TWI731300B - A novel polyvalent hpv vaccine composition - Google Patents

Abstract

Provided are a novel polyvalent human papillomavirus (HPV) DNA vaccine, a fusion protein used therein, and a polynucleotide encoding the fusion protein, and more specifically, a polyvalent HPV DNA vaccine composition including polynucleotides, which encode early protein antigen 6 (E6) of types 6, 11, 16, 18, 39, 45, and 56 HPV or immunogenic fragments thereof; and early protein antigen 7 (E7) of types 6, 11, 16, 18, 39, 45, and 56 human papillomavirus (HPV) or immunogenic fragments thereof, respectively.

Description

A new type of multivalent HPV vaccine component

The present invention is related to a new type of multivalent human papilloma virus (hereinafter referred to as HPV) DNA vaccine, and is a fusion protein for multivalent HPV DNA vaccine and a polynucleotide encoding the fusion protein.

Cervical cancer is one of the most fatal cancer types for women in the world (Einstein et al., Lancet Infect. Dis., 9: 347-356, 2009; Parkin and Bray, Vaccine 24(3S): 11-25, 2007), 75% of cases are caused by persistent infection with the most common high-risk HPV types (that is, HPV16 and HPV18) (Schiffman et al., Lancet, 370: 890-907, 2007; Forman et al. , Vaccine 30(5S): F12-23, 2012). The persistence of HPV infection is usually related to the lack of clear HPV-specific T cell immunity, and virus-specific T cells found in premalignant and malignant patients are usually reported as general dysfunction, and sometimes even suppressive. (Trimble, Cancer Immunol. Immunother. CII 59: 799-803, 2010). These results all point out that the functional damage of virus-specific T cells may be related to the occurrence of HPV-induced cervical cancer.

Cervical cancer occurs through high-risk HPV infection procedures, virus persistence, strain spread, and alienation of persistently infected cells, until premalignant lesions, and gradually transforming into invasive cancer (Schiffman et al., Lancet 370:890 -907, 2007). Stages 2 and 3 of premalignant cervical epithelioma, Especially those who are positive for HPV16 are regarded as high-grade lesions, and there may be a 30% chance of developing into invasive cancer (Moscicki et al., Vaccine 30(5S): F24-33, 2012). Therefore, there is an urgent need for effective therapeutic vaccines that can prevent serious complications of persistent HPV infection and eradicate HPV-related tumors.

There are currently two commercial HPV vaccines in Korea, a quadrivalent vaccine (Gardasil ^® ) and a bivalent vaccine (Cervarix ^® ). The 4-valent vaccine includes L1 VLPs of HPV types 6, 11, 16, and 18, while the 2-valent vaccine includes L1 VLPs of HPV types 16 and 18. Although HPV types 6 and 11 are the main causes of condyloma acuminatum, they are low-risk HPVs that are not related to cervical cancer. Therefore, from the perspective of preventing cervical cancer, both vaccines correspond to the prevention of types 16 and 18 A bivalent vaccine for type HPV.

At the same time, HPV E6 and E7 act like viral oncoproteins, functioning by linking and promoting the degradation of tumor suppressor proteins (that is, p53 and retinoblastoma (pRb)) (Yugawa and Kiyono, Rev. Med. Virol., 19: 97-113, 2009). These viral oncoproteins are regarded as ideal targets for therapeutic vaccines against CIN2/3 and cervical cancer, not only because these proteins can induce tumorigenesis, but also can be expressed by HPV-infected premalignant and malignant cells (Yugawa and Kiyono , Rev. Med. Virol., 19: 97-113, 2009). Because the recovery of cervical lesions is related to the existence of cellular immune response, rather than humoral immune response (Deligeoroglou et al., Infect. Dis. Obstet. Gynecol., 2013: 540850, 2013; Woo et al., Int.J. Cancer, 126: 133-141, 2010), a therapeutic vaccine that can selectively induce strong E6/E7 specific T cell immunity is highly anticipated by everyone.

Examples of vaccines currently under development that use HPV E6/E7 antigens include the components of DNA vaccines using HPV 16/18 E6/E7 antigens disclosed in Korean Patent Application No. 2017-0045254. However, the two commercial vaccine components and DNA vaccine components that use HPV 16/18 E6/E7 target only type 16 and 18 Type HPV, these belong to the high-risk group, so these vaccine components can only cover about 70% of all cervical cancers.

Therefore, there is an urgent need to develop a multivalent vaccine that can trigger an immune response to various HPV types at all risks, including cervical cancer.

The present invention provides a multivalent HPV DNA vaccine, which can effectively prevent and treat cervical cancer and other diseases caused by HPV infection by inducing an immune response to combat more types of HPV. However, the scope of application of the present invention is not limited to this either.

In the embodiment of the present invention, a multivalent HPV DNA vaccine component including polynucleotides is disclosed, which can be respectively 6, 11, 16, 18, 39, 45, and 56 types of HPV or its immune system. It encodes the early protein antigen 6 (E6) of the original fragment; and encodes the early protein antigen 7 (E7) of HPV types 6, 11, 16, 18, 39, 45, and 56 or immunogenic fragments thereof.

In other embodiments of the present invention, an antigen-linked fusion protein is disclosed, in which at least four types of HPV antigen units are connected by a linking sequence, and the antigen unit is an N-terminal fragment including E6 and E7 and C The polypeptides of the terminal fragments are selected from HPV groups including 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 HPV, with E6 (E6N) Derived from the sequence of the N-terminal fragment-E7 (E7C) and the C-terminal fragment-E7 (E7N) and the C-terminal fragment of E6 (E6C).

In other embodiments of the present invention, disclosed is a polynucleotide encoding a fusion protein.

In other embodiments of the present invention, disclosed is an expression vector in which the polynucleotide is operably linked to an expression control sequence.

In other embodiments of the present invention, disclosed is a 14-valent HPV DNA vaccine composition The part includes an expression vector in which the polynucleotide encoding the fusion protein is operably linked to a promoter, and multiple expression vectors to contain polynucleotides encoding all 14 E6/E7 antigen units.

In other embodiments of the present invention, a method for treating diseases caused by HPV infection is disclosed, including administering a multivalent HPV DNA vaccine component or a 14-valent HPV DNA vaccine component to an individual.

The foregoing and other technical content, features, and effects of the present invention will be clearly presented in the following detailed description of the preferred embodiment with reference to the drawings.

According to the point of view of the present invention, here is a HPV DNA vaccine component, which includes the early protein antigen 6 (E6) which can encode 6, 11, 16, 18, 39, 45 and 56 HPV or its immunogenic fragments respectively. ), and polynucleotides encoding the early protein antigen 7 (E7) of HPV types 6, 11, 16, 18, 39, 45, and 56 or immunogenic fragments, and E6 and E7 will not have wild-type functions.

According to the point of view of the present invention, the multivalent HPV DNA vaccine components may also include at least one that can encode at least one selected from the group consisting of HPV types 31, 33, 35, 51, 52, 58, and 59 or immunogenic fragments thereof. Early protein antigen 6 (E6); and at least one early protein antigen 7 (E7) selected from the group comprising HPV types 31, 33, 35, 51, 52, 58 and 59 or immunogenic fragments thereof, and wherein The other E6 and E7 will not have wild-type functions.

In the multivalent HPV DNA vaccine components, E6 and E7 may be divided into N-terminal fragments and C-terminal fragments, and expressed in the form of random mixed sequence E6/E7 antigen units, while E6/E7 mixed sequence antigens The unit may be a polypeptide, which will connect E6 and E6 in the order of the N-terminal fragment of E6 (E6N)-the C-terminal fragment of E7 (E7C)-the N-terminal fragment of E7 (E7N)-the C-terminal fragment of E6 (E6C) N-terminal fragment and C-terminal fragment of E7.

Among the multivalent HPV DNA vaccine components, at least two, at least three, or at least four HPV E6/E7 hybrid sequence antigen units may be expressed in the form of fusion proteins.

In the multivalent HPV DNA vaccine component, the E6/E7 hybrid sequenced antigen unit or fusion protein may also include a signal sequence, and the E6/E7 hybrid sequenced antigen unit or fusion protein may also include Flt3L.

Multivalent HPV DNA vaccine components may also include IL-7. The present inventors confirmed that when the multivalent HPV DNA vaccine is administered according to the embodiment of the present invention, the combination of the HPV DNA vaccine and IL-7 administration will significantly increase the anti-cancer effect (see FIG. 10B).

The multivalent HPV DNA of the present invention may also include at least one vaccine adjuvant acceptable to the pharmaceutical industry.

Among the multivalent HPV DNA vaccine components, the vaccine adjuvant may be a vaccine adjuvant used to stimulate the specific immune response of T lymphocytes, which contains IL-12 protein and IL-21 protein as effective ingredients, or contains coded IL -12 protein polynucleotide and IL-21 protein-encoding polynucleotide as effective ingredients.

Among the multivalent HPV DNA vaccine components, the vaccine adjuvant used to stimulate the specific immune response of T lymphocytes may consist of at least one component selected from the following groups: an IL-12 protein and an IL-21 protein, consisting of p35 Link (IL-12p35) and p40 link (IL-12p40); one to three vectors, each of which contains a pair of p35 link (IL-12p35) and p40 link (IL-12p35) that constitute IL-12 protein. -12p40) the encoded polynucleotide; A polynucleotide encoding IL-21 protein; and an mRNA molecule, each of which can encode IL-12p35, IL-12p40, and IL-21 proteins.

