WO2018009047A2 - A dna vaccine for preventing and treating hsv-2 infection - Google Patents

Abstract

The present disclosure relates to a novel DNA vaccine for preventing and treating herpes simplex virus-2 (HSV-2) infection, and provides a polynucleotide encoding a shuffled UL39 protein; a vector containing the polynucleotide; and a DNA vaccine composition containing the vector.

Description

A DNA VACCINE FOR PREVENTING AND TREATING HSV-2 INFECTION

This application claims priority to Korean Patent Application No. 10-2016-0125149 filed on September 28, 2016 and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which are incorporated by reference in their entirety.

The present disclosure relates to a novel DNA vaccine, and more particularly, to a DNA vaccine for preventing and treating herpes simplex virus-2 (HSV-2) infection.

Herpes simplex virus-2 (HSV-2) is a member of Herpetoviridae, a DNA virus that causes skin lesions, and 500 million people worldwide are infected with HSV-2 and it is estimated that 23 million people newly infected with HSV-2 will appear every year. In the United States, one in five adults is reported to be infected with HSV-2 and infection rates are also relatively high in major countries including BRIC countries (Brazil, Russia, India, and China). HSV-2 is infected through mucous membranes or damaged areas of the skin, causing symptoms such as virus necrosis in the infected areas during viral replication. As a specific disease, HSV-2 is a major cause of genital ulcers, which are small liquid-filled aggregates of blisters that cause sore scars when ruptured. HSV-2 also causes symptoms such as fever, overall sickness, muscle pain, urinary pain, vaginal discharge, etc. HSV-2 invades the dorsal root ganglia along the axons of the sensory neurons and undergoes latency for several years or a lifetime to repeat reactivation. Since HSV-2 is capable of human-to-human transmission even when it is in an asymptomatic state, it is very difficult to control the spread of the virus. In fact, the spread of serologically HSV-2-positive individuals increased by 30% from 1976 to 1994, and unless it prevents the spread of further infections, infection rates will increase to 39% for males and 49% for females by 2025. Furthermore, the fact that 60% of newborns delivered by women infected with HSV-2 die and 20% of them have a serious aftereffect in the nervous system or eyes, and HSV-2 infected people are three times more likely to become infected with HIV, has become a serious problem for global public health. In addition, HSV-2 infection is becoming an enormous economic burden. For example, the annual direct medical costs for the United States in 1996 for the treatment of HSV-2 infections were estimated to be US$ 283 million to US$ 984 million.

Unfortunately, to date there are no treatments that can fundamentally treat HSV-2 infection, and treatment options for symptoms are limited. Antiviral agents that inhibit viral replication (e.g., famciclovir, valaciclovir, or acyclovir) by interfering with viral DNA synthesis are used. When these agents are applied at the initial stage of viral infections, clinical symptoms can be alleviated or viral metastasis can be reduced, however, recurrences and transmission of viral infections cannot be prevented and are difficult to cure. For example, acyclovir may reduce HSV-2 transmission, but it cannot prevent latent infection of HSV-2 in the ganglion. Moreover, antiviral therapy causes many side effects including nausea, vomiting, and reduction of renal functions. Due to unmet medical needs for existing antiviral drugs, new and various vaccines for prevention and treatment of HSV-2 have been tried, but all of them were not effective. Conventional vaccines that contain inactivated viruses, reduced live viruses, modified live viruses, and cell culture-derived subunits were usually unsuccessful or very ineffective. The results of clinical trials of the three most recent candidate vaccines (i.e., the vaccine developed by Chiron, the vaccine developed by GlaxoSmithKline (GSK), and the vaccine developed by Vical) were shown to be not effective. The Chiron vaccine, which consists of a truncated form of gD and gB2 of HSV-2 conjugated with the adjuvant MF59, was shown to produce a high-titer antibody to HSV-2, but the effect of Chiron vaccine was temporary. The vaccine of GSK, containing alum and MPL as adjuvants and gD, also failed to prevent and treat HSV-2 infection. Finally, vaccines from Vical, containing the adjuvants Vaxfectin as an adjuvant and gD or UL46 and UL47, produced high titer antibody in animal experiments, however, in clinical trials, HSV-2 infection did not show any difference from that of the control group. From the previous case, it can be confirmed that high titer antibody response to HSV-2 is not sufficient for prevention and treatment of HSV-2 infection, and as a result, it is necessary to develop a therapeutic agent based on a new immune mechanism against HSV-2 infection.

In order to solve the various issues described above, the present disclosure provides a novel DNA vaccine capable of effectively preventing and treating HSV-2 infection. However, the present disclosure is not limited thereto.

In accordance with another exemplary embodiment, the present disclosure provides a polynucleotide encoding a shuffled UL39 protein comprising following five peptides: UL39-N1 peptide, corresponding to the amino acids at positions 14 to 154 of an amino acid sequence of UL39 protein of HSV-2, in which a transmembrane domain corresponding to the amino acids at positions 78 to 104 is deleted; UL39-C2 peptide, corresponding to the amino acids at positions 1117 to 1142 of the amino acid sequence of the UL39 protein; UL39-N2 peptide, corresponding to the amino acids at positions 155 to 227 of the amino acid sequence of the UL39 protein; UL39 N4-C1 peptide, corresponding to the amino acids at positions 399 to 1116 of the amino acid sequence of the UL39 protein; and UL39-N3 peptide, corresponding to the amino acids at positions 208 to 398 of the amino acid sequence of the UL39 protein, wherein the five peptides are randomly intermixed, but the shuffled UL39 protein does not comprise the original amino acid sequence of the UL39 protein.

In accordance with still another exemplary embodiment, the present disclosure provides a vector containing the polynucleotide.

In accordance with still another exemplary embodiment, the present disclosure provides an isolated host cell containing the vector.

