KR20230115528A - Recombinant Baculovirus vaccines comprising antigen ROP4 of Toxoplasma gondii - Google Patents

Abstract

The present invention relates to a recombinant baculovirus vaccine containing ROP4 (rhoptry protein 4) of Toxoplasma gondii. Specifically, the baculovirus vaccine has the advantage of generating selective antibodies through genetic engineering technology. In addition, it is non-pathogenic and safe, and no cytotoxicity or pathology is observed in mammalian cells or animal models. Based on this foundation, combining the inherent characteristics of human baculovirus without existing immunity can cause technological ripple effects by applying it to various infectious diseases besides Toxoplasma gondii.

Description

Recombinant Baculovirus vaccines comprising antigen ROP4 of Toxoplasma gondii}

The present invention relates to a recombinant baculovirus vaccine comprising ROP4 (rhoptry protein 4) of Toxoplasma gondii.

Toxoplasmosis is an obligare intercellular parasite and is a protozoan that causes toxoplasmosis in humans and animals worldwide. It is estimated that more than one-third of the world's population is infected with Toxoplasma gondii, and it is reported that infection rates in Korea range from 2 to 25% depending on the group.

When Toxoplasma gondii infects a mother, the trophozoite of Toxoplasma gondii can cross the placenta and infect the fetus. Infection with Toxoplasma gondii can lead to miscarriage or stillbirth in early fetuses, and congenital abnormalities such as visual impairment, hydrocephalus, and mental retardation can occur in mid-late fetuses despite normal delivery. In addition, Toxoplasma gondii, which infects healthy individuals, destroys cells of the immune system and reticuloendothelial system, and can cause diseases such as lymphadenitis, chorioretinitis, and encephalomyelitis. can be activated and cause meningitis or chorioretinitis.

Toxoplasmosis infection is usually treated by administering spiramycin or pyrimethamine alone, or by administering pyrimethamine and sulfa drugs in combination, which have been developed as preventive drugs for malaria. However, spiramycin is used for prophylaxis because of its low efficacy, and pyrimethamine does not have a therapeutic effect during pregnancy. In addition, concomitant administration of sulfa drugs such as pyrimethamine and sulfamethoxazole may cause bone marrow suppression and decrease the platelet count, and concomitant administration of folic acid may cause allergic reactions, renal impairment, blood disorders, and nausea. ), may cause side effects such as vomiting. Moreover, pyrimethamine or sulfadiazine, which are currently the most widely used anti-toxoplasmosis drugs, are gradually decreasing in their therapeutic effect due to increased tolerance of Toxoplasma gondii, so no effective human vaccine against Toxoplasma gondii infection has been developed. The situation is.

DNA, subunit or protein vaccines against Toxoplasma gondii have been studied, but these have not been successful. While immunization with DNA vaccines showed low immunogenicity, DNA vaccines containing the adjuvant IL-18 were able to induce humoral and cellular immune responses, resulting in limited protection. Therefore, finding a novel vaccine that is highly effective against Toxoplasmosis infection remains a major challenge for researchers, and therefore, it is essential to develop effective vaccines capable of protecting individuals from Toxoplasmosis infection using alternative methods.

Korean Registered Patent No. 10-2165100 (registered on October 6, 2020)

An object of the present invention is to provide a vaccine composition capable of inducing an immune response against Toxoplasma gondii, including a recombinant expression vector transfected with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4), and a method for preparing the same is providing

In order to achieve the above object, the present invention provides a vaccine capable of inducing an immune response against Toxoplasma gondii, including a recombinant virus transfected with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4) composition is provided.

In addition, the present invention comprises the steps of transfecting viral cells with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4); and culturing the virus cells to express ROP4.

The present invention relates to a recombinant baculovirus vaccine containing ROP4 (rhoptry protein 4) of Toxoplasma gondii, and specifically, the baculovirus vaccine has the advantage of selective antibody generation by genetic engineering technology. In addition, it is non-pathogenic and safe, and no cytotoxicity or pathology is observed in mammalian cells or animal models. Based on this foundation, combining the inherent characteristics of human baculovirus without existing immunity can cause technological ripple effects by applying it to various infectious diseases besides Toxoplasma gondii.

