HK1252391B - A dna vaccine for preventing and treating hsv-2 infection - Google Patents

Description

DNA vaccines for the prevention and treatment of HSV-2 infection

Technical Field

This application claims priority under 35 U.S.C. §119 to Korean Patent Application No. 10-2016-0125149, filed on September 28, 2016, and all rights arising therefrom, 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.

Background Art

Herpes simplex virus-2 (HSV-2), a member of the Herpesviridae family, is a DNA virus that causes skin lesions. Worldwide, 500 million people are infected with HSV-2, and 23 million new infections are estimated annually. In the United States, one in five adults is reportedly infected with HSV-2, and infection rates are also relatively high in most countries, including the BRIC countries (Brazil, Russia, India, and China). HSV-2 infects through damaged areas of the mucous membranes or skin, causing symptoms such as viral necrosis in the infected area during viral replication. As a specific disease, HSV-2 is the leading cause of genital ulcers, which are small, fluid-filled collections of blisters that, when ruptured, result in sore scars. HSV-2 also causes symptoms such as fever, overall sickness, muscle pain, painful urination, and vaginal discharge. HSV-2 invades the dorsal root ganglia along the axons of sensory neurons and undergoes a latent period that can last for years or life, allowing for repeated reactivation. Because HSV-2 can spread from person to person even when it is asymptomatic, it is very difficult to control the spread of the virus. In fact, the spread of serological HSV-2 positive individuals increased by 30% from 1976 to 1994. Unless the spread of further infection is prevented, the infection rate will increase to 39% for men and 49% for women by 2025. In addition, 60% of newborns born to women infected with HSV-2 die and 20% of them have serious sequelae in the nervous system or eyes, and HSV-2 infected people have a three times higher probability of contracting HIV, which has become a serious problem for global public health. In addition, HSV-2 infection is becoming a huge economic burden. For example, the annual direct medical expenses for treating HSV-2 infection in the United States in 1996 were estimated to be between $283 million and $984 million.

Summary of the Invention

Technical issues

Unfortunately, there is still no therapy that can fundamentally treat HSV-2 infection, and symptomatic treatment options are limited. Antiviral agents (such as famciclovir, valacyclovir or acyclovir) that inhibit viral replication by interfering with viral DNA synthesis have been used. When these drugs are applied in the early stages of viral infection, clinical symptoms can be alleviated or viral transfer can be reduced, but the recurrence and spread of viral infection cannot be prevented and it is difficult to cure. For example, acyclovir can reduce HSV-2 propagation, but it cannot prevent the latent infection of HSV-2 in ganglia. In addition, antiviral therapy causes many side effects, including nausea, vomiting and decreased renal function. Because existing antiviral drugs do not meet medical needs, new multiple vaccines for the prevention and treatment of HSV-2 have been attempted, but they are all ineffective. Conventional vaccines containing inactivated viruses, attenuated viruses (reduced live viruses), modified live viruses and cell culture-derived subunits are usually unsuccessful or have extremely low effectiveness. The clinical trial results of the latest three candidate vaccines (i.e., the vaccine developed by Chiron, the vaccine developed by GlaxoSmithKline (GSK), and the vaccine developed by Vical) showed that they were ineffective. The Chiron vaccine consists of gD and gB2, a truncated form of HSV-2 conjugated with the adjuvant MF59, which showed the production of high-titer antibodies against HSV-2, but the effect of the Chiron vaccine was temporary. The GSK vaccine contains alum and MPL as adjuvants as well as gD, which also failed to prevent and treat HSV-2 infection. Finally, the vaccine from Vical contains the adjuvant Vaxfectin as an adjuvant and gD or UL46 and UL47, which produced high-titer antibodies in animal experiments. However, in clinical trials, HSV-2 infection did not show any difference from the control group. It can be confirmed from the aforementioned cases that the high-titer antibody response to HSV-2 is not enough to prevent and treat HSV-2 infection. Therefore, it is necessary to develop a therapeutic agent based on a new immune mechanism against HSV-2 infection.

Technical solutions to problems

To address the above-mentioned various problems, the present disclosure provides a new DNA vaccine that can effectively prevent and treat HSV-2 infection.

However, the present disclosure is not limited thereto.

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

According to yet another exemplary embodiment, the present disclosure provides a vector comprising the polynucleotide.

