Vaccine having interleukin-21 as an antigen and an adjuvant

Abstract

Provision of vaccines containing antigens and IL-21. [Solution] A vaccine comprising an antigen and IL-21 is disclosed herein. Methods for enhancing the immune response in a subject are also disclosed herein. The methods may include administering the vaccine to a subject in need.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 058,304, filed on October 1, 2014, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a vaccine comprising an antigen and IL - 21, and a method of administering such a vaccine.

Background Art

[0003] Vaccines are used to stimulate an immune response in an individual and provide protection and / or treatment against specific diseases. Some vaccines contain antigens to induce an immune response. There are antigens that induce a strong immune response and antigens that induce a weak immune response. A weak immune response to an antigen can be enhanced by including an adjuvant in the vaccine. Adjuvants come in many different forms such as, for example, aluminum salts, oil emulsions, inactivated components of bacteria or other pathogens, cytokines, etc.

[0004] Cytokines are proteins made by cells that affect the behavior of other cells and, unlike many adjuvants, can regulate specific immune responses. One such cytokine is interleukin - 21 (IL - 21), which acts on lymphocytes and myeloid populations as well as epithelial cells and regulates innate and adaptive immune responses. IL - 21 has been shown to contribute to the functional differentiation of several CD4 + T cell subsets, promote the proliferation and functional responses of CD8 + T cells, and play a role in the development of B cell immunoglobulin responses. IL - 21 is produced by CD8 + T cell populations as well as CD4 + T cell populations, including follicular helper T (TFH) cells, type 17 helper T (Th17) cells, and natural killer T (NKT) cells.

[0005] In addition, vaccines are administered to many different tissues (e.g., intramuscular, intradermal, etc.) in many different ways (e.g., injection, oral, etc.). However, not all delivery methods are equal. While there are delivery methods that allow for higher compliance within a population of individuals, there are also delivery methods that can affect the immunogenicity and / or safety of the vaccine. Therefore, there remains a need in the art for the development of safe and more effective adjuvants that enhance the antigenic response regardless of antigen identity and route of administration.

Summary of the Invention

[0006] The present invention is directed to a vaccine comprising an antigen and IL-21. IL-21 may be encoded by a nucleotide sequence having at least about 95% identity to the nucleotide sequence set forth in SEQ ID NO: 3 and a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 3. IL-21 may be encoded by the nucleotide sequence set forth in SEQ ID NO: 3.

[0007] The antigen may be encoded by a first nucleic acid, and IL-21 may be encoded by a second nucleic acid. The second nucleic acid may further comprise an expression vector. The vaccine may further comprise the same encoded nucleic acid sequence and antigen peptide as the antigen, and the same encoded nucleic acid sequence and IL-21 peptide as the IL-21.

[0008] The antigen can be selected from the group consisting of human papillomavirus (HPV) antigen, human immunodeficiency virus (HIV) antigen, influenza antigen, Plasmodium falciparum antigen, Clostridium difficile antigen, and fragments thereof. The HPV antigen is selected from the group consisting of HPV16 E6 antigen, HPV16 E7 antigen, and combinations thereof. The HIV antigen is selected from the group consisting of EnvA, EnvB, EnvC, EnvD, B Nef-Rev, Gag, and any combination thereof. The influenza antigen may be selected from the group consisting of H1 HA, H2 HA, H3 HA, H5 Ha, BHA antigen, and any combination thereof. The Plasmodium falciparum antigen may include circumsporozoite antigen. The Clostridium difficile antigen may be selected from the group consisting of toxin A, toxin B, and combinations thereof.

[0009] The vaccine may further contain a pharmaceutically acceptable excipient.

[0010] The present invention also targets a method for enhancing an immune response in a subject in need of an immune response. The method can include administration of a vaccine containing an antigen and IL-21. IL-21 may be encoded by a nucleotide sequence having at least about 95% identity with the nucleotide sequence set forth in SEQ ID NO: 3 and a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 3. IL-21 may be encoded by the nucleotide sequence set forth in SEQ ID NO: 3.

[0011] Administration of the vaccine may include electroporation. Enhancement of the immune response in the subject may include enhancement of a cellular immune response, a humoral immune response, or both a cellular and humoral immune response in the subject.

[0012] The present invention further targets a nucleic acid molecule comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 3 and nucleotide sequences that are 95% or more identical to SEQ ID NO: 3. The nucleic acid molecule may be a plasmid.

Brief Description of the Drawings

[0013]

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[0014] The present invention relates to a vaccine that can be used to enhance an immune response against an antigen in a subject by using IL-21 as an adjuvant. IL-21 is a single-chain T cell-inducing cytokine.

[0015] In some examples, IL-21 can function as a universal adjuvant because it induces a higher immune response in a subject regardless of the antigen source or administration route compared to a vaccine containing the antigen alone. IL-21 can further increase the immune response to both viral and bacterial antigens, such as HIV antigens and Clostridium difficile antigens, respectively. IL-21 can further take the form of a DNA / peptide combination with an antigen, which is a nucleic acid / peptide combination that gives a greater immune response compared to DNA or peptide alone with an antigen. In some examples, IL-21 can further increase the immune response in both muscle tissue and skin tissue, as shown by an increase in interferon-γ (IFN-γ) production.

[0016] 1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0017] The terms "comprise", "include", "having", "has", "can", "contain", and their variants, as used herein, are intended to be open transitional terms, terms, or phrases that do not exclude the possibility of further acts or structures. The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements present herein, whether or not explicitly recited.

[0018] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0019] As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) that includes a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in a cell of an individual or mammal to which the nucleic acid is administered.

[0020] As used herein, "complement" or "complementary" means Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0021] As used interchangeably herein, "electroporation", "electropermeabilization", or "electrokinetic enhancement" ("EP") refers to the use of transmembrane electric field pulses that induce microscopic passages (pores) in biological membranes, the presence of which enables biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other side.

[0022] As used herein, "fragment" or "immunogenic fragment" means a nucleic acid sequence or a part thereof that encodes a polypeptide capable of inducing and / or enhancing an immune response in a mammal. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding the protein fragments described below. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences described below. In some embodiments, the fragment can comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, or at least 1000 nucleotides or more of at least one of the nucleic acid sequences described below.

[0023] As used herein, a fragment or immunogenic fragment also means a polypeptide sequence or a portion thereof that can induce and / or enhance an immune response in a mammal. The fragment may be a polypeptide fragment selected from at least one of the various amino acid sequences described below. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the proteins described below. In some embodiments, the fragment can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of at least one of the proteins described below.

[0024] As used herein, the term "gene construct" or "construct" refers to a DNA or RNA molecule that contains a nucleotide sequence encoding a protein. The coding sequence contains a start signal and a stop signal operably linked to regulatory elements including a promoter and a polyadenylation signal that can direct expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct or construct that contains the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence is expressed when present in the cells of an individual.

[0025] In the context of two or more nucleic acid or polypeptide sequences, as used herein, "identical" or "identity" means that a sequence has a certain percentage of residues that are identical in a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences in a particular region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment generates one or more gapped ends and the particular region being compared contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined using manual methods or computer sequence algorithms such as BLAST or BLAST 2.0.

[0026] As used herein, "immune response" means the activation of the host's immune system, such as that of a mammal, in response to the introduction of an antigen. The immune response can be in the form of a cellular response, a humoral response, or both.

[0027] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. A single-stranded representation also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the described single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complementary sequences. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. That is, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0028] The nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid can be either DNA, RNA, or a hybrid of both genomic and cDNA, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acid can be obtained by chemical synthesis or recombinant methods.

[0029] As used herein, "functionally linked" means that the expression of a gene is under the control of a spatially connected promoter. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between that promoter and the gene it controls within the gene from which the promoter is derived. As is known to those skilled in the art, variations in this distance can be accommodated without loss of promoter function.

[0030] As used herein, "peptide", "protein", or "polypeptide" can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0031] As used herein, "promoter" refers to a synthetic or naturally occurring molecule capable of causing, activating, or enhancing the expression of nucleic acids within a cell. A promoter may further include one or more specific transcriptional control sequences that enhance its expression and / or alter its spatial and / or temporal expression. A promoter may include distal enhancer or repressor elements that can be located thousands of base pairs from the transcription start site. Promoters can be derived from sources such as viruses, bacteria, fungi, plants, insects, and animals. A promoter can structurally or specifically control gene expression in response to the cell, tissue, or organ in which expression occurs, or the growth stage at which expression occurs, or external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0032] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of a protein or amino acid sequence described herein. A signal peptide / leader sequence typically directs the localization of a protein. The signal peptide / leader sequence used herein preferably promotes the secretion of the protein from the cell in which it is produced. The signal peptide / leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. The signal peptide / leader sequence is linked to the amino terminus of the protein.