1 shows the result of confirming the antibody reaction in serum collected after inoculation of Toxoplasma gondii ROP4 recombinant baculovirus vaccine into mice.
Figure 2 shows the result of confirming the antibody response in the mucosal tissue collected after inoculating the Toxoplasma gondii ROP4 recombinant baculovirus vaccine into mice.
Figure 3 shows the result of confirming immune cell activity through flow cytometry after inoculating a mouse with Toxoplasma gondii ROP4 recombinant baculovirus vaccine.
Figure 4 shows the results of confirming inflammatory cytokines in the brain after inoculating mice with Toxoplasma gondii ROP4 recombinant baculovirus vaccine and infecting mice with 50 times or more of the lethal dose of T oxoplasma gondii (ME49 strain).
Figure 5 is inoculated with Toxoplasma gondii ROP4 recombinant baculovirus vaccine into mice and infected with Toxoplasma gondii (ME49 strain) at least 50 times the lethal dose, then the number of Toxoplasma gondii cysts in brain tissue was measured, and the body weight and survival rate of the mice were measured. Indicates the checked result.

The present invention provides a vaccine composition capable of inducing an immune response against Toxoplasma gondii, including a recombinant virus transfected with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4).

Preferably, the virus may be a baculovirus, but is not limited thereto.

Preferably, the vaccine composition may be administered orally or nasally, but is not limited thereto.

In the present invention, " Toxoplasma gondii " is a gonosporidia belonging to the subclass of coccidia. Toxoplasmosis is an obligare intercellular parasite and is a protozoan that causes toxoplasmosis in humans and animals worldwide. Toxoplasma gondii is largely divided into oocyst, tachyzoit, infectious bradyzoit, schizont, and gametocyte. It goes through five stages of development. Toxoplasmosis is infected by ingesting water or vegetables contaminated by oocysts in cat feces, the definitive host, or by eating Toxoplasma gondii, which exists as cysts in meat, such as pigs, sheep, and cattle, which are intermediate hosts. can For example, the toxoplasma protozoan is Toxoplasma gondii It may be ME49 strain, but is not limited thereto.

Specifically, the ROP4 may consist of the amino acid sequence of SEQ ID NO: 1.

For example, the ROP4 Genebank Accession No. It may be an amino acid sequence represented by ABU24469.1.

In addition, the ROP4 may include a functional equivalent of a protein consisting of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the term “functional equivalent” refers to at least 70% or more, specifically 80% or more, more specifically 90% or more, most specifically, the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. As has 95% or more sequence homology, it means a protein having substantially the same physiological activity as the amino acid sequence of SEQ ID NO: 1.

In the present invention, the term "substantially equivalent physiological activity" means an activity capable of inducing a specific immune response against Toxoplasma gondii due to structural and functional homology with ROP4.

More specifically, the ROP4 may be encoded by the nucleic acid sequence of SEQ ID NO: 2. For example, the ROP4 Genebank Accession No. It may be a gene expressed as EU047558.1.

In the present invention, "vaccine composition" means a composition capable of preventing subsequent pathogen infection by administering a protein antigen having a vaccine effect. The vaccine composition may be a composition exhibiting antigenicity against Toxoplasma gondii.

Specifically, the vaccine composition may include any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immune stimulants, and combinations thereof.

Such pharmaceutically acceptable carriers are known in the art and include proteins, sugars, and the like. The carriers may be aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, including drinking water and buffered media, alcoholic/aqueous solutions, emulsions or suspensions. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers include electrolyte replenishers, liquids and nutritional supplements, such as those based on Ringer's dextrose. As preservatives and other additives, antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be further included. Preservatives may include, but are not limited to, formalin thimerosal, neomycin, polymyxin B and amphotericin B.

The vaccine composition may further include an adjuvant. The adjuvant refers to a compound or mixture that enhances the immune response and/or accelerates the rate of absorption after inoculation, and includes any absorption-promoting agent. Acceptable adjuvants are Freund's preservatives, Freund's incomplete preservatives, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluton polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, nitrophenol and the like, but are not limited thereto.

The vaccine composition may further include an immune stimulant. The immune stimulant may include artificially synthesized levamisole, isoprenosine, and cytokines. Examples of cytokines include interferon-α, interleukin-2, GM-CSF, and GCSF. The vaccine composition may be in the form of containing the virus-like particles or a concentrated solution thereof, or may be used in the form of a transformed host cell itself or a dry powder form of the transformed cell. In addition, the vaccine composition can be used together with other foods or food ingredients and can be appropriately used according to conventional methods.