According to yet another exemplary embodiment, the present disclosure provides an isolated host cell comprising the vector.

According to yet another exemplary embodiment, the present disclosure provides a composition comprising the polynucleotide or the vector or a pharmaceutically acceptable carrier.

According to yet another exemplary embodiment, the present disclosure provides a recombinant protein encoded by the polynucleotide.

According to yet another exemplary embodiment, the present disclosure provides an expression vector comprising one or more polynucleotides encoding two or more or all of the HSV-2 antigenic proteins selected from gB, gD, UL39, ICPO and ICP4 proteins.

According to yet another exemplary embodiment, the present disclosure provides a DNA vaccine composition comprising the polynucleotide, the vector, or the expression vector.

Advantageous Effects of the Invention

The DNA construct according to one embodiment of the present disclosure is capable of expressing recombinant UL39 and fusion proteins comprising the recombinant UL39 and conventional HSV-2 antigens, exhibits effective ability to protect against inflammation, and thus can be very effectively used as a vaccine for the prevention and treatment of HSV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic diagram illustrating the structure of a shuffled UL39 protein according to one embodiment of the present disclosure.

2 shows a schematic diagram illustrating an overview of animal experimental procedures using plasmid DNA designed to express a shuffled UL39 protein according to one embodiment of the present disclosure.

3 shows a graph illustrating the survival rate of animals infected with HSV-2 by administering plasmid DNA designed to express a shuffled UL39 protein according to the present disclosure and mock plasmid DNA as a negative control.

4 shows schematic diagrams illustrating the structures of each of the fusion proteins expressed by tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA according to one embodiment of the present disclosure.

Figure 5 shows a schematic diagram illustrating the experimental timeline for HSV-2 infection according to one embodiment of the present disclosure in the following groups: a group administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA, respectively; a group administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA together; and a group administered with mock plasmid DNA as a negative control.

6a is a graph showing the survival rates of the following experimental animal groups according to one embodiment of the present disclosure: a group infected with HSV-2 and administered with tPA-Flt3L-gB-UL39 plasmid DNA (-) and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA, respectively; a group co-administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA; a group administered with mock plasmid DNA as a negative control; and FIG6b is a graph showing the pathological scores of the experimental animal groups of FIG6a .

DETAILED DESCRIPTION

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

The term "shuffled" as used herein refers to mixing the order of domains when multiple domains are present in a particular protein. Specifically, the term "shuffled protein" (also known as "epitope-shuffled protein") as used herein refers to a recombinant protein in which the order of multiple epitopes is randomly mixed while maintaining the activity of the multiple epitopes to be recognized by the immune system, and is an immunogenic protein that has lost its original function as a protein but retained the activity of the epitopes.

The UL39 protein used to produce the shuffled UL39 protein can be RIR1_HHV2H registered as UniProt Accession No. P89462 as a standard sequence, and various variants having the functions of the UL39 protein (e.g., UniProte Accession Nos. G91261, A0A0E3Y5Z7, A0A0E3Y7N5, A0A120I2I0, A0A110B8A6, and A0A0E3Y758) can be used without any problem. When the length of any variant is different from that of the standard sequence, the positions corresponding to the positions of the standard sequence will be used.

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

The term "immune-enhancing peptide" as used herein refers to a peptide that activates cells associated with immune response (eg, dendritic cells, etc.) and thereby enhances the immune response.

The polynucleotide may further comprise a polynucleotide encoding a secretory signal peptide that induces 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 comprise a polynucleotide encoding one or two or more antigenic proteins of herpes simplex virus type 2 (HSV-2). The antigenic protein may be glycoprotein B (gB), glycoprotein D (gD), ICPO or ICP4.