[0033] As used herein, "subject" can mean a mammal that desires or requires to be immunized with the vaccine described herein. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0034] As used herein, "stringent hybridization conditions" can mean conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions can be selected to be about 5-10 °C lower than the thermal melting point (T m ) of a particular sequence at a defined ionic strength and pH. T m can be the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess at T m , 50% of the probes are occupied at equilibrium). Stringent conditions can be those where the salt concentration is less than about 1.0 M sodium ions, e.g., a concentration of 0.01-1.0 M sodium ions (or other salts) at pH 7.0-8.3, and the temperature is at least about 30 °C for short probes (e.g., about 10-50 nucleotides) and at least about 60 °C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times that of background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubation at 42 °C, and washing at 65 °C in 0.2× SSC and 0.1% SDS.

[0035] As used herein, "substantially complementary" may mean that in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, the first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the complementary sequence of the second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0036] As used herein, "substantially identical" can mean that the first and second amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or more amino acids. Also, substantially identical can mean that the first nucleic acid sequence and the second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or more nucleotides.

[0037] As used herein, "treatment" or "treating" means protecting an animal from a disease by means of preventing, suppressing, subduing, or completely eliminating the disease. Protection from a disease includes administering the vaccine of the present invention to the animal before the onset of the disease. Subduing a disease includes administering the vaccine of the present invention to the animal after induction of the disease but before its clinical appearance. Subduing a disease includes administering the vaccine of the present invention to the animal after the clinical appearance of the disease.

[0038] As used herein with respect to nucleic acids, "variant" means a nucleic acid that is (i) a portion or fragment of a reference nucleotide sequence, (ii) the complement of a reference nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to the reference nucleotide or its complement, or (iv) a nucleic acid that hybridizes to the reference nucleic acid, its complement, or a sequence that is substantially identical thereto, under stringent conditions.

[0039] A variant can be further defined as a peptide or polypeptide whose amino acid sequence differs by amino acid insertions, deletions, or conservative substitutions, but that retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to promote an immune response. A variant also means a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of one amino acid with another having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is typically recognized in the art as involving small-scale changes. These small-scale changes can be identified, in part, by considering the hydrophilicity index of the amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids having similar hydrophilicity indices can be substituted and the function of the protein can still be retained. In one aspect, amino acids having a hydrophilicity index of ±2 are substituted. Utilizing the hydrophilicity of amino acids can also clarify substitutions that result in a protein retaining its biological function. By considering the hydrophilicity of amino acids in a peptide, it is possible to calculate a useful metric that has been reported to correlate well with the maximum local average hydrophilicity of the peptide, i.e., antigenicity and immunogenicity. As understood in the art, substitution of amino acids having similar hydrophilicity values can result in a peptide retaining its biological activity, e.g., immunogenicity. The substitution can be carried out using amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by its specific side chain. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0040] The variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or a fragment thereof. The variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0041] As used herein, "vector" means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. The vector may contain or be capable of containing one or more heterologous nucleic acid sequences.

[0042] In the case of the recitation of numerical ranges herein, each numerical value having the same degree of precision intervening therebetween is explicitly contemplated. For example, in the range of 6 to 9, in addition to 6 and 9, the numerical values 7 and 8 are contemplated, and in the range of 6.0 to 7.0, the numerical values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0043] 2. Vaccine Vaccines comprising an antigen and an adjuvant are provided herein. The vaccines can enhance antigen presentation and the overall immune response to the antigen in an individual. The combination of the antigen and the adjuvant induces the immune system more efficiently than a vaccine containing the antigen alone. The vaccines can further induce an immune response when administered to different tissues such as muscle and skin.

[0044] The vaccines of the present invention can have the characteristics required of an effective vaccine, such as being safe so that the vaccine itself does not cause disease or death; having a protective effect against diseases resulting from exposure to pathogenic organisms such as viruses or bacteria; inducing neutralizing antibodies to prevent cell infection; inducing a protective T cell response against intracellular pathogens; and providing ease of administration, few side effects, biological stability, and low cost per dose. By combining an antigen and an adjuvant as described below, the vaccines can achieve some or all of these characteristics.

[0045] a. Adjuvant The vaccine can comprise an adjuvant and an antigen, as described below. The adjuvant can be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence can also include an additional sequence encoding a linker or tag sequence linked to the adjuvant by a peptide bond. The amino acid sequence can be a protein, a peptide, variants thereof, fragments thereof, or combinations thereof.

[0046] (1) IL-21 The adjuvant may be interleukin-21 (IL-21). IL-21 is a single-chain T cell-derived cytokine that has potent effects on B and T cell subsets, including natural killer (NK) cells and cytotoxic T cells (CD8+ T cells). Animal models of chronic infection suggest an important role for IL-21 in the control of T cell activity and viral replication and in patients with chronic viral infections such as HIV. IL-21 has been reported to significantly improve the cytotoxic CD8 T cell response. IL-21 has also been suggested to assist in the proliferation and differentiation of B cells.

[0047] IL-21, like IL-12, can stimulate IFN-γ production. IL-12 can activate naive T cells to induce IFN-γ production, and IL-21 can act on memory T cells to induce IFN-γ production. By including IL-21 in a vaccine, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 8-fold, and at least about 10-fold more IFN-γ production can be induced compared to a vaccine that does not contain IL-23. By including IL-21 in a vaccine, at least about 2-fold more IFN-γ production can be induced compared to a vaccine that does not contain IL-21. By including IL-21 in a vaccine, at least about 3-fold more IFN-γ production can be induced compared to a vaccine that does not contain IL-21.

[0048] IL-21 can enhance or boost the immune response against a target antigen. The antigen will be described in more detail later. In some examples, IL-21 can enhance the immune response against the antigen by about 75% to about 200%. Alternatively, IL-21 can enhance the immune response against the antigen by about 90% to about 130%. In still other alternative embodiments, IL-21 can enhance the immune response against the antigen by about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, or 130%.

[0049] In other embodiments, when the vaccines described herein are administered to a subject in need thereof, IL-21 can enhance or boost the immune response against the antigen by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0050] The nucleic acid encoding IL-21 may be derived from any number of organisms, such as mouse (Mus musculus), and human (Homo sapiens). The nucleic acid encoding IL-21 can be optimized with respect to codon usage frequency and the corresponding RNA transcript. For expression, codon optimization and RNA optimization of the nucleic acid encoding IL-21 can be performed. In some embodiments, the nucleic acid encoding IL-21 can include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding IL-21 can include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination. The nucleic acid encoding IL-21 can also include a nucleotide sequence encoding an IgE leader sequence. The IgE leader sequence may be located 5' of the IL-21 of the nucleic acid. In some embodiments, the nucleic acid encoding IL-21 does not or does not contain a nucleotide sequence encoding an IgE leader sequence. In another embodiment, the nucleic acid encoding IL-21 can include a nucleotide sequence encoding an HA tag (SEQ ID NO: 9). In yet another embodiment, the nucleic acid encoding IL-21 does not or does not contain a nucleotide sequence encoding an HA tag.

[0051] Mouse IL-21 may be an optimized nucleic acid sequence of SEQ ID NO: 1 encoding SEQ ID NO: 2. In some embodiments, Mouse IL-21 may be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO: 1. In other embodiments, Mouse IL-21 may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO: 2. Mouse IL-21 may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO: 2.

[0052] Human IL-21 may be an optimized nucleic acid sequence of SEQ ID NO: 3 that encodes SEQ ID NO: 4. In some embodiments, Human IL-21 may be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO: 3. In other embodiments, Human IL-21 may be a nucleic acid sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO: 4. Human IL-21 may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO: 4.

[0053] Some embodiments relate to fragments of SEQ ID NO: 1 and / or SEQ ID NO: 3. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 1 and / or SEQ ID NO: 3. In some embodiments, the fragment can comprise a sequence encoding a leader sequence, such as an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not comprise a coding sequence encoding a leader sequence.

[0054] Fragments of nucleic acids comprising nucleotide sequences having identity with fragments of SEQ ID NO: 1 and / or SEQ ID NO: 3 may be provided. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleic acid having at least 95% identity with SEQ ID NO: 1 and / or SEQ ID NO: 3. Some embodiments relate to fragments having at least 96% identity with fragments of the IL-21 nucleic acid sequences described herein. Some embodiments relate to fragments having at least 97% identity with fragments of the IL-21 nucleic acid sequences described herein. Some embodiments relate to fragments having at least 98% identity with fragments of the IL-21 nucleic acid sequences described herein. Some embodiments relate to fragments having at least 99% identity with fragments of the IL-21 nucleic acid sequences described herein. In some embodiments, the fragment comprises a sequence encoding a leader sequence, such as an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not comprise a coding sequence encoding a leader sequence.

[0055] Fragments of SEQ ID NO: 2 and / or SEQ ID NO: 4 may be provided. The fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 2 and / or SEQ ID NO: 4. In some embodiments, the fragment comprises a leader sequence, such as an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not comprise a leader sequence.

[0056] Fragments of proteins comprising amino acid sequences having identity to fragments of SEQ ID NO:2 and / or SEQ ID NO:4 may be provided. Such fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein having at least 95% identity to SEQ ID NO:2 and / or SEQ ID NO:4. Some embodiments relate to fragments having at least 96% identity to fragments of the IL-21 protein sequences described herein. Some embodiments relate to fragments having at least 97% identity to fragments of the IL-21 protein sequences described herein. Some embodiments relate to fragments having at least 98% identity to fragments of the IL-21 protein sequences described herein. Some embodiments relate to fragments having at least 99% identity to fragments of the IL-21 protein sequences described herein. In some embodiments, the fragment comprises a leader sequence, such as an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not comprise a leader sequence.