In the multivalent HPV DNA vaccine component, the IL-12p35 protein may have at least 90% sequence homology with the human IL-12p35 protein composed of the amino acid sequence of SEQ ID NO:1.

Among the multivalent HPV DNA vaccine components, the IL-12p40 protein may have at least 90% sequence homology with the human IL-12p40 protein composed of the amino acid sequence of SEQ ID NO: 2.

Among the multivalent HPV DNA vaccine components, the IL-21 protein may have at least 90% sequence homology with the human IL-21 protein composed of the amino acid sequence of SEQ ID NO: 3.

HPV DNA polyvalent vaccine composition, the vaccine adjuvant composition may be composed of at least one selected from the following group: i) one kind of MIP-1 α protein; ii) one kind of MIP-1α gene structure, in which the protein encoding MIP-1α The polynucleotide is operably linked to the promoter; iii) a composite gene structure in which the polynucleotide encoding the MIP-1α protein is operably linked to at least one of IL-12p35, IL-12p40 and IL-21 proteins, The internal ribosome entry site (IRES) or connecting peptide is encoded by the polynucleotide; and iv) an mRNA molecule encoding the MIP-1α protein.

In the multivalent HPV DNA vaccine component, the MIP-1α structure may be contained in an independent expression vector, or any one or more of one to three vectors, each of which includes: a separate code that can constitute IL A polynucleotide of a p35 link (IL-12p35) and a p40 link (IL-12p40) of the -12 protein; and a polynucleotide encoding the IL-21 protein.

In the multivalent HPV DNA vaccine component, the MIP-1α protein may have at least 90% sequence homology with the human MIP-1α protein composed of the amino acid sequence of SEQ ID NO: 10.

In other embodiments of the present invention, an antigen-linked fusion protein is disclosed, in which at least 4 types of HPV antigenic units are linked by a linker sequence, wherein the antigenic unit is an N-terminal fragment containing each of E6 and E7 and C E6 and E7 are derived from the N-terminal fragment of E6 (E6N)-the C-terminal fragment of E7 (E7C)-the N-terminal fragment of E7 (E7N)-the N-terminal fragment of E7 (E7N)-E6 (E6C) The sequence of the C-terminal fragment of) is selected from HPV types including 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 HPV.

The term "HPV" used here to represent human papillomavirus is a DNA-based virus with a diameter of 52-55nm, and it will transfer to the skin or the subcutaneous layer of any animal including humans. Retrospectively, more than 130 types of HPV have been discovered (zur Hausen H., Vaccine 24 Suppl 3: S3, 2006). HPV can be transferred through skin keratinocytes or mucous membranes. Most known HPVs do not show any signs in humans, but some HPVs may cause papilloma in humans. In addition, a small part of papilloma can also develop into cancer (that is, cervical cancer and testicular cancer). HPV 16 and HPV 18 are found in 70% of cervical cancer patients worldwide, and these HPVs are classified as high-risk groups. HPV DNA consists of 8,000 base pairs and is surrounded by a pentameric capsid protein rather than a non-lipid membrane. The capsid protein containing two structural proteins (that is, L1 and L2) is expressed during virus replication. Late in the cycle. In all HPV genomes, there are eight open reading frames (ORF), and each ORF is divided into three functional regions. Early proteins E1-E7 (that is, genes necessary for virus replication), L1-L2 (that is, a gene that expresses structural proteins that constitute viruses), and finally LCR to control virus replication and transcription.

The term "E6" as used herein represents an early expression protein necessary for HPV replication. It is linked to p53 and promotes the ubiquitination of p53, thereby inhibiting the function of p53 as a cancer tumor suppressor gene. In addition, E6 can also induce the breakdown of BAK, which is a protein that promotes apoptosis. In addition, E6 also plays the role of activating telomerase to start the main cell cycle.

The term "E7" used here represents an early expression protein necessary for HPV replication. It interacts with retinoblastoma (RB) and breaks down RB. Therefore, E2F, a transcription promoter inhibited by RB, will Was released. In addition, E7 also activates cyclin E and cyclin A, which act on the S phase of the cell cycle cycle, thereby starting the cell cycle of the host cell.

The term "immunogenic fragment" as used herein represents a fragment of a full-length antigen protein that can function as an antigen, that is, a fragment can induce an antigen-specific immune response.

In the fusion protein, the N-terminal fragment and the C-terminal fragment may overlap by 10 to 30 amino acids, and the antigen unit functions as an antigen because the antigen unit is a promiscuously ordered protein, but lacks the original wild-type E6 and E7 proteins The inherent function of (connecting p53 to pRb).

In the fusion protein, the antigen-linked fusion protein may additionally contain an fms tyrosine kinase-3 (Flt3) ligand (Flt3L) to its N-terminus, and the secretion signal sequence may be added here. The secretion signal sequence induces the secretion of recombinant proteins expressed as extracellular cells, and may be the tissue plasminogen activator (tPA) signal sequence, the herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or growth Hormone signal series.

The fusion protein may also contain polynucleotides encoding one or two or more immunity-enhancing peptides, and the immunity-enhancing peptides may be CD28, inducible costimulatory molecules (ICOS), fine Cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte associated protein (BTLA), death ligand 3 (DR3), 4-1BB, CD2, CD40 , CD30, CD27, signaling lymphocyte promoter molecule (SLAM), 2B4 (CD244), natural killer cell group 2, member D (NKG2D)/DNAX promoter protein 12 (DAP12), T cell immunoglobulin and mucin domain Contains protein 1 (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte promoter gene 3 (LAG3), B7-1, B7-H1, inducing glucocorticoid TNFR family-related protein (GITR), fms-like Flt3 ligand (Flt3 ligand), flagellin, herpes virus entry regulator (HVEM), or the cytoplasmic protein domain of OX40L [CD134(OX40) and CD252 ligand], or at least two of them The connection sequence.

In the fusion protein, the linking sequence is preferably a linking peptide, and the linking peptide may include (G ₄ S) _n (unit: SEQ ID NO: 32, n is an integer from 1 to 10), (GS) _n (n is An integer from 1 to 10), (GSSGGS) _n (unit: SEQ ID NO: 33, n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 34), EGKSSGSGSESKST (SEQ ID NO: 35), GSAGSAAGSGEF (SEQ ID NO: 36), (EAAAK) _n (unit: SEQ ID NO: 37, n is an integer from 1 to 10), CRRRRRREAEAC (SEQ ID NO: 38), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 39), GGGGGGGG (SEQ ID NO: 40), GGGGGG (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), PAPAP (SEQ ID NO: 43), (Ala-Pro) _n (n Is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 44), PLGLWA (SEQ ID NO: 45), TRHRQPRGWE (SEQ ID NO: 46), AGNRVRRSVG (SEQ ID NO: 47), RRRRRRR (SEQ ID NO: 48), GFLG (SEQ ID NO: 49), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 50), etc.

The term "fusion protein" as used herein represents a recombinant protein in which there are two or more Species of proteins or protein domains are responsible for specific functions when proteins are connected to each other. Traditionally, a connecting peptide with an elastic structure can be inserted between two or more proteins or protein domains, but any elastic connecting peptide that does not limit the inherent function of the polypeptide will be linked together and will not inhibit the possible use of the fusion protein. expression. Specific embodiments are as described above.

In other aspects of the present invention, a polynucleotide encoding a fusion protein is disclosed.

The polynucleotide may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to other aspects of the present invention, an expression vector in which a polynucleotide is operably linked to a control sequence is disclosed.

The term "operably linked to" as used herein means that the nucleic acid of interest (for example, in an in vitro transcription/transfer system or in a host cell) is linked to a control sequence so that the heterologous nucleic acid sequence can be expressed.

