KR20240120818A - Vaccine compositions comprising recombinant Zika virus envelope proteins expressed from plants and preparation methods thereof - Google Patents

Abstract

The present invention relates to a vaccine composition comprising, as an active ingredient, a Zika virus envelope protein comprising an amino acid sequence of SEQ ID NO: 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12; a recombinant vector for producing the recombinant Zika virus envelope protein in a plant; a transformant transformed with the vector; and a method for producing the recombinant Zika virus envelope protein.

Description

{Vaccine compositions comprising recombinant Zika virus envelope proteins expressed from plants and preparation methods thereof}

The present invention relates to a vaccine composition comprising a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment expressed in a plant, and a method for producing the same.

Zika virus is a flavivirus of the Flaviviridae family transmitted by mosquitoes, and is an infectious disease that causes Guillain-Barre syndrome and microcephaly in fetuses. Since the Zika virus first appeared in French Polynesia in 2013, its infectivity and pathogenicity have increased, raising concerns about its danger. After its first report in Brazil in May 2015, it has rapidly spread in Central and South America. As of June 1, 2016, there are 53 countries with recent outbreaks (since 2015), an estimated 440,000 to 1.3 million infections in Brazil alone in 2015, and approximately 240 confirmed cases of microcephaly as of May 26, with approximately 5,000 suspected and under-diagnosis cases. Given the continued spread of the infection, the market size is expected to be very large considering the possibility of an epidemic. However, as of now, there is no special preventive method for Zika virus infection other than aerosol insecticides to control mosquitoes, the vector of the disease.

Recently, the development of vaccines against Zika virus is in progress through the development of inactivated vaccines and recombinant protein expression using the E. coli expression system. However, existing vaccines represented by live or inactivated vaccines have the disadvantage of exhibiting toxicity, and in particular, live vaccines have concerns about side effects due to mutations. In addition, although the E. coli recombinant expression system has the advantages of low strain maintenance costs, simple medium composition and culture conditions, and simplification of the production process, there is a concern that it may cause side effects such as an immune response to a foreign protein. Therefore, there is a need for a Zika virus vaccine that minimizes the above side effects and has excellent immunogenicity and defensive efficacy.

Meanwhile, in the medical industry, there is a demand for the development of a system that can efficiently produce vaccine antigen proteins and mass-produce useful physiologically active substances in a short period of time to manufacture vaccines. Production of useful physiologically active substances using plants can eliminate various sources of contamination that may arise from the method of producing proteins by synthesizing them in animal cells or microorganisms, and is absolutely advantageous compared to the existing animal cell system in terms of facility technology and cost required for mass production when the demand for the relevant useful substances suddenly increases. Therefore, it is widely used in the medical industry, such as in vaccine development and therapeutic development, due to its advantage of enabling mass production at low cost in the shortest period of time.

Currently, plant-based production systems are being used commercially to synthesize foreign proteins, and the posttranslational modifications in plant cells are very similar to those in animal cells, allowing for the precise production of multimeric proteins. In the case of glycoproteins produced using plant systems, the amino acid residues that are glycosylated are identical to those in the original protein.

Therefore, the method of producing proteins, which are useful physiologically active substances, using plant expression systems has recently attracted attention because of its potential to replace the method of producing them using animal cells or microorganisms. Currently, vaccines being developed using plant expression systems mainly use proteins that perform the adhesion function essential for bacterial or viral mucosal infection, toxin proteins that exhibit bacterial toxicity, and structural proteins that form the structure of viruses as antigens in the case of pathogenic diseases, and in the case of non-pathogenic diseases, the main enzymes that produce disease-causing substances or causative substances are used as antigens. Research results so far have reported that recombinant antigen proteins derived from plants have a structure that has the same function as vaccines derived from animals, and various studies applying this are required.

KR 10-2017-0125484

The present invention was derived to solve the above-mentioned need for the development of a Zika virus vaccine and the problems in the prior art, and the present invention was completed by developing a recombinant Zika virus envelope protein fused with an aglycosylated human Fc (aghFc) fragment, which not only enables efficient production using plants but also exhibits high immunogenicity and virus neutralization ability, and a vaccine composition containing the same as an active ingredient.

Accordingly, the purpose of the present invention is to provide a recombinant vector for expressing the Zika virus envelope protein, comprising a polynucleotide encoding a gene for the Zika virus envelope protein consisting of an amino acid sequence of SEQ ID NO: 1; and a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

Another object of the present invention is to provide a transformant transformed with a recombinant vector according to the present invention.

Another object of the present invention is to provide a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc), which is produced using a 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 fused with an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc).

Another object of the present invention is to provide a vaccine composition comprising, as an active ingredient, a Zika virus envelope protein comprising an amino acid sequence of SEQ ID NO: 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

To achieve the above object, the present invention provides a recombinant vector for expressing the Zika virus envelope protein, comprising a polynucleotide encoding a gene for the Zika virus envelope protein consisting of an amino acid sequence of SEQ ID NO: 1; and a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

In one embodiment of the present invention, the recombinant vector may further include, but is not limited to, a polynucleotide encoding a peptide linker L consisting of an amino acid sequence of SEQ ID NO: 10.

In another embodiment of the present invention, the recombinant vector may further comprise a KDEL polynucleotide consisting of the base sequence of SEQ ID NO: 15, but is not limited thereto.

In another embodiment of the present invention, the recombinant vector may include, but is not limited to, a polynucleotide encoding an amino acid sequence of SEQ ID NO: 16 or a polynucleotide consisting of a base sequence of SEQ ID NO: 17.

In another embodiment of the present invention, the recombinant vector can be expressed in a plant, but is not limited thereto.

In addition, the present invention provides a transformant transformed with a recombinant vector according to the present invention.

In one embodiment of the present invention, the transformant may be a plant, but is not limited thereto.

In addition, the present invention provides a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc), which is produced using a recombinant vector according to the present invention.

In one embodiment of the present invention, the recombinant Zika virus envelope protein may be composed of the amino acid sequence of SEQ ID NO: 16, but is not limited thereto.

In addition, the present invention provides a method for producing a recombinant Zika virus coat protein fused with an aglycosylated human Fc fragment (aghFc), comprising the steps of: (a) transforming a plant with a recombinant vector according to the present invention; and (b) isolating and purifying a Zika virus coat protein and a recombinant Zika virus coat protein fused with an aglycosylated human Fc fragment (aghFc) from the plant.

In one embodiment of the present invention, the transformation in step (a) may be, but is not limited to, Agrobacterium-mediated transformation.

In another embodiment of the present invention, the purification in step (b) may be performed using a water-soluble fraction, but is not limited thereto.

In addition, the present invention provides a vaccine composition comprising, as an active ingredient, a Zika virus envelope protein comprising an amino acid sequence of SEQ ID NO: 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

In addition, the present invention provides a method for preventing or treating Zika virus infection, comprising a step of administering to a subject in need thereof a vaccine composition comprising, as an active ingredient, a recombinant Zika virus envelope protein fused with an amino acid sequence of SEQ ID NO: 1; and an aglycosylated human immunoglobulin Fc fragment (aghFc) having an amino acid sequence of SEQ ID NO: 12.

In addition, the present invention provides a composition comprising, as an active ingredient, a recombinant Zika virus envelope protein fused with an amino acid sequence of SEQ ID NO: 1; and an aglycosylated human immunoglobulin Fc fragment (aghFc) having an amino acid sequence of SEQ ID NO: 12, for use in the prevention or treatment of Zika virus infection.

In addition, the present invention provides a use for producing a vaccine for Zika virus infection, comprising a Zika virus envelope protein comprising an amino acid sequence of SEQ ID NO: 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) comprising an amino acid sequence of SEQ ID NO: 12.

In one embodiment of the present invention, KDEL (Lysine-aspartic acid-glutamic acid-leucine) may be further fused to the recombinant Zika virus envelope protein, but is not limited thereto.

In another embodiment of the present invention, L, consisting of an amino acid sequence of SEQ ID NO: 10, which is a peptide linker, may be further fused to the recombinant Zika virus envelope protein, but is not limited thereto.

In another embodiment of the present invention, the recombinant Zika virus envelope protein may be composed of the amino acid sequence of SEQ ID NO: 16, but is not limited thereto.

In another embodiment of the present invention, the vaccine composition may further comprise one or more of the following adjuvants, but is not limited thereto:

a) Alum;

b) MPL (monophosphoryl lipid A);

c) a mixture of alum and monophosphoryl lipid A (MPL);

d) Deacyl-lipooligosaccharide;

e) a mixture of diacyl-lipooligosaccharide and aluminum hydroxide; and

f) A mixture of diacyl-lipooligosaccharide, cationic liposome, and QS21.

In another embodiment of the present invention, the vaccine composition may comprise, but is not limited to, 1 to 30 μg of recombinant Zika virus envelope protein per dose.

In another embodiment of the present invention, the vaccine composition may satisfy one or more of the following characteristics, but is not limited thereto:

a) having protective activity against at least one species selected from the group consisting of Zika virus strain PRVABC59, Zika virus strain MR766 and Dengue virus type-2;

b) promoting the secretion of at least one selected from the group consisting of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), and interleukin-6 (IL-6); and

c) Induces the formation of maternal antibodies.

The recombinant Zika virus coat protein of the present invention is effectively expressed in plants by fusing an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc) to prevent an unintended immune response to the Fc fragment due to plant-specific glycosylation, and also acts as an antigen in the body, exhibiting high immunogenicity and virus neutralizing ability, and thus can be used as a novel Zika virus vaccine.

In addition, it was revealed that the immune cells of mice vaccinated with the vaccine composition containing the recombinant Zika virus envelope protein according to the present invention specifically increased the production of specific cytokines, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), and interleukin-6 (IL-6), in response to external stimulus antigens, thereby enhancing the immunological defense/prevention efficacy against Zika virus infection. Therefore, the vaccine composition of the present invention can be usefully used in the prevention of Zika virus infection.