[0057] b. Antigen As described above, the vaccine can comprise an antigen or a fragment or variant thereof and an adjuvant. The antigen can be anything that induces an immune response in a subject. Since purified antigens are usually not strongly immunogenic on their own, they are combined with an adjuvant as described above. The immune response induced by the antigen can be boosted or enhanced when combined with an adjuvant. Such an immune response can be a humoral immune response and / or a cellular immune response. In some embodiments, the combination of adjuvant and antigen can boost or enhance a cellular immune response in a subject. In other embodiments, the combination of adjuvant and antigen can boost or enhance a humoral immune response in a subject.

[0058] The antigen may be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence can also include an additional sequence encoding a linker or tag sequence linked to the antigen by a peptide bond. The amino acid sequence may be a protein, a peptide, variants thereof, fragments thereof, or combinations thereof.

[0059] The antigen may be contained in proteins, nucleic acids, or fragments thereof, variants thereof, or combinations thereof derived from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals. The antigen may be associated with autoimmune diseases, allergies, or asthma. In other embodiments, the antigen may be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV), or human immunodeficiency virus (HIV). As described below, the antigen of the vaccine may be selected from the group consisting of HIV antigens, Clostridium difficile antigens, and fragments thereof. The HIV antigen may be selected from the group consisting of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof.

[0060] Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. The antigen can induce a higher immune response when combined with an adjuvant as described above.

[0061] (1) Viral antigen The antigen may be a viral antigen, or a fragment thereof, or a variant thereof. The viral antigen may be derived from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen may be derived from a papillomavirus, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, variola virus (smallpox and cowpox), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex virus type 1 (oral herpes), herpes simplex virus type 2 (genital herpes), varicella zoster (chickenpox zoster, also known as shingles), cytomegalovirus (CMV), such as human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, chikungunya virus, Lassa fever virus, arenavirus, or an oncogenic virus.

[0062] (a) Hepatitis antigen IL-21 can be bound to or combined with a hepatitis virus antigen (i.e., hepatitis antigen), or a fragment thereof, or a variant thereof. The hepatitis antigen may be an antigen or immunogen derived from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and / or hepatitis E virus (HEV). In some embodiments, the hepatitis antigen may be a heterologous nucleic acid molecule(s) such as a plasmid(s) encoding one or more of the antigens derived from HAV, HBV, HCV, HDV, and HEV. The hepatitis antigen may be a full-length protein or an immunogenic fragment of the full-length protein.

[0063] The hepatitis antigen can include a consensus sequence and / or one or more modifications for improved expression. Genetic modifications including codon optimization, RNA optimization, and addition of a highly efficient immunoglobulin leader sequence to enhance the immunogenicity of the construct may be included in the modified consensus sequence. The consensus hepatitis antigen may include a signal peptide such as an immunoglobulin signal peptide such as IgE or IgG signal peptide, and in some embodiments, may include an HA tag. The immunogen can be designed to induce a broader and stronger cellular immune response than the corresponding codon-optimized immunogen.

[0064] The hepatitis antigen may be an antigen derived from HAV. The hepatitis antigen may be an HAV capsid protein, an HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.

[0065] The hepatitis antigen may be an antigen derived from HCV. The hepatitis antigen may be an HCV nucleocapsid protein (i.e., core protein), an HCV envelope protein (e.g., E1 and E2), an HCV non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.

[0066] The hepatitis antigen may be an antigen derived from HDV. The hepatitis antigen may be an HDV δ antigen, a fragment thereof, or a variant thereof.

[0067] The hepatitis antigen may be an antigen derived from HEV. The hepatitis antigen may be an HEV capsid protein, a fragment thereof, or a variant thereof.

[0068] The hepatitis antigen may be an antigen derived from HBV. The hepatitis antigen may be an HBV core protein, an HBV surface protein, HBV DNA polymerase, an HBV protein encoded by gene X, a fragment thereof, a variant thereof, or a combination thereof. The hepatitis antigen may be an HBV genotype A core protein, an HBV genotype B core protein, an HBV genotype C core protein, an HBV genotype D core protein, an HBV genotype E core protein, an HBV genotype F core protein, an HBV genotype G core protein, an HBV genotype H core protein, an HBV genotype A surface protein, an HBV genotype B surface protein, an HBV genotype C surface protein, an HBV genotype D surface protein, an HBV genotype E surface protein, an HBV genotype F surface protein, an HBV genotype G surface protein, an HBV genotype H surface protein, a fragment thereof, a variant thereof, or a combination thereof. The hepatitis antigen may be a consensus HBV core protein or a consensus HBV surface protein.

[0069] In some embodiments, the hepatitis antigen may be an HBV genotype A consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype A core protein, or an HBV genotype A consensus core protein sequence.

[0070] In other embodiments, the hepatitis antigen may be an HBV genotype B consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype B core protein, or an HBV genotype B consensus core protein sequence.

[0071] In still other embodiments, the hepatitis antigen may be an HBV genotype C consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype C core protein, or an HBV genotype C consensus core protein sequence.

[0072] In some embodiments, the hepatitis antigen may be an HBV genotype D consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype D core protein, or an HBV genotype D consensus core protein sequence.

[0073] In other embodiments, the hepatitis antigen may be an HBV genotype E consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype E core protein, or an HBV genotype E consensus core protein sequence.

[0074] In some embodiments, the hepatitis antigen may be an HBV genotype F consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype F core protein, or an HBV genotype F consensus core protein sequence.

[0075] In other embodiments, the hepatitis antigen may be an HBV genotype G consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype G core protein, or an HBV genotype G consensus core protein sequence.

[0076] In some embodiments, the hepatitis antigen may be an HBV genotype H consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype H core protein, or an HBV genotype H consensus core protein sequence.

[0077] In yet other embodiments, the hepatitis antigen may be an HBV genotype A consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype A surface protein, or an HBV genotype A consensus surface protein sequence.

[0078] In some embodiments, the hepatitis antigen may be an HBV genotype B consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype B surface protein, or an HBV genotype B consensus surface protein sequence.

[0079] In other embodiments, the hepatitis antigen may be an HBV genotype C consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype C surface protein, or an HBV genotype C consensus surface protein sequence.

[0080] In yet other embodiments, the hepatitis antigen may be an HBV genotype D consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype D surface protein, or an HBV genotype D consensus surface protein sequence.

[0081] In some embodiments, the hepatitis antigen may be an HBV genotype E consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype E surface protein, or an HBV genotype E consensus surface protein sequence.

[0082] In other embodiments, the hepatitis antigen may be an HBV genotype F consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype F surface protein, or an HBV genotype F consensus surface protein sequence.

[0083] In yet other embodiments, the hepatitis antigen may be an HBV genotype G consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype G surface protein, or an HBV genotype G consensus surface protein sequence.

[0084] In other embodiments, the hepatitis antigen may be an HBV genotype H consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype H surface protein, or an HBV genotype H consensus surface protein sequence.

[0085] (b) Human papillomavirus (HPV) antigen IL-21 can be conjugated or combined with a human papillomavirus (HPV) antigen, or a fragment or variant thereof. The HPV antigen may be derived from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. The HPV antigen may be derived from HPV types 6 and 11, which cause genital warts and are known to cause head and neck cancers.

[0086] The HPV antigen may be the HPV E6 or E7 domain of each HPV type. For example, in the case of HPV type 16 (HPV16), the HPV16 antigen can include the HPV16 E6 antigen, the HPV16 E7 antigen, fragments, variants, or combinations thereof. Similarly, the HPV antigen may be HPV 6 E6 and / or E7, HPV 11 E6 and / or E7, HPV 18 E6 and / or E7, HPV 31 E6 and / or E7, HPV 33 E6 and / or E7, HPV 52 E6 and / or E7, or HPV 58 E6 and / or E7, fragments, variants, or combinations thereof.

[0087] (c) RSV antigen IL-21 can be conjugated or combined with an RSV antigen, or a fragment or variant thereof. The RSV antigen may be the human RSV fusion protein (also referred to herein as "RSV F", "RSV F protein", and "F protein"), or a fragment or variant thereof. The human RSV fusion protein can be conserved between RSV subtypes A and B. The RSV antigen may be the RSV F protein from the RSV Long strain (GenBank AAX23994.1), or a fragment or variant thereof. The RSV antigen may be the RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. The RSV antigen may be a monomer, dimer, or trimer of the RSV F protein, or a fragment or variant thereof. The RSV antigen may be the consensus RSV F amino acid sequence, or a fragment or variant thereof. The RSV antigen may be an optimized nucleic acid encoding the RSV F amino acid sequence, or a fragment or variant thereof.