The term "control sequence" is a term that includes promoters, enhancers, and other control sequences (for example, polyadenylation signals). Control sequences include: one that will direct the expression of heterologous nucleic acid in many subject cells, one that will direct the expression of heterologous nucleic acid only in cells of special tissues (such as tissue-specific control sequences), and one that will direct specific signals to induce expression (E.g. induced control sequence). A common technique in the art is to be able to understand that the expression vector may depend on certain factors, such as the choice of the host cell to be transferred, the desired protein expression level, and so on. The expression vector of the present invention may be introduced into the host cell to express the fusion protein. Control sequences that allow expression in eukaryotic and prokaryotic cells are techniques well known to experts in the field. As described above, these control sequences include those that are responsible for initiating transcription under normal circumstances, and a polyadenylation signal that is responsible for the selective termination and stabilization of transcription. Other control sequences may include translation enhancement factors in addition to transcription control factors and/or native combination or heterologous promoter regions. For example, those that may include CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Labor Sarcoma virus), human elongation factor 1α promoter, inducing glucocorticoid MMTV promoter (Moloney mouse tumor virus), inducing metallothionein or inducing tetracycline promoter, or an amplifying agent, such as CMV amplifying agent or The control sequence of the SV40 amplifier is expressed in mammalian host cells. For the expression in the neuron, the neurofibril promoter, PGDF enlighten Movers, NSE promoters, PrP promoters, or thy-1 promoters will all be considered. These promoters are well known by experts in the field and are described in many literatures (Charron, J. Biol. Chem. 270: 25739-25745, 1995). For the expression of prokaryotic cells, they include lac promoter, tac Promoters, as well as many promoters including trp promoter, have been revealed. In addition to these factors that can initiate transcription, these control sequences may also include a transcription termination signal (such as the SV40 polyadenylation region or the TK polyadenylation region) that exists downstream of the polynucleotide according to the embodiment of the present invention. ). In the present invention, suitable expression vectors in the art are well known, such as Okayama-Berger cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (in vitro genes), pSPORT1 (GIBCO BRL), pGX27 ( Korean Patent No. 1442254), pX (Pagano (1992) Science 255, 1144-1147), yeast double hybrid vector, for example, pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), or eukaryotic Biological expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecule of the present invention, the vector may also include a polynucleotide encoding a secretion signal peptide, which is well known to experts in the art. In addition, depending on the expression system used, there is a leader sequence that can direct the fusion protein to the cell chamber according to the embodiment of the present invention, combined with the sequencing sequence of the polynucleotide, and the leader sequence is preferably directly secreted The translated protein or the protein that enters the peripheral cytoplasm or extracellular medium.

In addition, the vector of the present invention can also be formulated using, for example, standard recombinant DNA technology. Examples of standard recombinant DNA techniques include blunt-end and sticky-end joining, restriction enzyme therapy to provide appropriate ends, alkaline phosphatase therapy to remove phosphate groups to prevent improper bonding, and T4 DNA ligase production The enzyme ligation sequence and so on. According to the embodiments of the present invention, the vector of the present invention can be prepared by using chemical synthesis or genetic recombination technology or DNA encoding the fusion protein to obtain the DNA recombination encoding the signal peptide to obtain a vector containing appropriate control sequences. Vectors containing control sequences can be purchased or produced through commercial transactions. In the examples of the present invention, pGX27 (Korea Patent No. 1442254), a vector for formulating DNA vaccines, is used.

The expression vector according to the embodiment of the present invention may be an expression vector capable of expressing a fusion protein, and the expression vector may represent any form of plastid vector, viral vector, mucilage vector, Phage vectors, artificial human chromosomes, etc.

According to other viewpoints of the present invention, a 14-valent HPV DNA vaccine component is disclosed, which includes an expression vector in which a polynucleotide encoding a fusion protein is operably linked to a promoter, and includes all of the 14 types of E6. A multiple expression vector of polynucleotide of /E7 antigen unit.

The 14-valent HPV DNA vaccine component may include three expression vectors, the structure of which allows the polynucleotides encoding each of the three fusion proteins to link and replicate four to five E6/E7 antigen units of 14 HPVs.

The three expression vectors may be constructed as follows, but the structure of the three expression vectors is not limited to these three, and may also be a combination of different types: i) The first expression vector includes the first gene, and its architecture can encode the first fusion protein The first nucleic acid molecule is operably linked to the promoter, and the first fusion protein is the HPV16 E6/E7 antigen unit, where each of the 16 type HPV (HPV16) early antigen 6 (E6) and early antigen 7 (E7) The N-terminal fragment and the C-terminal fragment of E6 (16E6N) will be connected in the order of the N-terminal fragment of E6 (16E6N)-the C-terminal fragment of E7 (16E7C)-the N-terminal fragment of E7 (16E7N)-the C-terminal fragment of E6 (16E6C); A HPV18 E6/E7 antigen unit, in which the N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type 18 HPV (HPV18) will be the N-terminal fragment of E6 (18E6N)-E7 The C-terminal fragment of (18E7C)-the N-terminal fragment of E7 (18E7N)-the C-terminal fragment of E6 (18E6C) is connected in this order; a HPV35 E6/E7 antigen unit, in which type 35 HPV (HPV35) has each early antigen 6 (E6) and the N-terminal fragment and C-terminal fragment of early antigen 7 (E7) will be the N-terminal fragment of E6 (35E6N)-the C-terminal fragment of E7 (35E7C)-the N-terminal fragment of E7 (35E7N)-E6 (35E6C) The C-terminal fragments of) are connected in this order; a HPV45 E6/E7 antigen unit, in which the N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of type 45 HPV (HPV45) will be The N-terminal fragment of E6 (45E6N)-the C-terminal fragment of E7 (45E7C)-the N-terminal fragment of E7 (45E7N)-the C-terminal fragment of E6 (45E6C) are connected in this order; and a HPV58 E6/E7 antigen unit, in which The N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type 58 HPV (HPV58) will be the N-terminal fragment of E6 (58E6N)-the C-terminal fragment of E7 (58E7C)-E7 The N-terminal fragment of (58E7N)-C-terminal fragment of E6 (58E6C) is connected in this order by connecting peptides; ii) The second expression vector includes the second gene, and its structure allows the second nucleic acid molecule encoding the second fusion protein to be operably linked to the promoter, and the second fusion protein is the HPV31 E6/E7 antigen unit, Among them, the N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type 31 HPV (HPV31) will be the N-terminal fragment of E6 (31E6N)-the C-terminal fragment of E7 (31E7C) -The N-terminal fragment of E7 (31E7N)-The C-terminal fragment of E6 (31E6C) is connected in this order; a HPV33 E6/E7 antigen unit, in which type 33 HPV (HPV33) each has early antigen 6 (E6) and early antigen 7 The N-terminal fragment and C-terminal fragment of (E7) will be the N-terminal fragment of E6 (33E6N)-the C-terminal fragment of E7 (33E7C)-the N-terminal fragment of E7 (33E7N)-the C-terminal fragment of E6 (33E6C) Sequential connection; a HPV6 E6/E7 antigen unit, in which the N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type 6 HPV (HPV6) will be the N-terminal of E6 (6E6N) Fragment-C-terminal fragment of E7 (6E7C)-N-terminal fragment of E7 (6E7N)-C-terminal fragment of E6 (6E6C) This sequence connection; a HPV11 E6/E7 antigen unit, of which each type 11 HPV (HPV11) The N-terminal and C-terminal fragments of Early Antigen 6 (E6) and Early Antigen 7 (E7) will be the N-terminal fragment of E6 (11E6N)-the C-terminal fragment of E7 (11E7C)-the N-terminal fragment of E7 (11E7N)- The C-terminal fragment of E6 (11E6C) is connected in this order; and a HPV52 E6/E7 antigen unit, where each of the 52 type HPV (HPV52) early antigen 6 (E6) and early antigen 7 (E7) N-terminal fragments and C The N-terminal fragments of E6 (52E6N)-the C-terminal fragment of E7 (52E7C)-the N-terminal fragment of E7 (52E7N)-the C-terminal fragment of E6 (52E6C) are linked in this order by a connecting peptide; and The third expression vector includes the third gene, and its structure allows the third nucleic acid molecule encoding the third fusion protein to be operably linked to the promoter, and the third fusion protein is the HPV39 E6/E7 antigen unit, of which 39 The N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type HPV (HPV39) will be the N-terminal fragment of E6 (39E6N)-the C-terminal fragment of E7 (39E7C)-E7 The N-terminal fragment of (39E7N)-the C-terminal fragment of E6 (39E6C) are connected in this order; a HPV51 E6/E7 antigen unit, where each of the 51 type HPV (HPV51) early antigen 6 (E6) and early antigen 7 (E7 ) N-terminal fragment and C-terminal fragment will be E6 (51E6N) N-terminal fragment-E7 The C-terminal fragment of (51E7C)-the N-terminal fragment of E7 (51E7N)-the C-terminal fragment of E6 (51E6C) is connected in this order; a HPV56 E6/E7 antigen unit, in which type 56 HPV (HPV56) has each early antigen 6 (E6) and early antigen 7 (E7) N The N-terminal fragment and the C-terminal fragment of E6 (56E6N)-the C-terminal fragment of E7 (56E7C)-the N-terminal fragment of E7 (56E7N)-the C-terminal fragment of E6 (56E6C) are connected in this order; and a HPV59 E6/E7 antigen unit, of which the N-terminal fragment and C-terminal fragment of each early antigen 6 (E6) and early antigen 7 (E7) of type 59 HPV (HPV59) will be the N-terminal fragment of E6 (59E6N)-E7 ( The C-terminal fragment of 59E7C)-the N-terminal fragment of E7 (59E7N)-the C-terminal fragment of E6 (59E6C) are connected in this order by a connecting peptide.

In the vaccine component, the first fusion protein to the third fusion protein may have a secretion signal sequence and Flt3L added to the N-terminal, the secretion signal sequence is as described above.

The vaccine component may contain at least one vaccine adjuvant acceptable to the pharmaceutical industry. Aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virus particles, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a DL-α-tocopherol, dogfish A mixture of ene and polysorbate (an emulsifier), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs, ISCOMMATRIX, etc. may all be used as vaccine adjuvants.

The term "adjuvant" or "vaccine adjuvant" as used herein represents a pharmaceutical or immunological formulation for the purpose of improving the immune response of a vaccine.