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

Similarly, for antigenic proteins, when the standard sequence is gB, GB_HHV2H registered as UniProt Accession No. P08666 is used; when the standard sequence is gD, GD_HHV2H registered as UniProt Accession No. Q69467 is used; when the standard sequence is ICP0, ICP0_HHV2H registered as UniProt Accession No. P28284 is used; and when the standard sequence is ICP4, ICP4_HHV2H registered as UniProt Accession No. P90493 is used. The amino acid positions of the antigenic proteins are based on the positions indicated in the standard sequence. Various variants that retain the activity of the antigenic protein may also be used. When the length of these variants is different from that of the standard sequence, the amino acid positions corresponding to the amino acid positions of the standard sequence will be used. Examples of various variants may include: in the case of gB, variants with UniProt Accession Nos. P06763, Q69465, D6QV12, D6QV07, etc. may be used; in the case of gD, variants with UniProt Accession Nos. P03172, T1PZZ0, A0A0Y0QWV3, A0A110AVP3, A0A0Y0RM80, etc. may be used; in the case of ICP0, variants with UniProt Accession Nos. G9I221, G9I280, W0NW81, D6PUZ6, D6PUZ3, etc. may be used; in the case of ICP4, variants with UniProt Accession Nos. G9I282, A0A0E3Y5F0, A0A0Y0RBE8, A0A120I2I2, A0A0Y0RAK6, etc. may be used.

In addition, the polynucleotide can be replaced with codons having a high expression frequency in the host cell. As used herein, the term "codons replaced with codons having a high expression frequency in the host cell" or "optimized codons" means that when DNA is transcribed and translated into protein in the host cell, among the codons determining the amino acid, there are codons with a high preference according to the host, and the expression efficiency of the amino acid or protein encoded by the nucleic acid is improved by replacing the codons with a 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. In addition, the shuffled UL39 may sequentially include the UL39-N1 peptide of SEQ ID NO: 1, the UL39-C2 peptide of SEQ ID NO: 2, the UL39-N2 peptide of SEQ ID NO: 3, the UL39 N4-C1 peptide of SEQ ID NO: 4, and the UL39-N3 peptide of SEQ ID NO: 5, and in this case, the shuffled UL39 protein may include the amino acid sequence of SEQ ID NO: 6. However, changes in the order of the peptides may not cause any problems as long as they are not identical to the amino acid sequence of the original full-length UL39 protein.

According to another aspect of the present disclosure, a vector comprising the polynucleotide is provided.

According to one embodiment of the present disclosure, the vector can be an expression vector comprising a gene construct operably linked to a regulatory sequence, thereby allowing the polynucleotide to express the reorganized UL39 protein in a host cell. The expression vector can be in any form, including but not limited to plasmid vectors, viral vectors, cosmid vectors, phagemid vectors, artificial chromosomes, etc.

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

The term "regulatory sequence" as used herein is intended to include promoters, enhancers and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include: regulatory sequences that direct constitutive expression of a target nucleic acid in many host cells, regulatory sequences that direct target nucleic acid expression only in specific tissue cells (e.g., tissue-specific regulatory sequences), and regulatory sequences that direct expression induced by a specific signal (e.g., inducible regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector can vary depending on factors such as the choice of host cells to be transformed and the desired protein expression level. The expression vectors of the present disclosure can be introduced into host cells to express fusion proteins. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these regulatory sequences typically include regulatory sequences responsible for initiating transcription, and optionally include poly A signals responsible for terminating transcription and stabilizing transcripts. In addition to transcriptional regulatory elements, additional regulatory sequences may include translation enhancers and/or naturally associated or heterologous promoter regions. For example, possible regulatory sequences that enable expression in mammalian host cells may include: CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (weak sarcoma virus), human renal component 1 α-promoter (human renalelement 1 α-promoter), glucocorticoid-inducible MMTV-promoter (Moloney murine tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifiers such as CMV or SV40-amplifiers. For expression in neurons, it is believed that neurofilament-promoters, PGDF-promoters, NSE-promoters, PrP-promoters, or thy-1-promoters may be used. Such promoters are known in the art and are described in the literature (Charron, J. Biol. Chem . 270: 25739-25745, 1995). For prokaryotic expression, many promoters have been disclosed, including lac promoter, tac promoter, and trp promoter. In addition to factors capable of initiating transcription, regulatory sequences may also include transcription termination signals, such as, for example, SV40-poly-A or TK-poly-A sites located downstream of the polynucleotide according to one embodiment of the present disclosure. In the present disclosure, suitable expression vectors are well known in the art and include, for example, Okayama-Berg cDNA expression vector 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 λgt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the present disclosure, the vector may also additionally contain a polynucleotide encoding a secretion signal peptide. Such secretion signal peptides are well known to those skilled in the art. In addition, depending on the expression system used, a leader sequence that can direct the fusion protein to a specific intercellular organelle is linked to the coding sequence of the polynucleotide according to one embodiment of the present disclosure, preferably a leader sequence that can directly secrete the translated protein into the cytoplasm or extracellular matrix.

