DNA VACCINE COMPOSITION FOR PREVENTING AND TREATING HERPES ZOSTER, AND METHOD FOR ACTIVATING T CELLS FOR VZV ANTIGEN BY USING SAME

Abstract

The present invention provides a DNA vaccine composition for preventing and treating herpes zoster, containing: at least one type of plasmid containing the insertion site of a varicella-zoster virus (VZV)-derived gene encoding a VZV protein; and other pharmaceutically acceptable ingredients. The plasmid contains a plurality of plasmids containing the insertion site of heterologous genes which are different from each other. The plasmid contains: a first plasmid containing the insertion site of a first gene encoding IE-62 protein (SEQ ID NO: 1); a second plasmid containing the insertion site of a second gene encoding IE-63 protein (SEQ ID NO: 2); and a third plasmid containing the insertion site of a third gene encoding gE protein (SEQ ID NO: 3).

Claims

1. A plasmid DNA for preventing and treating herpes zoster, comprising an insertion site of a varicella-zoster virus (VZV)-derived gene encoding a VZV protein, wherein the plasmid DNA is directly administered into the body by an electroporation method to induce immune activity of T cells against a VZV antigen.

2. The plasmid DNA of claim 1, wherein the gene comprises one selected from the group consisting of a first gene encoding an IE-62 protein (SEQ ID NO: 1), a second gene encoding an IE-63 protein (SEQ ID NO: 2), and a third gene encoding a gE protein (SEQ ID NO: 3).

3. The plasmid DNA of claim 1, which comprises one selected from the group consisting of a plasmid DNA set forth in SEQ ID NO: 4, a plasmid DNA set forth in SEQ ID NO: 5, and a plasmid DNA set forth in SEQ ID NO: 6.

4. A DNA vaccine composition for preventing and treating herpes zoster, comprising: at least one plasmid containing an insertion site of a varicella-zoster virus (VZV)-derived gene encoding a VZV protein; and other pharmaceutically acceptable ingredients.

5. The DNA vaccine composition of claim 4, wherein the plasmid comprises a plurality of plasmids containing different insertion sites of heterologous genes.

6. The DNA vaccine composition of claim 4, wherein the plasmid comprises: a first plasmid containing an insertion site of a first gene encoding an IE-62 protein (SEQ ID NO: 1); a second plasmid containing an insertion site of a second gene encoding an IE-63 protein (SEQ ID NO: 2); and a third plasmid containing an insertion site of a third gene encoding a gE protein (SEQ ID NO: 3).

7. A method for activating T cells against a varicella-zoster virus (VZV) antigen, comprising: preparing a plasmid containing a VZV-derived gene encoding a VZV protein; and administering the plasmid into the body using an electroporation method.

Description

DESCRIPTION OF DRAWINGS

[0016] FIG. 1 is a diagram showing a vaccination schedule according to one exemplary embodiment of the present invention.

[0017] FIG. 2 shows graphs illustrating results of immunogenicity for experimental groups according to one exemplary embodiment of the present invention.

[0018] FIGS. 3 and 4 are graphs illustrating results of comparing immune responses of overlapping peptides of proteins in respective experimental groups.

[0019] FIGS. 5 to 7 are electrophoretic image of products expressed by respective plasmid genes.

BEST MODE

[0020] Hereinafter, a DNA vaccine for preventing and treating herpes zoster will be described in detail.

[0021] The DNA vaccine for preventing and treating herpes zoster according to the present invention is not a virus-based live vaccine but a DNA-based vaccine in which DNA itself is administered into the body. As a result, the DNA itself in the form of a plasmid, which is administered into the body to induce immune activity of T cells against a VZV antigen, serves as the vaccine.

[0022] The DNA vaccine encodes a VZV-derived antigen protein, and ultimately plays a role in forming the antigen protein in the body. The VZV-derived protein is preferably chosen in consideration of desired characteristics of the DNA vaccine. In this case, the protein may be chosen alone or in combination with plural types thereof.

[0023] According to one exemplary embodiment of the present invention, the protein may include proteins IE-62 (SEQ ID NO: 1) and IE-63 (SEQ ID NO: 2) and a glucoprotein gE (SEQ ID NO: 3), all of which may induce a cellular immune response, and the plasmid is designed so that a gene encoding such a protein can be inserted into the plasmid.