The vaccine composition includes a stabilizer, an emulsifier, aluminum hydroxide, aluminum phosphate, a pH adjuster, a surfactant, a liposome, an iscom adjuvant, a synthetic glycopeptide, an extender, carboxypolymethylene, a bacterial cell wall, a bacterial cell wall derivative, a bacterial vaccine, Animal poxvirus protein, viral subviral particle adjuvant, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldiamine It may further include one or more second adjuvants selected from the group consisting of octadecyl-ammonium bromide and mixtures thereof.

The vaccine composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injection solutions according to conventional methods. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used together.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate), sucrose or lactose, gelatin in the lecithin-like emulsifier. etc. can be used together. In addition to the above excipients, lubricants such as magnesium styrate and talc may also be used.

Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be used in addition to water and liquid paraffin, which are commonly used simple diluents. there is.

Sterile aqueous solutions, water-insoluble agents, suspensions, emulsions, and lyophilized preparations may be used for preparations for parenteral administration. Non-aqueous preparations and suspensions may be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

In the present invention, the term “vector” refers to a nucleic acid molecule used to transport linked nucleic acid fragments. As the vector, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments (ie, fragments insertable into the host cell genome by homologous recombination) may be used, but are not limited thereto. The plasmid is a type of vector and refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. In addition, viral vectors may have additional DNA ligated into the viral genome.

In the present invention, the term "expression vector" refers to a vector capable of directing the expression of a gene encoding a target protein operably linked thereto. Generally, expression vectors in the use of recombinant DNA technology are in the form of plasmids, so the terms plasmid and vector may be used interchangeably. However, it may also include other types of expression vectors that perform the same function, such as viral vectors.

Specifically, the vector may be a baculovirus vector. The baculovirus refers to a pathogenic virus that is not pathogenic to humans or vertebrates and has pathogenicity only to insects.

The expression vector is introduced into a host cell, and the host cell transformed by the introduced vector can produce the virus-like particle. In this case, the vector may include a promoter recognized by the host organism.

The nucleic acid sequence encoding the ROP4 may be operably linked to the promoter sequence. The "operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. That is, the gene encoding the virus-like particle can be operably linked to a promoter in the vector to regulate expression.

In addition, the expression vector may contain an appropriate marker gene necessary for selecting transformed host cells. The marker gene may be an antibiotic resistance gene or a fluorescent protein gene, and the antibiotic resistance gene may be selected from the group consisting of a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene, but is limited thereto It is not. The fluorescent protein gene is a yeast-enhanced green fluorescent protein (yEGFP) gene, a green fluorescent protein (GFP) gene, a blue fluorescent protein (BFP) gene, and a red fluorescent protein (red fluorescent protein, RFP) may be selected from the group consisting of genes, but is not limited thereto.

In the present invention, "transfection" means a method of delivering the vector into a microorganism or a specific cell, and when the target cell for transfection is a prokaryotic cell, it can be carried out by the CaCl2 method, the Hanhan method, the electroporation method, etc. . When the target cells for transfection are eukaryotic cells, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment may be used. It is not limited. In the case of transfection of fungi such as yeast, it may be generally performed by a transfection method using lithium acetate and heat shock and an electroporation method, but is not limited thereto.

In addition, the present invention comprises the steps of transfecting viral cells with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4); and culturing the virus cells to express ROP4.

Preferably, the ROP4 may consist of the amino acid sequence of SEQ ID NO: 1.

Preferably, the virus may be a baculovirus, but is not limited thereto.

Hereinafter, the present invention will be described in detail according to examples that do not limit the present invention. Of course, the following examples of the present invention are only intended to embody the present invention and are not intended to limit or limit the scope of the present invention. Therefore, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention.