In accordance with still another exemplary embodiment, the present disclosure provides a composition containing the polynucleotide or the vector or a pharmaceutically acceptable carrier.

In accordance with still another exemplary embodiment, the present disclosure provides a recombinant protein encoded by the polynucleotide.

In accordance with still another exemplary embodiment, the present disclosure provides an expression vector comprising one or more polynucleotides encoding two or more or all HSV-2 antigen proteins selected from the group consisting of gB, gD, UL39, ICP0, and ICP4 protein.

In accordance with still another exemplary embodiment, the present disclosure provides a DNA vaccine composition comprising the polynucleotide, the vector, or the expression vector.

The DNA construction according to an embodiment of the present disclosure beibg capable of expressing a shuffled UL39 and a fusion protein comprising the shuffled UL39 and the conventional HSV-2 antigens, exhibited an effective ability to protect against inflammation, and accordingly, it can be very efficiently used as a vaccine for preventing and treating HSV-2.

FIG. 1 represents a schematic diagram illustrating the structure of a shuffled UL39 protein in accordance with an embodiment of the present disclosure.

FIG. 2 represents a schematic diagram illustrating an outline of the procedure of an animal experiment using a plasmid DNA designed to express a shuffled UL39 protein in accordance with an embodiment of the present disclosure.

FIG. 3 represents a graph illustrating survival rates of animals infected with HSV-2 by administering a plasmid DNA designed to express a shuffled UL39 protein in accordance with an embodiment of the present disclosure and a mock plasmid DNA as a negative control.

FIG. 4 represents schematic diagrams illustrating each of the structures of fusion proteins expressed by tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA in accordance with an embodiment of the present disclosure.

FIG. 5 reprsents a schematic diagram illustrating an experimental schedule of HSV-2 infection in groups to which tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA were administered, respectively; a group to which tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA were co-administered; and a group to which mock plasmid DNA was administered as a negative control in accordance with an embodiment of the present disclosure.

FIG. 6a is a graph representing survival rate of experimental animal groups infected with HSV-2 and administrated with tPA-Flt3L-gB-UL39 plasmid DNA (-), and the tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA, respectively; a group co-administrated with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA; a group administrated with mock plasmid DNA as a negative control in accordance with an embodiment of the present disclosure and FIG. 6b is a graph representing pathology scores of the experimental animal groups of FIG. 6a.

In accordance with one aspect of the present disclosure, provided is a polynucleotide encoding a shuffled UL39 protein, wherein the shuffled UL39 protein comprises following five peptides: a UL39-N1 peptide which corresponds to the amino acids at positions 14 to 154 of an amino acid sequence of UL39 protein of HSV-2 and a transmembrane domain therein corresponding to the amino acids at positions 78 to 104 is deleted; a UL39-C2 peptide which corresponds to the amino acids at positions 1117 to 1142 of the amino acid sequence of the UL39 protein; a UL39-N2 peptide which corresponds to the amino acids at positions 155 to 227 of the amino acid sequence of the UL39 protein; a UL39 N4-C1 peptide which corresponds to the amino acids at positions 399 to 1116 of the amino acid sequence of the UL39 protein; and a UL39-N3 peptide which corresponds to the amino acids at positions 208 to 398 of the amino acid sequence of the UL39 protein, wherein the five peptides intermixed randomly but the shuffled UL39 protein does not comprise the original amino acid sequence of the UL39 protein.

As used herein, the term “shuffled” refers to mix the order of domains when there are multiple domains within a particular protein. In particular, as used herein, the term “shuffled protein”, also called “epitope shuffled protein”, refers to a recombinant protein in which the order of multiple epitopes is randomly intermixed while maintaining the activity of the multiple epitopes recognized by the immune system, and it is an immunogenic protein in which the original function as a protein is lost while retaining the activity of the epitopes.

The UL39 protein used for the production of the shuffled UL39 protein may be RIR1_HHV2H registered as UniProt registration number P89462 as a standard sequence, and a variety of variants (e.g., UniProt registration numbers G91261, A0A0E3Y5Z7, A0A0E3Y7N5, A0A120I2I0, A0A110B8A6, and A0A0E3Y758) possessing the function of UL39 protein may be used without any problem, and when the length of any of the variants is different from that of the standard sequence, a position corresponding to the position of the standard sequence will be applied.

The polynucleotide may further include a polynucleotide encoding one or two or more immunity-enhancing peptides, and the immunity-enhancing peptide may be a cytoplasmic domain of CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), natural-killer group 2, member D (NKG2D)/ DNAX-activating protein 12 (DAP12), T-Cell immunoglobulin and mucin domain containing protein 1 (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), fms-like tyrosine kinase 3 (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 L (ligand), or OX40L [ligand for CD134(OX40), CD252], or a linker of two or more thereof.

As used herein, the term “immunity-enhancing peptide” refers to a peptide which activates cells associated with an immune response (e.g., dendritic cells, etc.) and thereby increases the immune response.

The polynucleotide may further include a polynucleotide encoding a secretion signal peptide, and the secretion signal peptide induces the extracellular secretion of the shuffled UL39 protein and may be a signal sequence for tissue plasminogen activator (tPA), a signal sequence for herpes simplex virus glycoprotein D (HSV gD), or a signal sequence for growth hormone.

The polynucleotide may further include a polynucleotide which encodes one or two or more antigenic proteins of type 2 herpes simplex virus (HSV-2). The antigen protein may be glycoprotein B (gB), glycoprotein D (gD), ICPO, or ICP4.

In the above polynucleotide, the glycoprotein B may be a truncated form in which a signal sequence corresponding to the amino acids at positions 1 to 22 and a transmembrane domain corresponding to the amino acids at positions 772 to 792 are deleted; the glycoprotein D may be a truncated form in which a signal sequence corresponding to the amino acids at positions 1 to 25 and a transmembrane domain corresponding to the amino acids at positions 341 to 364 are deleted; the ICP0 may be a truncated form in which a nuclear localization signal (NLS) corresponding to the amino acids at positions 510 to 516 is deleted; and the ICP4 may be a truncated form in which the RS1.3 region corresponding to the amino acids at positions 767 to 1318 is deleted.