[0088] The post-fusion form of RSV F induces high-titer neutralizing antibodies in immunized animals and protects the animals from exposure to RSV. The present invention utilizes this immune response in the claimed vaccine. According to the present invention, the RSV F protein may be in the pre-fusion or post-fusion form.

[0089] The RSV antigen may also be the human RSV attachment glycoprotein (also referred to herein as "RSV G", "RSV G protein", and "G protein"), or a fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. The antigen may be the RSV G protein from the RSV Long strain (GenBank AAX23993), or a fragment or variant thereof. The RSV antigen may be the RSV G protein from RSV subtype B isolate H5601, RSV subtype B isolate H1068, RSV subtype B isolate H5598, RSV subtype B isolate H1123, or a fragment or variant thereof. The RSV antigen may be the consensus RSV G amino acid sequence, or a fragment or variant thereof. The RSV antigen may be an optimized nucleic acid encoding the RSV G amino acid sequence, or a fragment or variant thereof.

[0090] In other embodiments, the RSV antigen may be the human RSV non-structural protein 1 (“NS1 protein”), or a fragment or variant thereof. For example, the RSV antigen may be the RSV NS1 protein from the RSV Long strain (GenBank AAX23987.1), or a fragment or variant thereof. The human RSV antigen may also be the RSV non-structural protein 2 (“NS2 protein”), or a fragment or variant thereof. For example, the RSV antigen may be the RSV NS2 protein from the RSV Long strain (GenBank AAX23988.1), or a fragment or variant thereof. The RSV antigen may further be the human RSV nucleocapsid (“N”) protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV N protein from the RSV Long strain (GenBank AAX23989.1), or a fragment or variant thereof. The RSV antigen may be the human RSV phosphoprotein (“P”) protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV P protein from the RSV Long strain (GenBank AAX23990.1), or a fragment or variant thereof. The RSV antigen may also be the human RSV matrix (“M”) protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV M protein from the RSV Long strain (GenBank AAX23991.1), or a fragment or variant thereof.

[0091] In yet other embodiments, the RSV antigen may be a human RSV small hydrophobic ("SH") protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV SH protein from the RSV Long strain (GenBank AAX23992.1), or a fragment or variant thereof. The RSV antigen may also be a human RSV matrix protein 2-1 ("M2-1") protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV M2-1 protein from the RSV Long strain (GenBank AAX23995.1), or a fragment or variant thereof. The RSV antigen may further be a human RSV matrix protein 2-2 ("M2-2") protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV M2-2 protein from the RSV Long strain (GenBank AAX23997.1), or a fragment or variant thereof. The human RSV antigen may be the RSV polymerase L ("L") protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV L protein from the RSV Long strain (GenBank AAX23996.1), or a fragment or variant thereof.

[0092] In further embodiments, the RSV antigen can have a consensus amino acid sequence of the NS1, NS2, N, P, M, SH, M2-1, M2-2, or L proteins. The RSV antigen may be a human RSV protein or a recombinant antigen, such as any one of the proteins encoded by the human RSV genome.

[0093] In other embodiments, the RSV antigen may be, but is not limited to, the RSV F protein from the RSV Long strain, the RSV G protein from the RSV Long strain, the consensus RSV G amino acid sequence, an optimized nucleic acid encoding the RSV G amino acid sequence, the human RSV genome of the RSV Long strain, the consensus RSV F amino acid sequence, an optimized nucleic acid encoding the RSV F amino acid sequence, the RSV NS1 protein from the RSV Long strain, the RSV NS2 protein from the RSV Long strain, the RSV N protein from the RSV Long strain, the RSV P protein from the RSV Long strain, the RSV M protein from the RSV Long strain, the RSV SH protein from the RSV Long strain, the RSV M2-1 protein from the RSV Long strain, the RSV M2-2 protein from the RSV Long strain, the RSV L protein from the RSV Long strain, the RSV G protein from RSV subtype B isolate H5601, the RSV G protein from RSV subtype B isolate H1068, the RSV G protein from RSV subtype B isolate H5598, the RSV G protein from RSV subtype B isolate H1123, or a fragment thereof, or a variant thereof.

[0094] (d) Influenza antigen IL-21 can be conjugated or combined with an influenza antigen, or a fragment thereof, or a variant thereof. The influenza antigen is an antigen capable of inducing an immune response against one or more influenza serotypes in a mammal. The antigen can include the full-length translation product HA0, the subunit HA1, the subunit HA2, variants thereof, fragments thereof, or combinations thereof. The influenza hemagglutinin antigen can be a consensus sequence derived from multiple strains of influenza A serotype H1, a consensus sequence derived from multiple strains of influenza A serotype H2, a hybrid sequence containing a portion of two different consensus sequences derived from different sets of multiple strains of influenza A serotype H1, or a consensus sequence derived from multiple strains of influenza B. The influenza hemagglutinin antigen can be derived from influenza B.

[0095] The influenza antigen can also contain at least one antigenic epitope that can be effective against a specific influenza immunogen in which an immune response can be induced. The antigen can provide the entire repertoire of immunogenic sites and the epitopes present in intact influenza virus. The antigen can be a consensus hemagglutinin antigen sequence derived from the hemagglutinin antigen sequences of multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1 or serotype H2. The antigen can be a hybrid consensus hemagglutinin antigen sequence that can be obtained by combining two different consensus hemagglutinin antigen sequences or portions thereof. Each of the two different consensus hemagglutinin antigen sequences can be derived from different sets of multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1. The antigen can be a consensus hemagglutinin antigen sequence derived from the hemagglutinin antigen sequences of multiple influenza B virus strains.

[0096] In some embodiments, the influenza antigen may be an H1 HA, H2 HA, H3 HA, H5 HA, or BHA antigen. Alternatively, the influenza antigen may be a consensus hemagglutinin antigen comprising a consensus H1 amino acid sequence or a consensus H2 amino acid sequence. The consensus hemagglutinin antigen may be a synthetic hybrid consensus H1 sequence comprising portions of two different consensus H1 sequences derived from different sets of sequences from each other. An example of a consensus HA antigen that is a synthetic hybrid consensus H1 protein is a protein comprising the U2 amino acid sequence. The consensus hemagglutinin antigen may be a consensus hemagglutinin protein derived from a hemagglutinin sequence from an influenza B strain, such as a protein comprising a consensus BHA amino acid sequence.

[0097] The consensus hemagglutinin antigen may further comprise one or more additional amino acid sequence elements. The consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence on its N-terminus. The consensus hemagglutinin antigen may further comprise an immunogenic tag that is a unique immunogenic epitope detectable by an easily obtainable antibody. An example of such an immunogenic tag is a 9-amino acid influenza HA tag that can be ligated on the C-terminus of the consensus hemagglutinin. In some embodiments, the consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence on its N-terminus and an HA tag on its C-terminus.

[0098] The consensus hemagglutinin antigen may be a consensus hemagglutinin protein consisting of a consensus influenza amino acid sequence or a fragment and variant thereof. The consensus hemagglutinin antigen may be a consensus hemagglutinin protein comprising a non-influenza protein sequence and an influenza protein sequence or a fragment and variant thereof.

[0099] Examples of the consensus H1 protein include a protein that may consist of the consensus H1 amino acid sequence, or a protein that further includes an additional element such as an IgE leader sequence, or an HA tag, or both the IgE leader sequence and the HA tag.

[0100] Examples of the consensus H2 protein include a protein that may consist of the consensus H2 amino acid sequence, or a protein that includes an IgE leader sequence, or an HA tag, or both the IgE leader sequence and the HA tag.

[0101] Examples of the hybrid consensus H1 protein include a protein that may consist of the consensus U2 amino acid sequence, or a protein that includes an IgE leader sequence, or an HA tag, or both the IgE leader sequence and the HA tag.

[0102] Examples of the hybrid consensus influenza B hemagglutinin protein include a protein that may consist of the consensus BHA amino acid sequence, or the example may include an IgE leader sequence, or an HA Tag, or both the IgE leader sequence and the HA tag.

[0103] The consensus hemagglutinin protein can be encoded by a consensus hemagglutinin nucleic acid, its variant, or a fragment thereof. Unlike a consensus hemagglutinin protein that can be a consensus sequence derived from multiple different hemagglutinin sequences from different strains and variants, a consensus hemagglutinin nucleic acid refers to a nucleic acid sequence encoding a consensus protein sequence, and the coding sequence used can be different from the sequence used to encode a specific amino acid sequence among the multiple different hemagglutinin sequences from which the consensus hemagglutinin protein sequence is derived. The consensus nucleic acid sequence may be an optimized codon and / or optimized RNA. The consensus hemagglutinin nucleic acid sequence may contain a Kozak sequence in the 5' untranslated region. The consensus hemagglutinin nucleic acid sequence may contain a nucleic acid sequence encoding a leader sequence. The coding sequence of the N-terminal leader sequence is 5' of the hemagglutinin coding sequence. The N-terminal leader can promote secretion. The N-terminal leader may be an IgE leader or an IgG leader. The consensus hemagglutinin nucleic acid sequence can contain a nucleic acid sequence encoding an immunogenic tag. The immunogenic tag may be on the C-terminus of the protein, and the sequence encoding it is 3' of the consensus HA coding sequence. Since the immunogenic tag provides a unique epitope for which readily available antibodies exist, such antibodies can be used in assays to detect and confirm the expression of the protein. The immunogenic tag may be an HA tag at the C-terminus of the protein.