< Example 1> Toxoplasma gondii ( Toxoplasma gondii ) club protein 4 ( rhoptry protein 4; ROP4 ) expressing baculovirus vaccine manufacturing

Toxoplasma gondii ) A large amount of tachyzoite was collected from the peritoneal cavity on the 4th and 5th day after the mice were infected with tachyzoite by IP route. Toxoplasma gondii RNA was extracted with Invitrogen's RNA extraction kit, and Toxoplasma gondii antigen club protein (rhoptry protein 4; ROP4) was amplified by RT-PCR. Amplified ROP4 was incorporated into the pFastBac vector. Transformation was performed using DH10Bac competent cells, and it was confirmed that the bacmid DNA contained the ROP4 gene by colony PCR. For the production of baculovirus expressing ROP4, the construct confirmed by the colony PCR method was transfected into insect cell Sf9 at 27 ° C using Cellfectin II (Invitrogen) and Sf-900™ II SFM medium. ) to produce and scale up recombinant baculovirus. The amplified baculovirus titer was measured and used for animal experiments.

<Example 2> Verification of vaccine efficacy in animal models

Toxoplasma gondii ROP4 recombinant baculovirus vaccine was inoculated into mice, and antibody reactions were confirmed in serum collected.

Experimental method: For blood sample preparation, 200 μl blood was collected by orbital blood collection at 1 week and 3 weeks after rBV-ROP4 vaccination in mice, and serum was collected using a centrifuge at 3000 rpm, 4 ° C. for 20 minutes. A 96-well plate was coated with ME49 antigen (4 μg/ml) and reacted at 4° C. for 16 hours. The collected serum was diluted 1:50 and used, and toxoplasma gondii-specific IgG (FIG. 1A) and IgA (FIG. 1B) antibody reactions were measured at 490 nm using an ELISA reader.

Experimental results: It was confirmed that the rBV-ROP4 vaccinated group increased from Toxoplasma gondii to a high level compared to the non-immunized mouse group. High IgA antibody response in serum was rapidly increased at the time of the second inoculation in both oral and nasal routes.

Toxoplasma gondii ROP4 recombinant baculovirus vaccine was inoculated into mice, and antibody responses were confirmed in mucosal tissues collected.

Experimental method: Each mucosal sample acquisition was sacrificed at 16 dpi after infecting with about 50 times the lethal dose of Toxoplasma gondii 4 weeks after immunization. Intestine was cut at 5 cm from about 1-2 cm below the mouse and collected in a tube containing 500 μl of 1 × PBS. Fecal was collected in a tube by calculating 100 μl of 1 × PBS per 0.1 g. Vaginal samples were collected in tubes after repeated washing of the vaginal canal with 200 μl of 1×PBS. The collected mucosal samples were incubated at 37° C. for 1 hour, and then supernatants were obtained through centrifugation at 5000 rpm for 10 minutes. Collected mucosal samples were measured using ELISA. A 96-well plate was coated with ME49 antigen (4 μg/ml) and reacted at 4° C. for 16 hours.

Each mucosal sample intestine was diluted with 1× PBS at a ratio of 1:100, fecal 1:2, and vaginal 1:20, and reacted with the primary antibody at 37° C. for one hour. As secondary antibodies, HRP conjugated goat anti-mouse IgG and IgA (1:2000 dilution) were reacted at 37° C. for one hour and then measured at 490 nm using an ELISA reader.

Experimental results: Obtained mucosal samples were evaluated for antibody production in mucosal tissues of mice to evaluate immune induction. At 16 dpi, T. gondii specific IgG and IgA responses were observed in the vaccinated group than in the unimmunized Naive+Cha group. 2A and 2D: Immunization of mice with rBV-ROP4 in intestinal tissue induced higher amounts of T. gondii -specific IgG compared to oral route immunization against IgG and Naive + Cha group, mucosal An antibody response, IgA antibody, was induced at a high level. 2B and 2E: Increases in T. gondii IgG and IgA compared to Naive + Cha were observed in the vaccinated group in fecal tissues. 2C and 2F: Immunization of mice with rBV-ROP4 increased IgG and IgA antibody responses in vaginal tissues.

Mice were inoculated with Toxoplasma gondii ROP4 recombinant baculovirus vaccine, and immune cell activity was confirmed by flow cytometry.

