Vaccine with antigen and interleukin-21 as adjuvant

Abstract

Disclosed herein are vaccines comprising an antigen and IL-21. Also disclosed herein are methods for increasing an immune response in a subject. The methods can comprise administering the vaccine to the subject in need thereof.

Description

[0001] Citations of relevant applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 058,304, filed October 1, 2014, which is incorporated herein by reference. Technical Field

[0003] This invention relates to a vaccine comprising an antigen and IL-21, and a method of administering the vaccine. Background Technology

[0004] Vaccines are used to stimulate an individual's immune response to provide protection against and / or treatment for a specific disease. Some vaccines contain antigens that induce an immune response. Some antigens elicit a strong immune response, while others elicit a weak one. The weak immune response to an antigen can be enhanced by including an adjuvant in the vaccine. Adjuvants come in many different forms, such as aluminum salts, oil emulsions, sterile components of bacteria or other pathogens, cytokines, etc.

[0005] Cytokines are cellular proteins that influence the behavior of other cells, and unlike many adjuvants, they can modulate specific immune responses. One such cytokine is interleukin-21 (IL-21), which acts on lymphoid and bone marrow populations as well as epithelial cells to regulate innate and adaptive immune responses. IL-21 has been shown to contribute to the functional differentiation of several CD4+ T cell subsets, promote CD8+ T cell proliferation and functional responses, and play a role in the development of B cell immunoglobulin responses. IL-21 is produced by both CD8+ T cell populations and CD4+ T cell populations, including follicular helper T (TFH) cells, type 17 helper T (Th17) cells, and natural killer T (NKT) cells.

[0006] Vaccines are also administered to many different tissues (e.g., intramuscular, intradermal, etc.) in many different ways (e.g., injection, oral administration, etc.). However, not all delivery methods are equivalent. Some delivery methods allow for greater compliance within an individual population, while others can affect the immunogenicity and / or safety of the vaccine. Therefore, there remains a need in the art to develop safe and more effective adjuvants that enhance antigen responses regardless of antigen identity and route of administration. Summary of the Invention

[0007] This invention relates to vaccines comprising an antigen and IL-21. IL-21 may be encoded by a nucleotide sequence selected from the group consisting of: nucleotide sequences having at least about 95% identity with a nucleotide sequence as listed in SEQ ID NO:3, and nucleotide sequences as listed in SEQ ID NO:3. IL-21 may be encoded by a nucleotide sequence as listed in SEQ ID NO:3.

[0008] 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 also include an expression vector. The vaccine may also include an antigenic peptide having the same coding nucleic acid sequence as the antigen described above, and an IL-21 peptide having the same coding nucleic acid sequence as IL-21 described above.

[0009] Antigens can be selected from the following groups: human papillomavirus (HPV) antigen, human immunodeficiency virus (HIV) antigen, influenza antigen, Plasmodium falciparum antigen, Clostridium difficile antigen, and fragments thereof. HPV antigens can be selected from the following groups: HPV16E6 antigen, HPV16E7 antigen, and combinations thereof. HIV antigens can be selected from the following groups: Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof. Influenza antigens can be selected from the following groups: H1HA, H2HA, H3HA, H5HA, BHA antigens, and any combination thereof. Plasmodium falciparum antigens may include cyclosporine (CS) antigens. Clostridium difficile antigens can be selected from the following groups: toxin A, toxin B, and combinations thereof.

[0010] Vaccines may also contain pharmaceutically acceptable excipients.

[0011] The present invention also relates to a method for enhancing an immune response in subjects in need. The method may include administering a vaccine comprising an antigen and IL-21. IL-21 may be encoded by a nucleotide sequence selected from the group consisting of: nucleotide sequences having at least about 95% identity with the nucleotide sequence listed in SEQ ID NO:3, and nucleotide sequences listed in SEQ ID NO:3. IL-21 may be encoded by a nucleotide sequence listed in SEQ ID NO:3.

[0012] Vaccine administration may include electroporation. Enhancing the immune response in subjects may include enhancing cellular immune responses, humoral immune responses, or both.