In addition, the vaccine adjuvant may also be a vaccine adjuvant used to stimulate a specific immune response of T lymphocytes. It contains an IL-12 protein and an IL-21 protein as effective ingredients, or contains a polynucleus encoding an IL-12 protein. Glycolic acid and a polynucleotide encoding IL-21 protein are used as effective ingredients.

In particular, the vaccine adjuvant may contain at least one component selected from the group consisting of the following components: an IL-12 protein containing p35 link (IL-12p35) and p40 link (IL-12p40) and IL-21 protein ; One to three vectors, each of which contains a polynucleotide encoding the p35 link (IL-12p35) and p40 link (IL-12p40) that construct the IL-12 protein; a polynucleus encoding the IL-21 protein And an mRNA molecule, each of which encodes IL-12p35, IL-12p40, and IL-21 proteins.

In addition, it is possible that in the vaccine agents described above, parts of IL-12p35, IL-12p40 and IL-21 will also be included as proteins, while the other parts will be used as heterogeneous molecules. Like a mixture of expression vectors and/or mRNA molecules.

In particular, one to three vectors may contain a gene structure in which the polynucleotide is operably linked to a control sequence (such as a promoter) so that IL-12p35, IL-12p40, and IL-21 can all be expressed. Vaccine adjuvants may be constructed into one to three vectors, each of which can insert polynucleotides encoding IL-12p35, IL-12p40 and IL-21 into individual expression vectors (a triple vector system), or into One or two expression vectors (a single vector system or dual vector system). A specific example of such a single vector system to a triple vector system is as follows: i) There is a fourth expression vector including a fourth gene structure to a sixth gene structure, which can respectively encode IL-12p35, IL-12p40 and IL -21 protein polynucleotides, each of which can be operably linked to a promoter; ii) there is a fifth expression vector to the seventh expression vector containing the fourth gene structure to the sixth gene structure; iii) there is a first 8 expression vector and 9th expression vector, each includes two 4th gene structure to 6th gene structure, the others include a gene structure; iv) There is a fusion protein in which IL-21 is linked to IL-12p35 and Any one of IL-12p40; and the peptide between IL-12p35 and IL-12p40 is not included in the fusion protein; v) There is a 10th expression vector containing a 7th gene structure, where iv) can encode the fusion protein The polynucleotide can be operably linked to a promoter, and an eighth gene structure, where iv) the polynucleotide encoding the peptide can be operably linked to a promoter; vi) an 11th expression vector and a first 12 expression vectors, which respectively include the 7th expression vector and the 8th expression vector; vii) a 13th expression vector includes: a 9th gene structure, of which at least two can respectively encode IL-12p35, IL-12p40 and IL- The polynucleotide of the 21 protein can be operably linked to an IRES, and there is an option of the 10th gene structure, which is not included in the three polynucleotides The remaining polynucleotides in the 9th gene structure can be operably linked to a promoter; and viii) a 14th expression vector contains a 9th gene structure; and a 15th expression vector can optionally include the 10th gene structure; In the composition, IL-12p35 protein may be composed of amino acid sequence, at least 90%, preferably 95% of the sequence is homologous to human IL-12p35 composed of amino acid sequence SEQ ID NO:1, and it is also possible The use of IL-12p35 derived from non-humans (such as primates or apes) has a high degree of homology but does not induce any immune response in humans. The IL-12p40 protein may be composed of at least 90%, preferably 95% of the sequence and the amino acid sequence of human IL-12p40 which is composed of the amino acid sequence SEQ ID NO: 2, and it may also be used. IL-12p40 derived from non-humans (such as primates or apes) has a high degree of homology but does not induce any immune response in humans. It is also possible to use IL-12p35 and IL-12p40 as the sequences described in Korean Patent No. 0399728. The IL-21 protein may be composed of at least 90%, preferably 95% of the sequence and the amino acid sequence of human IL-21 which is composed of the amino acid sequence SEQ ID NO: 3, and it is also possible to use it. IL-21 derived from non-humans (such as primates or apes) has a high degree of homology but does not induce any immune response in humans.

In the vaccine component, the vaccine adjuvant may also include at least one component selected from the group consisting of the following components: i) a MIP-1α protein; ii) a MIP-1α gene structure, which encodes a polymer of MIP-1α protein The nucleotide can be operably linked to a promoter; iii) a composite gene structure in which the polynucleotide encoding the MIP-1α protein can be operably linked to the IL-encoding polynucleotide through the polynucleotide encoding the IRES or linker peptide. At least one of the polynucleotides of 12p35, IL-12p40 and IL-21 protein; and iv) an mRNA molecule encoding MIP-1α protein.

The polynucleotide encoding the MIP-1α protein can be operably linked to the polynucleotide encoding the IL-12p35, IL-12p40, and IL-21 protein by the polynucleotide encoding the linker peptide or IRES, or an independent Provided in the form of gene structure. In addition, the MIP-1α gene structure may also contain one to three In at least one body in the vector, these bodies include polynucleotides encoding the p35 link (IL-12p35) and the p40 link (IL-12p40) that construct the IL-12 protein, respectively; and one encoding IL-21 The protein polynucleotide, that is, the MIP-1α gene structure may be included in at least one selected from the group consisting of the fourth expression vector to the fifteenth expression vector described in the examples of vaccine adjuvants. In an expression vector.

In the vaccine component, the MIP-1α protein may be composed of at least 90%, preferably 95% of the sequence. These sequences have homology with the MIP-1α protein composed of the amino acid sequence SEQ ID NO: 10. It is also possible to use MIP-1α proteins derived from non-humans (such as primates or apes) that have a high degree of homology but do not induce any immune response in the human body.

Vaccine ingredients may also include IL-7.

Vaccine ingredients may also include pharmaceutically acceptable adjuvants, excipients or diluents in addition to carriers.

The term "pharmaceutical acceptable" as used herein represents a physiologically acceptable, but usually does not cause allergic reactions (such as gastrointestinal discomfort, dizziness, etc.) or similar reactions when used in humans. Examples of carriers, excipients and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, sodium alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl paraben, talc, magnesium stearate and mineral oil. It may also include fillers, anticoagulants, lubricants, moisturizers, fragrances, emulsifiers, preservatives, etc.

In addition to the adjuvants mentioned above, the vaccine ingredients may also include a traditionally used vaccine adjuvant. May use aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virus particles, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a DL-α tocopherol, Squalene and Polyxylenide 80 (an emulsifier)), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs, ISCOMMATRIX, etc.) are traditionally used as vaccine adjuvants.

In addition, the vaccine components according to the embodiments of the present invention may be formulated and produced by methods well known in the art, so that they can be quickly released, or maintained, or delayed when they are administered to mammals. The release of essential ingredients. These formulations may include powders, granules, tablets, emulsions, syrups, nebulizers, soft or hard gelatin capsules, injectable antiseptic solutions and antiseptic powders.

The vaccine components according to the present invention may be administered in various ways, including, for example, oral administration, non-digestive tract (for example, suppository, transdermal diffusion, intravenous injection, peritoneal injection, intramuscular injection, intralesional, intranasal, spinal canal), or It can be administered by implanting the device for continuous, continuous or repeated release. The number of medications can be one medication or several medications a day within the expected range, and the medication cycle time is not particularly limited.

The vaccine components according to the embodiments of the present invention may be administered by traditional system or typical medication methods (for example, intramuscular injection or intravenous injection). If DNA vaccine components are provided, electroporation may be the most preferred injection method for vaccine components. When electroporation is used, a method of injecting commercially available DNA drugs into the body (such as linporator ^{TM of} Italy IGEA, CUY21EDIT of JCBIO of South Korea, SP-4a of Swiss Supertech, OrbiJector ^® of SLVAXiGEN of South Korea, etc.) may be used.

The medication route according to the embodiment of the present invention may be administered through any traditional route, as long as the vaccine components can reach the target tissue. The route of administration may include, but is not limited to, non-digestive tract administration (for example, peritoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, and intrathecal injection).

In addition, the multivalent HPV DNA vaccine components according to the embodiments of the present invention may be combined with IL-7 protein or polynucleotide encoding IL-7 protein to be administered together. Traditionally, DNA drugs and protein drugs may be administered in separate formulations, but they may also be packaged in independent formulations and then administered through different routes, because they may have different routes of administration. For example, DNA vaccine ingredients may be Electroporation is administered by intramuscular injection, while IL-7 protein may be administered in the same way as general protein drugs, such as traditional intramuscular injection, intravenous injection, and peritoneal injection.

The vaccine components according to the embodiments of the present invention may be formulated in a suitable form with a carrier acceptable to the general pharmaceutical industry. Acceptable methods for the pharmaceutical industry may include, for example, water, suitable oils, physiological saline, parenteral vehicles (for example, aqueous glucose, ethylene glycol, etc.), and may also include stabilizers and preservatives. Suitable stabilizers may include antioxidants (such as sodium bisulfite, sodium sulfite, or antioxidants). Ascorbic acid). Suitable preservatives may include hydroxychloroaniline, methyl or paraben potassium esters and chlorobutanol. In addition, the ingredients according to the present invention may contain suspending agents, dissolution adjuvants, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH regulators, analgesics, and alleviating agents. Agents, antioxidants, etc., depending on the method of administration and preparation when necessary. Examples of suitable pharmaceutical industry-acceptable carriers and suitable formulation methods of the present invention, including the examples cited above, are described in detail in the literature [Remington's Pharmacy, New Edition].

