effector T cells (CD3 + , CD4 + , CD8 + ), exhausted T cells; PD-1 + ) and T reg cells (CD4 + , CD25 + , Foxp3 + ) were analyzed using flow cytometry.
- mice C57BL/6, female, 8 weeks old were inoculated with PBS or a vaccine candidate once (Day 0) and then the spleen was removed on the 7th day (Day 7) or 3 times after the inoculation (Day 42) on the 3rd day The spleen was removed (Day 45).
- the extracted spleen was placed in cold RPMI 1640 medium, the spleen was disrupted using a 5 ml syringe, and then flowed into a 40 ⁇ m nylon cell strainer (BD Bioscience) to separate spleen cells.
- the obtained spleen immune cells were suspended in MACs buffer (0.5% BSA + 2 mM EDTA in PBS) so that the cell concentration was 1 ⁇ 10 6 cells/ml.
- Effector T cells (CD3 + , CD4 + , CD8 + ) and exhausted T cells (PD-1 + ) were used with the antibodies shown in Table 1 below, and all antibodies used in the experiment were purchased from Biolegend. did After removing the supernatant by centrifuging the cell suspension at 4°C and 6,000 rpm for 5 minutes, 50 ⁇ l of an antibody diluted 1:250 was added to each group, followed by reaction at 4°C for 20 minutes. After 20 minutes, 500 ⁇ l of MACs buffer was added and centrifuged at 4°C and 6,000 rpm for 5 minutes. The supernatant was removed, fixed with 400 ⁇ l of 0.4% PFA (in PBS), and measured by flow cytometry.
- T reg cells (CD4 + , CD25 + , Foxp3 + ) were used with the antibodies shown in Table 1 below, and all antibodies used in the experiment were purchased from Biolegend.
- the cell suspension was centrifuged at 4°C and 6,000 rpm for 5 minutes to remove the supernatant, and 50 ⁇ l of CD4 + , CD25 + antibodies diluted 1:250 were added to each group, followed by reaction at 4°C for 20 minutes. . After 20 minutes, 500 ⁇ l of MACs buffer was added and centrifuged at 4°C and 6,000 rpm for 5 minutes.
- mice C57BL/6, female, 8 weeks old were inoculated three times (Day 0, 14, 42) of the vaccine candidate group by intramuscular injection, and then blood was collected at 2 days of age and serum was separated from the breeding mice.
- Each of the vaccine candidates Zika:Env1, or Zika:Env was dispensed at 0.1 ⁇ g/well in plates (Nunc-Immuno Plates; Thermo Scientific) and reacted at 4°C for more than 12 hours, followed by a wash buffer (0.05% Tween20 in PBS). ), and 200 ⁇ l of blocking buffer (1% BSA in PBS) was dispensed into each well and reacted at 37° C. for 2 hours to relieve non-specific binding.
- the serum obtained from the baby mouse was binary diluted from 1:100 with a dilution buffer (0.1% BSA in PBS) and dispensed into each well.
- a dilution buffer (0.1% BSA in PBS)
- 100 ⁇ l of Goat anti-Mouse IgG-heavy and light chain Antibody HRP Conjugated (Bethyl Laboratories) diluted 1:10,000 after washing with wash buffer to remove the serum sample was dispensed at 37°C. reacted for 1 hour.
- color was developed with TMB (Surmodics, USA) solution, and absorbance was measured at 450 nm.
- mice C57BL/6, female, 8 weeks old were inoculated with a vaccine candidate three times (Day 0, 14, 42) by intramuscular injection, and then the spleens were harvested at 30 days of age. Splenocytes were isolated from the spleen, stimulated with an antigen (vaccine candidate Zika:Env1, or Zika:Env), and cultured on an ELISPOT plate for 48 hours. As a negative control, spleen immune cells obtained by inoculating mice with PBS were used.
- IFN- ⁇ and IL-12 ELISPOT were performed using BD TM ELISPOT mouse IFN- ⁇ ELISPOT set (BD Life Sciences) and mouse IL-12 (p70) ELISpotBASIC (MABTECH), respectively, according to the manufacturer's instructions.