With respect to the antigen proteins as well, when the standard sequence was gB, GB_HHV2H registered as UniProt registration number P08666 was used; when the standard sequence was gD, GD_HHV2H registered as UniProt registration number Q69467 was used; when the standard sequence was ICP0, ICP0_HHV2H registered as UniProt registration number P28284 was used; and when the standard sequence was ICP4, ICP4_HHV2H registered as UniProt registration number P90493 was used. The amino acid positions of the antigen proteins are those which are indicated based on the standard sequence and a variety of variants retaining the activity of the antigenic proteins can also be used, and when the length of these variants differs from that of the standard sequence, the amino acid position corresponding to the amino acid position of the standard sequence will be applied. Examples of the variety of variants may include: in the case of gB, variants which have UniProt registration numbers of P06763, Q69465, D6QV12, D6QV07, etc., may be used; in the case of gD, variants which have UniProt registration numbers of P03172, T1PZZ0, A0A0Y0QWV3, A0A110AVP3, A0A0Y0RM80, etc., may be used; in the case of ICP0, variants which have UniProt registration numbers of G9I221, G9I280, W0NW81, D6PUZ6, D6PUZ3, etc., may be used; in the case of ICP4, variants which have UniProt registration numbers of G9I282, A0A0E3Y5F0, A0A0Y0RBE8, A0A120I2I2, A0A0Y0RAK6, etc., may be used.

In addition, the polynucleotide may be substituted with a codon having a high expression frequency in a host cell. As used herein, the terms “those replaced with codons of high expression frequency in a host cell” or “optimized codon” mean that, when DNA is transcribed and translated into a protein in a host cell, there are codons having high preference according to the host, among the codons that direct amino acids, and that the expression efficiency of the amino acid or protein encoded by the nucleic acid is increased by substituting with codons having high preference.

In the polynucleotide, the UL39-N1 peptide may include SEQ ID NO: 1; the UL39-C2 peptide may include SEQ ID NO: 2; the UL39-N2 peptide may include SEQ ID NO: 3; the UL39 N4-C1 peptide may include SEQ ID NO: 4; and the UL39-N3 peptide may include SEQ ID NO: 5. Additionally, the shuffled UL39 may sequentially include the UL39-N1 peptide of SEQ ID NO: 1, UL39-C2 peptide of SEQ ID NO: 2, UL39-N2 peptide of SEQ ID NO: 3, UL39 N4-C1 peptide of SEQ ID NO: 4, and UL39-N3 peptide of SEQ ID NO: 5, and in this case, the shuffled UL39 protein may include an amino acid sequence of SEQ ID NO: 6. However, the change in the sequence of each of the peptides may not cause any problem as long as it is not identical to the amino acid sequence of the original full length UL39 protein.

In accordance with another aspect of the present disclosure, there is provided a vector including the polynucleotide.

The vector in accordance with an embodiment of the present disclosure may be an expression vector which includes a gene construct operably linked to the regulatory sequence and thereby allows the polynucleotide to express the shuffled UL39 protein in a host cell. The expression vector may be in any form, without limitation, including plasmid vector, viral vector, cosmid vector, phagemid vector, artificial human chromosome, etc.

As used herein, the term “operably linked to” means that the nucleic acid sequence of interest (e.g., in an in vitro transcription/translation system or in a host cell) is linked to the regulatory sequence in such a way such that the nucleic acid can be expressed.

As used herein, the term “regulatory sequence” is meant to include promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those which direct the constitutive expression of a target nucleic acid in many host cells, those which instruct the expression of a target nucleic acid only in a specific tissue cell (e.g., tissue-specific regulatory sequences), and those which direct the expression to be induced by a particular signal (e.g., inducible regulatory sequences). Those skilled in the art will be able to understand that the design of the expression vector may vary depending on factors such as the selection of a host cell to be transformed and the desired level of protein expression, etc. The expression vector of the present disclosure may be introduced into a host cell to express the fusion protein. Regulatory sequences which enable expressions in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these regulatory sequences usually include regulatory sequences responsible for initiating transcription and, selectively, poly-A signals responsible for terminating transcription and stabilizing transcripts. Additional regulatory sequences may include translation-enhancing factors and/or native-combining or heterologous promoter regions in addition to transcriptional regulatory elements. For example, possible regulatory sequences enabling expression in mammalian host cells may include CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (low sarcoma virus), the human renal element 1 α-promoter, glucocorticoid-inducible MMTV-promoter (Moloney murine tumor virus), a metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV or SV40-amplifying agent. For the expression within neurons, it is considered that neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter, or thy-1-promoter may be used. Such promoters are known in the art and described in the literature (Charron, J. Biol . Chem. 270: 25739-25745, 1995). For prokaryotic expression, a number of promoters have been disclosed including lac promoter, tac promoter, and trp promoter. In addition to factors capable of initiating transcription, the regulatory sequences include a transcription termination signal such as the SV40-poly-A or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present disclosure. In the present disclosure, suitable expression vectors are well-known in the art, they include, for examples, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX27 (Korean Patent No. 1442254), pX (Pagano, Science 255, 1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris, Cell 75, 791-803, 1995) or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the present disclosure, the vector may additionally contain a polynucleotide encoding a secretion signal peptide. Such secretion signal peptides are well-known to those skilled in the art. Additionally, depending on the expression system used, a leader sequence that can direct the fusion protein to a certain intercellular organelle is linked to the coding sequence of the polynucleotide according to an embodiment of the present disclosure, and preferably a leader sequence capable of directly secreting a translated protein therefrom into the cytoplasmic periphery or extracellular matrix.