Figure 1 is a cleavage map showing the gene arrangement of the expression cassette for expression of two recombinant Zika virus envelope proteins in plants (Abbreviations, hereinafter the same: NB, new chaperone binding protein; Zika Env, Zika envelope protein ectodomain; hFc, Fc fragment of human immunoglobulin heavy chain; HDEL, histidine-aspartic acid-glutamic acid-leucine tag; L, peptide linker; aghFc, aglycosylated hFc; KDEL, Lysine-aspartic acid-glutamic acid-leucine tag).
Figure 2 is a drawing showing the expression of pTEX-NB:ZikaEnv:aghFc in plants and the results confirmed by Western blotting (T, total fraction; S, water-soluble fraction; P, pellet fraction).
Figure 3 is a diagram showing the results of comparing the solubility and expression levels of recombinant Zika virus envelope proteins ZikaEnv:aghFc and ZikaEnv:hFc by vector.
Figure 4 shows the results of isolating and purifying the recombinant Zika virus envelope protein ZikaEnv:aghFc from a plant. It is a drawing showing the results of Western blotting to confirm binding to resin during the process of isolating and purifying the recombinant Zika virus envelope protein ZikaEnv:aghFc from a plant (left) and the results of Coomassie staining after electrophoresis (SDS-PAGE) of the recombinant Zika virus envelope protein ZikaEnv:aghFc isolated and purified from the plant (right) (T: total plant extract, FT: flow-through).
Figure 5 shows the results of measuring antibody titers using indirect ELISA on serum isolated through orbital blood collection 7 to 14 days after vaccinating mice with vaccine candidates 1 to 3 times. A is a flow chart showing the vaccination and blood collection schedule, and B and C are graphs comparing the levels of antibodies recognizing ZikaEnv:aghFc and ZikaEnv:hFc in the sera of immunized mice by group (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 6 is a diagram showing the results of measuring the production of IFN-γ (A), TNF-α (B), IL-4 (C), and IL-6 (D) by ELISA analysis after 7 days (day 49) of the isolated spleen immune cells, which were stimulated with each Zika virus envelope protein antigen for 48 hours after each vaccination with three doses of the vaccine candidates in mice (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figures 7a to 7d are drawings showing the results of confirming the expression of CD4, CD8 T cells (CD4+, CD8+) producing IFN-γ and TNF-α by flow cytometry analysis after 7 days (day 49) of spleen immune cells isolated after 3 doses of each vaccine candidate were stimulated with each Zika virus envelope protein antigen for 12 hours. Specifically, Figures 7a and 7d show the results of confirming the expression of CD4 T cells, and Figures 7c and 7d show the results of confirming the expression of CD8 T cells.
Figure 8 shows the results of measuring antibody titers using indirect ELISA on serum isolated through orbital blood collection 7 to 14 days after vaccinating mice 1 to 3 times with vaccine candidates. A is a flow chart showing the vaccination and blood collection schedule, and B and C are graphs comparing the levels of antibodies recognizing ZikaEnv:aghFc and ZikaEnv:hFc in the serum of immunized mice by group (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 9 is a diagram showing the results of measuring the production of IFN-γ, TNF-α, IL-4, and IL-6 by ELISA analysis after 7 days (day 49) of the isolated spleen immune cells, which were stimulated with each Zika virus envelope protein antigen for 48 hours after each vaccination with three doses of the vaccine candidates in mice (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 10 is a diagram showing the results of confirming the expression of CD4, CD8 T cells (CD4+, CD8+) producing IFN-γ and TNF-α by flow cytometry analysis after 7 days (day 49) of spleen immune cells isolated from mice vaccinated three times with each vaccine candidate and stimulating them with each Zika virus envelope protein antigen for 12 hours.
Figure 11 shows the results of measuring antibody titers using indirect ELISA on serum isolated through orbital blood collection 7 to 14 days after vaccinating mice with vaccine candidates 1 to 3 times. A is a flow chart showing the vaccination and blood collection schedule, and B is a graph comparing the levels of antibodies recognizing ZikaEnv:aghFc in the serum of immunized mice by group (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 12 is a graph showing the results of measuring ZIKV envelope protein-specific IgG antibody titers using an ELISA kit (Alphadiagnostic, RV-403120-1) on serum isolated through orbital blood collection 7 to 14 days after vaccinating mice 1 to 3 times with the vaccine candidates (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 13 is a diagram showing the results of measuring neutralizing antibody titers using plaque reduction neutralization test (PRNT) on serum isolated through orbital blood collection 7 to 14 days after vaccinating mice 1 to 3 times with vaccine candidates (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 14 shows the results of measuring the production of IFN-γ, TNF-α, IL-4, and IL-6 by ELISA analysis after spleen immune cells isolated 7 days later (day 49) were stimulated with Zika virus envelope protein antigen for 48 hours after each vaccination with three doses of the vaccine candidates in mice (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 15 is a diagram showing the results of confirming the expression of CD4, CD8 T cells (CD4+, CD8+) producing IFN-γ and TNF-α by flow cytometry analysis after 7 days (day 49) of spleen immune cells isolated after vaccinating mice three times each with each vaccine candidate and stimulating them with the Zika virus envelope protein antigen for 12 hours.
Figure 16a is a diagram showing the results of confirming maternal IgG antibodies using indirect ELISA on serum isolated through orbital blood collection from offspring mice obtained by mating 7 days after inoculating mice three times with each vaccine candidate (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figure 16b is a diagram showing the results of confirming maternal transfer neutralizing antibody titers using plaque reduction neutralization test (PRNT) on serum isolated through orbital blood collection from offspring mice obtained by mating 7 days after inoculating mice three times with each vaccine candidate (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).
Figures 17 to 19 are drawings showing the results of observing the survival rate (Figure 17), weight change (Figure 18), and abnormal behavior (Figure 19) by inoculating mice three times with each vaccine candidate, mating them 7 days later, and challenging the resulting offspring with two types of Zika virus or dengue virus type 2.
Figure 20 is a diagram showing the results of measuring the amount of virus in the brain by qRT-PCR analysis after vaccinating mice three times with each vaccine candidate and challenging the offspring obtained by mating with two types of Zika virus or dengue virus type 2 7 days later (one-way ANOVA; *, p <0.05; **, p <0.01; ***, p < 0.001).

The present inventors conducted extensive research to develop a Zika virus vaccine that can minimize in vivo side effects and reduce production costs, and as a result, developed a recombinant Zika virus coat protein in which an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc) is fused to the Zika virus coat protein to prevent unintended immune responses to the Fc fragment due to plant-specific glycosylation, and completed the present invention by confirming that the developed recombinant Zika virus coat protein is not only effectively expressed in plants but also acts as an antigen in the body, exhibiting high immunogenicity and virus neutralizing ability.

Hereinafter, the present invention will be described in detail. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

The present invention can provide a recombinant vector for expressing Zika virus envelope protein, comprising a polynucleotide encoding a gene for Zika virus envelope protein consisting of an amino acid sequence of SEQ ID NO: 1; and a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

Vaccines to prevent viral diseases, including Zika virus infection, cannot be produced using bacteria due to problems with protein folding and glycosylation, and are mainly produced using animal cells.

However, the method of producing a vaccine using animal cells is not easy to manufacture because it requires a lot of cost for expanding facilities for mass production, and the vaccine is often expensive. In addition, inactivated virus vaccines manufactured using animal cells are not only difficult to store, but also have the disadvantage of being highly likely to be contaminated with viruses that can infect animals. However, the present invention complements these disadvantages by enabling the recombinant Zika virus envelope protein according to the present invention to be expressed in a plant. That is, unlike animal cells, plant cells have a very low possibility of being contaminated with viruses that can infect animals, and mass production is possible at any time as long as the cultivation area is secured. In addition, since long-term storage is possible through plants, stable and inexpensive vaccine production is possible.

Therefore, the recombinant vector for expressing the Zika virus envelope protein according to the present invention is a recombinant vector for plant expression that can be expressed in a plant.

In one specific embodiment of the present invention, the recombinant vector may additionally include a polynucleotide encoding NB (new chaperone binding protein), a polynucleotide encoding L, a polynucleotide encoding KDEL (Lys-Asp-Glu-Leu) tag, and the like.

The polynucleotide encoding the above-mentioned non-glycosylated human immunoglobulin Fc fragment (aghFc) is a gene comprising a base sequence of SEQ ID NO: 13, preferably a gene represented by SEQ ID NO: 13, but may include a base sequence having a sequence identity of 80% or more, more preferably 90% or more, more preferably 95% or more with the base sequence of SEQ ID NO: 13, and may be located at the 3' end of the polynucleotide sequence encoding the recombinant Zika virus envelope protein.

The polynucleotide encoding the above NB is a gene comprising the base sequence of SEQ ID NO: 3, preferably a gene represented by SEQ ID NO: 3, but may include a base sequence having a sequence homology of 80% or more, more preferably 90% or more, and more preferably 95% or more with the base sequence of SEQ ID NO: 3, and may be located at the 5' end of the polynucleotide sequence encoding the recombinant Zika virus coat protein. When the recombinant protein is expressed, the NB may have all or part of the amino acid sequence cut out, so that the entire amino acid sequence may be removed from the recombinant Zika virus coat protein of the present invention, or only some amino acids may remain.

The polynucleotide encoding the above L is a gene comprising a base sequence of SEQ ID NO: 11, preferably a gene represented by SEQ ID NO: 11, but may include a base sequence having a sequence identity of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence of SEQ ID NO: 11, and may be positioned between a polynucleotide encoding a gene of a Zika virus envelope protein as a peptide linker and a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aghFc).