[0104] (e) Human immunodeficiency virus (HIV) antigen IL-21 can be bound to or combined with an HIV antigen, or a fragment thereof, or a variant thereof. The HIV antigen can include a consensus sequence modified for the immunogen. Genetic modifications including codon optimization, RNA optimization, and addition of a highly efficient immunoglobulin leader sequence to enhance the immunogenicity of the construct may be included in the modified consensus sequence. The novel immunogen can be designed to induce a broader and stronger cellular immune response than the corresponding codon-optimized immunogen.

[0105] In some embodiments, the HIV antigen can be a subtype A consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the subtype A envelope protein, or a subtype A consensus envelope protein sequence.

[0106] In other embodiments, the HIV antigen can be a subtype B consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the subtype B envelope protein, or a subtype B consensus envelope protein sequence.

[0107] In still other embodiments, the HIV antigen can be a subtype C consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the subtype C envelope protein, or a subtype C consensus envelope protein sequence.

[0108] In further embodiments, the HIV antigen can be a subtype D consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the subtype D envelope protein, or a subtype D consensus envelope protein sequence.

[0109] In some embodiments, the HIV antigen may be a subtype B Nef-Rev consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the subtype B Nef-Rev protein, or a subtype B Nef-Rev consensus protein sequence.

[0110] In other embodiments, the HIV antigen may be a Gag consensus DNA sequence of subtype A, B, C, and D DNA sequence constructs, an IgE leader sequence linked to a consensus sequence for the Gag consensus subtype A, B, C, and D proteins, or a consensus Gag subtype A, B, C, and D protein sequence.

[0111] In yet other embodiments, the HIV antigen may be an MPol DNA sequence or an MPol protein sequence. The HIV antigen may be a nucleic acid or amino acid sequence of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or a combination thereof.

[0112] (2) Parasite antigen The antigen may be a parasite antigen or a fragment or variant thereof. The parasite may be a protozoan, a protozoon, or an ectoparasite. The protozoan (i.e., helminth) may be a platyhelminth (e.g., trematodes and cestodes), a hookworm, or a nematode (e.g., pinworms). The ectoparasite may be a louse, a flea, a tick, and a mite.

[0113] The parasite may be any parasite that causes the following diseases: Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, Balantidiasis, Baylisascaris procyonis, Chagas disease, Clonorchiasis, filariasis, cryptosporidiosis, diphyllobothriasis, Dracunculiasis, echinococcosis, elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsis buski, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, Isosporiasis, Kala-azar, Lyme disease, malaria, Opisthorchiasis, myiasis, onchocerciasis, pediculosis, schistosomiasis, sleeping sickness, Strongyloidiasis, taeniasis, Toxocariasis, Toxoplasmosis, trichinosis, and trichuriasis.

[0114] The parasite may be Acanthamoeba, Anisakis, Ascaris, Gasterophilus intestinalis, Balantidium coli, Pediculus humanus corporis, Diphyllobothrium latum, Leptotrombidium deliense, Trichinella spiralis, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Ancylostoma duodenale, Leishmania, Linguatula serrata, Clonorchis sinensis, Loa loa, Paragonimus westermani, Enterobius vermicularis, Plasmodium falciparum, Schistosoma mansoni, Strongyloides stercoralis, Cimex lectularius, Taenia solium, Toxoplasma gondii, Trypanosoma, Trichuris trichiura, or Wuchereria bancrofti.

[0115] (a) Malaria antigen IL-21 can be bound to or combined with a malaria antigen (i.e., PF antigen or PF immunogen), or a fragment thereof, or a variant thereof. The antigen may be derived from a parasite that causes malaria. The parasite that causes malaria may be Plasmodium falciparum. The Plasmodium falciparum antigen can include a circumsporozoite (CS) antigen.

[0116] In some embodiments, the malaria antigen may be a nucleic acid molecule such as a plasmid encoding one or more of the Plasmodium falciparum immunogens CS, LSA1, TRAP, CelTOS, and Ama1. The immunogen may be a full-length protein or an immunogenic fragment of a full-length protein. The immunogen can include a consensus sequence and / or modifications for improved expression.

[0117] In other embodiments, the malaria antigen may be a consensus sequence of TRAP, also referred to as SSP2, designed from a compilation of all full-length Plasmodium falciparum TRAP / SSP2 sequences (a total of 28 sequences) in the GenBank database. The consensus TRAP immunogen (i.e., ConTRAP immunogen) may include a signal peptide such as an immunoglobulin signal peptide like IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0118] In yet other embodiments, the malaria antigen may be CelTOS, which is also referred to as Ag2 and is a highly conserved Plasmodium antigen. The consensus CelTOS antigen (i.e., ConCelTOS immunogen) may include a signal peptide such as an immunoglobulin signal peptide like IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0119] In further embodiments, the malaria antigen may be Ama1, a highly conserved Plasmodium antigen. The malaria antigen may also, in some instances, be a consensus sequence of Ama1 (i.e., ConAmaI immunogen) that includes a signal peptide such as an immunoglobulin signal peptide like IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0120] In some embodiments, the malaria antigen may be a consensus CS antigen (i.e., consensus CS immunogen) that includes a signal peptide such as an immunoglobulin signal peptide like IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0121] In other embodiments, the malaria antigen may be a fusion protein comprising a combination of two or more of the PF proteins described herein. For example, the fusion protein may comprise two or more of a consensus CS immunogen, a ConLSA1 immunogen, a ConTRAP immunogen, a ConCelTOS immunogen, and a ConAma1 immunogen that are linked directly adjacent to each other or with a spacer or another amino acid in between. In some embodiments, the fusion protein comprises two PF immunogens. In some embodiments, the fusion protein comprises three PF immunogens. In some embodiments, the fusion protein comprises four PF immunogens. In some embodiments, the fusion protein comprises five PF immunogens.

[0122] Fusion proteins having two consensus PF immunogens may comprise CS and LSA1, CS and TRAP, CS and CelTOS, CS and Ama1, LSA1 and TRAP, LSA1 and CelTOS, LSA1 and Ama1, TRAP and CelTOS, TRAP and Ama1, or CelTOS and Ama1. Fusion proteins having three consensus PF immunogens may comprise CS, LSA1 and TRAP, CS, LSA1 and CelTOS, CS, LSA1 and Ama1, LSA1, TRAP and CelTOS, LSA1, TRAP and Ama1, or TRAP, CelTOS and Ama1. Fusion proteins having four consensus PF immunogens may comprise CS, LSA1, TRAP and CelTOS, CS, LSA1, TRAP and Ama1, CS, LSA1, CelTOS and Ama1, CS, TRAP, CelTOS and Ama1, or LSA1, TRAP, CelTOS and Ama1. Fusion proteins having five consensus PF immunogens may comprise CS or CS-alt, LSA1, TRAP, CelTOS and Ama1.

[0123] In some embodiments, the fusion protein comprises a signal peptide linked to the N-terminus. In some embodiments, the fusion protein comprises a plurality of signal peptides linked to the N-terminus of each consensus PF immunogen. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein. In some embodiments, the spacer between the PF immunogens of the fusion protein may be a proteolytic cleavage site. In some embodiments, the spacer may be a proteolytic cleavage site recognized by a protease found in the cells into which the vaccine is intended to be administered and / or taken up. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein, the spacer is a proteolytic cleavage site recognized by a protease found in the cells into which the vaccine is intended to be administered and / or taken up, and upon cleavage, the signal peptides of each consensus PF immunogen cause the fusion protein to comprise a plurality of signal peptides linked to the N-terminus of each consensus PF immunogen so as to move each consensus PF immunogen extracellularly.

[0124] (3) Bacterial antigen The antigen may be a bacterial antigen or a fragment or variant thereof. The bacterium may be any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydia, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Dictyoglomus, Deinococcus-Thermus, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotoga, and Verrucomicrobia.

[0125] The bacterium may be a Gram-positive bacterium or a Gram-negative bacterium. The bacterium may be an aerobic bacterium or an anaerobic bacterium. The bacterium may be an autotrophic bacterium or a heterotrophic bacterium. The bacterium may be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, a halophile, or a piezophile.

[0126] The bacterium may be Bacillus anthracis, an antibiotic-resistant bacterium, a pathogen, a food poisoning bacterium, an infectious bacterium, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. The bacterium may be Mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0127] (a) Mycobacterium tuberculosis antigen IL-21 can be bound to or combined with a Mycobacterium tuberculosis antigen (i.e., a TB antigen or a TB immunogen), or a fragment thereof, or a variant thereof. The TB antigen may be derived from the Ag85 family of TB antigens, such as Ag85A and Ag85B. The TB antigen may be derived from the Esx family of TB antigens, such as EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.