Additionally, the vectors of the present disclosure may be prepared by standard recombinant DNA techniques, and standard recombinant DNA techniques may include, for example, blunt end and adhesive end ligations, restriction enzyme treatment to provide appropriate ends, phosphorylation by alkaline phosphatase treatment to prevent inadequate binding, enzymatic linkage by T4 DNA ligase, etc. The vector of the present disclosure may be prepared by recombining the DNA encoding the signal peptide obtained by chemical synthesis or genetic recombinant technology and the DNA encoding the HSV-2 antigen protein of the present disclosure with a vector containing an appropriate regulatory sequence. The vector containing the regulatory sequence may be purchased commercially or manufactured, and in an embodiment of the present disclosure, pGX27, which is a vector for producing DNA vaccine was prepared and used.

As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains responsible for a specific function within a protein are linked so that each protein or domain is responsible for its intrinsic function. A linker having a flexible structure may conventionally be inserted between the two or more proteins or domains. Various linkers such as GS4 are known as such linkers.

According to another aspect of the present disclosure, an isolated host cell including the vector is provided.

As used herein, the term “host cell” includes prokaryotic cells or eukaryotic cells, and eukaryotic cells include higher eukaryotic cells including mammals as well as lower eukaryotic cells including fungi, yeasts, etc.

A host cell or non-human host subject transfected or transformed with a vector according to the present disclosure may be a host cell or a host subject that is genetically modified by the vector. As used herein, the term “genetically modified” means the polynucleotide or the vector according to an embodiment of the present disclosure introduced into one among a host cell, a host subject or predecessors/parents is present outside the genome of the host cell, the host subject, or the predecessors/parents. Furthermore, the polynucleotide or the vector according to an embodiment of the present disclosure may exist as an independent molecule outside the genome, preferably a replicable molecule such as an episome in the genetically-modified host cell or host subject. Alternately, it may be stably inserted into the genome of the host cell or host subject.

The host cell according to an embodiment of the present disclosure is a prokaryotic cell or eukaryotic cell. Suitable prokaryotic cells are those cells commonly used for cloning, such as E. coli or Bacillus subtilis. Additionally, eukaryotic cells include fungi, plant cells, and animal cells. Examples of suitable fungal cells are yeasts, preferably yeasts of the genus Saccharomyces, and most preferably S. cerevisiae. Examples of suitable animal cells may include insect cells, and preferably mammalian cells (e.g., HEK293, 293T, NSO, CHO, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, and C33A). The host cells, for example, CHO cells, can provide a post-translational modification of the shuffled UL39 protein according to an embodiment of the present disclosure, glycosylation of the shuffled UL39 protein at the accurate position, and secretion of functional molecules. Additionally, suitable cell lines known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC). According to the present disclosure, primary culture cells/cell cultures are considered to be able to function as host cells. These cells are particularly derived from insects (insects of the genus Drosophila or Blatta) or mammals (humans, pigs, mice, or rats). As described above, the primary cultured cells may be immune cells which include macrophages, monocytes, granulocytes, hematopoietic stem cells, lymphokine activated killer cells, gd cells, natural killer T cells (NKT cells), T cells, or natural killer cells (NK cells).

According to another aspect of the present disclosure, a composition containing the polynucleotide or the vector and a pharmaceutically acceptable carrier is provided.

In addition to the carrier, the composition may further contain a pharmaceutically acceptable adjuvant, excipient, or diluent.

As used herein, the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, etc., when administered to humans. Examples of the carrier, excipient, and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, hydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, fillers, anti-coagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc., may be additionally contained.

According to another aspect of the present disclosure, a recombinant protein encoded by the polynucleotide is provided.

According to another aspect of the present disclosure, an expression vector comprising one or more polynucleotides encoding two or more or all HSV-2 antigen proteins selected from the group consisting of gB, gD, UL39, ICPO, and ICP4 proteins, is provided.

With regard to the expression vector, the gB may be a polynucleotide in which a signal sequence corresponding to the amino acids at positions 1 to 22 and a transmembrane domain corresponding to the amino acids at positions 772 to 792 are deleted; the gD may be a polynucleotide in which a signal sequence corresponding to the amino acids at positions 1 to 25 and a transmembrane domain corresponding to the amino acids at positions 341 to 364 are deleted; the ICP0 may be a polynucleotide in which a nuclear localization signal (NLS) corresponding to the amino acids at positions 510 to 516 is deleted; and the ICP4 may be a polynucleotide in which the RS1.3 region corresponding to the amino acids at positions 767 to 1318 is deleted.

In the above expression vector, the UL39 may be a shuffled UL39 protein intermixed with internal domains, and the shuffled UL39 protein may be encoded by any of the polynucleotides described above.

Additionally, the expression vector may be prepared such that the gB, gD, UL39, ICP0, and ICP4 proteins are expressed as separate proteins or expressed in a single fusion protein form.

In the above expression vector, as described above, the shuffled UL39 protein is one in which UL39-N1 peptide of SEQ ID NO: 1, UL39-C2 peptide of SEQ ID NO: 2, UL39-N2 peptide of SEQ ID NO: 3, UL39 N4-C1 peptide of SEQ ID NO: 4, and UL39-N3 peptide of SEQ ID NO: 5 are randomly intermixed, but the shuffled UL39 protein may be one in which the peptides of SEQ ID NOS: 1 to 5 are sequentially connected. However, in this case, the shuffled UL39 protein excludes the original full length UL39 protein.

In the above expression vector, the gB, gD, UL39, ICP0, and ICP4 proteins may be expressed as separate proteins or expressed in a single fusion protein form.