The polynucleotide encoding the above KDEL tag is a gene comprising a base sequence of SEQ ID NO: 15, preferably a gene represented by SEQ ID NO: 15, but may include a base sequence having a sequence identity of 75% or more, more preferably 80% or more, more preferably 90% or more with the base sequence of SEQ ID NO: 15, and may be located at the 3' end of the polynucleotide sequence encoding the recombinant Zika virus envelope protein.

According to one embodiment of the present invention, a recombinant vector according to the present invention may be a polynucleotide encoding a gene of a Zika virus envelope protein consisting of an amino acid sequence of SEQ ID NO: 1; a polynucleotide encoding L consisting of an amino acid sequence of SEQ ID NO: 10; a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12; and a KDEL polynucleotide consisting of a base sequence of SEQ ID NO: 15, all of which are sequentially linked. When connected in the above order, that is, when including the expression cassette of ZikaEnv:aghFc shown in the cleavage map at the bottom of Fig. 1, the recombinant vector according to the present invention includes the base sequence of SEQ ID NO: 17, most preferably consists of the base sequence of SEQ ID NO: 17, but may include a base sequence having a sequence homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence of SEQ ID NO: 17.

In this specification, 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, and preferably means a vector manufactured so as to be capable of expressing a target antigen (in the present invention, a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc)). The “vector” refers to any medium for the introduction and/or transfer of a base into a host cell in vitro, in vivo, or in vivo, and may be a replicating unit (replicon) to which another DNA fragment can bind and cause replication of the bound fragment, and the “replication unit” refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an autonomous unit of DNA replication in vivo, that is, is capable of replicating by its own regulation.

The recombinant vector of the present invention preferably includes a promoter, which is a transcription initiation factor to which RNA polymerase binds, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site and a sequence for regulating the termination of transcription and translation, a terminator, etc., and may further include an NB gene, an L gene (peptide linker), an endoplasmic reticulum signal peptide (the same meaning as an endoplasmic reticulum targeting sequence) gene, a cloning site, etc. In addition, it may further include a selection marker gene, such as an Fc fragment of an aglycosylated human immunoglobulin (aghFc) and an antibiotic resistance gene for selecting a transformant.

In the present specification, a "peptide linker" is a peptide used to provide a physicochemical distance between domains in a recombinant protein, or for connection. The recombinant Zika virus envelope protein of the present invention may include a linker between the Zika virus envelope protein and the Fc fragment of the non-glycosylated human immunoglobulin heavy chain. The linker is not limited in type or number of amino acids as long as it can minimize an immune response, but is preferably 1 to 20 amino acids, and more preferably 1 to 10 amino acids. In one embodiment of the present invention, a peptide linker "L" consisting of an amino acid sequence of SEQ ID NO: 10 was used.

In this specification, the “Fc fragment of human immunoglobulin heavy chain (hFc)” refers to an Fc fragment in which only the heavy chain (H chain) portion is linked by an S-S bond when immunoglobulin is digested by papain and does not have an antigen-binding site, and 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: 4 or a human Fc fragment encoded by the base sequence of SEQ ID NO: 5, but is not limited thereto. In addition, the Fc fragment of the present invention includes a variant of the base sequence represented by SEQ ID NO: 5 within the scope of the present invention. Specifically, the gene may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more with the base sequence of SEQ ID NO: 5.

In the present specification, the term "aglycosylated human Fc (aghFc)" or "aglycosylated human immunoglobulin Fc fragment (aghFc)" or "aglycosylated human Fc fragment" refers to an Fc fragment from which a glycosylation site has been removed, and from which a conserved N-glycosylation site present in the Fc fragment has been removed in order to eliminate an unintended immune response to the Fc fragment due to plant-specific glycosylation. The Fc fragment (aghFc) of the aglycosylated human immunoglobulin heavy chain of the present invention is preferably an Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12 or an Fc fragment (aghFc) encoded by a base sequence of SEQ ID NO: 13, but is not limited thereto. In addition, the Fc fragment (aghFc) of the aglycosylated human immunoglobulin heavy chain of the present invention is an Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12 or an Fc fragment (aghFc) encoded by a base sequence of SEQ ID NO: 13, but is not limited thereto. A variant of the base sequence represented by 13 is included in the scope of the present invention. Specifically, the gene may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the base sequence of SEQ ID NO: 13.

The above “cloning site” refers to a site inserted for the purpose of connecting/separating each gene within a vector.

The above “endoplasmic reticulum signal peptide (ER signal sequence)” is not limited in type and amino acid sequence as long as it is a plant endoplasmic reticulum signal peptide known to those skilled in the art, and reference can be made to documents such as US 20130295065 and WO2009158716, for example. In one embodiment of the present invention, the endoplasmic reticulum signal peptide may be encoded by the base sequence of SEQ ID NO: 3 (NB; new chaperone binding protein), and after translation into a protein, part or all of the sequence may be removed.

The above selection marker genes may include, for example, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, Bleomycin, hygromycin, and chloramphenicol, and the aadA gene, and the above promoters may include, for example, pEMU promoter, MAS promoter, histone promoter, Clp promoter, cauliflower mosaic virus-derived 35S promoter, cauliflower mosaic virus-derived 19S RNA promoter, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory Virus Vaccine) promoter, etc. syncytial virus) promoter, EF-1α (Elongation factor-1 alpha) promoter, pEMU promoter, MAS promoter, histone promoter, Clp promoter, etc., and the terminator may include, for example, nopaline synthase (NOS), rice amylase RAmy1 A terminator, phaseolin terminator, terminator of the Octopine gene of Agrobacterium tumafaciens, rrnB1/B2 terminator of Escherichia coli, etc., but the above listed are examples and are not limited thereto.

In another aspect of the present invention, the present invention provides a transformant transformed with the recombinant vector described above. According to one embodiment of the present invention, the transformant may be a plant, but is not limited thereto.

In one specific embodiment of the present invention, the transformant may be preferably a microorganism such as Escherichia coli, Bacillus, Salmonella, yeast, an insect cell, an animal cell including a human, an animal such as a mouse, a rat, a dog, a monkey, a pig, a horse, a cow, Agrobacterium tumefaciens, a plant, and the like, and more preferably, food crops including rice, wheat, barley, corn, soybean, potato, red bean, oat, and sorghum; vegetable crops including Arabidopsis thale cress, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, and rapeseed; and fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, and bananas. and may be flowers including roses, carnations, chrysanthemums, lilies, and tulips, but is not limited thereto as long as it is an organism that can be transformed with the vector of the present invention.

In this specification, “transformation” refers to a general change in the genetic properties of an organism due to injected DNA, and a “transgenic organism” is an organism produced by injecting an external gene using a molecular genetic method, preferably an organism transformed by the recombinant expression vector of the present invention, and the organism is not limited to any living organism such as a microorganism, eukaryotic cell, insect, animal, or plant, and preferably includes, but is not limited to, Escherichia coli, Salmonella, Bacillus, yeast, animal cells, mouse, rat, dog, monkey, pig, horse, cow, Acrobacterium tumefaciens, or plants.

In the present specification, the “plant” may be used without limitation as long as it is a plant capable of mass-producing the recombinant Zika virus coat protein of the present invention, but more specifically, may be selected from the group consisting of tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper, and preferably tobacco. The tobacco in the present invention is a plant of the genus Nicotiana (Nicotiana genus) that can overexpress a protein, and is not particularly limited in type, and an appropriate variety can be selected to practice the present invention in accordance with the transformation method and the purpose of mass production of the protein. For example, varieties such as Nicotiana benthamiana L. or Nicotiana tabacum cv. xanthi can be used.

The above transformant can be produced by a method such as transformation, transfection, Agrobacterium-mediated transformation, particle gun bombardment, sonication, electroporation, and PEG (Polyethylen glycol)-mediated transformation, but there is no limitation as long as it is a method capable of injecting the vector of the present invention.

In another aspect of the present invention, the present invention can provide a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc), which is produced using a recombinant vector according to the present invention.

In addition, the recombinant Zika virus envelope protein according to the present invention may be a Zika virus envelope protein, L, an aglycosylated human Fc fragment (aghFc), and KDEL sequentially fused, and the recombinant Zika virus envelope protein thus obtained may be composed of the amino acid sequence of SEQ ID NO: 16; however, the present invention is not limited by the fusion order or amino acid sequence.

The above “fusion” includes both chemical and genetic fusion, and in the present invention, it preferably refers to genetic fusion. The above “genetic fusion” means a link formed by a linear covalent bond through genetic expression of a DNA sequence encoding a protein.

In the present invention, the recombinant Zika virus envelope protein is characterized by having high solubility.

In this specification, “solubility” means the degree to which a target protein or peptide can be dissolved in a solvent suitable for administration to the human body. Specifically, it may indicate the degree to which a solute is saturated with a given solvent at a specific temperature. Solubility can be measured by determining the saturated concentration of the solute, and for example, the solute can be added in excess to the solvent, stirred and filtered, and then the concentration can be measured using a UV spectrometer or HPLC, but is not limited thereto. High solubility is advantageous for the separation and purification of recombinant proteins, and has the advantage of maintaining the physiological activity or pharmacological activity of the recombinant protein by suppressing aggregation of the recombinant protein.

In another aspect of the present invention, the present invention can provide a method for producing a recombinant Zika virus coat protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc), comprising the steps of: (a) transforming a plant with the recombinant vector of any one of claims 1 to 4; and (b) isolating and purifying the Zika virus coat protein and the recombinant Zika virus coat protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) from the plant.

In one specific embodiment of the present invention, the purification in step (b) may be performed using a water-soluble fraction, but is not limited thereto.

In one specific embodiment of the present invention, the transformation in step (a) may be, but is not limited to, Agrobacterium-mediated transformation.