In addition, the vectors of the present disclosure can be prepared by standard recombinant DNA techniques, which may include, for example, blunt-end ligation and sticky-end ligation, restriction enzyme treatment to provide appropriate termini, phosphorylation by alkaline phosphatase treatment to prevent improper binding, enzymatic ligation by T4 DNA ligase, etc. The vectors of the present disclosure can be prepared by recombining DNA encoding a signal peptide (which is obtained by chemical synthesis or genetic recombination technology) and a DNA encoding an HSV-2 antigen protein of the present disclosure with a vector containing an appropriate regulatory sequence. Vectors containing regulatory sequences can be purchased commercially or prepared, and in one embodiment of the present disclosure, a vector pGX27 for producing a DNA vaccine is 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 specific functions are linked, such that each protein or domain is responsible for its inherent function. Typically, a linker having a flexible structure is 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 comprising the vector is provided.

The term "host cell" as used herein includes prokaryotic cells or eukaryotic cells, and eukaryotic cells include higher eukaryotic cells (including mammals) as well as lower eukaryotic cells (including fungi, yeast, etc.).

The host cell or non-human host object transfected or transformed using a vector according to the present disclosure can be a host cell or host object genetically modified by the vector. As used herein, the term "genetically modified" means that the polynucleotide or vector according to one embodiment of the present disclosure introduced into one of the host cell, host object, or ancestor/parental generation is present outside the genome of the host cell, host object, or ancestor/parental generation. In addition, the polynucleotide or vector according to one embodiment of the present disclosure can be present outside the genome as an independent molecule, preferably a replicable molecule in a genetically modified host cell or host object, such as an episome. Alternatively, it can be stably inserted into the genome of the host cell or host object.

The host cell according to one embodiment of the present disclosure is a prokaryotic cell or a eukaryotic cell. Suitable prokaryotic cells are those commonly used for cloning, such as Escherichia coli ( E. coli ) or Bacillus subtilis ( Bacillus subtilis ). In addition, eukaryotic cells include fungi, plant cells, and animal cells. Examples of suitable fungal cells are yeast, preferably yeast of the genus Saccharomyces , and most preferably Saccharomyces 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). Host cells such as CHO cells can provide: post-translational modification of the reorganized UL39 protein according to one embodiment of the present disclosure, glycosylation of the reorganized UL39 protein at precise locations, and secretion of functional molecules. In addition, suitable cell lines known in the art are available from cell line collections such as the American Type Culture Collection (ATCC). According to the present disclosure, it is believed that primary culture cells/cell cultures can serve as host cells. These cells are especially derived from insects (insects of the genus Drosophila or Blatta ) or mammals (human, pig, mouse or rat). As mentioned above, primary culture cells can be immune cells, including 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, there is provided a composition comprising the polynucleotide or vector and a pharmaceutically acceptable carrier.

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

The term "pharmaceutically acceptable" as used herein means that the composition is physiologically acceptable and generally does not cause allergic reactions such as gastrointestinal disorders and dizziness when administered to a human. Examples of carriers, excipients, and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anticoagulants, wetting agents, moistening agents, spices, emulsifiers, preservatives, etc. may be additionally included.

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 is provided, comprising one or more polynucleotides encoding two or more or all HSV-2 antigenic proteins selected from gB, gD, UL39, ICPO and ICP4 proteins.

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

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

Furthermore, expression vectors can be prepared so that the gB, gD, UL39, ICPO, and ICP4 proteins are expressed as separate proteins or as a single fusion protein.

In the above expression vector, as described above, although the shuffled UL39 protein is a protein in which the UL39-N1 peptide of SEQ ID NO: 1, the UL39-C2 peptide of SEQ ID NO: 2, the UL39-N2 peptide of SEQ ID NO: 3, the UL39 N4-C1 peptide of SEQ ID NO: 4, and the UL39-N3 peptide of SEQ ID NO: 5 are randomly mixed, the shuffled UL39 protein may be a protein in which the peptides of SEQ ID NOs: 1 to 5 are linked in sequence. However, in this case, the shuffled UL39 protein does not include the original full-length UL39 protein.