[0013] The present invention also relates to nucleic acid molecules comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO:3, and nucleotide sequences that are 95% or greater identical to SEQ ID NO:3. The nucleic acid molecule may be a plasmid. Attached Figure Description

[0014] Figure 1 A diagram of plasmid pVAX-mIL-21Opt is shown, which contains optimized nucleic acids encoding mouse IL-21.

[0015] Figure 2 The expression of IL-21 in the supernatant from transfected HEK 293T cells is shown.

[0016] Figure 3 The humoral immune response in mice immunized via the intramuscular route with plasmids encoding toxin A and toxin B antigens from Clostridium difficile was demonstrated using B-cell ELISpot assays.

[0017] Figure 4 This demonstrates the humoral immune response in mice immunized via the intramuscular route, as measured using ELISA.

[0018] Figure 5 The study demonstrates the cellular immune response in mice immunized via the intramuscular route with plasmids encoding EnvA and EnvC antigens from HIV, as measured using interferon-γ ELISpot.

[0019] Figure 6 The humoral immune response in mice immunized via the intramuscular route with plasmids encoding EnvA and EnvC antigens from HIV was demonstrated using ELISA. Detailed Implementation

[0020] This invention relates to vaccines that can be used to enhance the immune response of subjects to antigens by using IL-21 as an adjuvant. IL-21 is a single-chain, T-cell-derived cytokine.

[0021] In some cases, IL-21 can be used as a universal adjuvant because it elicits a higher immune response in subjects compared to vaccines containing a single antigen. IL-21 can also enhance immune responses to viral and bacterial antigens, such as HIV antigen and Clostridium difficile antigen, respectively. IL-21 can also be used with antigens in DNA / peptide combinations, which result in a greater nucleic acid / peptide immune response compared to the DNA or peptide of the antigen alone. In some cases, as demonstrated by increased interferon-γ (IFN-γ) production, IL-21 can also enhance immune responses in muscle and skin tissues.

[0022] 1. Definition

[0023] Unless otherwise defined, 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, this document (including the definitions) shall prevail. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and embodiments disclosed herein are exemplary only and are not intended to be restrictive.

[0024] As used herein, the terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and their variations are intended as open-ended conjunctions, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. This disclosure also covers other embodiments that “include the embodiments or elements presented herein,” “consist of the embodiments or elements presented herein,” and “consist primarily of the embodiments or elements presented herein,” whether or not explicitly stated.

[0025] As used herein, “adjuvant” means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen.

[0026] As used herein, “coding sequence” or “coding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal administering the nucleic acid.

[0027] As used herein, “complementary sequence” or “complementary” means Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0028] The terms “electroporation,” “electro-permeation,” or “electro-enhanced” (“EP”) used interchangeably in this article refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to flow from one side of the cell membrane to the other.

[0029] As used herein, “fragment” or “immunogenic fragment” means a nucleic acid sequence or portion thereof encoding a polypeptide capable of inducing and / or amplifying an immune response in mammals. The fragment may be a DNA fragment selected from at least one of a variety of nucleotide sequences encoding protein fragments described below. The fragment may 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 listed below. In some embodiments, the fragment may comprise at least 20 or more, at least 30 or more, at least 40 or more, at least 50 or more, at least 60 or more, at least 70 or more, at least 80 or more, at least 90 or more, at least 100 or more, at least 150 or more, at least 200 or more, at least 250 or more, at least 300 or more, at least 350 or more, at least 400 or more, at least 450 or more, at least 500 or more, at least 550 or more, at least 600 or more, at least 650 or more, at least 700 or more, at least 750 or more, at least 800 or more, at least 850 or more, at least 900 or more, at least 950 or more, or at least 1000 or more nucleotides of at least one of the nucleic acid sequences listed below.

[0030] As used herein, a fragment or immunogenic fragment also means a polypeptide sequence or portion thereof capable of inducing and / or enhancing an immune response in mammals. A fragment may be a polypeptide fragment selected from at least one of the various amino acid sequences listed below. A fragment may contain 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 listed below. In some embodiments, the fragment may contain 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.