Experimental method: Splenocytes and mesenteric lymph node cells were prepared in a single cell suspension of spleen cells (1×10 ⁶ cells/mouse) and lymph node cells (1×10 ⁵ cells/mouse) in 5% CO ₂ for 2 hours for flow cytometric analysis. They were stimulated with T. gondii ME49 antigen (4 μg/ml) at 37°C. After stimulation, flow cytometry was performed to evaluate the proliferation of CD4 ⁺ and CD8 ⁺ T cells as well as germinal center B cells in the spleen and mesenteric lymph nodes of mice. Each antibody was purchased from BD Biosciences (Franklin Lakes NJ USA) and Invitrogen (Waltham MA USA) and then stained with a fluorescently conjugated antibody. Stained cells were acquired using an Accuri C6 flow cytometer and analyzed with C6 Accuri software (BD Biosciences Franklin Lakes NJ USA).

Experimental Results: Induction of CD4 ⁺ and CD8 ⁺ T cell populations in MLN was similar between groups vaccinated with Naive + Cha and rBV-ROP4 vaccines by oral route. However, when rBV-ROP4 was administered intranasally, T cell proliferation was activated (FIGS. 3A and 3B). Germinal center B cell responses observed in spleen cells and MLN cell responses were high in all vaccinated groups. T. gondii infection in unimmunized mice induced a germinal center B cell response, but this was dramatically increased upon immunization with the vaccine. Both immune pathways contributed to the high induction response of germinal center B cells in both spleen and MLN (Fig. 3C and Fig. 3D).

< Example 3> Toxoplasma recombinant baculovirus Vaccine defense efficacy

After inoculating mice with Toxoplasma gondii ROP4 recombinant baculovirus vaccine and infecting mice with more than 50 times the lethal dose of T oxoplasma gondii (ME49 strain), inflammatory cytokines were identified in the brain.

Experimental method: At 16 dpi, the mouse brain tissue was individually homogenized in 500 μl of 1 × PBS, and then the supernatant was collected through a centrifuge. The pro-inflammatory cytokines IFN-gamma and IL-6 were measured using the BD OptEIA ELISA kit (BD Biosciences Franklin Lakes NJ USA) in the brain supernatant of mice infected with T. gondii using a standard curve generated using an ELISA meter at 570 nm. Cytokine concentrations were calculated using

Experimental results: In order to determine the production of pro-inflammatory cytokines IFN-gamma and IL-6, brain supernatants were collected at 16 dpi after infection with a lethal dose of Toxoplasma gondii to determine the inflammatory response (FIGS. 4A and 4B). Compared to the non-immunized infection control group (Naive+Cha), a significantly lower level of IFN-gamma was detected in the brain supernatant in the group immunized with rBV-ROP4 (Immunization). It was confirmed that a low level of the pro-inflammatory cytokine IL-6 was detected in the vaccination group compared to the non-immunized infection control group (Naive + Cha) (FIG. 4B).

After inoculating mice with Toxoplasma gondii ROP4 recombinant baculovirus vaccine and infecting mice with more than 50 times the lethal dose of Toxoplasma gondii (ME49 strain), the number of Toxoplasma gondii cysts in brain tissue was measured, and the body weight and survival rate of mice were confirmed. .

Experimental method: After homogenizing mouse brain tissue with 1x PBS at 16 dpi, turbidity with 44% percoll was stacked on top of 67% percoll, using density gradient centrifugation at 12100 rpm, 4℃, 20 minutes, and only the middle layer was obtained from the three layers. The red blood cells were removed using the RBC solution. Then, 1× PBS was added and used for 20 minutes at 6000 rpm through a centrifuge to remove other solutions and obtain only Toxoplasma gondii. The collected cysts were measured by counting the numbers in 5 different areas on a slide glass through a microscope (Leica DMi8, Leica, Wetzlar, Germany) and calculating the average. Body weight and survival rate were measured at 5-day intervals after Toxoplasma gondii infection, and animal experiments were conducted when the body weight of the non-immunized infected control group lost less than 75%.

Experimental results: After infecting non-immunized mice and rBV-ROP4 vaccine immunized mice with a very high amount of Toxoplasma gondii, the lethal dose was more than 50 times higher than in previous studies, the brains were collected and the number of cysts was confirmed (Fig. 5A). In the group vaccinated with rBV-ROP4 vaccine, more than 6000 cysts were detected in the non-immunized control group (Naive + Cha). However, in the vaccination group, the number of cysts was less than about 2000, which was more than three times lower. Therefore, at the time of body weight measurement, the infected control group had a high weight loss of 75% on day 16. On the other hand, the weight loss rate of the vaccinated group was protected by more than 80%. In the survival rate result, the vaccination group showed a survival rate of 100%. However, the survival rate of the non-immunized infected control group was 0%.