In the above expression vectors, gB, gD, UL39, ICPO and ICP4 proteins can be expressed as separate proteins or in the form of a single fusion protein.

In the above-mentioned expression vector, the polynucleotide may further comprise a polynucleotide encoding a secretory signal peptide, and as described above, the secretory 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 immune enhancing peptide is the cytoplasmic domain of CD28, inducible costimulatory molecule (ICOS), cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte-associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signal transducing lymphocyte activation molecule (SLAM), 2B4 (CD244), natural killer cell family 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, herpes virus entry mediator (HVEM), CD40 L (ligand) or OX40L [a ligand of CD134 (OX40), CD252] or a connecting peptide of two or more thereof. The immune enhancing peptide may preferably be Flt3 ligand, and the Flt3 ligand may have the amino acid sequence of SEQ ID NO: 9.

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

Specifically, the DNA vaccine composition may contain: a first expression vector comprising a first gene construct, wherein 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, wherein 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 comprise 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 comprise an immune-enhancing peptide, and the immune-enhancing peptide is the same as described above.

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

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

As used herein, the term "adjuvant" refers to a pharmaceutical or immunological substance administered for the purpose of enhancing the immune response of a vaccine.

The adjuvant can be aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosomes, 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, ISCOM, or ISCOMMATRIX.

In addition, the vaccine composition according to one embodiment of the present disclosure can be formulated using methods known in the art to allow for rapid release, sustained release, or extended release of the active ingredient after administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard capsules, sterile injectable solutions, and sterile powders.

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

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

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

The vaccine composition according to one embodiment of the present disclosure can be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include, for example, water, suitable oil, saline, aqueous carriers (such as glucose aqueous solution, ethylene glycol aqueous solution, etc.) for parenteral administration, and the like, and may additionally include stabilizers and preservatives. Examples of suitable stabilizers may include antioxidants, such as sodium bisulfite, sodium sulfite, and ascorbic acid. Examples of suitable preservatives may include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. In addition, when needed according to administration method or preparation, compositions according to the present disclosure may suitably include suspending agents, solubilizing agents, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH regulators, analgesics, buffers, antioxidants, etc. Pharmaceutically acceptable carriers and formulations suitable for the present disclosure, including those exemplified above, are described in the literature [Remington's Pharmaceutical Sciences, latest edition].

The dosage of the vaccine composition for a patient depends on many factors, including the patient's height, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs to be administered concurrently. Pharmaceutically active DNA can be administered in an amount of 100 ng/kg to 10 mg/kg of body weight (kg), more preferably 1 μg/kg to 500 μg/kg (body weight), and most preferably 5 μg/kg to 50 μg/kg (body weight), and can be administered in a unit dose of 10 μg, 100 μg, 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 the dosage can be adjusted to take into account the above factors.

Example

Hereinafter, the present disclosure is described 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 can be implemented in various other forms. 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 belongs.

Example 1: Preparation of shuffled UL39 plasmid DNA

Based on the inventors' previous study that UL39 (ICP10), one of the HSV-2 antigens, induces CD4 ⁺ T cell and CD8 ⁺ T cell responses (Posavad et al. , Mucosal Immunol . 126, 2015), the inventors of the present disclosure investigated whether a shuffled construct mixed with the internal domain of UL39 could act as a vaccine to induce an immune response.

To this end, the present inventors designed a shuffled UL39 antigen as a mixed protein of five fragments of the UL39 antigen (N1: UL39 _{14-154 (Δ78-104)} , C2: UL39 _1117-1142 , N2: UL39 _155-227 , N4-C1: UL39 _399-1116 , N3: UL39 _208-398 ) based on the pGX27 plasmid vector (Korean Patent No. 1442254) that has been modified to enhance expression capacity. They also prepared a plasmid vector containing a gene construct encoding the shuffled UL39 antigen and named this plasmid vector shuffled-UL39 plasmid DNA.

Specifically, shuffled-UL39 plasmid DNA was prepared by inserting the following gene construct into the pGX27 plasmid vector. The gene construct comprises a polynucleotide (SEQ ID NO: 7) encoding UL39-N1, UL39-C2, UL39-N2, UL39-N4-C1 and UL39-N3 (SEQ ID NO: 6) in a sequentially linked order, which is based on the form of 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) ( Figure 1 ).