[0031] As used herein, “gene construct” or “construct” refers to a DNA or RNA molecule containing a nucleotide sequence encoding a protein. The coding sequence includes start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to a gene construct or construct containing necessary regulatory elements operatively linked to a coding sequence encoding a protein such that, when the gene construct is present in the cells of an individual, the coding sequence will be expressed.

[0032] As used herein in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identical" means that the sequences have a specified percentage of identical residues in a specified region. This percentage can be calculated by: optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions of identical residues in the two sequences to produce a number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to produce the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region being compared contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, not the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0033] As used herein, “immune response” refers to the activation of the host’s immune system (e.g., the mammalian immune system) in response to the introduction of an antigen. Immune responses can take the form of cellular responses, humoral immune responses, or both.

[0034] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the described single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially the same nucleic acid and its complement. Single strands provide probes that can hybridize with the target sequence under strict hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under strict hybridization conditions.

[0035] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA, genomic DNA, cDNA, RNA, or a hybrid, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained through chemical synthesis or recombinant methods.

[0036] As used herein, "operably linked" means that gene expression is controlled by a promoter spatially linked to it. Under its control, the promoter can be positioned at the 5' (upstream) or 3' (downstream) of the gene. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene from which it originates. As is known in the art, this distance can be varied without losing promoter function.

[0037] As used herein, “peptide,” “protein,” or “polypeptide” can refer to a sequence of linked amino acids and can be natural, synthetic, or a combination of natural and synthetic modifications.

[0038] As used herein, "promoter" refers to a synthetic or naturally derived molecule that confers, activates, or enhances the expression of nucleic acids in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. Promoters may also contain distal enhancers or repressor elements, which may be located approximately several thousand base pairs from the transcription start site. Promoters can be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the constitutive or differential expression of genomic components relative to the cells, tissues, or organs in which expression occurs, or relative to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the phage T7 promoter, phage T3 promoter, SP6 promoter, lactose operon-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.

[0039] The terms “signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be attached to the N-terminus of a protein or amino acid sequence listed herein. The signal peptide / leader sequence typically indicates the location of a protein. The signal peptide / leader sequence used herein preferably facilitates the secretion of the protein from the cell that produced it. The signal peptide / leader sequence is often cleaved from the remainder of the protein, which is frequently referred to as the mature protein after secretion from the cell. The signal peptide / leader sequence is attached to the N-terminus of the protein.

[0040] As used herein, “subject” can refer to a mammal that wants or needs to be immunized with the vaccine described herein. A mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0041] As used herein, “strict hybridization conditions” can refer to conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize with a second nucleic acid sequence (e.g., a target) as in a complex mixture of nucleic acids. Strict conditions are sequence-dependent and vary under different conditions. Strict conditions can be selected by choosing a specific sequence with a thermal melting point (T0) at a defined ionic strength pH. m It will be about 5-10℃ lower. mIt can be that a probe that is 50% complementary to the target at the stated temperature hybridizes to the target sequence (because the target sequence is in excess, so at T...). m The temperature (under specified ionic strength, pH, and nucleic acid concentration) at equilibrium (where 50% of the probe is occupied). Rigorous conditions can be conditions where the salt concentration is below about 1.0 M sodium ions at pH 7.0 to 8.3, such as about 0.01–1.0 M sodium ion concentration (or other salts), 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). Rigorous conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal can be at least 2 to 10 times that of background hybridization. Exemplary rigorous hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42°C; or 5x SSC, 1% SDS, incubated at 65°C, washed at 65°C with 0.2x SSC and 0.1% SDS.

[0042] As used herein, “substantially complementary” means that the first sequence is identical to the complementary sequence of the second sequence 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, 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%, or that the two sequences hybridize under strict hybridization conditions.