- the IFN- ⁇ capture antibody was diluted 1:200 and added at a time of 100 ⁇ l/well, followed by reaction at 4° C. for 16 hours. After washing 3 times with a wash buffer, 200 ⁇ l/well of a blocking reagent (RPMI1640, 10% FBS) was added and reacted at room temperature for 2 hours. After washing 3 times with wash buffer, splenocytes were aliquoted at 5 ⁇ 10 5 cells/well, and antigens (vaccine candidates Zika:Env1, Zika:Env) were treated at a concentration of 1 ⁇ g/ml at 37°C, 5% Incubated in a CO 2 incubator for 48 hours.
- a blocking reagent RPMI1640, 10% FBS
- the detection antibody was diluted 1:250 and 100 ⁇ l/well was added, followed by reaction at room temperature for 2 hours.
- streptavidin (streptavidin)-HRP was diluted 1:100 and added at a time of 100 ⁇ l/well, followed by reaction at room temperature for 1 hour.
- the color development reaction was observed using the BD AEC substrate set (BD Life Sciences), the reaction was stopped by washing the wells with DDW, and then dried in the air. The spots in each well were counted and analyzed.
- the IL-12 capture antibody was diluted to 15 ⁇ g/ml and added at 100 ⁇ l/well, followed by reaction at 4° C. for 16 hours. After washing 4 times with PBS, 200 ⁇ l/well of a blocking reagent (RPMI1640, 10% FBS) was added and reacted at room temperature for 1 hour. After washing 4 times with wash buffer, splenocytes were aliquoted at 5 ⁇ 10 5 cells/well, and antigens (vaccine candidates Zika:Env1, Zika:Env) were treated at a concentration of 1 ⁇ g/ml at 37°C, 5% Incubated in a CO 2 incubator for 48 hours.
- a blocking reagent RPMI1640, 10% FBS
- the detection antibody was diluted to 1 ⁇ g/ml, added at 100 ⁇ l/well, and reacted at room temperature for 2 hours. Then, after washing 3 times with a wash buffer, streptavidin-HRP was diluted 1:100, 100 ⁇ l/well was added, and reacted at room temperature for 1 hour. After washing with PBS 5 times, the color development reaction was observed using the BD AEC substrate set (BD Life Sciences), the wells were washed with DDW to stop the reaction, and after drying in the air, the spots in each well were counted and analyzed. .
- mice C57BL/6, female, 8 weeks of age were inoculated 3 times (Day 0, 14, 42) of the vaccine candidate group by intramuscular injection, and then the spleen was harvested at 30 days of age.
- Splenocytes were isolated from the spleen and aliquoted at 5 ⁇ 10 5 cells/well in a 96-well cell culture plate, and re-treated with an antigen (vaccine candidate Zika:Env1, or Zika:Env) corresponding to a concentration of 1 ⁇ g/ml. After stimulation 48 hours later, cytokine production was analyzed by ELISA analysis.
- cytokines IFN- ⁇ , IL-12, TNF- ⁇
- mouse IFN- ⁇ ELISA MAX TM standard set mouse IL-12 ELISA MAX TM standard set
- mouse TNF- ⁇ ELISA MAX TM standard set Biolegend
- the capture antibody corresponding to each cytokine was diluted 1:200 and put into a plate (Nunc-Immuno Plates; Thermo Scientific) by 100 ⁇ l/well, and then reacted at 4° C. for 16 hours. After washing 4 times with wash buffer, it was reacted with 10% FBS (in PBS), a blocking reagent, for 1 hour. After washing again 4 times with wash buffer, 100 ⁇ l of each cell culture solution was added and reacted at room temperature for 2 hours. After washing 4 times with wash buffer, 100 ⁇ l/well of the detection antibody diluted 1:200 was added and reacted at room temperature for 2 hours.
- mice In mice (C57BL/6, female, 8 weeks old), vaccine candidate (Zika:Env1, or Zika:Env) 10-50 ⁇ g/mouse and the adjuvant Alum 50-500 ⁇ g/mouse + MPL 20 ⁇ g/mouse was mixed in PBS so that the total volume for each mouse was 140 ⁇ l, inoculated 3 times (Day 0, 14, 42) by intramuscular injection, and then mated.
- Two Zika virus Puerto Rico-derived strain PRVABC59
- Kenya-derived strain MR766 Kenya-derived strain MR766)
- dengue virus type 2
- the amount of each inoculated virus is summarized in Table 2 below, and subcutaneous inoculation was performed.