[0024] The plasmid DNA is administered so that the plasmid DNA is mixed with a liquid in the body. For this purpose, the plasmid DNA of the present invention may be designed to be introduced into the body with high efficiency using an electroporation method. Only when such an electroporation method is used, in vivo delivery efficiency may be maximized, thereby maximizing therapeutic efficacy. A site of the body to which the plasmid DNA is administered using the electroporation method includes a region of muscle, etc., but the present invention is not particularly limited thereto. For example, the site of the body may be determined in consideration of various factors such as the age of a subject to which the plasmid DNA is administered, the type of a disease, concurrent diseases, etc. An electroporation system (CELLECTRA commercially available from Inovio Pharmaceuticals Inc.) may be used as electroporation equipment used to realize the electroporation method.

[0025] The genes, for example, a first gene, a second gene and a third gene which may encode and express the IE-62, IE-63 and gE proteins, respectively, are incorporated into the plasmid using a conventional method. However, to maximize the medicinal effect and administration efficiency, plasmids set forth, respectively, in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 may be designed in this example to maximize the efficiency of the DNA vaccine. The respective plasmids may be used alone, but are preferably included in the DNA vaccine in a combination of two or more different plasmids. For example, the DNA vaccine containing all the three plasmids may be contemplated in one exemplary embodiment of the present invention. Meanwhile, different types of plasmids which are not specified in this example may also be newly introduced.

[0026] The plasmid may be administered into the body by means of an injectable drug, etc. In this case, the plasmid should be prepared with a high concentration and high purity to improve delivery efficiency and immune response efficiency. Also, the ease in mass production should be ensured in a commercial aspect. The plasmid according to one exemplary embodiment of the present invention has a foundation to be mass-produced with such a high concentration and high purity.

[0027] The DNA vaccine for preventing and treating herpes zoster according to one exemplary embodiment of the present invention is a vaccine for liquids, that is, injectable drugs, which includes at least one plasmid DNA as described above. Thus, the DNA vaccine includes the plasmid DNA and a liquid ingredient. Here, the liquid ingredient may be pure water. In addition to the water, the DNA vaccine composition according to one exemplary embodiment of the present invention may further include pharmaceutically acceptable functional additives. In this case, such additives may be introduced at a level known in the related art.

[0028] The DNA vaccine composition may, for example, include all the three plasmids as described above.

[0029] Hereinafter, the efficiency of the plasmid DNA according to one exemplary embodiment of the present invention will be described based on the specific contents of experiments.

[Experiment 1] Evaluation of Immunogenicity

[0030] Screening of VZV Gene Candidates Usable in DNA Vaccine

[0031] Genes of proteins IE-62 and IE-63 and a glucoprotein gE, all of which may induce a cellular immune response, were screened from 67 genes encoding proteins in the VZV genome. The type of the VZV genome was divided into a total of 5 clades, and there was no significant difference in amino acid sequences of the IE-62, IE-63 and gE between the clades. Plasmids were constructed using sequences of the aforementioned IE-62, IE-63 and gE genes. The amino acid sequences of the proteins are as set forth in SEQ ID NOs: 1 to 3, and the DNA sequences of the constructed plasmids are as set forth in SEQ ID NOs: 4 to 6. In this example, the DNA vaccine included the plasmid and a liquid ingredient (i.e., water), but did not include other ingredients.

[0032] Experiment on Mice

[0033] The IE-62, IE-63 and gE genes were prepared as candidate materials for the DNA vaccine, and administered into C57BL/6 mice. Thereafter, splenocytes were removed from the mice to evaluate immunogenicity of T cells against a VZV antigen. A total of 5 groups were used for this experiment, and each group consisted of five mice, as follows.

[0034] 1) Neg: A group in which mice are vaccinated with a vector (n=5)

[0035] 2) IE62: A group in which mice are vaccinated with an IE-62 DNA vaccine (n=5)

[0036] 3) IE63: A group in which mice are vaccinated with an IE-63 DNA vaccine (n=5)

[0037] 4) gE: A group in which mice are vaccinated with a gE DNA vaccine (n=5)

[0038] 5) IE62+IE63+gE: A group in which mice are vaccinated with a mixture of IE-62, IE-63 and gE DNA vaccines

[0039] The vaccination was performed three times at an interval of 2 weeks using an electroporation system (CELLECTRA commercially available from Inovio Pharmaceuticals Inc.). In this case, an amount of the DNA was 30 g/mice upon every vaccination, and the mixed group was vaccinated at 30 g for each DNA (a total amount of 90 g). After 12 days of the vaccination, splenocytes were removed to perform an immuoassay. A vaccination schedule is shown in FIG. 1. FIG. 1 is a diagram showing a vaccination schedule according to one exemplary embodiment of the present invention.