[0043] As used in this article, "substantially similar" can mean that the first and second amino acid sequences are similar in the order of amino acids 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. The regions containing 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% of amino acids are identical. "Roughly the same" can also refer to the first and second nucleic acid sequences being in the range 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. 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% of the regions containing 400, 500, 600, 700, 800, 900, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are identical.

[0044] As used herein, "treatment" or "treating" can mean protecting an animal from disease or completely eliminating disease through prevention, suppression, or containment. Disease prevention involves administering the vaccine of the present invention to the animal before the onset of disease. Disease suppression involves administering the vaccine of the present invention to the animal after the onset of disease but before its clinical manifestation. Disease containment involves administering the vaccine of the present invention to the animal after the clinical manifestation of disease.

[0045] As used herein, “variant” for nucleic acids means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a portion of a reference nucleotide sequence; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a substantially identical sequence under stringent conditions.

[0046] A variant can be further defined as a peptide or polypeptide that differs in its amino acid sequence through the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. Representative examples of “biological activity” include the ability to bind to specific antibodies or promote an immune response. A variant also means a protein having a substantially identical amino acid sequence to a reference protein that retains at least one biological activity. Conserved substitution of amino acids, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor variations. These minor variations can be identified in part by taking into account the hydrophilicity index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of the amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. In the case of peptides, considering the hydrophilicity of amino acids allows for the calculation of the peptide's maximum local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. As understood in the art, substitution with amino acids of similar hydrophilicity values ​​can produce peptides that retain biological activity (e.g., immunogenicity). Substitution can be performed with amino acids whose hydrophilicity values ​​are in the range of ±2. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biocompatible amino acid substitutions are understood to depend on the relative similarity of these amino acids, and particularly on the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0047] Variants can be nucleic acid sequences that are substantially identical across the entire length of the gene sequence or a fragment thereof. The nucleic acid sequences can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical across the entire length of the gene sequence or a fragment thereof. Variants can also be amino acid sequences that are substantially identical across the entire length of the amino acid sequence or a fragment thereof. The amino acid sequences can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical across the entire length of the amino acid sequence or a fragment thereof.

[0048] As used herein, "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. A vector can contain or include one or more heterologous nucleic acid sequences.

[0049] For the purposes of describing numerical ranges in this article, each intermediate number with the same precision is explicitly included. For example, for the range of 6–9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range of 6.0–7.0, the numbers 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 included.

[0050] 2. Vaccine

[0051] This article describes vaccines that contain both antigens and adjuvants. Vaccines can increase antigen presentation and the overall immune response to antigens in subjects. The combination of antigen and adjuvant is more effective at inducing the immune system than vaccines containing only antigens. When administered to different tissues, such as muscle and skin, vaccines can further induce an immune response.

[0052] The vaccine of the present invention can possess the characteristics required of an effective vaccine, such as safety so that the vaccine itself does not cause disease or death; protection against disease caused by exposure to live pathogens (such as viruses or bacteria); induction of neutralizing antibodies to prevent cellular infection; induction of protective T cells against intracellular pathogens; and provision of ease of administration, few side effects, biostability, and low cost per dose. Some or all of these characteristics can be achieved by combining antigens and adjuvants as discussed below.

[0053] adjuvant

[0054] Vaccines may contain adjuvants and antigens as described below. Adjuvants may be nucleic acid sequences, amino acid sequences, or combinations thereof. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence may also contain additional sequences encoding adapter sequences or tag sequences linked to the adjuvant via peptide bonds. The amino acid sequence may be a protein, peptide, variants thereof, fragments thereof, or combinations thereof.

[0055] (1)IL-21

[0056] Adjuvants can include interleukin-21 (IL-21). IL-21 is a single-chain, T-cell-derived cytokine with potent activity against B and T cell subsets, including natural killer (NK) cells and cytotoxic T cells (CD8+ T cells). Animal models of chronic infection have demonstrated the crucial role of IL-21 in T cell activity and viral replication control, as well as in patients with chronic viral infections such as HIV. IL-21 has been reported to critically improve cytotoxic CD8+ T cell responses. IL-21 has also been shown to aid B cell proliferation and differentiation.