In the above expression vector, the polynucleotide may further comprise a polynucleotide encoding a secretion signal peptide, and as described above, the secretion signal peptide may be a signal peptide for tissue plasminogen activator (tPA), a signal peptide for herpes simplex virus glycoprotein D (HSV gD), or a signal peptide for growth hormone.

In the above expression vector, the immunity-enhancing peptide is a cytoplasmic domain of CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), natural-killer group 2, member D (NKG2D)/ DNAX-activating protein 12 (DAP12), T-Cell immunoglobulin and mucin domain containing protein 1 (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), fms-like tyrosine kinase 3 (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 L (ligand), or OX40L [ligand for CD134(OX40), CD252], or a linker of two or more thereof. The immunity-enhancing peptide may preferably be an Flt3 ligand and the Flt3 ligand may have an amino acid sequence of SEQ ID NO: 9.

According to another aspect of the present disclosure, a DNA vaccine composition comprising the polynucleotide, the vector, or the expression vector described above is provided.

Specifically, the DNA vaccine composition may include a first expression vector comprising a first gene construct, in which a first polynucleotide encoding a first fusion protein including gB and UL39 is operably linked to a promoter; and a second expression vector comprising a second gene construct, in which a second polynucleotide encoding a second fusion protein comprising gD, ICP0, and ICP4 is operably linked to a promoter.

In the DNA vaccine composition, the first fusion protein and/or the second fusion protein may further include a secretion signal peptide, and the secretion signal peptide is the same as described above.

In the DNA vaccine composition, the first fusion protein and/or the second fusion protein may further include an immunity-enhancing peptide, and the immunity-enhancing peptide is the same as described above.

In the DNA vaccine composition, the UL39 may be a shuffled UL39 in which internal domains are intermixed.

The DNA vaccine composition may include at least one pharmaceutically acceptable adjuvant.

As used herein, the term “adjuvant” refers to a pharmaceutical or immunological agent that is administered for the purpose of enhancing the immune response of a vaccine.

The adjuvant may be aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80, which is an emulsifier), CpG, Flagellin, Poly I: C, AS01, AS02, ISCOMs, or ISCOMMATRIX.

Additionally, the vaccine composition according to an embodiment of the present disclosure may be formulated using a method known in the art to allow rapid release, or sustained or delayed release of an active ingredient upon its administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders.

The vaccine composition according to an embodiment of the present disclosure may be administered by a variety of routes including, for example, oral, parenteral (e.g., suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, intradermal, and intraspinal routes, and additionally, may be administered using an implantable device for continuous or repeated release. The number of administrations may be once or several times a day within a desired range but the administration period is not particularly limited thereto.

The vaccine composition according to an embodiment of the present disclosure may be administered by conventional systemic or topical administration (e.g., intramuscular injection or intravenous injection), but most preferably by means of an electroporator. The electroporators to be used may include an electric perforator for injecting commercially-available DNA drugs (e.g., Glinporator^TM of IGEA of Italy, CUY21EDIT of JCBIO of Korea, or SP-4a of Supertech of Switzerland, etc.)

With regard to the administration routes, the vaccine composition according to an embodiment of the present disclosure may be administered via any conventional route as long as it can reach the target tissue. Such administration route may be, parenteral administration (e.g., intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and intrasynovial administration), but not limited thereto.

The vaccine composition according to an embodiment of the present disclosure may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration (e.g., aqueous glucose, glycols, etc.), etc., and may additionally contain a stabilizer and a preservative. Examples of suitable stabilizers may include antioxidants such as sodium hydrogen sulfite, sodium sulfite, and ascorbic acid. Examples of suitable preservatives may include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Additionally, when necessary according to the administration method or formulations, the composition according to the present disclosure may appropriately include a suspension, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffering agent, an antioxidant, etc. Pharmaceutically acceptable carriers and formulations suitable for the present disclosure, including those exemplified above, are described in the literature [Remington's Pharmaceutical Sciences, recent edition].

The dosage for a patient of the vaccine composition differs depending on many factors, including the patient’s height, body surface area, age, a particular compound to be administered, sex, time and route of administration, general health conditions, and other drugs to be administered simultaneously. Pharmaceutically active DNA may be administered in an amount of 100 ng/body weight (kg) to 10 mg/body weight (kg), more preferably 1 ㎍/kg to 500 ㎍/kg (body weight), and most preferably 5 ㎍/kg to 50 ㎍/kg (body weight), and may be administered in a unit dose of 10 ㎍, 100 ㎍, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg, and dosages may be adjusted considering the above factors.

Hereinafter, the present disclosure is explained in more detail below through Examples and Experimental Examples. However, the present disclosure is not limited to these Examples and Experimental Examples described below, but may be implemented in various other forms, and the following Examples and Experimental Examples are provided to fully illustrate the scope of the present disclosure to those of ordinary skill in the art to which the present disclosure pertains.

Example 1: Preparation of shuffled-UL39 plasmid DNA

The inventors of the present disclosure, based on their previous study that UL39 (ICP10), one of the HSV-2 antigens, induces CD4⁺ T cells and CD8⁺ T cellular responses (Posavad et al., Mucosal Immunol. 126, 2015), have investigated whether the shuffled construct in which the internal domains of UL39 are intermixed could function as a vaccine to induce an immune response.

To this end, the inventors designed a shuffled UL39 antigen that is an intermixed protein of 5 split pieces (N1: UL39_{14-154( Δ78 -104)}, C2: UL39_1117-1142, N2: UL39_155-227, N4-C1: UL39_399-1116, N3: UL39_208-398) of UL39 antigen based on the pGX27 plasmid vector (Korean Patent No. 1442254) which is improved so as to enhance the expression ability, and a plasmid vector containing a gene construct encoding the shuffled UL39 antigen was prepared and the plasmid vector was designated as shuffled-UL39 plasmid DNA.