In another aspect of the present invention, the present invention can provide a vaccine composition comprising, as an active ingredient, a Zika virus envelope protein comprising an amino acid sequence of SEQ ID NO: 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of SEQ ID NO: 12.

The present invention relates to a vaccine composition containing a recombinant Zika virus coat protein having an amino acid sequence of sequence number 1. In one embodiment of the present invention, a recombinant plant expression vector capable of expressing the Zika virus coat protein, which is a major immunogenic antigenic region of Zika virus, is produced and cloned, and the vector is introduced into Agrobacterium strain GV3101 or LBA4404, and Nicotiana benthamiana is infiltrated with the Agrobacterium into which the vector has been introduced, thereby producing the recombinant Zika virus coat protein from Nicotiana benthamiana. The recombinant Zika virus coat protein induces an immune response, such as an antibody response to an antigen similar to that when the virus penetrates a living body and activation of various immune molecules capable of directly destroying the pathogen, thereby providing a preventive strategy against Zika virus.

The binding site of the aglycosylated human immunoglobulin Fc fragment (aghFc) may be directly linked to the C-terminus of a peptide or protein intended for expression or synthesis in plant cells, or may be added (or linked) indirectly, i.e., with other peptides or protein(s) intervening.

In one specific embodiment of the present invention, the recombinant Zika virus envelope protein may be a polypeptide consisting of an amino acid sequence of SEQ ID NO: 10, i.e., further fused with a peptide linker, L. The binding site of L may be directly linked to the C-terminus of a peptide or protein intended for expression or synthesis in a plant cell, or may be added (or linked) indirectly, i.e., with other peptide or protein(s) interposed therebetween.

In one specific embodiment of the present invention, the recombinant Zika virus envelope protein may be a polypeptide consisting of an amino acid sequence of SEQ ID NO: 14, i.e., further fused with a KDEL tag. The binding site of the KDEL may be directly linked to the C-terminus of a peptide or protein intended for expression or synthesis in a plant cell, or may be added (or linked) indirectly, i.e., with other peptide or protein(s) intervening therebetween.

In one specific embodiment of the present invention, the recombinant Zika virus envelope protein may be composed of an amino acid sequence of SEQ ID NO: 16. For example, the recombinant Zika virus envelope protein may be a Zika virus envelope protein, L, an aglycosylated human Fc fragment (aghFc), and a KDEL tag sequentially fused.

In one specific embodiment of the present invention, the vaccine composition may additionally comprise one or more of the following adjuvants, but is not limited thereto:

a) Alum;

b) MPL (monophosphoryl lipid A);

c) a mixture of alum and monophosphoryl lipid A (MPL);

d) Deacyl-lipooligosaccharide;

e) a mixture of diacyl-lipooligosaccharide and aluminum hydroxide; and

f) A mixture of diacyl-lipooligosaccharide, cationic liposome, and QS21.

The term “adjuvant” as used herein refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase or influence the immune response. Typically, it usually means a carrier or auxiliary substance for an immunogen and/or other pharmaceutically active substances or compositions. Typically, the term “adjuvant” should be interpreted in a broad sense and refers to a wide range of substances or stratagerms that can enhance the immunogenicity of an antigen incorporated into the adjuvant or administered together with the adjuvant. In addition, an adjuvant can be divided into, but is not limited to, an immune potentiator, an antigen delivery system, or a combination thereof. Examples of widely known adjuvants include aluminum hydroxide, Freud's complete or incomplete adjuvant, DEAE dextran, levamisole, PCG, and poly I:C or poly A:U, alum, monophosphoryl lipid A (MPL), a mixture of alum and monophosphoryl lipid A (MPL), CIA06 (a mixture of deacyl-lipooligosaccharide, and aluminum hydroxide), and CIA09A (a mixture of deacyl-lipooligosaccharide, cationic liposome, and QS21). In one embodiment of the present invention, CIA09A was used as the adjuvant, but is not limited thereto.

In the present invention, the vaccine composition comprises 50 to 2,500 μg, 50 to 2,000 μg, 50 to 1,500 μg, 50 to 1,000 μg, 50 to 500 μg, 50 to 300 μg, 50 to 100 μg, 100 to 2,000 μg, 100 to 1,500 μg, 100 to 1,000 μg, 100 to 500 μg, 100 to 300 μg, 300 to 1,500 μg, 300 to 1,000 μg, 300 to 500 μg, 500 to 600 μg, 500 to 800 μg, 500 to 1,000 μg, 500 to It may contain, but is not limited to, 1500 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, or 500 μg of alum.

In the present invention, the vaccine composition may contain, but is not limited to, 5 to 100 μg, 5 to 80 μg, 5 to 50 μg, 5 to 30 μg, 5 to 20 μg, 5 to 10 μg, 10 to 80 μg, 10 to 50 μg, 10 to 30 μg, 10 to 20 μg, 20 to 30 μg, 20 to 40 μg, 20 to 50 μg, 20 to 80 μg, 20 to 100 μg, 5 μg, 10 μg, 15 μg, 18 μg, or 20 μg of MPL per single administration dose.

In addition, in the present invention, when the vaccine composition contains 5 μg of the recombinant Zika virus envelope protein, the alum may be contained in a weight of 10 to 500 μg, 30 to 500 μg, 50 to 500 μg, 70 to 500 μg, 100 to 500 μg, 150 to 500 μg, 200 to 500 μg, 300 to 500 μg, 400 to 500 μg, 450 to 500 μg, 480 to 500 μg, 490 to 500 μg, and the MPL may be contained in a weight of 4 to 20 μg, 5 to 20 μg, 7 to 20 μg, 10 to 20 μg, 15 to 20 μg, 18 to 20 μg, but is not limited thereto. It is not.

In the present invention, the vaccine composition is administered in a single dose of 50 to 700 ul, 50 to 600 ul, 50 to 500 ul, 50 to 400 ul, 50 to 300 ul, 50 to 200 ul, 50 to 100 ul, 100 to 700 ul, 200 to 700 ul, 300 to 700 ul, 400 to 700 ul, 400 to 600 ul, 450 to 600 ul, 500 to 600 ul, 500 to 550 ul, 500 to 540 ul, 500 to 520 ul, 500 to 510 ul, 50 ul, 100 ul, It may contain, but is not limited to, 200 ul, 300 ul, 400 ul, or 500 ul of CIA06 (a mixture of Deacyl-lipooligosaccharide and Aluminum hydroxide).

In the present invention, the vaccine composition is administered in a single dose of 50 to 700 ul, 50 to 600 ul, 50 to 500 ul, 50 to 400 ul, 50 to 300 ul, 50 to 200 ul, 50 to 100 ul, 100 to 700 ul, 200 to 700 ul, 300 to 700 ul, 400 to 700 ul, 400 to 600 ul, 450 to 600 ul, 500 to 600 ul, 500 to 550 ul, 500 to 540 ul, 500 to 520 ul, 500 to 510 ul, 50 ul, 100 ul, It may contain, but is not limited to, 200 ul, 300 ul, 400 ul, or 500 ul of CIA09A (a mixture of Deacyl-lipooligosaccharide, Cationic liposome, and QS21).

The term “Zika virus” as used herein refers to a virus belonging to the family Flaviviridae having a genome of about 11 kilobases of single-stranded positive-sense RNA, wherein the RNA genome encodes seven nonstructural proteins and three structural proteins. The three major viral structural proteins include capsid (C), pre-membrane (prM), and envelope (E), and the seven nonstructural proteins include one long open reading frame (ORF) encoding NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

In the present invention, the “envelope protein (EnV)” included in the structural protein is responsible for the entry of Zika virus and is the main target of neutralizing antibodies. Up to now, it has been found that a monoclonal antibody that neutralizes a number of potent flavivirus types targets domain-Ⅲ of the envelop protein. Therefore, the Zika virus envelop protein including the domain-Ⅲ exhibits immunogenicity when administered to an individual and can greatly contribute to the formation of antibodies capable of fighting Zika virus infection.

The above Zika virus coat protein includes a functional equivalent of the amino acid sequence represented by SEQ ID NO: 1 within the scope of the present invention, and the functional equivalent means a polypeptide having a sequence homology of at least 60% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more with the amino acid sequence as a result of addition, substitution, or deletion of an amino acid, and exhibiting substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1, and is not limited thereto as long as it is an amino acid sequence of the Zika virus coat protein that can be stably produced using a plant.

In the present invention, the vaccine composition may contain, but is not limited to, 1 to 30 μg, 1 to 25 μg, 1 to 20 μg, 1 to 15 μg, 1 to 10 μg, 1 to 5 μg, 1 to 3 μg, 3 to 20 μg, 3 to 15 μg, 3 to 10 μg, 3 to 8 μg, 3 to 5 μg, 5 to 8 μg, 5 to 10 μg, 5 to 15 μg, 5 to 20 μg, 5 to 25 μg, 5 to 30 μg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg of recombinant Zika virus envelope protein per single administration dose.

The term “antigen” as used herein refers to all substances that induce an immune response in the body, and is preferably not limited to viruses, compounds, bacteria, pollen, cancer cells, or some peptides or proteins thereof, or any substance that can induce an immune response in the body.

The term “vaccine” as used herein refers to a biological preparation containing an antigen that induces an immune response in a living organism, and an immunogen that induces immunity in a living organism by injection or oral administration to a human or animal to prevent infection. The animal is a human or a non-human animal, and the non-human animal refers to, but is not limited to, pigs, cows, horses, dogs, goats, sheep, etc.

In one specific embodiment of the present invention, the vaccine composition may satisfy one or more of the following characteristics, but is not limited thereto:

a) having protective activity against at least one species selected from the group consisting of Zika virus strain PRVABC59, Zika virus strain MR766 and Dengue virus type-2;

b) promoting the secretion of one or more cytokines selected from the group consisting of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), and interleukin-6 (IL-6); and

c) Induces the formation of maternal antibodies.