The dosage of vaccine components for a patient may vary depending on many factors, including the patient’s height, body surface area, age, special combination of drugs used, gender, time and route of administration, general health status, and concurrent use Other drugs. Pharmaceutically effective DNA may be 100ng/body weight (kg)-10mg/body weight (kg), preferably from 1μg/kg to 500μg/kg (body weight), preferably from 5μg/kg to 50μg/kg (Weight) to take the medicine, and the dosage may need to be adjusted by considering various factors.

In addition, the vaccine components of the present invention may be administered in therapeutically effective amounts.

The term "therapeutically effective amount" used here represents a sufficient amount to treat a disease under a reasonable benefit/risk ratio when applied in medical treatment, and the effective dose level may be determined based on the following factors, including the type of object and the severity of the disease , Age, gender, drug activity, drug sensitivity, medication time, medication route and dissolution rate, length of treatment time, factors including concurrent medication and other factors well known in the medical field. The vaccine component of the present invention may be administered at a dose of 0.1 mg/kg to 1 g/kg, and better practice is to use a dose from 1 mg/kg to 500 mg/kg, and the dosage may be 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, Unit doses of 6 mg, 7 mg, 8 mg, etc. are used. At the same time, the dosage of the drug may need to be adjusted appropriately according to the age, gender and health of the patient.

According to other viewpoints of the present invention, a method for treating diseases caused by HPV infection is disclosed, including medication, for individuals, multivalent HPV DNA vaccine components, or 14-valent HPV DNA vaccine components.

In the above treatment methods, the diseases caused by HPV infection may be squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumor (NET), clear cell carcinoma, villous adenocarcinoma ( VGA), non-cancerous malignancies, melanoma, lymphoma, or cervical intraepithelial tumor Change (CIN).

In the above treatment methods, the vaccine components may be administered by electroporation in vivo.

In order to solve the shortcomings that traditional HPV vaccines cannot be widely used in HPV infection diseases such as cervical cancer, the present invention formulates a multivalent HPV DNA vaccine composed of 3 plastids, of which a multivalent DNA vaccine covers all 6 , 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 types of HPV, which are mainly prevalent in cervical intraepithelial neoplasia (CIN), children belonging to high-risk groups Cervical cancer, vulvar intraepithelial neoplasia (VIN), vulvar cancer, anogenital warts, and condyloma acuminatum, each HPV hybrid sequence E6/E7 can be expressed in the form of a single giant fusion protein of every 4 or 5 types. And perform experiments using multivalent HPV DNA vaccines. As a result, it can be confirmed from experiments that the multivalent HPV DNA vaccine induces specific immune responses of T cells to the two representative HPV types HPV16 and HPV18 and other HPV types. These results are very exciting. The use of proteins that can express multiple promiscuous sequencing antigens The expression vector can achieve these results, in which there are several promiscuous sequenced proteins, instead of a single promiscuous sequenced protein, which will only be connected by the linking sequence, and the promiscuous sequenced antigen protein does not even need to use the original antigen protein. In addition, it is surprising that the multivalent HPV DNA vaccine according to the embodiments of the present invention not only induces a specific immune response to HPV16 and HPV18 T cells similar to the traditional bivalent vaccine, but even uses only a quarter of the traditional bivalent vaccine. A small dose of one, but in cases of types 6, 11, 39, 45 and 56, the multivalent HPV DNA vaccine can also induce a higher T cell specific immune response than HPV16 or HPV18. Therefore, the multivalent HPV DNA vaccine according to the embodiments of the present invention can be used very efficiently as a vaccination vaccine that can prevent 7 kinds of HPV at the same time. In addition, the multivalent HPV DNA vaccine can also be effectively used as a preventable vaccine. The 33, 35, 51, 52, 58 and 59 groups select at least one infection in the high-risk group, and a vaccine for the treatment of infectious diseases.

Later, the present invention will be explained in more detail through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples described below, but can be implemented in various other forms. The following examples and experimental examples can expose the present invention so as to fully and completely convey the scope of the present invention to those experts in the field to which the present invention belongs.

Example 1: Preparation of a multivalent HPV DNA vaccine

In order to formulate a multivalent HPV DNA vaccine, the present invention obtains polynucleotides that can encode N-terminal fragments and C-terminal fragments of various types of E6 and E7 obtained by PCR. This is to express E6 and E7 antigens in the form of promiscuous sequenced proteins. The E6 and E7 early expression proteins of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 that belong to the high-risk group cannot display wild-type E6 and E7 The linked polynucleotides will be obtained in the connection method disclosed in Figure 1 and Table 1, thereby preparing three gene structures. Each of the three gene structures is inserted into the pGX27 vector to prepare a HPV DNA structure , Each of the three gene structures is named BD-14A, BD-14B and BD-14C, and the components containing these three vectors are named BD-14. The reason why the structure of the multivalent HPV DNA vaccine should be formulated into a triple vector system is that pGX-27 will have insufficient capacity when the size of the inserted gene structure is too large.

As shown in Figure 1 and Table 1, the structure of various HPV types E6 and E7 antigens in which the antigen is divided into N-terminal fragments and C-terminal fragments is constructed. The partial sequences (20 aa) of these fragments overlap respectively, and then there is a fusion polypeptide. Among them, the C-terminal fragment of E7 (E7C) is connected to the N-terminal end of E6 (E6N), and there is a fusion polypeptide, and the C-terminal fragment of E6 (E6C) is connected to the N-terminal fragment of E7 (E7N). ) The 5 connecting peptides are connected again, allowing 4 to 5 antigen units of each type to link (GS) 5 to the connecting sequence to be contained in a single carrier. Each type of seed type is inserted into an expression vector shown as an example in Table 1, but these are only for the purpose of schematic illustration, and in fact may be formulated in any other order.

Example 2: Preparation of human IL-2 and IL-21 expression vectors

2-1: Single carrier system

The present invention designs a single vector system that can express IL-12 and IL-21 in the form of a single vector.

For this purpose, the present invention specially formulated a gene structure, by linking the human IL-12 protein (that is, the hIL-12p35 polypeptide composed of the amino acid sequence SEQ ID NO:1 and the amino acid sequence SEQ ID NO:1). The iL-12p40 polypeptide consisting of NO: 2) is derived from one of the two subunits of the polynucleotide (SEQ ID NO: 4 and 5) to EMCV, and has the internal ribosome entry site of the nucleic acid sequence SEQ ID NO: 6 ( IRES); Then sequentially link the RSV promoter (pRSV) of SEQ ID NO: 7 to a polynucleoside that can encode the human IL-21 protein (hIL-21) composed of the amino acid sequence SEQ ID NO: 3 Acid (SEQ ID NO: 8) to the 3'end of the polynucleotide encoding hIL-12p40 polypeptide; then insert the gene structure into the multiple replication site of the pGX-27 vector (Korean Patent No. 1442254), thereby A vector according to an embodiment of the present invention is formulated. The vector formulated in this way is called "hBD-121" (Figure 2A).

2-2: Dual carrier system

The present invention designs a dual vector system to express IL-12 and IL-21 into independent vectors.

The dual vector system is formulated as follows. A polynucleotide encoding hIL-12p35 polypeptide (SEQ ID NO: 1) and a polynucleotide encoding hIL-12p40 polypeptide (SEQ ID NO: 2) will be linked to a nucleic acid sequence SQE ID NO: 6 EMCV-IRES; and its composition will be inserted into the multiple replication sites of the pGX-27 vector; then in a similar manner, it encodes human IL-21 composed of the amino acid sequence SEQ ID NO: 3 The polynucleotide (SEQ ID NO: 8) of the protein (hIL-21) will also be inserted into the multiple replication sites of pGX-27; thereby, a vector expressing each IL-12 and IL-21 can be obtained. The vectors formulated in this way are called "'hBD-12" and "hBD-21".

2-3: Triple carrier system

Because IL-12 is a dimeric protein composed of hIL-12p35 polypeptide and hIL-12p40 polypeptide, hIL-12p35 polypeptide and hIL12p40 polypeptide can be expressed by independent vectors. In this way, according to the embodiment of the present invention, hIL-12p35 polypeptide, hIL-12p40 polypeptide and IL-21 may be expressed by any of three independent construction vectors, and for convenience, this will be named " , The triple carrier system".

The triple carrier system may be formulated as follows.

Polynucleotides (SEQ ID NOS: 4, 5 and 8) encoding hiL-12p35 polypeptide, hIL-12p40 polypeptide and hIL-21 will be inserted into various replication sites of the pGX-27 vector to prepare a triple vector system .