- Example 1 Recombinant Zika virus envelope protein (Zika virus Envelope protein) plant expression vector preparation
- a recombinant plant expression vector was prepared so that the Zika virus envelope protein could be expressed in plants.
- the gene information on the Zika virus envelope protein was obtained, and the gene (SEQ ID NO: 2) was synthesized with a sequence optimized for expression in Nicotiana benthamiana.
- the vector thus constructed contains the nucleotide sequence of SEQ ID NO: 11.
- Example 2 Confirmation of expression of recombinant Zika virus envelope protein
- Each of the two plant expression vectors prepared in Example 1 above was transformed into Agrobacterium strain LBA4404 by electroporation.
- Transformed Agrobacterium in 5 ml of YEP (Yeast Extract Peptone) liquid medium yeast extract 10 g, peptone 10 g, NaCl 5 g, kanamycin 50 mg/L, rifampicin 25 mg/L
- YEP Yeast Extract Peptone
- liquid medium yeast extract 10 g, peptone 10 g, NaCl 5 g, kanamycin 50 mg/L, rifampicin 25 mg/L
- 1 ml of the primary culture solution was inoculated into 50 ml of fresh YEP medium, and cultured with shaking at 28° C. for 6 hours.
- Agrobacteria cultured in this way were collected by centrifugation (7,000 rpm, 4°C, 5 minutes), and then infiltration buffer [10 mM MES (pH 5.7), 10 mM MgCl 2 , 200 ⁇ M acetosyringon] was resuspended at a concentration of OD 1.0 at a wavelength of 600 nm.
- Agro-infiltration was performed by injecting the agrobacterial suspension into the back of the Nicotiana benthamiana leaf using a syringe with the needle removed.
- the protein in the aqueous fraction (S) and the protein in the pellet (P) fraction contained in the solution were mixed with the whole extract before centrifugation ( total; T) and confirmed by Western blotting. More specifically, 30 ⁇ l of each fraction was mixed with SDS sample buffer and then heated. And after electrophoresis on a 10% SDS-PAGE gel to separate proteins by size, move the separated proteins to a PVDF membrane, and then pass through a blocking step using 5% skim milk, to bind to hFc After binding to the secondary antibody, the ECL solution was treated according to the method provided by the manufacturer to confirm the recombinant Zika:Env protein. The results are shown in FIG. 2 .
- the recombinant Zika virus envelope protein expression vector of the present invention can effectively express the recombinant Zika virus envelope protein in plants, and the recombinant Zika virus envelope protein prepared using the vector has high water solubility (water solubility). ), it was confirmed that separation and purification were easy, and aggregation of the recombinant protein was inhibited, and thus it was effective in maintaining the physiological or pharmacological activity of the recombinant protein.
- Example 3 Isolation and purification of recombinant Zika virus envelope protein
- a protein extraction solution [50 mM Sodium phosphate (pH 8.0), 300 mM NaCl, 100 mM Sodium sulfite, 0.5% Triton X -100, 1.5% PVPP] 1 L was added and the tissue was disrupted with a blender, followed by centrifugation at 10,000 rpm at 4° C. for 40 minutes to recover the protein extract.
- affinity chromatography was performed with a column filled with Protein A agarose resin.
- the recombinant Zika:Env protein was eluted with an elution solution [100 mM sodium citrate (pH 3.0), 300 mM NaCl].
- an elution solution [100 mM sodium citrate (pH 3.0), 300 mM NaCl].
- a neutralizing solution [1.5 M Tris-Cl (pH 8.8)] was added to pH 7.4 for protein stabilization.
- the elution solution containing the recombinant Zika:Env protein was replaced with a storage solution [50 mM Tris-Cl (pH 7.4), 300 mM NaCl] using a UF (ultrafiltration) system using a 50 kDa membrane filter and the buffer was replaced and concentrated. carried out.
- the binding of the recombinant Zika virus envelope protein to the resin was confirmed by western blotting (see Fig. 3), and the separated and purified recombinant Zika virus envelope protein was electrophoresed (SDS-PAGE) followed by Coomassie staining. It was confirmed that separation and purification were successful through (Coomassie staining) (see FIG. 4).