These results support that the rBV-ROP4 vaccine shows a high vaccine protection against Toxoplasma gondii at a very good level, even though the rBV-ROP4 vaccine infects with Toxoplasma gondii at a lethal dose of more than 50 times.

On the other hand, 1) In a previous study related to VLP-ROP4, 450 cysts were infected, and about 600 cysts were detected in the non-immunized control group. The number of cysts was confirmed. However, although the present invention infected a very high amount of Toxoplasma gondii with about 50 times the lethal dose, the vaccination group confirmed a 3-fold or more lower number of cysts compared to the non-immunized infected control group.

2) Virus-like particle (VLP) vaccine is less effective as a vaccine due to protein denaturation by gastric acid when inoculated via the oral route, but the recombinant baculovirus (rBV) used in this study is a vaccine despite oral route vaccination showed a high level of protective efficacy.

3) Vaccination by the oral route and the nasal route does not use a needle, so the vaccination is convenient, and the safety of the medical staff and the patient's skin are not damaged. The preventive effect is expected to be very high.

In conclusion, (1) Toxoplasma gondii baculovirus vaccine efficacy evaluation study was conducted by the present inventors at home and abroad for the first time. Compared to the VLPs vaccine research that has been conducted so far, the baculovirus vaccine has many advantages (economical, fast manufacturing process, simple manufacturing and purification process).

(2) Both the oral and nasal vaccines used in the present invention cause mucosal immunity and systemic immunity to block the infection of the digestive mucosal pathway of Toxoplasma gondii, and antibodies due to systemic immunity when the worms migrate and settle. Acts on worms and inhibits the growth of worms.

(3) Oral vaccine is a very convenient and safe route that does not require a nurse and does not require a needle, so it is painless and can be purchased and consumed at a pharmacy without going to a hospital or public health center. is the path

Normally, when the vaccine is taken orally, the vaccine protein antigenicity is destroyed by the action of gastric acid (pH2-3) in the stomach, so the vaccine efficacy cannot be seen. Nevertheless, in the present invention, high vaccine efficacy was confirmed by oral route inoculation of baculovirus expressing Toxoplasma gondii antigen.