[0128] In some embodiments, the TB antigen may be a heterologous nucleic acid molecule, such as a plasmid, encoding one or more of the Mycobacterium tuberculosis immunogens from the Ag85 family and the Esx family. The immunogen may be a full-length protein or an immunogenic fragment of the full-length protein. The immunogen can contain a consensus sequence and / or modifications for improved expression. The consensus immunogen may contain an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may contain an HA tag.

[0129] (b) Clostridium difficile antigen IL-21 can be conjugated or combined with a Clostridium difficile antigen (i.e., a CD antigen or CD immunogen), or a fragment thereof, or a variant thereof. The CD antigen can be toxin A or toxin B. In some embodiments, the CD antigen can be a heterologous nucleic acid molecule such as a plasmid encoding toxin A, toxin B, or both toxin A and toxin B. The CD antigen may be a full-length protein or an immunogenic fragment of a full-length protein. The CD antigen can include a consensus sequence and / or modifications for improved expression. The CD antigen can include a signal peptide such as an immunoglobulin signal peptide such as an IgE or IgG signal peptide, and in some embodiments, can include an HA tag.

[0130] (4) Fungal antigen The antigen may be a fungal antigen or a fragment or variant thereof. The fungus may be of the species Aspergillus, Blastomyces dermatitidis, Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exophiala, or Cladosporium.

[0131] c. Vector The vaccine can comprise one or more vectors containing one or more heterologous nucleic acids encoding an antigen and an adjuvant. The one or more vectors may be capable of expressing the antigen and the adjuvant. The one or more vectors may generally be expression constructs, which are plasmids commonly used for introducing specific genes into target cells. Once the expression vector enters the cell, the protein encoded by the gene is produced by the ribosomal complex, which is the transcription and translation machinery of the cell. Plasmids are often genetically engineered to contain regulatory sequences that act as enhancer and promoter regions and result in efficient transcription of the gene on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and thus proteins.

[0132] The vector may have expression signals such as a strong promoter, a strong stop codon, regulation of the distance between the promoter and the cloned gene, and insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

[0133] (1) Expression vector The vector may be a circular plasmid or a linear nucleic acid. Circular plasmids and linear nucleic acids can direct the expression of specific heterologous nucleotide sequences in suitable target cells. The vector can have a promoter operably linked to a nucleotide sequence encoding an antigen or a nucleotide sequence encoding an adjuvant, which may be operably linked to a termination signal. The vector can also contain sequences necessary for proper translation of the nucleotide sequence. The vector containing the target nucleotide sequence may be chimeric, i.e., at least one of its components is heterologous to at least one of the other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to a certain external stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0134] (2) Circular and linear vectors The vector may be a circular plasmid that can transform a target cell by integration into the cell genome or can exist extrachromosomally (e.g., autonomous replication of a plasmid having an origin of replication).

[0135] The vector may be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing heterologous DNA encoding an antigen or an adjuvant and enabling the cell to translate the sequence into an antigen recognized by the immune system, or may be an adjuvant.

[0136] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that can be efficiently delivered to a subject via electroporation and expresses one or more desired antigens and / or one or more desired adjuvants. The LEC may be any linear DNA that does not have any phosphate backbone. The DNA can encode one or more antigens and / or one or more adjuvants. The LEC may contain a promoter, an intron, a stop codon, and / or a polyadenylation signal. Expression of the antigen or adjuvant may be controlled by a promoter. The LEC may not contain any antibiotic resistance gene and / or phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression or desired adjuvant expression.

[0137] The LEC may be derived from any plasmid that can be linearized. The plasmid may be capable of expressing an antigen and / or an adjuvant. The plasmid may be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that is capable of expressing DNA encoding an antigen or encoding an adjuvant and enabling the cell to translate the sequence into an antigen recognized by the immune system, or an adjuvant.

[0138] The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR may each be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0139] (3) Promoters, introns, stop codons, and polyadenylation signals The vector may be a promoter. The promoter may be any promoter capable of driving gene expression and regulating the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase that transcribes the antigen sequence or adjuvant sequence described herein. The choice of promoter used to direct the expression of the heterologous nucleic acid depends on the particular application. The promoter may be located at a distance from the transcription start site in the vector approximately the same as the distance from the transcription start site in its natural environment. However, variations in this distance may be tolerated without loss of promoter function.

[0140] The promoter may be operably linked to a nucleic acid sequence encoding an antigen and may be signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The promoter may be operably linked to a nucleic acid sequence encoding an adjuvant and may be signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.

[0141] The promoter may be a CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown to be effective for expression in eukaryotic cells.

[0142] The vector may include an enhancer and an intron having functional splice donor and acceptor sites. The vector may contain a transcription termination site downstream of the structural gene to provide efficient termination. The termination site may be obtained from the same gene as the promoter sequence or from a different gene.

[0143] d. Vaccine excipients and other components The vaccine may further contain a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients can be functional molecules such as vehicles, adjuvants other than IL-21, carriers, or diluents. Pharmaceutically acceptable excipients may be transfection promoters, including surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs containing monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters.

[0144] The transfection promoter can be a polyanion, polycation, or lipid including poly-L-glutamate (LGS). The transfection promoter can be poly-L-glutamate, and poly-L-glutamate can be present in the vaccine at a concentration of less than 6 mg / ml. The transfection promoter can also include surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene. Hyaluronic acid can also be integrated with the gene construct and used or administered. The DNA plasmid vaccine may contain a transfection promoter such as a lipid, lecithin liposome or other liposomes known in the art such as a DNA liposome mixture (see, for example, International Patent No. 09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0145] Pharmaceutically acceptable excipients can be adjuvants in addition to IL-21. The additional adjuvant may be another gene expressed in a separate plasmid or delivered as a protein combined with the plasmid described above in the vaccine. The adjuvant can be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine in epithelium (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 containing IL-15 lacking a signal sequence and optionally containing a signal peptide derived from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0146] In addition to IL-21, other genes that may be useful as adjuvants include MCP-1, MIP-1a, MIP-1b, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular endothelial growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and genes encoding functional fragments thereof.

[0147] The vaccine may further contain a gene vaccine adjuvant as described in U.S. Patent Application No. 021,579, filed April 1, 1994, which is incorporated herein by reference in its entirety.

[0148] Vaccines can be formulated according to the mode of administration used. Injectable vaccine pharmaceutical compositions can be sterilized and can be pyrogen-free and particulate-free. Isotonic formulations or solutions can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may contain a vasoconstrictor. Examples of isotonic solutions can include phosphate buffered saline. The vaccine may further contain stabilizers such as gelatin and albumin. Stabilizers such as LGS or polycations or polyanions can stabilize the formulation for extended periods at room temperature or ambient temperature.

[0149] 3. Method of vaccination The present invention also targets a method for enhancing an immune response in a subject by different routes of administration of a vaccine. Enhancement of the immune response can be used to treat and / or prevent a disease in a subject.

[0150] The method can include administering to a subject a vaccine disclosed herein. The subject administered the vaccine can have an enhanced or boosted immune response compared to a subject administered the antigen alone. In some embodiments, the immune response in the subject administered the vaccine can be enhanced by about 18% to about 650%. Alternatively, the immune response in the subject administered the vaccine can be enhanced by about 45% to about 260%. In yet other alternative embodiments, the immune response in the subject administered the vaccine can be enhanced by about 93% to about 130%.

[0151] In other embodiments, the administered vaccine can enhance or boost the immune response in the subject by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0152] The dosage of the vaccine may be 1 μg to 10 mg of the active ingredient per kg of body weight per hour, or may be 20 μg to 10 mg of the ingredient per kg of body weight per hour. The vaccine can be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of times of taking the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0153] a. Administration The vaccine can be formulated according to standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered by such techniques in dosages well known to those skilled in medical technology, taking into account factors such as the age, sex, weight, and condition of a particular patient, as well as the route of administration. The subject may be a mammal such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

[0154] The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccine can be administered in an amount sufficient to induce an immune response. In therapeutic administration, the vaccine is administered to a subject in need thereof in an amount sufficient to induce a therapeutic effect. The appropriate amount to achieve this is defined as the "therapeutically effective dosage". The amount effective for this use depends, for example, on the specific composition of the vaccine administration schedule, the mode of administration, the stage and severity of the disease, the general health of the patient, and the judgment of the prescribing physician.

[0155] Vaccines can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996); Felgner et al. (U.S. Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997) (the entire contents of all of these are incorporated herein by reference). The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. Those skilled in the art know that the selection of a pharmaceutically acceptable carrier containing physiologically acceptable compounds depends, for example, on the route of administration of the expression vector.

[0156] Vaccines can be delivered via a variety of routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For certain vaccine DNAs, the vaccine can be delivered to the interstitial space of an individual's tissue (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055) (the entire contents of these are incorporated herein by reference). The vaccine can also be administered to muscle, or via intradermal or subcutaneous injection, or can be administered transdermally (such as by iontophoresis). Epidermal administration of the vaccine can also be used. Epidermal administration can include mechanically or chemically stimulating the outermost layer of the epidermis to stimulate an immune response to an irritant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference in their entirety).