- the recombinant Zika virus envelope protein of the present invention is not only effectively expressed in plants, but also has high water solubility, so it is easy to isolate and purify, and also acts as an antigen in the body to have high immunogenicity and virus neutralization ability. Therefore, it could be confirmed that it can be used as a novel Zika virus vaccine composition.
- the effects of two types of Zika virus envelope protein and adjuvant (immune adjuvant) combined were evaluated in several items to search for the most effective vaccine composition.
- Example 4 Candidate selection of vaccines and adjuvants containing recombinant Zika virus envelope protein
- Zika virus vaccine candidates (Zika:Env1 and Zika:Env) expressed in plants (Nicotiana benthamiana) and adjuvant candidates include Alum, which induces a Th2-type immune response and enhances antigen presentation;
- MPL Monophosphoryl-lipid A
- TLR toll-like receptor
- the vaccine candidate group (Zika:Env1, Zika:Env) was confirmed to show the shape of a virus like particle (VLP) using transmission electron microscopy (TEM), but as can be seen in FIG. None of the envelope proteins showed the form of VLPs.
- Antibodies recognizing vaccine candidates Zika:Env1 and Zika:Env were measured using a self-constructed Indirect ELISA method to evaluate whether a target level of antibody is produced during vaccine administration.
- Zika virus antibody in serum isolated from mice 7 to 14 days later (Day-1, 7, 28, 49) after intramuscular injection of PBS or vaccine candidates 1-3 times (Day 0, 14, 42) into mice was measured (refer to the flowchart of FIG. 6a).
- Vaccine candidates are inoculated 1-3 times (Day 0, 14, 42), and after 7 to 14 days (Day-1, 7, 28, 49), the obtained serum is diluted at a ratio of 1:100 or 1:400
- the induction of Th1 and Th2 cell-mediated immune responses was confirmed by comparative analysis of the IgG1 and IgG2c subtypes of the antibody through the constructed Indirect ELISA method.
- Neutralizing antibodies produced during vaccine administration were measured using a Plaque reduction neutralization test (PRNT) (see Table 2). After 7 days of inoculation (Day 49) by intramuscular injection of PBS or vaccine candidates into mice 1-3 times (Day 0, 14, 42), blood was collected from mice and serum was separated. The serum of 5 mice of the same group was pooled and used, and after immobilization at 56°C for 30 minutes, the control serum and the experimental group serum were diluted at the same dilution ratio, respectively.
- PRNT Plaque reduction neutralization test
- neutralizing antibody induction levels against the MR766 Zika virus strain were evaluated as 200 and 100 in Group 2 and 4, respectively, in the Zika:Env1 inoculated group, and 400 and 400 in the Zika:Env inoculated group, Group 3, Group 5, and Group 6, respectively. It was observed that more neutralizing antibodies were induced when evaluated as 1600 and 3200.
- the neutralizing antibody induction level against the PRVABC59 Zika virus strain was also evaluated to be less than 100 and less than 100 in Group 2 and Group 4 inoculated with Zika:Env1, respectively, and Group 3, Group 5, and Group 6 inoculated with Zika:Env, respectively. It was observed that more neutralizing antibodies were induced at 400, 800, and 3200.
- the neutralizing antibody induction level against MR766 strain and PRVABC59 strain was 3200 in group 6, which was mixed inoculated with Zika:Env 10 ⁇ g and Alum 500 ⁇ g + MPL 20 ⁇ g, which was significantly higher than that of other groups.
- lymphocytes expressing IFN- ⁇ or IL-12 were measured by ELISPOT assay.
- IFN- ⁇ and IL-12 ELISPOT were performed using BD TM ELISPOT mouse IFN- ⁇ ELISPOT set (BD Life Sciences) and mouse IL-12 (p70) ELISpotBASIC (MABTECH), respectively, according to the manufacturer's instructions.
- Group 2 Zika:Env1 50 ⁇ g + Alum 50 ⁇ g
- group 6 Zika:Env 10 ⁇ g + Alum 500 ⁇ g + MPL 20 ⁇ g
- the IL-12 secreting cells were similar to the IFN- ⁇ secreting cell test results when the vaccine candidate group was inoculated once, and when the vaccine candidate group was inoculated twice, the highest IL- Twelve secretory cells were observed (see FIG. 11 ). However, when the vaccine candidate group was inoculated three times, all experimental groups (groups 2-6) did not show a significant difference compared with group 1, the control group.