Example 3: Preparation of human IL-12, IL21 and MIP-α expression vectors

The gene structure can encode any one of the hIL-12p35 polypeptide composed of the amino acid sequence SEQ ID NO: 1 and the hIL-12p40 polypeptide composed of the amino acid sequence SEQ ID NO: 2 ( SEQ ID NOS: 4 and 5) to the EMCV-derived internal ribosome entry site (IRES) with the nucleic acid sequence SEQ ID NO: 6; and the human EF-1α promoter ( pEF-1α) and the polynucleotide (SEQ ID NO: 11) that can encode the human protein MIP-1α (hMIP-1α) consisting of the amino acid sequence SEQ ID NO: 10, are sequentially linked to SEQ ID NO: 7 RSV promoter (pRSV) and polynucleotide (SEQ ID NO: 8) that can encode human IL-21 protein consisting of amino acid sequence SEQ ID NO: 3; and insert the gene structure into pGX-27 Multiple replication sites of the vector; the vector prepared therefrom was named "'hBD-121A" (Figure 2B).

Example 4: Preparation of IL-12 and IL-21 expression vectors for mice

A gene structure is prepared by combining the encoding mouse IL-12 protein (that is, the mIL-12p35 polypeptide composed of the amino acid sequence SEQ ID NO: 12 and the mIL-12p40 polypeptide composed of the amino acid sequence SEQ ID NO: 13). (Polypeptide) the polynucleotides (SEQ ID NOS: 14 and 15) of any one of the two subunits are linked to the EMCV-derived internal ribosome entry site (IRES) with the nucleic acid sequence of SEQ ID NO: 6; The RSV promoter (pRSV) of SEQ ID NO: 7 and the IL-21 protein (mIL-21) encoding the amino acid sequence SEQ ID NO: 16 are linked to the 3'of the polynucleotide encoding the mIL-12p40 polypeptide End; then insert the gene structure into the multiple replication sites of the pGX-27 vector to match The vector according to the embodiment of the present invention was prepared; the vector prepared therefrom was named "mBD-121" (Figure 2A).

Example 5: Preparation of expression vectors for mouse IL-12, IL-21 and MIP-1α

A gene structure is prepared by combining a polynucleoside encoding any one of the mIL-12p35 polypeptide composed of the amino acid sequence SEQ ID NO: 12 and the mIL-12p40 polypeptide composed of the amino acid sequence SEQ ID NO: 13 Acid (SEQ ID NOS: 14 and 15), linked to the EMCV-derived internal ribosome entry site (IRES) having the nucleic acid sequence of SEQ ID NO: 6; and the human EF- which sequentially will consist of the nucleic acid sequence of SEQ ID NO: 9 1α promoter (pEF-1α) and polynucleotides (SEQ ID NO: 19) that can encode the human protein MIP-1α (hMIP-1α) consisting of the amino acid sequence SEQ ID NO: 18, are sequentially linked to The 3'end of the polynucleotide encoding the mIL-12p40 polypeptide is then sequentially connected to the RSV promoter (pRSV) of SEQ ID NO: 7 and the mouse IL-21 composed of the amino acid sequence of SEQ ID NO: 16 Protein (mIL-21) polynucleotide (SEQ ID NO: 17); then the gene structure was inserted into various replication sites of pGX-27; the vector prepared by this was named "mBD-121A" (Figure 2B) .

Experimental example 1: Analysis of BD-14 expression

1-1: ELISA test

In order to confirm whether BD-14 (that is, the multivalent HPV DNA vaccine prepared according to the embodiment of the present invention in Example 1) can be expressed normally when introduced into mammalian cells, the present invention is converted with BD-14 (that is, BD14A). , BD-14B and BD-14C) mammalian cells, and will use the Flt3L specific antigen contained in each structure to analyze the existence of protein expression.

Especially the COS-7 cell line will be inoculated in each 100mm petri dish, and cultured for 16 hours, and converted with an empty vector (simulated plastid DNA), while BD14A, BD-14B and BD-14C plastid DNA will be Formulated in Example 1 with liposomes. Each transformant will be cultured in an incubator (37°C, CO ₂ ) for 3 days, and the culture supernatant of COS-7 cells will be restored under various conditions and used as a sample. Since the protein present in each sample is the form of Flt3L and protein binding, the protein was quantified with Flt3L ELISA reagent (Figure 3A).

As shown in Figure 3A, confirm all BD14A, BD-14B and BD-14C All have good expressions.

1-2: Western blot analysis

The present invention uses the cell lysate contained in Experimental Example 1-1 to perform SDS-PAGE electrophoresis, converts to nylon membrane, and uses anti-Flt3L antibody to perform Western blot analysis (Abcam, Cat# ab52648) (Figure 3B).

As shown in Figure 3B, it was confirmed that all BD14A, BD-14B, and BD-14C can express the fusion protein in the expected size.

Experimental example 2: Analysis of immune response in BD-14

2-1: Analysis of T cell specific immune response

According to the embodiment of the present invention, the inventors used BD14 to administer medicine on experimental animals, and used the method of counting the number of spleen cells showing immune response for each antigen to check whether BD-14 can induce a specific immune response of T cells. In particular, C57BL/6 mice (that is, experimental animals) will be divided into a group with empty carrier (control group) (n=5) for medication, and a group with traditional bivalent HPV DNA vaccine medication (Korea Patent Gazette Issue No. 10-2017 -0045254) (n=5), and a group of BD-14 medications according to an embodiment of the present invention (n=5). Then every 2μg of plastid DNA (0.67μg for BD-14A to BD-14C) was administered twice to the femoral muscle by in vivo electroporation at two-week intervals. The animal was sacrificed two weeks after the last medication, and its spleen was taken out, and then the number of splenic immune cells that responded to each E6/E7 antigen was calculated by ELISPOT analysis (Figure 4A and 4B).

As shown in Figure 4B, in the BD-14 example according to the experimental example of the present invention, all types of HPV E6/E7 antigens successfully elicited a specific immune response to T cells, although the specific immune response of T cells to HPV 35 , 52 and 59 types are relatively weak, but at the same time, there is no significant difference in the response ability of HPV 16 and 18 E6/E7 compared with the traditional bivalent vaccine. These results confirm that although the E6/E7 antigen ratio of HPV type 16 and type 18 of BD-14 is only one third of the traditional bivalent HPV DNA vaccine in the example of the present invention, the E6/E7 type of HPV type 16 and type 18 The antigen still has an equivalent effect. In particular, it was confirmed that HPV types 6, 11, 39, 45, and 56 have stronger reactions than those caused by HPV types 16 and 18.

2-2: Analysis of anti-tumor effect

The present invention uses tumor animal mimics to perform in vivo anti-cancer activity analysis to confirm whether BD-14 according to the embodiment of the present invention is effective in the prevention and treatment of cancer caused by HPV infection.

In particular, C57BL/6 mice (i.e. experimental animals) will be divided into a group with empty carrier (control group) (n=8) for medication, and a group with traditional bivalent HPV DNA vaccine medication (Korea Patent Gazette Issue No. 10-2017 -0045254) (n=8), and a group of BD-14 medications according to the embodiment of the present invention (n=8). Then, derived from the lung epithelial cells of C57BL/6 mice and converted to TC-1 cells expressing the E6/E7 antigen of HPV16, 5×10 ⁵ cells were subcutaneously injected into the back to induce tumors. Three days after the cancer cells were administered, the in vivo electroporation method was used at two-week intervals, using OrbiJector ^® (SLVAXiGEN, South Korea) with 2μg each of plastid DNA (0.67μg for BD-14A to BD-14C) for femoral muscle Take the medicine twice. Tumor size and survival rate of experimental animals will be checked at 3 to 4 days from the 7th day of tumor injection (Figures 5A and 5C).

As shown in Figure 5B, the BD-14 according to the embodiment of the present invention not only significantly reduced the tumor size compared with the control group, but also exhibited anti-tumor effects similar to the traditional bivalent HPV DNA vaccine. In addition, as shown in FIG. 5C, compared with the results of tumor size analysis, the BD14 medication group according to the embodiment of the present invention also showed a higher survival rate than the traditional bivalent HPV DNA vaccine medication group. Based on a comprehensive review of these results, it can be confirmed that the BD-14 vaccine component according to the embodiment of the present invention is only one-third of the high-risk group (such as HPV16 or HPV18), but the BD-14 vaccine component is similar to the traditional one. Compared with the bivalent DNA vaccine, not only does it show equivalent or better anti-cancer performance, but the BD-14 vaccine is also a highly innovative vaccine component that induces a specific immune response of T cells and even targets other types of HPV. , Can increase the ability to prevent cervical cancer by 90% or more.

Experimental example 3: Effect analysis of BD-121 as a vaccine adjuvant

3-1: ELISA test

The present invention respectively converts the hBD-121 structure and mBD-121 structure according to the embodiment of the present invention in Examples 2 and 4 into cells, and checks whether IL-12 and IL-21 are normally expressed in the converted cells. In particular, the COS-7 cell line will be inoculated in each 100mm culture dish and cultured for 16 hours, using an empty vector (simulated plastid DNA), hBD-121 plastid DNA prepared in Example 2-1 and Example 4 The prepared mBD-121 plastid DNA was transformed with liposomes. Each transformant will be cultured in an incubator (37°C, CO ₂ ) for 3 days, and the culture supernatant of COS-7 cells will be restored under various conditions and used as a sample. The IL-12 and IL-21 proteins present in each sample will use antibodies that specifically identify IL-12 and IL-21 (IL-12: R&D Systems, Cat# D1200, IL-21: BioLegend, Cat # 433808) was quantified by ELISA assay (Figure 6A and Figure 6B).