Specifically, the shuffled-UL39 plasmid DNA was prepared by inserting a gene construct, which includes a polynucleotide (SEQ ID NO: 7) encoding the sequentially-linked form in the order of UL39-N1, UL39-C2, UL39-N2, UL39-N4-C1, and UL39-N3 (SEQ ID NO: 6) based on the form divided into UL39-N1 (SEQ ID NO: 1), UL39-C2 (SEQ ID NO: 2), UL39-N2 (SEQ ID NO: 3), UL39-N4-C1 (SEQ ID NO: 4), and UL39-N3 (SEQ ID NO: 5), into the pGX27 plasmid vector (FIG. 1).

Experimental Example 1: Confirmation of defense efficacy against HSV -2 infection by shuffled-UL39 plasmid DNA

The inventors of the present disclosure, in order to confirm whether the shuffled-UL39 plasmid DNA according to an embodiment of the present disclosure is effective in defending against HSV-2 infection in an infected animal model in vivo, the ability to protect against HSV-2 infection was evaluated after the administration of the plasmid DNA vaccine.

Specifically, the C57BL/6 mice were divided into a group administered with mock plasmid DNA and a group administered with the shuffled-UL39 plasmid DNA, respectively. The corresponding plasmid DNA (4 ㎍) was administered intramuscularly by electroporation two times at 2-week intervals into each group, and 2 weeks after the final administration, the mice were infected with HSV-2 virus (1×10⁴ pfu) through an intravaginal route (FIG. 2 and Table 1). After infection, viabilities of HSV-2 infected groups were evaluated by monitoring the survival of each group of mice for 10 days (FIG. 3).

Group	Vaccine	No. of Experimental Animals	Dose (㎍)	Route (Method of Administration)
Control Group	Mock Plasmid		8	4	Intramuscular (Electroporation)
Experimental Group	Shuffled UL39	8	4	Intramuscular(Electroporation)

As a result, as can be confirmed in FIG. 3, in the group administered with mock plasmid DNA, most of the mice died on day 8 of the HSV-2 virus infection, whereas the group administered with the shuffled-UL39 plasmid DNA showed a survival rate of 40% until day 10 of the HSV-2 virus infection and significant improvement in the survival rate was observed.

Example 2: Preparation of plamid DNA expressing HSV -2 complex antigen

The inventors of the present disclosure have confirmed the possibility of UL39 as a DNA vaccine from the results of Experimental Example 1, and as a result, have prepared plasmid DNA which is recombined with other antigen proteins.

Specifically, based on the pGX27 plasmid vector used in Example 1, tPA-Flt3L-gB-UL39 plasmid DNA comprising a polynucleotide (SEQ ID NO: 12) encoding tPA-Flt3L-gB-UL39 fusion protein(SEQ ID NO: 11), in which various kinds of HSV-2 antigens, i.e., glycoprotein B (gB_{23 -904( Δ772 -792)}, SEQ ID NO: 10) whose signal sequence (gB_{1 -22}) and transmembrane domain (gB_{772 -792}) are removed, and shuffled UL39 (SEQ ID NO: 6) used in Example 1 are linked; and tPA-Flt3L-gD-IPC0-ICP4 plasmid DNA comprising a polynucleotide (SEQ ID NO: 17) encoding tPA-Flt3L-gD-IPC0-ICP4 fusion protein (SEQ ID NO: 16), in which glycoprotein D(gD_{6 -393( Δ341 -364)}, SEQ ID NO: 13) whose signal sequence (gD_{1 -25}) and transmembrane domain (gD_{341 -364}) are removed, infected cell polypeptide 0 (ICP0_{Δ 510-516}, SEQ ID NO: 14) whose nuclear localization signal (NLS, ICP0_{510 -516}) is removed, and infected cell polypeptide 4 (ICP4_{Δ767 -1318}, SEQ ID NO: 15) whose RS1.3 part (ICP4_767-1318) is removed are linked were prepared, respectively. Both plasmids, for the efficient expression, are in a from in which the codon-optimized tPA secretion signal peptide (SEQ ID NO: 8) and the immune system activating protein FMS-like tyrosine kinase 3 ligand (Flt3L, SEQ ID NO: 9) are added to the N-terminus (FIG. 4).

Experimental Example 2: Confirmation of defense efficacy against HSV -2 infection by tPA- Flt3L -gB-UL39 and tPA- Flt3L - gD - ICP0 - ICP4 plasmid DNA

In order to confirm whether the tPA-Flt3L-gB-UL39 plasmid DNA and the tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA prepared in Example 2 have an ability to protect against HSV-2 infection, the present inventors evaluated their abilities to protect against HSV-2 infection after the administration of the vaccines.

Specifically, the C57BL/6 mice were divided into groups administered with mock plasmid DNA, tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA, respectively, and a group co-administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA. Each group was intramuscularly administered twice with 4 ㎍ each of the corresponding plasmid DNA (for the co-administration, 4 ㎍ each was administered) at two-week intervals by in vivo electroporation, and two weeks thereafter, infected through an intravaginal route with HSV-2 virus (1×10⁴ pfu) (FIG. 5 and Table 2). The survival rate and pathology score of each group of mice were monitored daily for 20 days after the infection to evaluate the ability to protect against HSV-2 infection (FIG. 6a and 6b). The pathology score was scored according to the description in the literature published by (Oh et al., Proc . Natl . Acad . Sci . USA. 113(6): E762-E771, 2016; pathology score "0", no symptom; "1", a mild level of genital erythema and edema; "2", an intermediate level of genital inflammation; "3", purulent genital damage; "4", hind limb paralysis; "5", death).