- the secretion of IFN- ⁇ and TNF- ⁇ during one-time inoculation of the vaccine candidate group was similar to the ELISPOT test results in Group 2 (Zika:Env1 50 ⁇ g + Alum 50 ⁇ g) and Group 6 (Zika:Env 10). ⁇ g + Alum 500 ⁇ g + MPL 20 ⁇ g). There was no significant difference between the groups in IL-4, but when group 6 was not sensitized, the highest IL-12 secretion was observed, confirming that overall immunogenicity was increased.
- TNF- ⁇ secretion was decreased in the 3 dose group, but in group 5 (Zika:Env 50 ⁇ g + Alum 50 ⁇ g + MPL 20 ⁇ g), and group 6 (Zika:Env 10 ⁇ g + Alum 500 ⁇ g + MPL 20 ⁇ g) It was found that the amount of IFN- ⁇ secretion increased by about 4 to 19 times compared to the 2 inoculation group (see FIG. 16 ).
- CD4 + , CD8 + T cells (CD3 + , CD4 + , CD8 + ) were measured using flow cytometry in splenocytes isolated from mice inoculated with each vaccine candidate group. .
- Maternal-transferred antibodies in serum isolated from blood obtained from blood collection of baby mice born to mothers vaccinated with the vaccine candidate group on the second day of birth were measured using a self-constructed indirect ELISA method.
- FIGS. 20A and 20B it was observed that the serum of baby mice born to mothers vaccinated with the vaccine candidate showed a higher level of antibody titer compared to the negative control group (PBS; Group 1). was able to measure In particular, it was confirmed that the Zika:Env inoculation group (groups 3, 5, and 6) significantly increased the maternal transfer antibody compared to the Zika:Env1 inoculation group (groups 2 and 4).
- Lymphocytes expressing IFN- ⁇ or IL-12 in spleen immune cells isolated by excising the spleen of baby mice born to mothers vaccinated with the vaccine candidate group on the 30th day of birth were measured by ELISPOT analysis.
- IFN- ⁇ and IL-12 ELISPOT were performed using BD TM ELISPOT mouse IFN- ⁇ ELISPOT set (BD Life Sciences) and mouse IL-12 (p70) ELISpotBASIC (MABTECH), respectively, according to the manufacturer's instructions.
- IFN- ⁇ -producing immune cells were overall increased in the baby mice born to the vaccine candidate group compared to the baby mice born to the negative control group (PBS; group 1).
- PBS negative control group
- group 6 it was confirmed that the number of IFN- ⁇ producing immune cells increased more than 8 times compared to the PBS inoculation group during antigen restimulation.
- IL-12 secretion was also confirmed that the IL-12 production was significantly increased upon antigen restimulation in baby mice born in groups 3, 4, and 5 than in baby mice born in the control group. It was found that the IL-12 secretion was increased about 400 times more than the group (see B of FIG. 23 ).
- TNF- ⁇ secretion it was confirmed that the amount of TNF- ⁇ secretion was significantly increased upon antigen restimulation in baby mice born in groups 2, 4, 5, and 6 than in baby mice born in the control group, especially in the case of group 4, which was not sensitized.
- IL-12 it was confirmed that the overall immunogenicity was increased by confirming that the TNF- ⁇ production was increased by about 400 times or more than that of the control group.
- TCID 50 tissue culture infective from serum obtained 2 days after challenge inoculation with two Zika viruses (MR766, PRVABC59 strain) or dengue virus (DENV type 2) at 2 days of age, pups born to mothers vaccinated with vaccine candidates. dose 50) to measure the virus content in the serum.
- the sera of 5 mice of the same group were pooled and used, and after non-assimilation at 56°C for 30 minutes, the sera of the control group and the experimental group were diluted by decimal from 10 1 to 10 3 , respectively.
- the diluted serum was inoculated into Vero cells or Vero 76 cells and cultured at 37° C., 5% CO 2 in an incubator, while observing the presence or absence of CPE to calculate a TCID 50 value.