As shown in the results in Figure 6A, when hIL-12 was introduced in a DNA amount of 4μg, the protein expression level in the sample introduced into hBD-121 plastid DNA was greater than 4,000 pg/mL, so it was confirmed that these proteins were normally expressed. When hIL-21 was introduced in 4μg of DNA, the protein expression level in the sample was as high as close to 200ng/mL, so it was confirmed that these proteins can be expressed at a high level. In addition, as shown in Fig. 6B, the mouse structure shows a result similar to the human structure. At the same time, the two proteins in the control group introduced into the empty vector were not expressed at all, thus confirming that the vaccine adjuvant expression system of the present invention functions normally.

3-2: Western blot analysis

The present invention uses the cell lysate obtained from the experimental example 3-1 to perform SDS-PAGE electrophoresis, converts to nylon membrane, and then uses anti-IL-12A antibody, anti-IL-12B antibody and anti-IL-21 antibody to perform western blot analysis (Not shown in the picture).

Both IL-12 and IL-21 can be transfected with BD-121 plastid DNA according to embodiments of the present invention.

Experimental example 4: Analysis of the effect of BD-121A as an adjuvant of Epidemiology

4-1: Analysis of T cell specific immune response

The present invention performs an analysis of whether BD-121A as a vaccine adjuvant can improve the function of the BD-14 vaccine.

For this purpose, the inventors administered the BD-14 according to the embodiment of the present invention and the mBD-121A formulated in Example 5 to experimental animals, and used the method of counting the number of spleen cells showing immune responses to each antibody to check whether the medication would induce T cell specific immune response. In particular, C57BL/6 mice (that is, experimental animals) will be divided into one group with empty carrier (control group) for medication (n=3), one group with only BD-14 medication (n=5), and one group with basis The BD-14 and mBD-121A of the invention examples are used together (n=5), and then the group that only uses BD-14 will use each plastid DNA (BD-14A, BD-14B and BD-14C) The drug was administered in a dose of 1.3 μg, while the group that was co-administered with BD-14 and mBD-121A used each plastid DNA in a dose of 1 μg, and OrbiJector ^® (SLVAXiGEN, South Korea) was used at one time by in vivo electroporation. On the femoral muscle head. Two weeks after the treatment, the animals were bullied and the spleen was taken out, and then the ELISPOT analysis was used to calculate the number of immune cells in the spleen that reacted to various E6/E7 antibodies (Figures 7A and 7B).

As shown in the results of Figure 7B, it was confirmed that mBD-121A according to the embodiment of the present invention, compared with BD-14 alone, enhanced the E6/E7 specific T cell responses of various HPV types, especially HPV 16 (that is, high Risk group) showed that T cell immune response increased by at least 2 times, and HPV 31, 33, 51 and 58 showed a significant increase in immune response. These results confirm that BD-121A according to Examples of the present invention is a very effective vaccine adjuvant for multivalent HPV DNA vaccines.

4-2: Analysis of anti-tumor performance

In particular, C57BL/6 mice (that is, experimental animals) will be divided into a group with empty carrier (control group) (n=10) for medication, and a group with traditional bivalent HPV DNA vaccine medication (Korea Patent Gazette Issue No. 10-2017 -0045254) (n=13), and a group of BD-14 and BD-121 medications according to the embodiment of the present invention (n=13). Then, the TC-1 cells used in Experimental Example 2-2 were subcutaneously injected into the back in the number of ^{5×10 5 cells to induce tumors.} Seven days after the injection of TC-1 cells, use OrbiJector ^® (SLVAXiGEN, South Korea) with 4μg of plastid DNA (1μg each for BD-14A to BD-14C) using in vivo electroporation at two-week intervals to administer the femoral muscle. twice. Tumor size and survival rate of experimental animals will be checked at 3 to 4 days from the 7th day of tumor injection (Figures 8A and 8C).

As shown in the confirmation results of Figures 8B and 8C, it is confirmed that the multivalent vaccine according to the embodiment of the present invention shows a significant anti-cancer performance compared with the negative control group (groups administered with PBS), and is compared with the positive control group (that is, The traditional bivalent vaccine) has equivalent or similar or better anti-cancer performance. Consider the resistance The amount of the original (HPV16 E6/E7) is only about a quarter of that of the traditional bivalent vaccine. The above results mean that the antigen shows similar anti-cancer performance. In addition, although there are many kinds (number) of antigens administered at the same time, it can also induce various immune responses, and the anti-cancer performance of the special type (HPV16) will not be impaired.

Experimental example 5: Analysis of the mechanism of vaccine action

In order to examine the mechanism by which the multivalent HPV vaccine according to the embodiment of the present invention may function, the present inventors prepared the experiment by removing CD-4 T cells and CD-8 T cells with anti-CD4 or anti-CD8 antibodies. Animals, and a comparative analysis on the anticancer effect of the HPV DNA vaccine according to the embodiment of the present invention was performed.

In particular, the experiment will divide C57BL/6 mice (that is, experimental animals) into four groups for execution: one group is treated with an empty carrier as the control group (n=9), and one group is treated with the vaccine according to the embodiment of the present invention plus the vaccine Adjuvant (BD14A+BD121A) was used as the control group (i.e. subtype antibody (isotype)) (n=9), one group was treated with anti-CD4 antibody (n=9); and the other group was treated with anti-CD8 antibody Medication (n=9). The experimental schedule is as follows: The TC-1 cancer cells of experimental example 2-2 ^{were inoculated subcutaneously 7 times with 5×10 5} cells per animal, and the antibodies (isotype, anti-CD4 and anti-CD8 antibodies) were From the day of inoculation of cancer cells on the first day, at 7-day intervals, 200μg/injection/mouse was administered intraperitoneally each time, and the vaccine components according to the embodiments of the present invention (BD-14A and BD-121) ) Starting from the 3rd day after cancer cell inoculation, with 7 days as an interval, the femoral muscle was intramuscularly injected 3 times by electroporation at a dose of 8 μg/injection/mouse (Figure 9A). The tumor size and survival rate of the experimental animals were checked at 3 to 4 days from the 9th day of tumor inoculation (Figures 9B to 9C).

As shown in Figures 9B and 9C, it was observed that the anti-cancer effect in the CD4 T cell removed mice was maintained, and the anti-cancer effect in the CD8 T cell removed mice was compared with that of the control group (simulation body and subtype Antibody) is much lower. These results show that the multivalent HPV DNA vaccine according to the examples of the present invention exhibits anti-cancer immunity by CD8 T cells, and these results are very important to the known fact that CD8 T cells are very important for the effect of anti-cancer therapeutic vaccines. stable.

Experimental example 6: Effect analysis of co-administration with IL-7

Interleukin 7 (IL-7) is a cytokine that is well known for promoting the alienation of pluripotent stem cells into lymphoptic cells, and it plays an important role in the formation of B cells and T cells. Although IL-7 is The well-known role is to promote the malignant transformation of blood cancer (that is, acute lymphoblastic leukemia or T-cell lymphoma). In general solid tumors, IL-7 is known to interfere with the dynamic balance of CD8 and CD4 cells, thus reducing CD4. ⁺ CD25 ⁺ Foxp3 ⁺ regulates the proportion of T cells, and clinical phase 1 and phase 2 trials are already underway for certain cancers. From the results of experimental example 5, IL-7 tends to affect the balance between CD4 T cells and CD8 T cells, and cause the increase of CD8 T cells. Therefore, the present inventors assumed that IL-7 combined with the HPV DNA vaccine of the present invention may have a synergistic effect when co-administered. In order to confirm this hypothesis, the inventors used the vaccine according to the examples of the present invention alone or in combination with IL-7, and analyzed its anti-cancer effect.

In particular, the experiment will divide C57BL/6 mice (ie experimental animals) into three groups for execution: one group is treated with an empty carrier as the control group (n=8), and one group is treated with the vaccine according to the embodiment of the present invention (BD14A) Medication; one group is administered with the vaccine and IL-7 (BD14A+IL-7) according to the embodiment of the present invention. The TC-1 cancer cells of Experimental Example 2-2 were ^{injected into the vagina in an amount of 1×10 5} cells per animal to prepare tumor-induced in situ tumor mimics, and the vaccine (4μg) or vaccine (4μg) and IL-7 (50μg) was administered 3 times intramuscularly from the day of cancer cell inoculation (that is, on the 7th, 14th, and 28th day), and it will be administered at seven-day intervals from the day of cancer cell inoculation The tumor volume was measured (Figure 10A).

As shown in the results confirmed in Fig. 10B, it can be confirmed that the administration of BD14 significantly improved the anticancer effect when compared with the grouping of PBS administration when performed with the in-situ tumor mimic. This confirms that the results are consistent with those obtained in experiments using subcutaneously injected ectopic tumors. More interestingly, when BD14 and IL-7 are used together, the anticancer effect is significantly improved compared to when BD14 is used alone. This indicates that it is possible to co-administer the HPV DNA vaccine according to the embodiment of the present invention and cytokines having a different mechanism of action from T cells.