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

To confirm whether the shuffled-UL39 plasmid DNA according to one embodiment of the present disclosure effectively protects against HSV-2 infection in vivo in an infected animal model, the inventors of the present disclosure evaluated the ability to protect against HSV-2 infection after administration of the plasmid DNA vaccine.

Specifically, C57BL/6 mice were divided into two groups, one receiving mock plasmid DNA and the other receiving recombinant-UL39 plasmid DNA. Each group received the corresponding plasmid DNA (4 μg) intramuscularly via electroporation twice, two weeks apart. Two weeks after the last administration, the mice were infected intravaginally with HSV-2 (1 x 10 ⁴ pfu) (Figure 2 and Table 1). Following infection, the survival rate of mice in each group was monitored for 10 days to assess the viability of the HSV-2-infected groups (Figure 3).

[Table 1]

Group vaccine Number of experimental animals Dosage (μg) Route (method of administration) control group mock plasmid 8 4 Intramuscular (electroporation) Experimental group Restructured UL39 8 4 Intramuscular (electroporation)

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

Example 2: Preparation of plasmid DNA expressing HSV-2 composite 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 therefore, prepared plasmid DNA recombined with other antigenic proteins.

Specifically, based on the pGX27 plasmid vector used in Example 1, the following were prepared: tPA-Flt3L-gB-UL39 plasmid DNA, comprising a polynucleotide (SEQ ID NO: 12) encoding a tPA-Flt3L-gB-UL39 fusion protein (SEQ ID NO: 11), in which various types of HSV-2 antigens (i.e., glycoprotein B (gB 23-904 ( _Δ772-792 ) from which the signal sequence (gB _1-22 ) and the transmembrane domain (gB _772-792) were removed, SEQ ID NO: 10) and the shuffled UL39 used in Example 1 (SEQ ID NO: 6)) were linked; and tPA-Flt3L-gD-IPC0-ICP4 plasmid DNA, comprising a polynucleotide (SEQ ID NO: 16) encoding a tPA-Flt3L-gD-IPC0-ICP4 fusion protein (SEQ ID NO: 17) were prepared. 17), in which the signal sequence (gD _1-25 ) and transmembrane domain (gD _341-364 ) of glycoprotein D were removed (gD _{6-393 (Δ341-364)} , SEQ ID NO: 13), the nuclear localization signal (NLS, ICP0 _510-516 ) of infected cell polypeptide 0 (ICP0 _Δ510-516 , SEQ ID NO: 14), and the RS1.3 portion (ICP4 _767-1318 ) of infected cell polypeptide 4 (ICP4 _Δ767-1318 , SEQ ID NO: 15) were removed. For efficient expression, both plasmids were in a form in which a codon-optimized tPA secretion signal peptide (SEQ ID NO: 8) and immune system activation protein FMS-like tyrosine kinase 3 ligand (Flt3L, SEQ ID NO: 9) were added to the N-terminus (Figure 4).

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

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

Specifically, 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, 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 4 μg of each corresponding plasmid DNA twice at two-week intervals via in vivo electroporation. Two weeks thereafter, the mice were infected intravaginally with HSV-2 (1×10 ⁴ pfu) ( FIG5 and Table 2 ). Survival rates and pathology scores of the mice in each group were monitored daily for 20 days after infection to evaluate their ability to protect against HSV-2 infection ( FIG6A and 6B ). Pathological scoring was performed according to the description in the published literature (Oh et al., Proc. Natl. Acad. Sci. USA. 113(6): E762-E771, 2016; pathological score "0", asymptomatic; "1", mild genital erythema and edema; "2", moderate genital inflammation; "3", purulent genital lesions; "4", hind limb paralysis; "5", death).