- Table 4 The results are shown in Table 4 below.
- infectious virus was detected in the sera obtained 2 days after challenge with the MR766 strain in all groups except for mice born to mothers of group 6 (Zika:Env 10 ⁇ g + Alum 500 ⁇ g + MPL 20 ⁇ g).
- the blood virus level was 10 2.5 TCID 50 /ml in baby mice born to mothers inoculated with PBS, the control group. It was measured, and it was confirmed that the amount of virus in the blood was reduced to 10 2 , 10 2.3 TCID 50 /ml in baby mice born to mothers belonging to groups 3, 5, and 6, respectively. In addition, it was confirmed that the baby mice born to mothers of groups 2 and 4 did not detect the virus in the blood, so that the offspring born from mothers vaccinated with the vaccine candidate took over the mother's antibody and exhibited protective ability against the PRVABC59 strain.
- the results of the comparative examples are summarized in Table 5 below.
- the number means the ranking, and the closer to 1 in the relevant area, the better the effect.
- the IgG2c ratio was about 1.6-2.5 times in the group mixed with MPL (groups 4-6) when the vaccine candidate group was administered once, Th1-cell-mediated immune response was high, but the IgG2c/IgG1 ratio was observed to be close to 1 when the vaccine candidate group was repeatedly inoculated 2-3 times, and the IgG2c/IgG1 ratio at the time of single inoculation in the group containing only Alum (Group 2-3) was conversely.
- Th2 cell-mediated immune response was high with this 0.2-0.4, but the IgG2c/IgG1 ratio was close to 1 when the vaccine mixture was repeatedly inoculated 2-3 times.
- IFN- ⁇ and IL-12 secreting cells were most likely to be observed in groups 4-6, which is a group mixed with MPL as a result of a single inoculation with the vaccine candidate group, unlike the group in which MPL was not mixed.
- groups 4-6 which is a group mixed with MPL as a result of a single inoculation with the vaccine candidate group, unlike the group in which MPL was not mixed.
- group 6 Zika:Env 10 ⁇ g + Alum 500 ⁇ g + MPL 20 ⁇ g
- the highest levels of IFN- ⁇ and IL-12 secretory cells were observed, and the ELISA result was also The highest levels of IFN- ⁇ and TNF- ⁇ secretion were observed in groups 2 and 6.
- the ratio of effector T cells did not show a significant difference between each group at the first and third vaccinations of the vaccine candidate group, and the T cell population following repeated vaccination of the vaccine did not show any change.
- the activity of T cells was increased during inoculation with the vaccine candidate group.
- the population did not increase upon repeated inoculation in all vaccine candidate groups, and thus effector T cell depletion or activation inhibition did not appear.
- the recombinant Zika virus envelope protein of the present invention is not only effectively expressed in plants, but also has high water solubility, so it is easy to isolate and purify, and also acts as an antigen in the body to exhibit high immunogenicity and virus neutralization ability. Therefore, it can be used as a novel Zika virus vaccine.
- immune cells of mice inoculated with the recombinant Zika virus envelope protein-containing vaccine composition according to the present invention are interferon-gamma (IFN- ⁇ ), interleukin-12 (IL- 12) and tumor necrosis factor-alpha (TNF- ⁇ ) production was specifically increased, and it was found that the immunological defense/preventive efficacy against Zika virus infection was increased. Therefore, the vaccine composition of the present invention can be usefully used for the prevention of Zika virus infection.
- IFN- ⁇ interferon-gamma
- IL-12 interleukin-12
- TNF- ⁇ tumor necrosis factor-alpha
- the present invention can verify an expression system with more excellent immune efficacy by referring to the results of directly applying a recombinant Zika virus vaccine composition developed using a plant cell-derived expression system to an animal model, which is a recombinant Zika virus that can be applied to humans. It can be used for the development of vaccines for other mosquito-borne infectious diseases similar to Zika virus while urging the development of a Zika virus vaccine, so it is expected to have great industrial use value.
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Abstract
Description
Claims (29)
- 서열번호 1의 아미노산 서열을 포함하는 재조합 지카바이러스(Zika virus) 외피단백질; 및 애주번트(adjuvant)로서 알럼(alum), MPL(monophosphoryl lipid A) 또는 이들의 조합을 유효성분으로 포함하는, 백신 조성물.