(4) Oral vaccines are also safer and more convenient than nasal vaccines. The nasal route may cause some discomfort for the vaccine material to enter the nasal cavity, and there is a possibility of vaccine loss during the ingestion process without medical personnel's help, which may affect vaccine efficacy.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Recombinant Baculovirus vaccines comprising antigen ROP4 of Toxoplasma gondii <130> ADP-2021-0871 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 578 <212> PRT <213> Toxoplasma gondii <400> 1 Met Gly His Pro Thr Ser Phe Gly Gln Pro Ser Cys Leu Val Trp Leu 1 5 10 15 Ala Ala Ala Phe Leu Val Leu Gly Leu Cys Leu Val Gln Gln Gly Ala 20 25 30 Gly Arg Gln Arg Pro His Gln Trp Lys Ser Ser Glu Ala Ala Leu Ser 35 40 45 Val Ser Pro Ala Gly Asp Ile Val Asp Lys Tyr Ser Arg Asp Ser Thr 50 55 60 Glu Gly Glu Asn Thr Val Ser Glu Gly Glu Ala Glu Gly Ser Arg Gly 65 70 75 80 Gly Ser Trp Leu Glu Gln Glu Gly Val Glu Leu Arg Ser Pro Ser Gln 85 90 95 Asp Ser Gln Thr Gly Thr Ser Thr Ala Ser Pro Thr Gly Phe Arg Arg 100 105 110 Leu Leu Arg Arg Leu Arg Phe Trp Arg Arg Gly Ser Thr Arg Gly Ser 115 120 125 Asp Asp Ala Ala Glu Val Ser Arg Arg Thr Arg Val Pro Leu His Thr 130 135 140 Arg Leu Leu Gln His Leu Arg Arg Val Ala Arg Ile Ile Arg His Gly 145 150 155 160 Val Ser Ala Ala Ala Gly Arg Leu Phe Gly Arg Val Arg Gln Val Glu 165 170 175 Ala Glu Arg Pro Gln Pro Val Phe Thr Glu Gly Asp Pro Pro Asp Leu 180 185 190 Glu Thr Asn Ser Leu Tyr Tyr Arg Asp Lys Val Pro Gly Gln Gly Ile 195 200 205 Ile Gln Glu Ile Leu Arg Gln Lys Pro Gly Ile Ala His His Pro Glu 210 215 220 Ser Phe Ser Val Val Ala Ala Asp Glu Arg Val Ser Arg Thr Leu Trp 225 230 235 240 Ala Glu Gly Gly Val Val Arg Val Ala Ser Glu Leu Gly Gln Pro Gly 245 250 255 Arg Val Leu Val Arg Gly Arg Arg Ile Gly Leu Phe Arg Pro Gly Met 260 265 270 Gln Phe Glu Ala Thr Asp Gln Ala Thr Gly Glu Pro Met Thr Ala Leu 275 280 285 Val Gly His Thr Val Leu Glu Ala Thr Ala Arg Asp Val Asp Ser Met 290 295 300 Arg Asn Glu Gly Leu Ala Val Gly Leu Phe Gln Lys Val Lys Asn Pro 305 310 315 320 Tyr Leu Ala Asn Arg Tyr Leu Arg Phe Leu Ala Pro Phe Asp Leu Val 325 330 335 Thr Ile Pro Gly Lys Pro Leu Val Gln Lys Ala Lys Ser Arg Asn Glu 340 345 350 Val Gly Trp Val Lys Asn Leu Leu Phe Leu Leu Pro Pro Thr His Val 355 360 365 Asp Met Glu Thr Phe Val Asp Glu Ile Gly Arg Phe Pro Gln Glu Asp 370 375 380 Arg Pro Leu Ala Asp Ala Ala Arg Leu Tyr Leu Thr Val Gln Ala Val 385 390 395 400 Arg Leu Val Ala His Leu Gln Asp Glu Gly Val Val His Gly Lys Ile 405 410 415 Met Pro Asp Ser Phe Cys Leu Lys Arg Glu Gly Gly Leu Tyr Leu Arg 420 425 430 Asp Phe Gly Ser Leu Val Arg Ala Gly Ala Lys Val Val Val Pro Ala 435 440 445 Glu Tyr Asp Glu Tyr Thr Pro Pro Glu Gly Arg Ala Ala Ala Arg Ser 450 455 460 Arg Phe Gly Ser Gly Ala Thr Thr Met Thr Tyr Ala Phe Asp Ala Trp 465 470 475 480 Thr Leu Gly Ser Val Ile Phe Leu Ile Trp Cys Ser Arg Ala Pro Asp 485 490 495 Thr Lys Ser Gly Tyr Glu Tyr Ser Val Glu Phe Phe Phe Ser Arg Cys 500 505 510 Arg Arg Val Pro Glu Asn Val Lys Leu Leu Val Tyr Lys Leu Ile Asn 515 520 525 Pro Ser Val Glu Ala Arg Leu Leu Ala Leu Gln Ala Ile Glu Thr Pro 530 535 540 Glu Tyr Arg Glu Met Glu Glu Gln Leu Ser Ala Ala Ser Arg Leu Tyr 545 550 555 560 Ser Gly Asp Gly Thr Leu Thr Gly Gly Asp Asp Asp Met Pro Pro Leu 565 570 575 Glu Thr <210> 2 <211> 2039 <212> DNA <213> Toxoplasma gondii <400> 2 tccggcatgg aaaaaatgcg tctagctaac cgcgggcccc aatgtcgcac ggccttggtc 