In one embodiment of the present invention, the vaccine composition may have a protective effect against an Asian-lineage Zika virus strain or an African-lineage Zika virus strain. Suitable Zika virus strains of the present invention include, without limitation, viruses from the African and/or Asian lineages. Several lineages within the African and Asian lineages of Zika virus have already been identified. For example, series MR 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD 7117, ArD 9957, ArD 30101, ArD 30156, ArD 30332, HD 78788, ArD 127707, ArD 127710, 127984, ArD 127988, ArD 127994, ArD 128000, ArD 132912, 132915, ArD 141170, ArD 142623, ArD 149917, ArD 149810, ArD 149938, ArD 157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA 27101, ArA 27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506/96, ArA 975-99, ArA 982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria68, Malaysia66, Kedougou84, Suriname, MR1429, PRVABC59, ECMN2007, DakAr41524, H/PF/2013, R103451, 103344, 8375, JMB-185, ZIKV/H, The vaccine composition of the present invention may be protective against one or more suitable strains of Zika virus known in the art, including Zika virus strain PRVABC59, Zika virus strain MR766. In some embodiments, the vaccine composition of the present invention was found to be protective against Zika virus strain PRVABC59, Zika virus strain MR766.

In one specific embodiment of the present invention, the vaccine composition exhibits the highest neutralizing antibody, cellular immune response, maternal antibody formation ability and protective efficacy when administered once to three times at intervals of 14 to 28 days.

Meanwhile, the “vaccine composition” of the present invention can be used by being formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injectable solutions, respectively, according to conventional methods. When formulating, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the lecithin-like emulsifier with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, insoluble preparations, suspensions, emulsions, and lyophilized preparations. Insoluble preparations and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The routes of administration of the vaccine composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred. The term “parenteral” as used herein 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 age, weight, sex, physical condition, etc. of the subject. The amount required to induce an immune protective response in the subject without any particular side effects may vary depending on the optional presence of the recombinant protein used as the immunogen and the excipient.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

Example Ⅰ. Production of a recombinant Zika virus envelope protein plant expression vector

1. Creating pTEX-NB:ZikaEnv:aghFc vector

As shown in Fig. 1, a recombinant plant expression vector capable of expressing the Zika virus coat protein in plants was produced. More specifically, genetic information on the Zika virus coat protein was obtained, and a gene (SEQ ID NO: 2) was synthesized with a sequence optimized for expression in Nicotiana benthamiana.

The ZikaEnv:hFc expression vector, which fuses human IgG Fc to the Zika virus envelope protein, uses Zika:Env disclosed in Korean Patent Application No. 10-2020-0103211 as is, and the vector produced in this way contains the base sequence of sequence number 9.

To obtain the ZikaEnv:aghFc recombinant antigen, a polynucleotide encoding NB (new chaperone binding protein) (SEQ ID NO: 3), a polynucleotide encoding Zika virus coat protein (SEQ ID NO: 2), a polynucleotide encoding L (SEQ ID NO: 11), a polynucleotide encoding an aglycosylated human immunoglobulin heavy chain Fc fragment with a conserved N-glycosylation site deleted (SEQ ID NO: 13), and a polynucleotide encoding KDEL (Lys-Asp-Glu-Leu) peptide (SEQ ID NO: 15) were sequentially linked to construct a recombinant Zika virus coat protein plant expression vector between the MacT promoter and the RD29B terminator of the pTEX vector, which is a plant transient expression vector. The vector constructed in this way includes the base sequence of SEQ ID NO: 17.

2. Confirmation of pTEX-NB:ZikaEnv:aghFc expression in plants

The vector plasmid constructed in the above Ⅰ-1. was transformed into Agrobacterium (LBA4404 strain), cultured, and then injected into tobacco ( Nicotiana benthamiana ) leaves using a needleless syringe. Four days later, the tobacco leaves were harvested and expression was confirmed by western blot analysis. As a result, as shown in Fig. 2, it was confirmed that ZikaEnv:aghFc was mostly present in the water-soluble compartment (S).

From the above results, it was predicted that the vector produced in the above Ⅰ-1. can effectively express a recombinant Zika virus coat protein in a plant, and that the recombinant Zika virus coat protein produced using the vector has high solubility, so that separation and purification are easy, and that aggregation of the recombinant protein is suppressed, so that it is effective in maintaining the physiological activity or pharmacological activity of the recombinant protein.

Furthermore, the expression of ZikaEnv:aghFc and ZikaEnv:hFc in plants was compared.

When expressing an external gene in a plant, a silencing suppressor is essential, so the silencing suppressor, TCV-CP, must be expressed together with the target gene. Therefore, when expressing the commonly used pCAMBIA1300 vector, Agrobacterium transformed with ZikaEnv:hFc and Agrobacterium transformed with the TCV-CP expression vector must be mixed and injected into the leaves. On the other hand, pTEX, a vector dedicated to temporary expression, contains an expression cassette that can express TCV-CP, so it can be temporarily expressed with a single Agrobacterium culture. In addition, the pCAMBIA1300 vector and pTEX vector have different promoters and terminators that can express the target gene, so expression comparison is necessary in each vector backbone. Therefore, the expression of a total of four vectors was compared: NB-ZikaEnv:hFc and NB-ZikaEnv:aghFc using the pCAMBIA1300 vector, and pTEX-NB:ZikaEnv:hFc and pTEX-NB:ZikaEnv:aghFc using the pTEX vector.

As a result, as shown in Fig. 3, it was confirmed that there was no difference in the solubility of the expressed antigen with the pCAMBIA1300 vector, and that there was no difference between the pCAMBIA1300 vector and the pTEX vector. In addition, it was confirmed that there was no significant difference in the expression levels of ZikaEnv:aghFc and ZikaEnv:hFc.

Example II. Isolation and purification of recombinant Zika virus envelope protein

A recombinant Zika virus envelope protein was expressed in a large amount of tobacco leaves using a vacuum infiltration method. Specifically, whole plants (tobacco) were immersed in an Agrobacterium suspension buffer, and then Agrobacterium was injected using a vacuum transformation method. The tobacco injected with Agrobacterium was harvested, frozen in liquid nitrogen, and then crushed in a blender with an extraction buffer and centrifuged to remove debris to extract total protein from plants expressing ZikaEnv:aghFc. After binding to protein A agarose resin for 1 hour, the resin was washed with a wash buffer, and then eluted and neutralized to isolate the recombinant Zika virus envelope protein. Since a large amount of buffer was used during elution, the antigen concentration of the eluate was very low, so concentration was performed after elution.

As shown in Figure 4, the binding of the recombinant Zika virus envelope protein to the resin was confirmed through Western blotting, and the successful separation and purification of the purified recombinant Zika virus envelope protein was confirmed through Coomassie staining after electrophoresis (SDS-PAGE).

Example III. Selection of vaccine and adjuvant candidates containing recombinant Zika virus envelope protein

Recombinant subunit type vaccines are safer than killed and attenuated vaccines, but they are easily decomposed in the body, which can cause problems with sustained potency, such as failure to continuously present antigens. Therefore, adjuvants are essential. The Zika virus vaccine candidates ZikaEnv:aghFc and ZikaEnv:hFc expressed in the plant tobacco ( Nicotiana benthamiana ), the adjuvant candidate Alum, which induces Th2-type immune responses and enhances antigen-presenting, and Monophosphoryl-lipid A (MPL; EcML), a TLR 4 agonist known to induce Th1-type immune responses, are mixed, and the detoxified LOS (dLOS; EyeGene Inc.), a non-toxic and highly effective TLR4 agonist adjuvant, is mixed with aluminum hydroxide. CIA06 (Deacyl-lipooligosaccharide (dLOS) + Aluminum hydroxide; EyeGene Inc.), dLOS mixed with cationic liposomes and immunopotentiator QS21, and CIA09A mixed with deacyl-lipooligosaccharide (dLOS), cationic liposomes, and QS21 were selected and experiments were conducted with the vaccine candidates with the combinations shown in Table 1 (each combination was divided into groups 1 to 12). The doses of the immunopotentiator candidates used were Alum 500 ug/mouse, EcML 20 ug/mouse, CIA06 and CIA09A 50 ul/mouse, and CIA06 and CIA09A were used according to the manufacturer's instructions (mouse dose 50 ul, human dose 500 ul).

Group Vaccine (10 μg/mouse) Adjuvant 1 - - 2 - Alum + EcML 3 - CIA06 4 - CIA09A 5 ZikaEnv:hFc - 6 ZikaEnv:hFc Alum + EcML 7 ZikaEnv:hFc CIA06 8 ZikaEnv:hFc CIA09A 9 ZikaEnv:aghFc - 10 ZikaEnv:aghFc Alum + EcML 11 ZikaEnv:aghFc CIA06 12 ZikaEnv:aghFc CIA09A

1. Endotoxin measurement test

To confirm the safety of the vaccine candidates, the endotoxin level of the antigen in one dose of the vaccine was measured through an endotoxin test, an in vitro test method using LAL (Limulus Amebocyte Lysate) reagent. The endotoxin test was performed using GenScript's Toxin Sensor Chromogenic LAL Endotoxin Assay kit (GenScript, USA) according to the manufacturer's experimental method. As a result, as shown in Table 2, it was confirmed that the vaccine candidates, the antigens ZikaEnv:aghFc and ZikaEnv:hFc, were less than 20EU/mL, which is the recommended level for recombinant subunit vaccine.