- 제1항에 있어서,상기 재조합 지카바이러스 외피단백질은 서열번호 4의 아미노산 서열로 이루어진 폴리펩티드가 융합된 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 재조합 지카바이러스 외피단백질은 서열번호 6의 아미노산 서열로 이루어진 폴리펩티드가 융합된 것을 특징으로 하는, 백신 조성물.
- 제3항에 있어서,상기 재조합 지카바이러스 외피단백질은 서열번호 8의 아미노산 서열로 이루어진 폴리펩티드가 더 융합된 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 재조합 지카바이러스 외피단백질은 서열번호 10 또는 서열번호 12의 아미노산 서열을 포함하는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 알럼은 재조합 지카바이러스 외피단백질 중량 대비 1 : 1 내지 50 (외피단백질 : 알럼) 비율로 포함되는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 MPL은 재조합 지카바이러스 외피단백질 중량 대비 1 : 0.4 내지 2 (외피단백질 : MPL) 비율로 포함되는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 알럼 및 MPL의 조합은 재조합 지카바이러스 외피단백질 중량 대비 1 : 1 내지 50 : 0.4 내지 2 (외피단백질 : 알럼 : MPL) 비율로 포함되는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 백신 조성물은 1회 내지 3회 접종되는 것을 특징으로 하는, 백신 조성물.
- 제9항에 있어서,상기 접종은 14 내지 28일 간격으로 이루어지는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 백신 조성물은 지카바이러스 PRVABC59 스트레인, 지카바이러스 MR766 스트레인 및 뎅기 바이러스(Dengue virus) 타입-2로 이루어진 군에서 선택되는 1종 이상에 대한 방어능이 있는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 백신 조성물은 인터페론-감마(IFN-γ), 인터루킨-12(IL-12) 및 종양괴사인자-알파(TNF-α)로 이루어진 군에서 선택된 어느 하나 이상의 분비를 촉진시키는 것을 특징으로 하는, 백신 조성물.
- 제1항에 있어서,상기 백신 조성물은 모체 이행 항체(maternal antibody)의 형성을 유도하는 것을 특징으로 하는, 백신 조성물.
- 서열번호 2의 염기서열로 이루어진 지카바이러스 외피단백질의 유전자 서열을 포함하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항에 있어서,상기 벡터는 서열번호 3의 염기서열을 더 포함하는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항에 있어서,상기 벡터는 서열번호 4의 아미노산 서열을 코딩하는 폴리뉴클레오티드; 또는 서열번호 5의 염기서열을 더 포함하는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항에 있어서,상기 벡터는 서열번호 6의 아미노산 서열을 코딩하는 폴리뉴클레오티드; 또는 서열번호 7의 염기서열을 더 포함하는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제17항에 있어서,상기 벡터는 서열번호 8의 아미노산 서열을 코딩하는 폴리뉴클레오티드; 또는 서열번호 9의 염기서열을 더 포함하는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항에 있어서,상기 벡터는 서열번호 11 또는 서열번호 13의 염기서열을 포함하는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항 내지 제19항 중 어느 한 항에 있어서,상기 재조합 벡터는 식물체에서 발현되는 것을 특징으로 하는, 지카바이러스 외피단백질 발현용 재조합 벡터.
- 제14항 내지 제19항 중 어느 한 항의 재조합 벡터로 형질전환된, 형질전환체.
- 제21항에 있어서,상기 형질전환체는 식물체인 것을 특징으로 하는, 형질전환체.
- 제14항 내지 제19항 중 어느 한 항의 재조합 벡터를 이용하여 제조된, 재조합 지카바이러스 외피단백질.
- 제23항에 있어서,상기 재조합 지카바이러스 외피단백질은 서열번호 10 또는 서열번호 12의 아미노산 서열로 이루어진 것을 특징으로 하는, 재조합 지카바이러스 외피단백질.
- (a) 제21항의 형질전환체를 배양하는 단계; 및(b) 상기 형질전환체 또는 배양액으로부터 재조합 지카바이러스 외피단백질을 분리 및 정제하는 단계를 포함하는, 재조합 지카바이러스 외피단백질의 생산 방법.