[0057] Similar to IL-12, IL-21 can stimulate IFN-γ production. While IL-12 activates naive T cells to induce IFN-γ production, IL-21 acts on memory T cells to induce IFN-γ production. Compared to vaccines without IL-21, vaccines containing IL-21 can induce IFN-γ production at least approximately 1.5 times, at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 8 times, and at least approximately 10 times. Compared to vaccines without IL-21, vaccines containing IL-21 can induce IFN-γ production at least approximately 2 times. Compared to vaccines without IL-21, vaccines containing IL-21 can induce IFN-γ production at least approximately 3 times.

[0058] IL-21 can increase or enhance a subject's immune response to an antigen. The antigen is described in more detail below. In some cases, IL-21 can increase the immune response to an antigen by approximately 75% to approximately 200%. Alternatively, IL-21 can increase the immune response to an antigen by approximately 90% to approximately 130%. In other alternative embodiments, IL-21 can increase the immune response to the antigen by approximately 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%.

[0059] In other embodiments, when the vaccine described herein is administered to subjects in need, IL-21 can increase or enhance the immune response to the antigen by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0060] The nucleic acid encoding IL-21 can be derived from any number of organisms, such as mice (Mus musculus) and humans (Homo sapiens). The nucleic acid encoding IL-21 can be optimized regarding codon usage and corresponding RNA transcripts. Codon and RNA optimization can be performed on the nucleic acid encoding IL-21 for expression. In some embodiments, the nucleic acid encoding IL-21 may contain a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. The nucleic acid encoding IL-21 may contain multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency. The nucleic acid encoding IL-21 may also include a nucleotide sequence encoding an IgE leader sequence. The IgE leader sequence may be located at the 5' of IL-21 in the nucleic acid. In some embodiments, the nucleic acid encoding IL-21 contains no or no nucleotide sequence encoding an IgE leader sequence. In other embodiments, the nucleic acid encoding IL-21 may include a nucleotide sequence encoding an HA tag (SEQ ID NO: 9). In other implementations, the nucleic acid encoding IL-21 does not contain or does not contain a nucleotide sequence encoding the HA tag.

[0061] Mouse IL-21 may be an optimized nucleic acid sequence SEQ ID NO:1, which encodes 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 listed 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 listed 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 listed in SEQ ID NO:2.

[0062] Human IL-21 may be an optimized nucleic acid sequence SEQ ID NO:3, which 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 listed in SEQ ID NO:3. In other embodiments, human 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 listed in SEQ ID NO:4.

[0063] Some embodiments involve fragments of SEQ ID NO:1 and / or SEQ ID NO:3. The fragment may contain 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 may include a sequence encoding a leader sequence, such as an immunoglobulin leader sequence, like an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence for a leader sequence.

[0064] Fragments of nucleic acids having nucleotide sequences identical to those of SEQ ID NO:1 and / or SEQ ID NO:3 may be provided. These fragments may contain 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 the nucleic acids having 95% or greater identity with those of SEQ ID NO:1 and / or SEQ ID NO:3. Some embodiments involve fragments having 96% or greater identity with the IL-21 nucleic acid sequence described herein. Some embodiments involve fragments having 97% or greater identity with the IL-21 nucleic acid sequence described herein. Some embodiments involve fragments having 98% or greater identity with the IL-21 nucleic acid sequence described herein. Some embodiments involve fragments having 99% or greater identity with the IL-21 nucleic acid sequence described herein. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as an immunoglobulin leader sequence, like the IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence for a leader sequence.

[0065] Fragments of SEQ ID NO:2 and / or SEQ ID NO:4 may be provided. The fragment may contain 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 contains a leader sequence, such as an immunoglobulin leader sequence, like an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence.