As described above, BD-14 (that is, the multivalent HPV DNA vaccine according to the embodiment of the present invention), although it has a complicated structure, can still express E6/E7 hybrid sequenced proteins normally. (That is, the antigen protein contained therein) and can successfully induce the actual T cell specific immune response of various types of HPV E6/E7 antigens. As a result of the analysis of the anti-cancer effect of a cancer model expressing HPV16 E6/E7 antigen, BD-14 showed that it has the same or better anti-cancer effect compared with the traditional bivalent DNA vaccine. In addition, the co-administration of the multivalent HPV DNA vaccine according to the embodiment of the present invention and BD-121A (that is, the vaccine adjuvant according to the embodiment of the present invention) will significantly increase the T cell specific immune response of HPV E6/E7 antigen, and also Will show a more significant anti-cancer effect.

Therefore, the vaccine component containing the multivalent HPV DNA vaccine according to the embodiment of the present invention, the DNA vaccine, and BD-121 or BD-121A as vaccine adjuvants may be effectively used to prevent various HPV infections, and to treat diseases such as cervical cancer. HPV infection symptoms such as the risk of fatal diseases.

Although the new multivalent HPV vaccine components have been described with reference to specific examples, those are only suitable for the purposes illustrated in the diagram. Therefore, professionals familiar with the field understand that various modifications and changes can be made without departing from the spirit and scope of the present invention defined by the additional patent application scope. Therefore, the actual protection scope of the present invention should be determined by the accompanying patent application scope. The scope of technology is determined.

Figure 1 is a series of schematic diagrams showing the structures of three fusion proteins (BD-14A, BD-14B and BD-14C) included in the multivalent HPV DNA vaccine according to the examples; Figure 2A is a schematic diagram showing the structures according to the example The structure of the vaccine adjuvant BD-121; Fig. 2B is a schematic diagram showing the structure of the vaccine adjuvant BD-121A according to the examples; Fig. 3A is a series of diagrams showing how to use each BD-14A, BD-14B And BD-14C plastid according to the ELISA test of the expression level of COS-7 converted fms tyrosine kinase-3 (hereinafter referred to as Flt3L); Fig. 3B is an image showing the destruction of cells according to Fig. 3A The cell lysate was analyzed by immunoblotting, using an anti-Flt3L antibody; Figure 4A shows the vaccination schedule of the experiment of the immunoreaction analysis of the multivalent HPV DNA vaccine (BD-14) according to the example; Figure 4B is a graph showing the results of enzyme-linked immunospot analysis, in which spleen cells extracted from mice vaccinated with the multivalent HPV DNA vaccine (BD-14) according to the examples and the traditional bivalent HPV DNA vaccine are analyzed These vaccines will specifically respond to E6/E7 of various types of HPV; Figure 5A shows the experiment based on the analysis of the anti-cancer effect of the multivalent HPV DNA vaccine (BD-14) against cancers induced by HPV. Vaccination schedule; Figure 5B is a graph showing the volume change of tumor tissue in mice with tumor xenotransplantation over time. These mice were worn with empty bodies (pGX27), multivalent according to the examples HPV DNA vaccine (BD-14), and the traditional bivalent HPV DNA vaccine for vaccination; Figure 5C is a chart showing the survival rate recorded based on the time of tumor xenotransplantation of mice. These mice were vaccinated with empty bodies (pGX27) and implemented according to Example of the multivalent HPV DNA vaccine (BD-14) and the traditional bivalent HPV DNA vaccine for vaccination; Figure 6A is a series of charts showing how to use the vaccine adjuvant (that is, a hBD- 121 structure) ELISA test results of the concentration of IL-12 and IL-21 in the culture supernatant transferred to COS-7 cells; Figure 6B is a series of graphs illustrating the use of vaccine adjuvants ( That is to say, an ELISA test result of the concentration of IL-12 and IL-21 in the culture supernatant of a mBD-121 structure) transferred to COS-7 cells. Figure 7A shows the antibody used to confirm the composition of the DNA vaccine according to the example. Vaccination schedule plan for cancer effect; Figure 7B is a chart showing that when the multivalent HPV DNA vaccine (BD-14) is used alone or in combination with the vaccine adjuvant (BD-121A) according to the examples, some special The results of enzyme-linked immunospot analysis on spleen cells exhibiting immune responses to various HPV E6/E7; Figure 8A shows the vaccination for comparing the anti-cancer effects between the DNA vaccine components of the example and the traditional bivalent vaccine components Time course plan; Figure 8B is a chart showing the comparison results of the volume changes of tumor tissue over time when the multivalent HPV DNA vaccine components according to the examples are used; Figure 8C is a chart showing that When the multivalent HPV DNA vaccine components according to the examples are administered, the survival rate of experimental animals is compared over time; Figure 9A shows the vaccination time course of the animal experiment confirming the mechanism of action of the multivalent HPV DNA vaccine components according to the examples Plan; Figure 9B is a chart showing that when the multivalent HPV DNA vaccine components are administered according to the embodiment, the experimental animals are treated with anti-CD4 antibodies and anti-CD8 antibodies to remove CD4 T cells and CD8 T cells, Changes in tumor volume over time in this situation; Figure 9C shows the survival rate of the experimental animals when the multivalent HPV DNA vaccine components are administered to the experimental animals according to the embodiment, after a certain period of time, the CD4 T cells and CD8 are removed by administering anti-CD4 antibodies and anti-CD8 antibodies, respectively. T cell; Figure 10A shows the vaccine inoculation schedule of the animal experiment used to check the anti-cancer effect when the HPV DNA vaccine component according to the embodiment is combined with IL-7 medicine; and Figure 10B is a series of charts showing When the HPV DNA vaccine component according to the embodiment is administered alone or in combination with IL-7, the results of the examination regarding the change in the volume of cancer cells over time.

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Claims (14)

A multivalent human papillomavirus (HPV) DNA vaccine component, which contains polynucleotides that can encode 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, respectively Early protein antigen 6 (E6) of type HPV or immunogenic fragments thereof; and early protein of type 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 Antigen 7 (E7) or its immunogenic fragments, of which E6 and E7 do not have wild-type functions; wherein E6 and E7 are divided into N-terminal fragments and C-terminal fragments respectively, and expressed in the form of E6/E7 hybrid sequence antigen units; wherein E6 /E7 hybrid sequence antigen unit is a polypeptide, the N-terminal fragment and C-terminal fragment of E6 and E7 are the N-terminal fragment of E6 (E6N)-the C-terminal fragment of E7 (E7C)-the N-terminal fragment of E7 (E7N)-E6 The sequence of the C-terminal fragments of (E6C) are connected to each other. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 1, wherein at least two E6/E7 hybrid sequence antigen units of the human papilloma virus are linked to each other in the form of an expressed fusion protein. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 2, wherein the E6/E7 hybrid sequence antigen unit or fusion protein further contains a signal sequence. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 2, wherein the E6/E7 hybrid sequence antigen unit or fusion protein also contains Flt3L. The multivalent human papillomavirus (HPV) DNA vaccine component as described in claim 1, which also contains IL-7. The multivalent human papillomavirus (HPV) DNA vaccine component as described in claim 1, which also contains at least one vaccine adjuvant acceptable to the pharmaceutical industry. The multivalent human papillomavirus (HPV) DNA vaccine component as described in claim 6, wherein the vaccine adjuvant is a vaccine adjuvant used to simulate the specific immune response of T lymphocytes, including IL-12 protein and IL-21 Protein as an effective ingredient, or contains a polynucleotide encoding IL-12 protein or a polynucleotide encoding IL-21 protein as an effective ingredient. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 7, wherein the vaccine adjuvant used to simulate the specific immune response of T lymphocytes contains at least one component selected from the following group: a p35 IL-12 protein and IL-21 protein composed of link (IL-12p35) and p40 link (IL-12p40); one to three vectors, each of which contains a p35 chain that encodes a constructable IL-12 protein, respectively Polynucleotides that bind (IL-12p35) and p40 (IL-12p40); a polynucleotide encoding IL-21 protein; and mRNA molecules, each of which encodes IL-12p35, IL-12p40, and IL -21 protein. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 7, wherein the IL-12p35 protein is human IL-12p35 composed of the amino acid sequence SEQ ID NO:1. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 7, wherein the IL-12p40 protein is human IL-12p40 composed of the amino acid sequence SEQ ID NO: 2. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 7, wherein the IL-21 protein is human IL-21 composed of the amino acid sequence SEQ ID NO: 3. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 7, wherein the vaccine adjuvant further comprises at least one component selected from the following group: i) a MIP-1 α protein; ii) a MIP -1 α gene structure, in which the polynucleotide encoding MIP-1 α protein can be operably linked to the promoter; iii) a composite gene structure in which the polynucleotide encoding MIP-1 α protein can encode ribosomes The polynucleotide or linker peptide of the entry site (IRES) can be operably linked to at least one of IL-12p35, IL-12p40 and IL-21 proteins; and iv) an mRNA molecule encoding MIP-1α protein. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 12, wherein the MIP-1 α gene structure is contained in a separate expression vector, or one to three types each contain a protein that encodes and constructs IL-12 Any one or more polynucleotides in the p35 link (IL-12p35) and p40 link (IL-12p40) vector; and a polynucleotide encoding IL-21 protein. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 13, wherein the MIP-1 α protein is a human MIP-1 α protein composed of the amino acid sequence SEQ ID NO: 10.

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