- 제25항에 있어서,상기 단계 (b)의 정제는 수용성 분획을 사용하여 정제하는 것을 특징으로 하는, 재조합 지카바이러스 외피단백질의 생산 방법.
- 서열번호 1의 아미노산 서열을 포함하는 재조합 지카바이러스 외피단백질; 및 애주번트로서 알럼, MPL 또는 이들의 조합을 유효성분으로 포함하는 백신 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 지카바이러스 감염증의 예방 또는 치료 방법.
- 서열번호 1의 아미노산 서열을 포함하는 재조합 지카바이러스 외피단백질; 및 애주번트로서 알럼, MPL 또는 이들의 조합을 유효성분으로 포함하는 조성물의, 지카바이러스 감염증의 예방 또는 치료 용도.
- 서열번호 1의 아미노산 서열을 포함하는 재조합 지카바이러스 외피단백질; 및 애주번트로서 알럼, MPL 또는 이들의 조합의, 지카바이러스 감염증에 이용되는 백신을 생산하기 위한 용도.
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BR112023003065A BR112023003065A2 (pt) | 2020-08-18 | 2021-08-11 | Composição de vacina compreendendo uma proteína envelope recombinante de zika virus e método de preparação da mesma |
US18/041,703 US20230293665A1 (en) | 2020-08-18 | 2021-08-11 | Vaccine composition comprising plant-expressed recombinant zika virus envelope protein and preparation method therefor |
MX2023002062A MX2023002062A (es) | 2020-08-18 | 2021-08-11 | Composicion de vacuna que comprende proteina de envoltura de virus del zika recombinante expresada en plantas y metodo de preparacion de la misma. |
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KR1020200103211A KR102557824B1 (ko) | 2020-08-18 | 2020-08-18 | 식물 발현 재조합 지카바이러스 외피단백질을 포함하는 백신 조성물 및 이의 제조방법 |
KR10-2020-0103211 | 2020-08-18 |
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US20140127749A1 (en) * | 2011-04-21 | 2014-05-08 | Arizona Borad Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizo | Methods of protein production and compositions thereof |
US9833505B1 (en) * | 2016-07-18 | 2017-12-05 | Variation Biotechnologies Inc. | Vaccine compositions for treatment of Zika virus |
KR20200093723A (ko) * | 2019-01-28 | 2020-08-06 | 주식회사 바이오앱 | 당화된 Ag85A 단백질을 포함하는 결핵 예방용 백신 조성물 및 이의 제조방법 |
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KR20170125484A (ko) | 2016-05-04 | 2017-11-15 | 서울대학교산학협력단 | 지카바이러스 감염 질환의 예방 또는 치료용 약제학적 조성물 |
KR102200773B1 (ko) * | 2018-09-19 | 2021-01-12 | 주식회사 바이오앱 | 돼지의 Fc 단편과 융합된 항원 및 이를 포함하는 백신 조성물 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140127749A1 (en) * | 2011-04-21 | 2014-05-08 | Arizona Borad Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizo | Methods of protein production and compositions thereof |
US9833505B1 (en) * | 2016-07-18 | 2017-12-05 | Variation Biotechnologies Inc. | Vaccine compositions for treatment of Zika virus |
KR20200093723A (ko) * | 2019-01-28 | 2020-08-06 | 주식회사 바이오앱 | 당화된 Ag85A 단백질을 포함하는 결핵 예방용 백신 조성물 및 이의 제조방법 |
Non-Patent Citations (2)
Title |
---|
DATABASE Protein 25 July 2016 (2016-07-25), ANONYMOUS: "Fc IgG1 heavy chain constant region, partial [Homo sapiens]", XP055499927, retrieved from NCBI Database accession no. AEV43323.1 * |
WANG XINYI, TAI WANBO, ZHANG XIAOLU, ZHOU YUSEN, DU LANYING, SHEN CHUANLAI: "Effects of Adjuvants on the Immunogenicity and Efficacy of a Zika Virus Envelope Domain III Subunit Vaccine", VACCINES, vol. 7, no. 4, 27 October 2019 (2019-10-27), XP055901580, DOI: 10.3390/vaccines7040161 * |
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KR102557824B1 (ko) | 2023-07-20 |
KR20220022260A (ko) | 2022-02-25 |
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