[Table 2]

Group vaccine Number of mice Dosage (μg) Route of administration (method of administration) control group simulation 10 8 Intramuscular (electroporation) Experimental Group 1 gB-UL39 (BD-02B) 10 4 + 4 (analog) Intramuscular (electroporation) Experimental Group 2 gD-ICP0/ICP4 10 4 + 4 (analog) Intramuscular (electroporation) Experimental Group 3 gD-ICP0/ICP4 + gB-UL39 10 4 + 4 Intramuscular (electroporation)

As shown in Figure 6a, all animals in the group given mock plasmid DNA died on the 10th day of HSV-2 virus infection. In contrast, significantly improved protection against HSV-2 virus was confirmed in the groups given tPA-Flt3L-gB-UL39 plasmid DNA or tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA. The mice in the group given tPA-Flt3L-gB-UL39 plasmid DNA showed a 40% survival rate, while the mice in the group given ICPO-ICP4 plasmid DNA showed a 100% survival rate. Mice co-administered with tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA also showed a 100% survival rate.

6b, the group administered with mock plasmid DNA showed a high pathology score, while the group administered with tPA-Flt3L-gB-UL39 plasmid DNA showed a significantly lower pathology score than the group administered with mock plasmid DNA, but showed a higher pathology score than the group administered with tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA. Furthermore, the group administered with both tPA-Flt3L-gB-UL39 plasmid DNA and tPA-Flt3L-gD-ICP0-ICP4 plasmid DNA showed a significantly improved pathology score compared to the groups administered with the individual plasmid DNAs.

As described above, DNA that can express the shuffled UL39 according to one embodiment of the present disclosure and DNA that can express a fusion protein combining the shuffled UL39 antigen and the conventional HSV-2 antigen show effective ability to protect against inflammation, and therefore they can be used very effectively as vaccines for the prevention and treatment of HSV-2.

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

Industrial Applicability

The polynucleotides according to the present disclosure can be used to prepare DNA vaccine compositions for preventing and treating HSV-2 infection.

Sequence Listing Free Text

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO: 14 is the amino acid sequence of ICP0 _Δ510-516 protein.

SEQ ID NO: 15 is the amino acid sequence of ICP4 _Δ767-1318 protein.

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

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

Claims (34)