[0040] Preparation of Antigen for Experiments

[0041] To systematically measure the entire immune response to proteins encoded by candidate genes, a series of overlapping peptides (OLPs) were designed and prepared. Each of the overlapping peptides was designed to consist of 15 amino acids with 10 amino acids overlapping with its adjacent peptides. Also, the overlapping peptides were designed to contain full-length amino acid sequences of the IE-62, IE-63 and gE proteins. As a result, 261 IE-62 overlapping peptides, 54 IE-63 overlapping peptides and 124 gE overlapping peptides were prepared. The 37 or 38 consecutive IE-62 overlapping peptides were mixed to prepare a total of 7 peptide mixtures, which were then used. The 27 consecutive IE-63 overlapping peptides were mixed to prepare a total of 2 peptide mixtures, which were then used. The 40 or 41 consecutive IE-63 overlapping peptides were mixed to prepare a total of 3 peptide mixtures, which were then used. 5% DMSO was used as the negative control, and a mixture of phorbol myristate acetate (PMA) and ionomycin was used as the positive control. Peptide mixtures prepared by mixing the overlapping peptides are listed in the following Table 1.

TABLE-US-00001 TABLE 1 Number of mixed Antigen SEQ ID NOs of overlapping Proteins names amino acids peptides IE-62 IE62-1 1-37 37 IE62-2 28-74 37 IE62-3 75-111 37 IE62-4 112-143 37 IE62-5 149-185 37 IE62-6 186-223 38 IE62-7 224-261 38 IE-63 IE63-1 1-27 27 IE63-2 28-54 27 gE gE -1 1-27 41 gE -2 28-54 40 gE -3 1-41 41

[0042] A lysate prepared after MRC-5 cells were infected with VZV was used as the antigen, and an MRC-5 cell lysate was used as a negative antigen corresponding to the lysate.

[0043] IFN- ELISPOT Assay Protocol Splenocytes were seeded at 110.sup.6 cells/well to perform an ELISPOT assay. When the splenocytes were stimulated with a mixture of overlapping peptides, the overlapping peptides were administered so that a concentration of each of the overlapping peptides reached 1 g/ml per well, and, when the splenocytes were stimulated with a lysate antigen, the lysate antigen was administered so that a concentration of the lysate antigen reached 50 g/ml per well. In this case, this experiment was repeated in triplicate. The assay results obtained through the stimulation with the overlapping peptide antigen was calculated, as follows: Mean of OLP pools-specific spot forming cells (SFCs)-Mean of 5% DMSO-specific SFCs. The assay results obtained through the stimulation with the lysate antigen was calculated, as follows: Mean of VZV-specific SFCs-Mean of MRC-5-specific SFCs. The maximum value of SFCs per well was calculated to be 500.

[0044] Experimental Results of Immunogenicity

[0045] The results of antigen evaluation with respect to the respective vaccines showed that no immune response was induced with respect to the stimulus of overlapping peptides in the Neg-treated group used as the negative control. It was revealed that an immune response was induced with respect to the overlapping peptide of the corresponding protein in the IE-62-, IE-63- and gE-treated groups in which each of the IE-62, IE-63 and gE DNA vaccines were administered alone. It was revealed that an immune response was induced with respect to the overlapping peptides of the three proteins in the IE62+IE63+gE-treated group in which all the IE-62, IE-63 and gE DNA vaccines were administered. When a DNA vaccine of a candidate material was administered, an immune response was induced with respect to the overlapping peptides specific to the candidate material. FIG. 2 shows graphs illustrating results of immunogenicity for experimental groups according to one exemplary embodiment of the present invention.

[0046] FIGS. 3 and 4 are graphs illustrating results of comparing immune responses of overlapping peptides of proteins in respective experimental groups. Referring to FIG. 3, the immune responses of the overlapping peptides of the corresponding proteins were compared in the groups in which a single DNA vaccine was administered and the group in which a mixture of three DNA vaccines was administered. As a result, it was revealed that, when the mixture of three DNA vaccines was administered, the immune response to IE-62 decreased by 43.3%, there was no difference in the immune response to IE-63, and the immune response to gE decreased by 9.6%, compared to when the single DNA vaccine was administered.

[0047] Validity of Selection of Antigen

[0048] Referring to FIG. 4, the splenocytes were stimulated with the lysate antigen in each of the experimental groups. As a result, it was confirmed that no immune response was induced in the Neg-treated group, but a strong immune response was induced in the group in which the DNA vaccine of the candidate material was administered. From these result, it can be seen that the proteins such as IE-62, IE-63, gE and the like were expressed in the cells infected with VZV, and an immune response occurred in the cells infected with VZV when the immune response to IE-62, IE-63 and gE selected as the candidate materials was induced in this experiment.