Type Vaccine Endotoxin level(EU/mL) Antigen ZikaEnv:hFc 3.25 ZikaEnv:aghFc 3.10

2. Selection of adjuvant

2-1. Confirmation of antibody production following vaccination

To evaluate whether the target level of antibodies was produced upon vaccination, antibodies recognizing the vaccine candidates ZikaEnv:hFc and ZikaEnv:aghFc were measured using an indirect ELISA method developed in-house. The vaccine candidates in Table 1 were vaccinated into mice by intramuscular injection 1-3 times (Day 0, 14, 42), and antigen-specific antibodies in the serum isolated from the mice 7 to 14 days after vaccination (Day 7, 28, 49) were measured.

As shown in Fig. 5, the antibody titer measurement results showed that when the vaccine candidates in Table 1 were administered 1 to 3 times, the antibody titer significantly increased in the adjuvant mixed group (groups 6-8, 10-12) compared to the negative control group (PBS; group 1), and no difference was observed between the adjuvant mixed groups.

Meanwhile, the vaccine-only groups (groups 5 and 9) also showed a significant increase in antibody titer compared to the negative control group, but the difference between vaccine candidates was greater than that in the adjuvant mixed group, confirming that the adjuvant mixed group effectively and rapidly produces antigen-specific antibodies.

2-2. Confirmation of neutralizing antibody induction ability following vaccination

The neutralizing antibodies produced when the vaccine was administered were measured using the Plaque reduction neutralization test (PRNT). The vaccine candidates in Table 1 were intramuscularly injected into 8-week-old C57BL/6 mice 1-3 times (Day 0, 14, 42), and blood was collected from the mice 7 days after vaccination (Day 49) and the serum was separated. The sera of five mice in the same group were pooled and used, and after inactivation at 56℃ for 30 minutes, the control (PBS) serum and the test group sera were diluted at the same dilution ratio. Two types of ZIKV (MR766, PRVABC59 strains) with confirmed titers (PFU/ml) were diluted so that about 50 plaques/well were produced in the virus control, and then mixed 1:1 with the diluted serum and incubated at 37℃ for 30 minutes. After 30 minutes of virus reaction, the serum and virus reaction solution were inoculated onto Vero cells or Vero 76 cells, allowed to adsorb for 2 hours, and then overlay media (1% Sea plaque agar or 1.4% methyl cellulose) was added and cultured at 37℃, 5% _CO2 for 5 or 14 days. The number of plaques was confirmed by staining with 1% Crystal violet, and the PRNT ₅₀ was calculated through % inhibition.

As a result, as shown in Table 3, the neutralizing antibody induction level for the MR766 ZIKV strain was confirmed to be the highest in groups 8 and 12 containing CIA09A, with a neutralizing antibody titer of 640. Similarly, the neutralizing antibody induction level for the PRVABC59 ZIKV strain was also confirmed to be the highest in groups 8 and 12 containing CIA09A, with a neutralizing antibody titer of 640 and 320, respectively. Group 11, which mixed the ZikaEnv:aghFc antigen and CIA06, also showed the second highest neutralizing antibody induction ability after CIA09A, with a neutralizing antibody titer of 320 for the MR766 strain and PRVABC59 strain.

Group Injection Ag Adjuvant PRNT ₅₀ Titer MR766 PRVABC59 1 - - <10 <10 2 - Alum + EcML <10 <10 3 - CIA06 <10 <10 4 - CIA09A <10 <10 5 ZikaEnv:hFc - <10 10 6 ZikaEnv:hFc Alum + EcML 160 80 7 ZikaEnv:hFc CIA06 160 160 8 ZikaEnv:hFc CIA09A 640 640 9 ZikaEnv:aghFc - <10 10 10 ZikaEnv:aghFc Alum + EcML 160 40 11 ZikaEnv:aghFc CIA06 320 320 12 ZikaEnv:aghFc CIA09A 640 320

2-3. Cellular immunity induction ability measurement test

A. Confirmation of cytokine production

To analyze the effects of vaccine candidates and adjuvants on the secretion of major cytokines that activate immunogenicity, such as IFN-γ, TNF-α, IL-4, and IL-6, mice (C57BL/6, Female, 8 weeks old) were intramuscularly injected with the vaccine candidates in Table 1 three times (Day 0, 14, 42), and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the spleen and seeded into a 96-well cell culture plate at 5 × 10 ⁵ cells/well. After restimulation with antigens (ZikaEnv:hFc, ZikaEnv:aghFc) at a concentration of 1 μg/ml, cytokine production was analyzed using an ELISA assay 48 hours later. Measurement of cytokines (IFN-γ, TNF-α, IL-4, IL-6) secreted into cell culture medium was performed using ELISA MAX™ Deluxe Set Mouse IFN-γ, ELISA MAX™ Deluxe Set Mouse TNF-α, ELISA MAX™ Deluxe Set Mouse IL-4, ELISA MAX™ Deluxe Set Mouse IL-6 (Biolegend, USA) according to the manufacturer's experimental method.

As a result, as shown in Fig. 6, the secretion amounts of representative Th1 cytokines, IFN-γ and TNF-α, were measured to be the highest in groups 8 and 12 to which the immune enhancer CIA09A was added, and in particular, it was confirmed that the cytokine production was significantly increased in group 12, which included the ZikaEnv:aghFc antigen, compared to the control group (group 1: Mock).

It was also confirmed that the secretion amount of IL-4, a Th2 cytokine, and IL-6, which induces differentiation of naive T cells into Th17, significantly increased in groups 8 and 12 to which the immune enhancer CIA09A was added compared to the control group.

From the above results, it was inferred that when CIA09A adjuvant was mixed, not only the Th1 cell response of the vaccine but also Th2 and Th17 cell responses were induced. Therefore, it was judged that when CIA09A adjuvant was used together, not only the Th1 cell response capable of dealing with pathogens would increase, but also Th2 and Th17 cell responses could be induced.

B. Intracellular cytokine staining

To analyze the T cell response to antigen stimulation at the cell level, CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α were analyzed in spleen cells isolated from mice that were vaccinated three times with the vaccine candidates in Table 1. Specifically, 8-week-old C57BL/6 mice were intramuscularly vaccinated three times (Day 0, 14, 42) with the vaccine candidates in Table 1, and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the spleen and seeded at 5 × 10 ⁵ cells/well in a 96-well cell culture plate. After 12 hours of treatment with antigen (ZikaEnv:aghFc) and Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; Invitrogen Life Technologies, USA) at a concentration of 1 μg/ml, the cells were stained with each antibody (CD4, CD8, IFN-γ, TNF-α) and analyzed using flow cytometry.

As a result, as shown in Figs. 7a to 7d, similar to the results of evaluating the production amount of cytokines, it was confirmed that the expression of CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α was measured to be the highest in group 12 containing CIA09A. Therefore, it was confirmed that not only the secretion amount of IFN-γ and TNF-α but also the secretory cells increased when CIA09A was added.

Example IV. Determination of Zika virus vaccine antigen amount

In order to determine the antigen dose indicating the efficacy of the vaccine, the immunogenicity induced by administering the adjuvant CIA09A (EyeGene Inc.) selected in Example Ⅲ and the Zika virus vaccine candidates (ZikaEnv:hFc, ZikaEnv:aghFc) at different doses (5, 10, 20, 30 μg) was evaluated. The adjuvant and vaccine candidates were combined as shown in Table 4 for the vaccine candidates.

1. Comparison of antibody production according to vaccination

To confirm whether the vaccine candidates in Table 4 can produce the target level of antibodies, we measured antibodies recognizing the vaccine candidates ZikaEnv:hFc and ZikaEnv:aghFc using our self-developed Indirect ELISA method. Specifically, mice were injected intramuscularly with PBS or the vaccine candidates in Table 4 1-3 times (Day 0, 14, 42), and antigen-specific antibodies in the serum isolated from mice 7 to 14 days later (Day 7, 28, 49) were measured.

As a result, as shown in Figure 8, when the vaccine candidates were administered 1-3 times, the antibody titer significantly increased in all groups compared to the negative control group, but there was no difference according to the antigen dose.

2. Comparison of neutralizing antibody induction ability according to vaccination

The neutralizing antibodies produced when the vaccine was administered were measured using the Plaque reduction neutralization test (PRNT). Specifically, 8-week-old C57BL/6 mice were intramuscularly vaccinated with the vaccine candidates in Table 4 1-3 times (Day 0, 14, 42), and blood was collected from the mice 7 days after vaccination (Day 49) and the serum was separated. The sera of five mice in the same group were pooled and used, and after inactivation at 56℃ for 30 minutes, the sera of the control group and the sera of the vaccine candidates were diluted at the same dilution ratio. Two types of Zika virus (ZIKV) (MR766, PRVABC59 strains) with confirmed titers (PFU/ml) were diluted and prepared so that about 50 plaques/well were produced in the virus control, and then mixed 1:1 with the diluted serum and incubated at 37℃ for 30 minutes. After 30 minutes of virus reaction, the serum and virus reaction solution were inoculated onto Vero cells or Vero 76 cells, allowed to adsorb for 2 hours, and then overlay media (1.4% methyl cellulose) was added and cultured at 37℃ and 5% _CO2 for 5 or 14 days. The number of plaques was confirmed by staining with 1% Crystal violet, and the PRNT ₅₀ was calculated through % inhibition.

As a result, as shown in Table 5, the neutralizing antibody induction level for the MR766 ZIKV strain was confirmed to be the highest at 640 in groups 2 and 6 containing 5 μg of antigen. The neutralizing antibody induction level for the PRVABC59 ZIKV strain was confirmed to be the highest at 320 in groups 2 and 3 containing 5 and 10 μg of antigen in the ZikaEnv:hFc group, and at 320 in group 6 containing 5 μg of antigen in the ZikaEnv:aghFc group.