1. A polynucleotide encoding a modified UL39 protein, wherein the modified UL39 protein comprises the following five peptides: The UL39-N1 peptide, composed of the amino acid sequence represented by SEQ ID NO: 1; The UL39-C2 peptide, composed of the amino acid sequence represented by SEQ ID NO: 2; The UL39-N2 peptide, composed of the amino acid sequence represented by SEQ ID NO: 3; The UL39 N4-C1 peptide, composed of the amino acid sequence represented by SEQ ID NO: 4; and The UL39-N3 peptide, composed of the amino acid sequence represented by SEQ ID NO: 5, The UL39-N1 peptide, the UL39-C2 peptide, the UL39-N2 peptide, the UL39 N4-C1 peptide, and the UL39-N3 peptide are connected in sequence. 2. The polynucleotide of claim 1, further comprising a polynucleotide encoding one, two or more immune-enhancing peptides. 3. The polynucleotide of claim 2, wherein the immune-enhancing peptide is the cytoplasmic domain of CD28, inducible co-stimulatory molecule (ICOS), cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte-associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signal transduction lymphocyte activation molecule (SLAM), 2B4 (CD244), natural killer cell family 2 member D (NKG2D)/DNAX-activating protein 12 (DAP12), protein 1 containing T-cell immunoglobulin and mucin domains (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family-associated protein (GITR), and fms-like tyrosine kinase 3. (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 ligand or OX40 ligand or two or more of these linking peptides. 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 more antigenic proteins of herpes simplex virus-2 (HSV-2). 7. The polynucleotide of claim 6, wherein the antigenic 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 amino acids 1-22 and a transmembrane domain corresponding to amino acids 772-792 are missing. 9. The polynucleotide of claim 7, wherein the glycoprotein D is a truncated form in which a signal peptide corresponding to amino acids 1-25 and a transmembrane domain corresponding to amino acids 341-364 are missing. 10. The polynucleotide of claim 7, wherein the ICP0 is a truncated form in which the nuclear localization signal (NLS) corresponding to amino acids 510-516 is missing. 11. The polynucleotide of claim 7, wherein the ICP4 is a truncated form in which the RS1.3 region corresponding to amino acids 767-1318 is omitted. 12. The polynucleotide according to any one of claims 1-11, wherein the polynucleotide is a codon-optimized polynucleotide. 13. The polynucleotide according to any one of claims 1-11, wherein the UL39 protein is a polynucleotide consisting of the amino acid sequence represented by SEQ ID NO: 6. 14. A vector comprising a polynucleotide according to any one of claims 1-11. 15. The vector of claim 14, wherein the vector is an expression vector in which the polynucleotide is operatively linked to the promoter. 16. An isolated host cell comprising the vector of claim 14 or 15. 17. A composition comprising a polynucleotide according to any one of claims 1-11 or a carrier according to claim 14 or 15, and a pharmaceutically acceptable carrier. 18. A recombinant protein encoded by a polynucleotide according to any one of claims 1-11. 19. An expression carrier comprising Polynucleotides encoding the ICP4 protein of HSV-2; Polynucleotides encoding the gB protein of HSV-2; Polynucleotides encoding the gD protein of HSV-2; The polynucleotide encoding the reorganized UL39 protein of claim 1; and Polynucleotides encoding the ICP0 protein of HSV-2. 20. The expression vector of claim 19, wherein the gB is a truncated form in which the signal sequence corresponding to amino acids 1-22 and the transmembrane domain corresponding to amino acids 772-792 are missing. 21. The expression vector of claim 19, wherein the gD is a truncated form in which the signal sequence corresponding to amino acids 1-25 and the transmembrane domain corresponding to amino acids 341-364 are missing. 22. The expression vector of claim 19, wherein the ICP0 is a truncated form in which the nuclear localization signal (NLS) corresponding to amino acids 510-516 is missing. 23. The expression vector of claim 19, wherein the ICP4 is a truncated form in which the RS1.3 region corresponding to amino acids 767-1318 is missing. 24. The expression vector according to any one of claims 19-23, wherein the gB, gD, shuffled UL39, ICP0 and ICP4 proteins are expressed as individual proteins or as a fusion protein. 25. The expression vector according to any one of claims 19-23, wherein the polynucleotide further comprises a polynucleotide encoding a secretion signal peptide. 26. The expression vector of claim 25, 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. 27. The expression vector according to any one of claims 19-23, wherein the polynucleotide further comprises a polynucleotide encoding an immune-enhancing peptide. 28. The expression vector of claim 27, wherein the immune-enhancing peptide is the cytoplasmic domain of CD28, inducible co-stimulatory molecule (ICOS), cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte-associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signal transduction lymphocyte activation molecule (SLAM), 2B4 (CD244), natural killer cell family 2 member D (NKG2D)/DNAX-activating protein 12 (DAP12), protein 1 containing T-cell immunoglobulin and mucin domains (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family-associated protein (GITR), and fms-like tyrosine kinase 3. (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 ligand or OX40 ligand or two or more of these linking peptides. 29. A DNA vaccine composition comprising an expression vector according to any one of claims 19-23. 30. The DNA vaccine composition of claim 29, comprising: A first expression vector comprising a first gene construct, the first gene construct containing a first polynucleotide operatively linked to a promoter to encode a first fusion protein comprising gB and shuffled UL39; and A second expression vector containing a second gene construct, the second gene construct containing a second polynucleotide encoding a second fusion protein including gD, ICP0 and ICP4, which is operatively linked to a promoter. 31. The DNA vaccine composition of claim 30, wherein the first fusion protein and/or the second fusion protein further comprises a secretion signal peptide. 32. The DNA vaccine composition of claim 31, wherein the secretory 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. 33. The DNA vaccine composition of claim 31, wherein the first fusion protein and/or the second fusion protein further comprises an immune-enhancing peptide. 34. The DNA vaccine composition of claim 33, wherein the immune-enhancing peptide is a cytoplasmic domain of CD28, an inducible co-stimulatory molecule (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), B-lymphocyte and T-lymphocyte-associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signal transduction lymphocyte activation molecule (SLAM), 2B4 (CD244), natural killer cell family 2 member D (NKG2D)/DNAX-activating protein 12 (DAP12), protein 1 containing T-cell immunoglobulin and mucin domains (TIM1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3 (LAG3), B7-1, B7-H1, glucocorticoid-induced TNFR family-associated protein (GITR), and fms-like tyrosine kinase 3. (Flt3) ligand, flagellin, herpesvirus entry mediator (HVEM), CD40 ligand or OX40 ligand or two or more of these linking peptides.