[0049] Experimental Evaluation

[0050] In a laboratory animal model, the DNA vaccines prepared using the genes encoding the IE-62, IE-63 and gE proteins were evaluated and analyzed. As a result, it was revealed that the T cells exhibited sufficient immunogenicity when the DNA vaccines administered alone or in combination thereof. The immunogenicity of the T cells was analyzed through the stimulation of the VZV lysate antigen. As a result, it was judged that it was reasonable to select the IE-62, IE-63 and gE as the candidate materials for the vaccines.

[Experiment 2] Evaluation of In Vitro Protein Expression

[0051] Criteria for Expression Confirmation [0052] Transfection: RD cells (110.sup.6 cells/T75 flask), 30 g of pDNA, Lipofectamine 2000, cell harvesting after 24 hours of transfection [0053] RD cells: human rhabdomyosarcoma [0054] Cell lysis: 210.sup.6 cells/90 l of RIPA buffer with Protease Inhibitor Cocktail [0055] SDSPAGE: LDS sample buffer with Reducing agent (DTT), 30 l loading (19.5 l of lysate), 4 to 12% Bis-Tris gel, MES running buffer at 165 V for 35 minutes [0056] Protein transfer: NC membrane, Dry blotting at 20 V for 1 minute.fwdarw.at 23 V for 4 minutes.fwdarw.at 25 V for 2 minutes [0057] Protein detection: 1) Blocking for 1 hour, 2) Antibody reaction (1:100) for an hour and a half (HA-probe HRP (SantaCruz, Cat. No.: sc-805HRP), Goat anti-VZV IE62 (SantaCruz, Cat. No.: sc-17525), Donkeyanti-goat Ig GHRP (SantaCruz, Cat. No.: sc-2020), 3), and HRP chromogenic substrate (Invitrogen, Cat. No.: WP20004))

[0058] Evaluation of Expression Confirmation

[0059] FIGS. 5 to 7 are electrophoretic image of products expressed by the respective plasmid genes.

[0060] Referring to FIG. 5, the plasmid encoding an IE-62 gene was introduced into RD cells derived from human rhabdomyosarcoma using Lipofectamine 2000. After 24 hours, the cells were lysed to extract proteins, and the proteins were then subjected to Western Blotting using an antibody for detecting an expressed antigen. An expressed protein of the plasmid encoding the IE-62 gene was detected using anti-VZV IE62 (SantaCruz, Cat. No.: SC-2020), and visualized using a HRP chromogenic substrate (Invitrogen, Cat. No.: WP20004). As a result, it was revealed that the product size of the insert gene expressed from the plasmid encoding the IE-62 gene was identical to the predicted molecular weight of the insert gene, indicating that the product of the gene introduced into the RD cells was the IE-62 antigen protein of VZV.

[0061] Referring to FIG. 6, the plasmid encoding an IE-63 gene was introduced into RD cells derived from human rhabdomyosarcoma using Lipofectamine 2000. After 24 hours, the cells were lysed to extract proteins, and the proteins were then subjected to Western Blotting using an antibody for detecting an expressed antigen. An expressed protein of the plasmid encoding the IE-63 gene was detected using HA-probe HRP (SantaCruz, Cat. No.: SC-805HRP), and visualized using a HRP chromogenic substrate (Invitrogen, Cat. No.: WP20004). As a result, it was revealed that the product size of the insert gene expressed from the plasmid encoding the IE-63 gene was identical to the predicted molecular weight of the insert gene, indicating that the product of the gene introduced into the RD cells was the IE-63 antigen protein of VZV.

[0062] Referring to FIG. 7, the plasmid encoding a gE gene was introduced into RD cells derived from human rhabdomyosarcoma using Lipofectamine 2000. After 24 hours, the cells were lysed to extract proteins, and the proteins were then subjected to Western Blotting using an antibody for detecting an expressed antigen. An expressed protein of the plasmid encoding the gE gene was detected using HA-probe HRP (SantaCruz, Cat. No.: SC-805HRP), and visualized using a HRP chromogenic substrate (Invitrogen, Cat. No.: WP20004). As a result, it was revealed that the product size of the insert gene expressed from the plasmid encoding the gE gene was identical to the predicted molecular weight of the insert gene, indicating that the product of the gene introduced into the RD cells was the gE antigen protein of VZV.