Group Injection Ag Antigen amount (μg) PRNT ₅₀ Titer MR766 PRVABC59 1 - - <10 <10 2 ZikaEnv:hFc 5 640 320 3 10 320 320 4 20 160 160 5 30 160 80 6 ZikaEnv:aghFc 5 640 320 7 10 320 160 8 20 320 160 9 30 160 160

3. Comparison of cellular immunity induction ability

3-1. Evaluation of cytokine production

To analyze the effect of vaccination with the vaccine candidates in Table 4 on the secretion of IFN-γ, TNF-α, IL-4, and IL-6, which are major cytokines that activate immunogenicity, mice (C57BL/6, Female, 8 weeks old) were intramuscularly injected with the vaccine candidates in Table 4 three times (Day 0, 14, 42), and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the removed spleens and seeded into a 96-well cell culture plate at 5 × 10 ⁵ cells/well. After restimulation with the vaccine candidate antigens (ZikaEnv:hFc, ZikaEnv:aghFc) at a concentration of 1 μg/ml, cytokine production was analyzed using an ELISA assay 48 hours later. Measurement of cytokines (IFN-γ, TNF-α, IL-4, IL-6) secreted into cell culture medium was performed using ELISA MAX™ Deluxe Set Mouse IFN-γ, ELISA MAX™ Deluxe Set Mouse TNF-α, ELISA MAX™ Deluxe Set Mouse IL-4, ELISA MAX™ Deluxe Set Mouse IL-6 (Biolegend, USA) according to the manufacturer's experimental method.

As a result, as shown in Fig. 9, the production of representative Th1 cytokines, IFN-γ and TNF-α, was measured to be the highest in groups 2 and 6 containing 5 μg of antigen, and in particular, it was confirmed that the cytokine production significantly increased in groups 6-9 containing ZikaEnv:aghFc antigen compared to the control group (group 1: Mock). However, when the antigen amount was 20 μg or more, the cytokine production was found to decrease.

In addition, the results of measuring the production of IL-4, a Th2 cytokine, and IL-6, which induces differentiation of naive T cells into Th17, showed that IL-4 cytokine production was significantly increased in all groups compared to the control group (Group 1: Mock), and there was no difference between the groups. IL-6 cytokine production increased in Group 2 containing 5 μg of ZikaEnv:hFc and Groups 6-9 containing ZikaEnv:aghFc antigen, suggesting that when 5-10 μg of antigen is administered, not only the Th1 cell response capable of dealing with pathogens increases, but also Th2 and Th17 cell responses can be induced.

3-2. Intracellular cytokine staining

To analyze the T cell response to antigen stimulation at the cell level, we analyzed the changes in CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α, which can determine whether individual cells have enhanced immunity, in spleen cells isolated from mice that were vaccinated three times with the vaccine candidates in Table 4. Specifically, 8-week-old C57BL/6 mice were intramuscularly vaccinated three times (Day 0, 14, 42) with the vaccine candidates in Table 4, and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the spleen and seeded at 5 × 10 ⁵ cells/well in a 96-well cell culture plate. After treatment with the vaccine candidate antigen (ZikaEnv:aghFc) and Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; Invitrogen Life Technologies, USA) at a concentration of 1 μg/ml for 12 hours, surface and intracellular staining was performed with each antibody (CD4, CD8, IFN-γ, TNF-α), and analysis was performed using flow cytometry.

As a result, as shown in Fig. 10, no significant difference was observed according to the amount of antigen, but it was confirmed that the expression of CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α increased in the group containing the ZikaEnv:aghFc antigen compared to ZikaEnv:hFc.

Example V. Confirmation of the effectiveness of the Zika virus vaccine

In order to determine the antigen amount indicating the efficacy of the vaccine candidate in the antigen amount determination test of Example Ⅵ, the vaccine candidate was administered to mice at different doses (5, 10, 20, 30 μg), and the highest immune function was induced at the lowest dose. Therefore, in the efficacy evaluation test of the Zika virus vaccine, the target antigen amount of 5 μg was used as a starting point, and 1 μg and 10 μg were added, and the experiment was conducted with the vaccine candidate group of the combination as shown in Table 6.

1. Comparison of antibody production according to vaccination

To evaluate whether the target level of antibodies is produced upon vaccination, we measured antibodies recognizing the vaccine candidate ZikaEnv:aghFc using an indirect ELISA method we developed ourselves. Specifically, mice were intramuscularly injected with PBS or the vaccine candidates in Table 6 1-3 times (Day 0, 14, 42), and antigen-specific antibodies in the serum isolated from the mice 7 to 14 days after vaccination (Day 7, 28, 49). For the antibody measurement method, the experiment was conducted by referring to a related paper and calculating the titer as a value greater than three times the average OD450 value of the blank of the MaxiSorp 96-well plate, with the standard for a positive judgment.

As a result, as shown in Figure 11, antibody titers significantly increased in all vaccine candidate groups compared to the negative control group when 1-3 times of vaccination was administered, but there was no difference according to antigen dose.

Furthermore, to evaluate whether the target level of antibodies was produced when the vaccine was administered, antibodies recognizing the ZIKV envelope protein were measured using a commercially available ELISA kit (Alphadiagnostic, RV-403120-1) according to the manufacturer's experimental method.

As a result, as shown in Fig. 12, it was confirmed that the antibody titer significantly increased in all vaccine candidate groups compared to the negative control group when the vaccine candidates were administered 1-3 times, and the highest antibody titer was shown in group 3 containing 5 μg of antigen protein.

2. Comparison of neutralizing antibody induction ability according to vaccination

2-1. Confirmation of neutralizing antibody induction ability following vaccination

The neutralizing antibodies produced when the vaccine was administered were measured using the Plaque reduction neutralization test (PRNT). Specifically, 8-week-old C57BL/6 mice were intramuscularly vaccinated with the vaccine candidates in Table 6 1-3 times (Day 0, 14, 42), and blood was collected from the mice 7 days after vaccination (Day 49) and the serum was separated. The separated serum was inactivated at 56°C for 30 minutes, and the control serum and vaccine candidate serum were diluted at the same dilution ratio. Two types of Zika virus (ZIKV) (MR766, PRVABC59 strains) and dengue virus (DENV)-type 2 with confirmed titers (PFU/ml) were diluted so that about 50 plaques/well were produced in the virus control, and then mixed 1:1 with the diluted serum and incubated at 37°C for 30 minutes. After 30 minutes of virus reaction, the serum and virus reaction solution were inoculated onto Vero cells or Vero 76 cells, and then allowed to adsorb for 2 hours. Overlay media (1.4% methyl cellulose) was added and cultured at 37°C, 5% _CO2 for 5 or 14 days. The number of plaques was confirmed by staining with 1% Crystal violet, and the PRNT ₅₀ was calculated through % inhibition.

As a result, as shown in Fig. 13, the neutralizing antibody induction level for the MR766 ZIKV strain was confirmed to be the highest at approximately 480 in group 3 containing 5 μg of antigen protein. The neutralizing antibody induction level for the PRVABC59 ZIKV strain was also confirmed to be the highest at approximately 288 in group 3 containing 5 μg of antigen protein.

3. Comparison of cellular immunity induction ability

3-1. Evaluation of cytokine production

To analyze the effect of vaccination with the vaccine candidates in Table 6 on the secretion of IFN-γ, TNF-α, IL-4, and IL-6, which are major cytokines that activate immunogenicity, mice (C57BL/6, Female, 8 weeks old) were intramuscularly injected with the vaccine candidates in Table 6 three times (Day 0, 14, 42), and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the spleen and seeded into a 96-well cell culture plate at 5 × 10 ⁵ cells/well. After restimulation with the vaccine candidate antigen (ZikaEnv:aghFc) at a concentration of 1 μg/ml, the cell culture fluid was obtained for 48 hours, and cytokine production analysis was performed using an ELISA assay. Measurement of cytokines (IFN-γ, TNF-α, IL-4, IL-6) secreted into cell culture medium was performed using ELISA MAX™ Deluxe Set Mouse IFN-γ, ELISA MAX™ Deluxe Set Mouse TNF-α, ELISA MAX™ Deluxe Set Mouse IL-4, ELISA MAX™ Deluxe Set Mouse IL-6 (Biolegend, USA) according to the manufacturer's experimental method.

As shown in Figure 14, the results of measuring the production of representative Th1 cytokines, IFN-γ and TNF-α, showed high secretion amounts in all vaccine groups, and in particular, the cytokine production was measured to be the highest in group 2 containing 1 μg of antigen.

As a result of measuring the production of IL-4, a Th2 cytokine, and IL-6, which induces differentiation of naive T cells into Th17, the cytokine production significantly increased in all groups compared to the control group (Group 1: Mock), and there was almost no difference between the groups.

3-2. Intracellular cytokine staining

To analyze the T cell response to antigen stimulation at the cell level, we analyzed the changes in CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α, which can determine whether individual cells have enhanced immunity, in spleen cells isolated from mice that were vaccinated three times with the vaccine candidates in Table 6. Specifically, 8-week-old C57BL/6 mice were intramuscularly vaccinated three times (Day 0, 14, 42) with the vaccine candidates in Table 6, and the spleens were removed from the mice 7 days after vaccination (Day 49). Splenocytes were isolated from the spleen and seeded at 5 × 10 ⁵ cells/well in a 96-well cell culture plate. After treatment with the vaccine candidate antigen (ZikaEnv:aghFc) and Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; Invitrogen Life Technologies, USA) at a concentration of 1 μg/ml for 12 hours, surface and intracellular staining was performed with each antibody (CD4, CD8, IFN-γ, TNF-α), and analysis was performed using flow cytometry.

As a result, as shown in Fig. 15, it was confirmed that the expression of CD4+ T cells or CD8+ T cells producing IFN-γ and TNF-α increased in group 3 containing 5 μg of antigen protein compared to the control group (group 1).