[0066] Fragments of proteins having an amino acid sequence identical to the fragments of SEQ ID NO:2 and / or SEQ ID NO:4 may be provided. The fragments may contain 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 the protein having 95% or greater identity with SEQ ID NO:2 and / or SEQ ID NO:4. Some embodiments involve fragments having 96% or greater identity with the IL-21 protein sequence described herein. Some embodiments involve fragments having 97% or greater identity with the IL-21 protein sequence described herein. Some embodiments involve fragments having 98% or greater identity with the IL-21 protein sequence described herein. Some embodiments involve fragments having 99% or greater identity with the IL-21 protein sequence described herein. In some embodiments, the fragment includes a leader sequence, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some implementations, the fragment does not contain a leader sequence.

[0067] b. Antigen

[0068] Vaccines may contain antigens, fragments or variants thereof, as discussed above, and adjuvants. The antigen can be any substance that induces an immune response in a subject. Purified antigens are generally not strongly immunogenic on their own and are therefore combined with adjuvants as described above. When combined with adjuvants, the immune response induced by the antigen can be enhanced or increased. The immune response can be a humoral immune response and / or a cellular immune response. In some embodiments, the combination of adjuvant and antigen can enhance or increase the subject's cellular immune response. In other embodiments, the combination of adjuvant and antigen can enhance or increase the subject's humoral immune response.

[0069] An antigen can be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, its variants, fragments, or a combination thereof. The nucleic acid sequence may also contain additional sequences encoding adapter sequences or tag sequences linked to the antigen via peptide bonds. The amino acid sequence can be a protein, a peptide, its variants, fragments, or a combination thereof.

[0070] Antigens can be contained in proteins, nucleic acids, or fragments thereof, variants thereof, or combinations thereof from any number of organisms (e.g., viruses, parasites, bacteria, fungi, or mammals). Antigens can be associated with autoimmune diseases, allergies, or asthma. In other embodiments, antigens can 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 can be selected from the group consisting of: HIV antigen, Clostridium difficile antigen, and fragments thereof. HIV antigens can be selected from the group consisting of: Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof.

[0071] Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. When combined with adjuvants as described above, antigens can elicit an even stronger immune response.

[0072] (1) Viral antigens

[0073] Antigens can be viral antigens, fragments thereof, or variants thereof. Viral antigens can originate from a virus belonging to one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens can originate from papillomaviruses, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), smallpox virus (severe and mild smallpox), 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, and Ebola virus. Herpes simplex virus (HSV), Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex virus 1 (oral herpes), herpes simplex virus 2 (genital herpes), herpes zoster (varicella-zoster virus, also known as chickenpox), cytomegalovirus (CMV) (e.g., human CMV), Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, chikungunya virus, lassa virus, sand virus, or carcinogenic virus.

[0074] (a) Hepatitis antigen

[0075] IL-21 can be conjugated or combined with hepatitis virus antigens (i.e., hepatitis antigens) or fragments thereof or variants thereof. Hepatitis antigens can be antigens or immunogens 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, hepatitis antigens can be heterologous nucleic acid molecules (such as plasmids) encoding one or more antigens from HAV, HBV, HCV, HDV, and HEV. Hepatitis antigens can be the full-length protein or an immunogenic fragment.

[0076] The hepatitis antigen may contain a common sequence and / or one or more modifications to improve expression. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences to increase the immunogenicity of the construct, may be included in the modified common sequence. The common hepatitis antigen may contain a signal peptide, such as an immunoglobulin signal peptide (e.g., IgE or IgG signal peptide), and in some embodiments, may contain an HA tag. Immunogens can be engineered to elicit a stronger and broader cellular immune response than a correspondingly codon-optimized immunogen.

[0077] Hepatitis antigens can be antigens derived from HAV. Hepatitis antigens can be HAV capsid proteins, HAV non-structural proteins, fragments thereof, variants thereof, or combinations thereof.

[0078] Hepatitis antigens can be antigens derived from HCV. Hepatitis antigens can be HCV nucleocapsid proteins (i.e., core proteins), HCV envelope proteins (e.g., E1 and E2), HCV non-structural proteins (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), fragments thereof, variants thereof, or combinations thereof.

[0079] Hepatitis antigens can be antigens derived from HDV. Hepatitis antigens can be HDVδ antigens, fragments thereof, or variants thereof.