Group	Vaccine	No. of Mice	Dose (㎍)	Administration Route (Administration Method)
Control Group	mock	10	8	Intramuscular (Electroporation)
Experimental Group 1	gB-UL39 (BD-02B)	10	4 + 4 (mock)	Intramuscular (Electroporation)
Experimental Group 2	gD-ICP0/ICP4	10	4 + 4 (mock)	Intramuscular (Electroporation)
Experimental Group 3	gD-ICP0/ICP4 +gB-UL39	10	4 + 4	Intramuscular (Electroporation)

As a result, as can be seen in FIG. 6a, all of the animals in the group administered with mock plasmid DNA died on day 10 of infection with HSV-2 virus. In contrast, in the groups administered with tPA-Flt3L-gB-UL39 plasmid DNA or tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA, a significantly improved ability to protect against HSV-2 virus was confirmed. The mice in the group administered with tPA-Flt3L-gB-UL39 plasmid DNA showed 40% survival rate and those in the group administered with ICP0-ICP4 plasmid DNA showed 100% survival rate. The mice co-administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA also showed 100% survival rate.

Furthermore, as can be seen in FIG. 6b, the group administered with mock plasmid DNA showed a high pathology score, and the group administered with tPA-Flt3L-gB-UL39 plasmid DNA showed a significantly lower pathology score than that of the group administered with mock plasmid DNA but a higher pathology score than that of the group administered with tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA. Meanwhile, the group co-administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA showed a significantly improved pathology score compared to each group administered with each plasmid DNA.

As described above, the DNA which can express the shuffled UL39 according to an embodiment of the present disclosure and the DNA which can express the fusion protein, in which the shuffled UL39 antigen and the conventional HSV-2 antigen were combined, exhibited an effective ability to protect against inflammation, and accordingly, they can be very efficiently used as a vaccine for preventing and treating HSV-2.

Although the DNA vaccine for preventing and treating HSV-2 infection has been described with reference to the specific embodiments, it is not limited thereto. Therefore, it will be readily understood by those skilled in the art that various modifications and changes can be made thereto without departing from the spirit and scope of the present disclosure defined by the appended claims.

The polynucleotide according to the present disclosure can be used for preparing a DNA vaccine composition for preventing and treating HSV-2 infection.

SEQ ID NO: 1 is an amino acid sequence of UL39-N1 peptide.

SEQ ID NO: 2 is an amino acid sequence of UL39-C2 peptide.

SEQ ID NO: 3 is an amino acid sequence of UL39-N2 peptide.

SEQ ID NO: 4 is an amino acid sequence of UL39-N4-C1 peptide.

SEQ ID NO: 5 is an amino acid sequence of UL39-N3 peptide.

SEQ ID NO: 6 is an amino acid sequence of a shuffled UL39 protein.

SEQ ID NO: 7 is a polynucleotide sequence encoding a shuffled UL39 protein.

SEQ ID NO: 8 is an amino acid sequence of tPA secretion signal peptide.

SEQ ID NO: 9 is an amino acid sequence of Flt3 ligand (Flt3L).

SEQ ID NO: 10 is an amino acid sequence of gB_{23 -904( Δ772 -792)} peptide.

SEQ ID NO: 11 is an amino acid sequence of tPA-Flt3L-gB-UL39 fusion protein.

SEQ ID NO: 12 is a polynucleotide sequence encoding tPA-Flt3L-gB-UL39 fusion protein.

SEQ ID NO: 13 is an amino acid sequence of gD_{6 -393( Δ341 -364)} protein.

SEQ ID NO: 14 is an amino acid sequence of ICP0_{Δ510 -516} protein.

SEQ ID NO: 15 is an amino acid sequence of ICP4_{Δ767 -1318} protein.

SEQ ID NO: 16 is an amino acid sequence of tPA-Flt3L-gD-IPC0-ICP4 fusion protein.

SEQ ID NO: 17 is a polynucleotide sequence encoding tPA-Flt3L-gD-IPC0-ICP4 fusion protein.

Claims (35)

1. A polynucleotide encoding a shuffled UL39 protein, wherein the shuffle UL39 protein comprises following five peptides:

   UL39-N1 peptide, corresponding to the amino acids at positions 14 to 154 of an amino acid sequence of UL39 protein of HSV-2, in which the transmembrane domain corresponding to the amino acids at positions 78 to 104 is deleted;

   UL39-C2 peptide, corresponding to the amino acids at positions 1117 to 1142 of the amino acid sequence of the UL39 protein;

   UL39-N2 peptide, corresponding to the amino acids at positions 155 to 227 of the amino acid sequence of the UL39 protein;

   UL39 N4-C1 peptide, corresponding to the amino acids at positions 399 to 1116 of the amino acid sequence of the UL39 protein; and

   UL39-N3 peptide, corresponding to the amino acids at positions 208 to 398 of the amino acid sequence of the UL39 protein,