4. Confirmation of maternal transfer antibodies

4-1. Maternal IgG antibody evaluation

Maternal antibodies in the serum were isolated from the blood obtained from pups born to mothers who had been vaccinated three times with the vaccine candidates in Table 6 on the second day after birth, and measured using an indirect ELISA method we developed ourselves. The antibody measurement method was conducted by referring to a related paper, and the standard for a positive judgment was calculated as a titer that was greater than three times the average OD450 value for the blank of a MaxiSorp 96-well plate.

As a result, as shown in Fig. 16a, it was confirmed that antigen-specific IgG antibody titers were significantly increased in pups born from the vaccine candidate group compared to the control group. In particular, when group 3 containing 5 μg of antigen protein was vaccinated, the highest maternal transferred antibody titers were measured.

4-2. Evaluation of maternal transfer neutralizing antibodies

The blood of pups born from mothers who were vaccinated three times with the vaccine candidates in Table 6 was collected on the second day after birth, and the serum was separated from the blood obtained. The sera of five mice in the same group were pooled, and maternal transfer neutralizing antibodies in the serum were measured using the plaque reduction neutralization test (PRNT).

As shown in Fig. 16b, the neutralizing antibody titers for two types of Zika viruses (MR766, PRVABC59) were significantly increased in the pups born from the vaccine candidate group compared to the control group, and in particular, the highest maternal-transferred neutralizing antibody titer was induced when group 3 was inoculated with 5 μg of antigen protein.

5. Confirmation of the protective effect of vaccine candidates against Zika virus and dengue virus challenge

To evaluate the protective efficacy of the vaccine, pups born from dams that were vaccinated three times (Day 0, 14, 42) with the vaccine candidates in Table 6 were infected with dengue virus (DENV type 2) at 2 days of age to evaluate the cross-protection efficacy with two types of Zika virus (MR766, PRVABC59) and 5 mice in the same group were measured for the survival rate, body weight, and abnormal behavior. The Zika virus and dengue virus used are shown in Table 7.

Virus name,
strain Distribution center Virus titer
(TCID ₅₀ /ml) Dose of vaccination
(TCID ₅₀ /mouse) Uganda ZIKV strain, MR766 BEI resource 10 ^8.1 10 ⁶ Puerto Rico ZIKV strain, PRVABC59 BEI resource 10 ^7.4 10 ⁶ dengue virus,
type 2 National pathogen
Resource Bank 10 ^7.4 10 ⁶

As a result of measuring the survival rate, as shown in Fig. 17, when the pups born from the control mice were challenged with the MR766 strain, all the mice died by the 8th day of infection, and one mouse in group 2 containing 1 μg of ZikaEnv:aghFc died on the 14th day of infection. When infected with PRVABC59, the control mice gradually died, and only one mouse out of five survived on the 21st day, and one mouse out of group 2 containing 1 μg of ZikaEnv:aghFc died on the 2nd day of infection. Finally, when infected with DENV type 2 to evaluate the cross-protection efficacy, two mice died on the 2nd day of infection and one mouse died on the 10th day, for a total of three out of five mice. In group 2 containing 1 μg of ZikaEnv:aghFc, one mouse died on the second and sixth days after infection, and two out of five mice died. In contrast, in group 3 containing 5 μg of ZikaEnv:aghFc and group 4 containing 10 μg, all mice survived against all viruses, confirming protection against ZIKV and DENV.

As a result of measuring the body weight gain rate, as shown in Fig. 18, when the pups born from the control mice were challenged with two types of ZIKV and DENV, it was confirmed that the body weight of the surviving mice decreased compared to the vaccine candidate group in addition to the mice that died. In group 2 containing 1 μg of ZikaEnv:aghFc, some mice died when infected with two types of ZIKV and DENV, but it was confirmed that the body weight of the surviving mice was no different from that of the other groups.

ZIKV targets neural progenitor cells, which can affect gene expression, neurogenesis, differentiation of neuronal development, and induce apoptosis, which can cause various neurological abnormalities including microcephaly. Therefore, since neurological lesions induced by ZIKV can lead to abnormal behaviors, abnormal behaviors were measured by expressing the clinical symptoms observed after infection with two types of ZIKV and DENV as a *clinical score (0-3) (*Clinical score; 0 point: normal, 1 point: staggering gait, paralysis of front or hind limbs, inability to stand properly (wide stance), 2 points: 25% weight loss or dyspnea, 3 points: death).

As a result of the abnormal behavior measurement, as shown in Fig. 19, similar to the previous survival rate, abnormal behavior was observed only in the control group and the vaccine candidate group including ZikaEnv:aghFc 1 ug, and no abnormal behavior was observed in the vaccine candidate groups including 5 μg and 10 μg.

6. Measurement of the amount of virus in the organ (brain) after challenge vaccination

To evaluate the cross-protection efficacy against two types of Zika viruses (MR766, PRVABC59) in pups born from dams vaccinated three times (on Days 0, 14, and 42) with the vaccine candidates in Table 6, pups were challenged with dengue virus (DENV type 2) in Table 7 at 2 days of age. The MR766-vaccinated group was euthanized on dpi 6, and the PRVABC59 and DENV-vaccinated groups were euthanized on dpi 12. The brains of the pups were obtained and stored at -70℃. The weight of the mouse brains was measured, and 500 μl of PBS was added to the tube, and homogenized using a homogenizer. ZIKV and DENV RNA were extracted from tissue and blood samples according to the directions of an RNA/DNA extraction kit (Intron, Korea). RNA was reverse transcribed into cDNA using the AMPIGENE ^® cDNA Synthesis kit (ENZ-KIT 106-0200), and real-time PCR was performed using ZIKV-specific primers (forward (SEQ ID NO: 18): 5′-CGCAGGATCATAGGTGATGAAG-3′, reverse (SEQ ID NO: 19): 5′-CCTGACAACACTAAAATTGGTGC-3′) or DENV-specific primers (forward (SEQ ID NO: 20): 5′-TTAGAGGAGACCCCTCCC-3′, reverse (SEQ ID NO: 21): 5′-TCTCCTCTAACCTCTAGTCC-3′) and standard DNA.

As a result, as shown in Fig. 20, when ZIKV and DENV were inoculated into pups born from control mother mice, ZIKV and DENV were detected in the brains of the pups, but it was observed that the virus copy number was significantly reduced in pups born from mother mice vaccinated with the post-vaccination group.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Attach electronic file of sequence list

Claims (17)

A recombinant vector for expressing Zika virus envelope protein, comprising a polynucleotide encoding a gene for a Zika virus envelope protein having an amino acid sequence of SEQ ID NO: 1; and a polynucleotide encoding an aglycosylated human immunoglobulin Fc fragment (aglycosylated human Fc; aghFc) having an amino acid sequence of SEQ ID NO: 12.
In the first paragraph,
A recombinant vector for expressing the Zika virus envelope protein, characterized in that the recombinant vector further comprises a polynucleotide encoding a peptide linker L consisting of an amino acid sequence of sequence number 10.
In the first paragraph,
A recombinant vector for expressing the Zika virus envelope protein, characterized in that the recombinant vector further comprises a KDEL polynucleotide consisting of a base sequence of sequence number 15.
In the first paragraph,
A recombinant vector for expressing the Zika virus envelope protein, characterized in that the recombinant vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 16 or a polynucleotide consisting of the base sequence of SEQ ID NO: 17.
In any one of claims 1 to 4,
A recombinant vector for expressing the Zika virus envelope protein, characterized in that the recombinant vector is expressed in a plant.
A transformant transformed with a recombinant vector according to any one of claims 1 to 4.
In Article 6,
A transformant, characterized in that the transformant is a plant.
A recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc), produced using the recombinant vector of any one of claims 1 to 4.
In Article 8,
The recombinant Zika virus envelope protein is characterized in that the recombinant Zika virus envelope protein consists of the amino acid sequence of sequence number 16.
(a) a step of transforming a plant with a recombinant vector of any one of claims 1 to 4; and
(b) a method for producing a recombinant Zika virus coat protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc), comprising the step of isolating and purifying a Zika virus coat protein from the plant; and a recombinant Zika virus coat protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc).
In Article 10,
A method for producing a recombinant Zika virus envelope protein, characterized in that the transformation in step (a) is Agrobacterium-mediated transformation.
A vaccine composition comprising, as an active ingredient, a Zika virus envelope protein comprising an amino acid sequence of sequence number 1; and a recombinant Zika virus envelope protein fused with an aglycosylated human immunoglobulin Fc fragment (aghFc) consisting of an amino acid sequence of sequence number 12.
In Article 12,
A vaccine composition characterized in that KDEL (Lysine-aspartic acid-glutamic acid-leucine) is further fused to the recombinant Zika virus envelope protein.
In Article 12,
A vaccine composition, characterized in that the recombinant Zika virus envelope protein is composed of an amino acid sequence of sequence number 16.
In Article 12,
The vaccine composition is characterized in that it further comprises one or more of the following adjuvants:
a) Alum;
b) MPL (monophosphoryl lipid A);
c) a mixture of alum and monophosphoryl lipid A (MPL);
d) Deacyl-lipooligosaccharide;
e) a mixture of deacyl-lipooligosaccharide and aluminum hydroxide; and
f) A mixture of deacyl-lipooligosaccharide, cationic liposome, and QS21.
In Article 12,
A vaccine composition, characterized in that the vaccine composition comprises 1 to 30 μg of recombinant Zika virus envelope protein per single administration dose.
In Article 12,
The above vaccine composition is characterized by satisfying one or more of the following characteristics:
a) having protective activity against at least one species selected from the group consisting of Zika virus strain PRVABC59, Zika virus strain MR766 and Dengue virus type-2;
b) promoting the secretion of at least one selected from the group consisting of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), and interleukin-6 (IL-6); and
c) Induces the formation of maternal antibodies.