60 aagggacggc gcgacctgca gagacgggaa tccgagccaa gcgctgagtg tccttgcttc 120 tggctgagcc gccatcctgc ggcaccgtcc atgactgcgc acagcatccc gggagtatgg 180 tgattgtgtc agtcttattt cttttgaagg cacttcagat gtgtacttca gaatgttgtt 240 acgataattg cggatgcagc tgccacctct gaacggttga tttggttgtg agtcctccca 300 acatggggca ccctacctct ttcggacagc cgtcgtgtct tgtctggcta gctgcggcat 360 tccttgttct ggggctttgc ctcgtccagc aaggcgctgg cagacagcgg cctcaccagt 420 ggaagagctc ggaagccgct ttgagtgtca gtccggcagg agacatcgtg gacaagtatt 480 ctcgtgattc cactgaggga gaaaacactg tctcagaggg ggaagctgag ggcagtcggg 540 ggggttcatg gctggagcag gagggggttg aattaaggtc gccttcacag gacagccaga 600 caggaacctc gaccgcgtcc cccacaggct tcagaaggtt actcaggcgt ttgcgttttt 660 ggcgtcgtgg gagtacacgc ggatccgatg atgcagcaga agtttccagg aggactcggg 720 ttcctctgca tacccgtctg cttcagcatt tgcggcgagt agcgagaatc atccgacacg 780 gggtttcagc ggccgctggc agattgttcg ggcgagttcg gcaagttgag gcggaacgtc 840 cacaacccgt tttcactgaa ggtgatcctc cggatctcga gacgaattcc ttgtattatc 900 gcgacaaggt tccaggacag gggataatcc aggagatcct caggcagaag ccgggaatcg 960 cacaccaccc cgagtcattt agtgtggttg ctgctgacga gagagtatca cggactctgt 1020 gggctgaggg cggggttggg agagttgctt cagaattggg ccaacctggg agagtgctcg 1080 tgaggggggg acgaatcggt ttgtttcgcc caggaatgca gtttgaagca acagaccagg 1140 cgacaggaga accgatgact gcgcttgttg gccatactgt gcttgaggcc acggcaagag 1200 acgtagattc gatgcgcaac gaaggattag ctgttgggtt gtttcaaaag gtgaagaacc 1260 catatctagc taacaggtat ctaagatttc tggccccgtt cgatttggtg acgatcccgg 1320 ggaagccatt agttcagaaa gctaagtcac gtaatgaggt tggctgggtc aaaaatctgt 1380 tgtttctgct tcctccaact catgtcgata tggaaacgtt tgtcgacgag attggtcgat 1440 ttccacaaga ggaccgcccc ctagctgatg ccgctcgatt gtatcttact gttcaggcgg 1500 tgaggttggt agcgcacctc caagacgaag gagtggtgca cgggaagata atgcctgatt 1560 cgttttgttt gaagcgtgag gggggcctgt acttgcgtga ctttggctct ctggtgagag 1620 cgggcgcaaa agtggtggtg cccgcggaat atgatgagta tacgccgcca gaaggtcgtg 1680 cggccgcccg aagtcgtttc ggctctgggg cgaccacgat gacgtacgca ttcgacgcct 1740 ggacactggg ttcggttata tttttaattt ggtgctctcg agctcccgat acaaaatcgg 1800 ggtacgaata cagtgtcgaa ttcttttttt cgaggtgccg gcgggtgcca gagaacgtga 1860 aacttctggt ttacaagttg attaaccctt ctgtcgaggc ccgcctgctc gccttgcaag 1920 cgatagagac tccggaatac agggagatgg aagaacagct gtccgctgct tcgcgcctgt 1980 actcaggtga tgggacgttg acagggggag acgatgacat gccaccactg gaaacgtga 2039

Claims (8)

A vaccine composition capable of inducing an immune response against Toxoplasma gondii comprising a recombinant virus transfected with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (ROP4). The vaccine composition according to claim 1, wherein the ROP4 is composed of the amino acid sequence of SEQ ID NO: 1. The vaccine composition according to claim 1, wherein the ROP4 is encoded by the nucleic acid sequence of SEQ ID NO: 2. The vaccine composition according to claim 1, wherein the virus is a baculovirus. The vaccine composition according to any one of claims 1 to 4, wherein the vaccine composition is administered orally or nasally. transfecting viral cells with a recombinant expression vector expressing Toxoplasma gondii antigen club protein 4 (rhoptry protein 4; ROP4); and
A method for producing a vaccine capable of inducing an immune response against Toxoplasma gondii, comprising culturing the virus cells to express ROP4. The method of claim 6, wherein the ROP4 is composed of the amino acid sequence of SEQ ID NO: 1. The method of claim 6, wherein the virus is a baculovirus.