   wherein the five peptides are randomly intermixed, but the shuffled UL39 protein does not have the original amino acid sequence of the UL39 protein.
2. The polynucleotide of claim 1, further comprising a polynucleotide encoding one or two or more immunity-enhancing peptides.
3. The polynucleotide of claim 2, wherein the immunity-enhancing peptide is a cytoplasmic domain of CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), natural-killer group 2, member D (NKG2D)/DNAX-activating protein 12 (DAP12), T-Cell immunoglobulin and mucin domain containing protein 1 (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), fms-like tyrosine kinase 3 (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 L (ligand), or OX40L [ligand for CD134(OX40), CD252], or a linker of two or more thereof.
4. The polynucleotide of claim 1, further comprising a polynucleotide encoding a secretion signal peptide.
5. The polynucleotide of claim 4, wherein the secretion signal peptide is a signal peptide for tissue plasminogen activator (tPA), a signal peptide for herpes simplex virus glycoprotein D (HSV gD), or a signal peptide for growth hormone.
6. The polynucleotide of claim 1, further comprising a polynucleotide encoding one or two or more antigen proteins of herpes simplex virus-2 (HSV-2).
7. The polynucleotide of claim 6, wherein the antigen protein is glycoprotein B (gB), glycoprotein D (gD), ICP0, or ICP4
8. The polynucleotide of claim 7, wherein the glycoprotein B is a truncated form in which a signal peptide corresponding to the amino acids at positions 1 to 22 and a transmembrane domain corresponding to the amino acids at positions 772 to 792 are deleted.
9. The polynucleotide of claim 7, wherein the glycoprotein D is a truncated form in which a signal peptide corresponding to the amino acids at positions 1 to 25 and a transmembrane domain corresponding to the amino acids at positions 341 to 364 are deleted.
10. The polynucleotide of claim 7, wherein the ICP0 is a truncated form in which a nuclear localization signal (NLS) corresponding to the amino acids at positions 510 to 516 is deleted.
11. The polynucleotide of claim 7, wherein the ICP4 is a truncated form in which the RS1.3 region corresponding to the amino acids at positions 767 to 1318 is deleted.
12. The polynucleotide of claim 1, wherein the shuffled UL39 protein is a polypeptide in which the UL39-N1 peptide, the UL39-C2 peptide, the UL39-N2 peptide, the UL39 N4-C1 peptide, and the UL39-N3 peptide are sequentially linked.
13. The polynucleotide according to any one of claims 1 to 12, wherein the polynucleotide is a codon-optimized, polynucleotide.
14. The polynucleotide according to any one of claims 1 to 12, comprising:

    the UL39-N1 peptide consists of SEQ ID NO: 1;

    the UL39-C2 peptide consists of SEQ ID NO: 2;

    the UL39-N2 peptide consists of SEQ ID NO: 3;

    the UL39 N4-C1 peptide consists of SEQ ID NO: 4; and

    the UL39-N3 peptide consists of SEQ ID NO: 5.
15. The polynucleotide according to any one of claims 1 to 12, wherein the shuffled UL39 protein is a polynucleotide consists of an amino acid sequence represented by SEQ ID NO: 6.
16. A vector comprising the polynucleotide according to any one of claims 1 to 12.
17. The vector of claim 16, wherein the vector is an expression vector in which the polynucleotide operably linked to a promoter.
18. An isolated host cell comprising the vector of claim 16 or 17.
19. A composition comprising the polynucleotide according to any one of claims 1 to 12 or the vector of claim 16 or 17 and a pharmaceutically acceptable carrier.
20. A recombinant protein encoded by the polynucleotide according to any one of claims 1 to 12.
21. An expression vector comprising one or more polynucleotides encoding two or more or all HSV-2 antigen proteins selected from the group consisting of gB, gD, UL39, ICP0, and ICP4 proteins.
22. The expression vector of claim 21, wherein the gB is a truncated form in which a signal sequence corresponding to the amino acids at positions 1 to 22 and a transmembrane domain corresponding to the amino acids at positions 772 to 792 are deleted.
23. The expression vector of claim 21, wherein the gD is a truncated form in which a signal sequence corresponding to the amino acids at positions 1 to 25 and a transmembrane domain corresponding to the amino acids at positions 341 to 364 are deleted.
24. The expression vector of claim 21, wherein the ICP0 is a truncated form in which a nuclear localization signal (NLS) corresponding to the amino acids at positions 510 to 516 is deleted.
25. The expression vector of claim 21, wherein the ICP4 is a truncated form in which the RS1.3 region corresponding to the amino acids at positions 767 to 1318 is deleted.
26. The expression vector of claim 21, wherein the UL39 is a shuffled UL39 in which internal domains are intermixed.
27. The expression vector of claim 26, wherein the shuffled UL39 protein comprises following five peptides:

    UL39-N1 peptide, corresponding to the amino acids at positions 14 to 154 of an amino acid sequence of UL39 protein of HSV-2, in which a transmembrane domain corresponding to the amino acids at positions 78 to 104 is deleted;

    UL39-C2 peptide, corresponding to the amino acids at positions 1117 to 1142 of the amino acid sequence of the UL39 protein;

    UL39-N2 peptide, corresponding to the amino acids at positions 155 to 227 of the amino acid sequence of the UL39 protein;

    UL39 N4-C1 peptide, corresponding to the amino acids at positions 399 to 1116 of the amino acid sequence of the UL39 protein; and

    UL39-N3 peptide, corresponding to the amino acids at positions 208 to 398 of the amino acid sequence of the UL39 protein,

    wherein the five peptides are randomly intermixed, but the UL39 shuffled protein does not have the original amino acid sequence of the UL39 protein.
28. The expression vector of claim 26, wherein the shuffled UL39 is encoded by the polynucleotide of claim 1.
29. The expression vector according to any one of claims 21 to 28, wherein the gB, gD, UL39, ICP0 and ICP4 proteins are expressed as separate proteins or expressed in the form of one fusion protein.
30. The expression vector according to any one of claims 21 to 28, wherein the polynucleotide further comprises a polynucleotide encoding a secretion signal peptide.
31. The expression vector of claim 30, wherein the secretion signal peptide is a signal sequence for tissue plasminogen activator (tPA), a signal sequence for herpes simplex virus glycoprotein D (HSV gD), or a signal sequence for growth hormone.
32. The expression vector according to any one of claims 21 to 28, wherein the polynucleotide further comprises a polynucleotide encoding an immunity-enhancing peptide.
33. The expression vector according to any one of claims 21 to 28, wherein the polynucleotide further comprises a polynucleotide encoding an immunity-enhancing peptide.
34. A DNA vaccine composition comprising the expression vector according to any one of claims 21 to 28.
35. The DNA vaccine composition of claim 34, comprising:

    a first expression vector comprising a first gene construct including a first polynucleotide encoding a first fusion protein comprising gB and UL39 is operably linked to a promoter; and

    a second expression vector comprising a second gene construct including a second polynucleotide encoding a second fusion protein comprising gD, ICP0, and ICP4 is operably linked to a promoter.

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