[0157] The vaccine can also be formulated for administration via the nasal cavity. When the carrier is solid, a formulation suitable for nasal administration can include, for example, a coarse powder having a particle size in the range of about 10 to about 500 microns, which is administered in a manner of inhalation through the nose, i.e., rapidly inhaled through the nasal cavity from a container of powder maintained near the nose. The formulation may be by nasal spray, nasal drops, or aerosol administration by nebulizer. The formulation can include an aqueous or oily solution of the vaccine.

[0158] The vaccine may be a liquid formulation such as a suspension, syrup, or elixir. The vaccine can also be a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., administration of an injection), such as a sterile suspension or emulsion.

[0159] The vaccine can be incorporated into liposomes, microparticles, or other polymeric matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993) (the contents of which are incorporated herein by reference in their entirety). Liposomes may consist of phospholipids or other lipids and can be non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to prepare and administer.

[0160] The vaccine can be administered by electroporation using methods and the like described in U.S. Patent No. 7,664,545, the content of which is incorporated herein by reference. The electroporation may be by methods and / or devices described in U.S. Patent Nos. 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the content of which is incorporated herein in its entirety by reference. The electroporation may be performed by a minimally invasive device.

[0161] A minimally invasive electroporation device ("MID") may be a device for injecting the aforementioned vaccine and related fluids into body tissue. The device may comprise a hollow needle, a DNA cassette, and fluid delivery means, and the device is configured to be used by activating the fluid delivery means to inject DNA into the body tissue simultaneously (e.g., automatically) while the needle is inserted into the body tissue. This has the advantage of providing a more uniform distribution of the fluid in the body tissue due to the ability to gradually inject the DNA and related fluids while the needle is inserted. The pain experienced during the injection can be reduced because the injected DNA is distributed over a larger area.

[0162] The MID can inject the vaccine into the tissue without using a needle. The MID can inject the vaccine as a small stream or jet, and with such force, the vaccine penetrates the tissue surface and enters the underlying tissue and / or muscle. The motive power for the small stream or jet may be provided by the expansion of a compressed gas such as carbon dioxide passing through a micro-aperture in an instant. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655, U.S. Patent No. 6,520,950, U.S. Patent No. 7,171,264, U.S. Patent No. 6,208,893, U.S. Patent No. 6,009,347, U.S. Patent No. 6,120,493, U.S. Patent No. 7,245,963, U.S. Patent No. 7,328,064, and U.S. Patent No. 6,763,264 (the content of each of which is incorporated herein by reference).

[0163] The MID may comprise a syringe that forms a high-speed jet of liquid without pain. Such needleless syringes are commercially available. Examples of needleless syringes that can be utilized herein include those described in U.S. Patent Nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310 (the content of each of which is incorporated herein by reference).

[0164] A desired vaccine in a form suitable for direct or indirect electrical transport is typically introduced (e.g., injected) into the tissue to be treated using a needleless syringe by contacting the syringe with the tissue surface to initiate delivery of the jet of the agent with sufficient force to penetrate the vaccine into the tissue. For example, when the tissue to be treated is mucosa, skin, or muscle, the agent is emitted toward the mucosal surface or skin surface, respectively, with sufficient force to penetrate through the stratum corneum into the dermis layer or into the underlying tissue and muscle.

[0165] Needleless injectors are well-suited for delivering vaccines to all types of tissue, particularly the skin and mucosa. In some embodiments, the needleless injector may be used to propel a liquid containing the vaccine onto the surface and into the skin or mucosa of the subject. Representative examples of the various types of tissue that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, mid-pharynx, lip, larynx, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testicle, skin, mucosal tissue, ovary, blood vessel, or any combination thereof.

[0166] The MID may have a needle electrode for electroporating the tissue. For example, generating pulses between multiple pairs of electrodes in a multi-electrode array set in a rectangular or square pattern provides improved results over pulsing between a pair of electrodes. For example, U.S. Patent No. 5,702,359, titled "Needle Electrodes for Mediated Delivery of Drugs and Genes," discloses an array of needles capable of applying pulses to multiple pairs of needles during a therapeutic treatment. In its use as incorporated herein by reference in its entirety, the needles are arranged in a circular array but have connectors and switching devices that allow pulsing between opposing needle electrodes. A pair of needle electrodes may be used to deliver a recombinant expression vector to a cell. Such devices and systems are described in U.S. Patent No. 6,763,264, the content of which is incorporated herein by reference. Alternatively, a single needle similar to a conventional injection needle may be used to allow injection and electroporation of DNA, applying a lower voltage pulse than that delivered by currently used devices, thus reducing the electrical stimulation experienced by the patient, and a single-needle device may be used.

[0167] The MID may comprise one or more electrode arrays. The array may comprise two or more needles of the same diameter or different diameters. The needles may be spaced uniformly or non-uniformly. The needles may be from 0.005 inches to 0.03 inches, from 0.01 inches to 0.025 inches, or from 0.015 inches to 0.020 inches. The needles may have a diameter of 0.0175 inches. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more apart.

[0168] The MID may be composed of a pulse generator and a vaccine syringe having two or more needles for delivering a vaccine and an electroporation pulse in a single step. The pulse generator may enable flexible programming of the pulse and injection parameters, as well as comprehensive recording and storage of electroporation and patient data, via a personal computer operated by a flash card. The pulse generator may be capable of delivering various voltage pulses over a short period. For example, the pulse generator may be able to deliver 15 voltage pulses three times over a period of 100 ms. An example of such a MID is the Elgen 1000 system by Inovio Biomedical Corporation, described in U.S. Patent No. 7,328,064 (the content of which is incorporated herein by reference).

[0169] The MID may be the CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) device and system, a modular electrode system that facilitates the introduction of macromolecules such as DNA into cells of selected tissues within the body or in plants. The modular electrode system may comprise a plurality of needle electrodes, a hypodermic needle, an electrical connector that provides electrical conductivity connection from a programmable constant electrode pulse controller to the plurality of needle electrodes, and a power source. The operator can grip the plurality of needle electrodes mounted on a support structure and firmly insert them into selected tissues within the body or in plants. Next, the macromolecule is delivered by a hypodermic needle to the selected tissue. The programmable constant current controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the macromolecule into the cells between the plurality of electrodes. By limiting the power dissipation within the tissue by the constant current pulse, cell death due to overheating of the cells is minimized. The Cellectra device and system are described in U.S. Patent No. 7,245,963, the content of which is incorporated herein by reference.

[0170] The MID may be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may comprise a device that provides a hollow needle and fluid delivery means, the device being configured to operate the fluid delivery means to inject the desired vaccine described herein into the body tissue simultaneously (e.g., automatically) while the needle is being inserted into the body tissue. The ability to gradually inject fluid while the needle is inserted provides the advantage of a more uniform distribution of the fluid through the body tissue. Also, the pain experienced during injection is thought to be reduced because the volume of fluid being injected is distributed over a larger area.

[0171] Furthermore, the automatic injection of the fluid facilitates the automatic monitoring and recording of the actual amount of fluid being injected. This data may be stored by the control device for documentation purposes if needed.

[0172] It should be understood that the injection rate may be either linear or non-linear, and that the injection can be carried out after the needle has been inserted through the skin of the subject to be treated and while they are being further inserted into the body tissue.

[0173] Suitable tissues into which a fluid can be injected by the device of the present invention include tumor tissue, skin or liver tissue, but may also be muscle tissue.

[0174] The device further comprises needle insertion means for guiding the insertion of the needle into the body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that both the needle insertion and the fluid injection can be controlled so that the insertion rate can match the desired injection rate. This also makes the device easier for the user to operate. Optionally, means may be provided for automatically inserting the needle into the body tissue.

[0175] The user can select the time to start the fluid injection. However, ideally, the injection is started when the tip of the needle reaches the muscle tissue, and the device may include means for sensing when the needle has been inserted to a depth sufficient to start the fluid injection. This means that it can prompt the fluid injection to start automatically when the needle reaches the desired depth (usually the depth at which the muscle tissue begins). The depth at which the muscle tissue begins may be interpreted, for example, as the insertion depth of the needle of the present invention, such as a value of 4 mm, which is considered sufficient for the needle to pass through the skin layer.

[0176] The sensing means may comprise an ultrasonic probe. The sensing means may comprise means for sensing changes in impedance or resistance. In this case, although such means may not be able to record the depth of the needle in the body tissue by itself, rather, it is configured to sense changes in impedance or resistance when the needle moves from different types of body tissue into the muscle. Any of these alternative means provides a relatively accurate and simple-to-operate means for sensing that injection can commence. The depth of needle insertion may be further recorded as required and can be used to control the injection of fluid such that the volume of fluid injected is determined when the depth of needle insertion is recorded.

[0177] The device may further comprise a base for supporting the needle and a housing for receiving the base therein, and the base is movable relative to the housing such that when the base is in a first rearward position relative to the housing, the needle retracts within the housing, and when the base is in a second forward position within the housing, the needle extends from the housing. This is advantageous for the user as the housing can be placed on the patient's skin and then the needle can be inserted into the patient's skin by moving the housing relative to the base.

[0178] As described above, it is desirable to achieve a controlled fluid injection rate such that when the needle is inserted into the skin, the fluid is uniformly distributed along the length of the needle. The fluid delivery means may comprise piston drive means configured to inject the fluid at a controlled rate. The piston drive means can be actuated, for example, by a servo motor. However, the piston drive means may also be actuated by a base that is axially movable relative to the housing. It should be understood that alternative means of fluid delivery may be provided. Thus, for example, a sealed container that can be compressed for fluid delivery at a controlled or uncontrolled rate may be provided in place of a syringe and piston system.

[0179] The aforementioned device can be used for all kinds of injections. However, since it is assumed to be particularly useful in the field of electroporation, it may further comprise means for applying a voltage to the needle. This enables the needle to be used not only for injection but also as an electrode during electroporation. This is particularly advantageous because it means that an electric field is applied to the same region as the fluid to be injected. Conventionally, in electroporation, it has been very difficult to accurately align the previously injected fluid and the electrode, so users tend to inject a larger volume of fluid than required over a wider area and apply the electric field over a higher area to ensure overlap between the injected substance and the electric field. Using the present invention, it is possible to reduce both the volume of the fluid to be injected and the magnitude of the applied electric field while achieving good compatibility between the electric field and the fluid.

[0180] The present invention has a plurality of aspects shown by the following non-limiting examples.

[0181] 3. Examples Example 1 Expression of IL-21 For the expression of IL-21, a plasmid encoding the IL-21 gene (i.e., pVAX-mIL-21 Opt) was constructed (Figure 1). The DNA sequence of IL-21 was codon-optimized and RNA-optimized before insertion into the plasmid.

[0182] The plasmid was transfected into HEK 293T cells to confirm the expression of IL-21. The cell supernatant was analyzed by ELISA. The results showed that IL-21 was expressed in HEK 293T cells (Figure 2).

[0183] Example 2 Measurement of IL-21-enhanced IgG and IgA serum titers Mice were used as a model system to determine whether IL-21 can function as an adjuvant when the vaccine is administered via the intramuscular route. The vaccine contained toxin A and toxin B antigens derived from Clostridium difficile and IL-21, which were encoded by their respective plasmids.

[0184] Specifically, a group of mice was immunized using plasmid pVAX-mIL-21 Opt (described above in Figure 1 and Example 1) and a plasmid encoding the aforementioned toxin A and toxin B antigens derived from Clostridium difficile. A second group of mice was immunized with only the plasmid encoding the toxin A and toxin B antigens. A third group of mice was immunized with only the empty control plasmid pVAX. The mice were immunized via the intramuscular route using electroporation. Circulating antigen-specific IgG and IgA antibody-secreting cells were then analyzed in the blood of the immunized animals to determine the effect of the IL-21 adjuvant.

[0185] As shown in the upper panel of Figure 4, the IL-21 adjuvant increased the total amount of serum anti-toxin A IgG compared to immunization with the antigen alone, and a robust titer was seen at a 1:2000 dilution in the IL-21 group, but not in the group that received only the antigen. When serum IgA was analyzed, it was pointed out that the presence of the IL-21 adjuvant resulted in a detectable level of antigen-specific IgA showing a titer at a 1:2000 dilution, while immunization with the antigen alone did not show a robust signal compared to naive animals (Figure 4, lower panel).

[0186] The above data showed that IL-21 has the ability to function as an adjuvant when administered via the intramuscular route because it increased the humoral immune response against toxin A and toxin B antigens derived from Clostridium difficile. The above data also showed that IL-21 can function as an adjuvant together with bacterial antigens.

[0187] Example 3 IL-21 enhanced the cellular and humoral immune responses against HIV antigens. The IL-21 adjuvant was also administered in combination with plasmids encoding the EnvA and EnvC antigens derived from HIV. By including IL-21 in the vaccine, both the cellular and humoral immune responses against EnvC were enhanced. The vaccine contained the EnvA and EnvC antigens derived from HIV and IL-21. The EnvA antigen, EnvC antigen, and IL-21 were encoded by separate plasmids.

[0188] Specifically, the EnvA antigen was a consensus protein (SEQ ID NO: 6) encoded by the nucleotide sequence set forth in SEQ ID NO: 5. This nucleotide sequence set forth in SEQ ID NO: 5 was incorporated into a plasmid.

[0189] The EnvC antigen was a consensus protein (SEQ ID NO: 8) encoded by the nucleotide sequence set forth in SEQ ID NO: 7. This nucleotide sequence set forth in SEQ ID NO: 7 was incorporated into a plasmid.

[0190] Specifically, a group of mice was immunized using the plasmid pVAX-mIL-21 Opt (described above in Figure 1 and Example 1) and the plasmids encoding the aforementioned EnvA and EnvC antigens. A second group of mice was immunized with the plasmids encoding the EnvA and EnvC antigens only. A third group of mice was immunized using the empty control plasmid pVAX. The mice were immunized via the intramuscular route using electroporation. The cellular immune responses in the immunized groups of mice were examined using an interferon-γ ELISpot assay.

[0191] As shown in Figure 5, immunization with IL-21 enhanced the cellular immune response against the EnvC antigen by more than two-fold compared to the antigen alone. Therefore, these data suggested that IL-21 could function as an adjuvant in muscle tissue because it increased the cellular immune responses against the EnvA and EnvC antigens.

[0192] After the third immunization against the EnvA protein by ELISA, the antibody response was measured in the blood of immunized animals (Figure 6). At a dilution of 1:400, the average O.D. of HIV EnvA / C was approximately 0.6, while the IL-21 adjuvant group had an average O.D. reading of 1.0.

[0193] Taking these data together, it is suggested that IL-21 exerts a novel adjuvant activity in the form of an increase in the frequency of antibody-secreting cells, the amount of IgG produced and class switching, an increase in the amount of IgA produced, and an increase in IFN-γ secretion when included in a DNA vaccine.

[0194] It should be understood that the above detailed description and the accompanying examples are illustrative only and should not be construed as limitations on the scope of the invention, which is defined only by the appended claims and their equivalents.

[0195] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Without limitation, such changes and modifications, including those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

Claims

1. A vaccine comprising an antigen or a nucleotide sequence encoding said antigen and IL-21, wherein: a) said antigen is selected from the group consisting of human papillomavirus (HPV) antigen, human immunodeficiency virus (HIV) antigen, influenza antigen, malaria parasite antigen, C. difficile antigen, and fragments thereof; and b) IL-21 is selected from the group consisting of: an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence set forth in SEQ ID NO: 4 and the amino acid sequence set forth in SEQ ID NO: 4 and an amino acid sequence selected from the group consisting of; or c) a combination of the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, said vaccine.

2. IL-21 is selected from the group consisting of: a nucleotide sequence having at least about 95% identity to the nucleotide sequence set forth in SEQ ID NO: 3 and the nucleotide sequence set forth in SEQ ID NO: 3 and is encoded by a nucleotide sequence selected from the group consisting of; IL-21 is selected from the group consisting of: an amino acid sequence having at least about 95% identity to the full length of the amino acid sequence set forth in SEQ ID NO: 4 and the amino acid sequence set forth in SEQ ID NO: 4 and an amino acid sequence selected from the group consisting of; or any combination thereof, the vaccine according to claim 1.

3. The vaccine according to claim 2, wherein IL-21 is encoded by the nucleotide sequence set forth in SEQ ID NO: 3; IL-21 comprises the amino acid sequence set forth in SEQ ID NO:

4.

4. The vaccine according to claim 1, wherein said antigen is encoded by a first nucleic acid and IL-21 is encoded by a second nucleic acid.

5. The vaccine according to claim 4, further comprising a nucleic acid sequence and an antigen peptide encoded by the same antigen as claimed in claim 4, and a nucleic acid sequence and an IL-21 peptide encoded by the same IL-21 as claimed in claim 4.

6. The vaccine according to claim 4, wherein the second nucleic acid further comprises an expression vector.

7. The vaccine according to claim 1, wherein the HPV antigen is selected from the group consisting of HPV16 E6 antigen, HPV16 E7 antigen, and combinations thereof.

8. The vaccine according to claim 1, wherein the HIV antigen is selected from the group consisting of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof.

9. The vaccine according to claim 1, wherein the influenza antigen is selected from the group consisting of H1 HA, H2 HA, H3 HA, H5 HA, BHA antigen, and any combination thereof.

10. The vaccine according to claim 1, wherein the Plasmodium falciparum antigen comprises a circumsporozoite (CS) antigen.

11. The vaccine according to claim 1, wherein the Clostridium difficile antigen is selected from the group consisting of toxin A, toxin B, and combinations thereof.

12. The vaccine according to claim 1, further comprising a pharmaceutically acceptable excipient.

13. The vaccine according to claim 1, for enhancing an immune response in a subject in need of an immune response.

14. The vaccine according to claim 13, wherein administration of the vaccine to the subject comprises electroporation.

15. The enhancement of the immune response in the subject is the vaccine according to claim 13, which includes a cellular immune response, a humoral immune response, or both a cellular and a humoral immune response in the subject.

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