[0080] Hepatitis antigens can be antigens derived from HEVs. Hepatitis antigens can be HEV capsid proteins, fragments thereof, or variants thereof.

[0081] Hepatitis antigens can be antigens derived from HBV. Hepatitis antigens can be HBV core protein, HBV surface protein, HBV DNA polymerase, HBV protein encoded by gene X, its fragments, its variants, or combinations thereof. Hepatitis antigens can be HBV genotype A core protein, HBV genotype B core protein, HBV genotype C core protein, HBV genotype D core protein, HBV genotype E core protein, HBV genotype F core protein, HBV genotype G core protein, HBV genotype H core protein, HBV genotype A surface protein, HBV genotype B surface protein, HBV genotype C surface protein, HBV genotype D surface protein, HBV genotype E surface protein, HBV genotype F surface protein, HBV genotype G surface protein, HBV genotype H surface protein, its fragments, its variants, or combinations thereof. Hepatitis antigens can be shared HBV core proteins or shared HBV surface proteins.

[0082] In some implementations, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype A, an IgE leader sequence linked to a common sequence of the HBV genotype A core protein, or a common core protein sequence of HBV genotype A.

[0083] In other embodiments, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype B, an IgE leader sequence linked to a common sequence of the HBV genotype B core protein, or a common core protein sequence of HBV genotype B.

[0084] In other embodiments, the hepatitis antigen may be an HBV genotype C common core DNA sequence construct, an IgE leader sequence linked to a common sequence of the HBV genotype C core protein, or an HBV genotype C common core protein sequence.

[0085] In some implementations, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype D, an IgE leader sequence linked to a common sequence of the HBV genotype D core protein, or a common core protein sequence of HBV genotype D.

[0086] In other embodiments, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype E, an IgE leader sequence linked to a common sequence of the HBV genotype E core protein, or a common core protein sequence of HBV genotype E.

[0087] In some implementations, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype F, an IgE leader sequence linked to a common sequence of the HBV genotype F core protein, or a common core protein sequence of HBV genotype F.

[0088] In other embodiments, the hepatitis antigen may be an HBV genotype G common core DNA sequence construct, an IgE leader sequence linked to a common sequence of the HBV genotype G core protein, or an HBV genotype G common core protein sequence.

[0089] In some implementations, the hepatitis antigen may be a common core DNA sequence construct of HBV genotype H, an IgE leader sequence linked to a common sequence of the HBV genotype H core protein, or a common core protein sequence of HBV genotype H.

[0090] In other embodiments, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype A, an IgE leader sequence linked to the common sequence of the surface protein of HBV genotype A, or a common surface protein sequence of HBV genotype A.

[0091] In some implementations, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype B, an IgE leader sequence linked to the common sequence of the HBV genotype B surface protein, or the common surface protein sequence of HBV genotype B.

[0092] In other embodiments, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype C, an IgE leader sequence linked to the common sequence of the surface protein of HBV genotype C, or a common surface protein sequence of HBV genotype C.

[0093] In other embodiments, the hepatitis antigen may be a common surface DNA sequence construct of HBV genotype D, an IgE leader sequence linked to a common sequence of HBV genotype D surface protein, or a common surface protein sequence of HBV genotype D.

[0094] In some implementations, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype E, an IgE leader sequence linked to the common sequence of the HBV genotype E surface protein, or the HBV genotype E common surface protein sequence.

[0095] In other embodiments, the hepatitis antigen may be a common surface DNA sequence construct of HBV genotype F, an IgE leader sequence linked to a common sequence of HBV genotype F surface protein, or a common surface protein sequence of HBV genotype F.

[0096] In other embodiments, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype G, an IgE leader sequence linked to the common sequence of the surface protein of HBV genotype G, or a common surface protein sequence of HBV genotype G.

[0097] In other embodiments, the hepatitis antigen may be a construct of the common surface DNA sequence of HBV genotype H, an IgE leader sequence linked to the common sequence of the surface protein of HBV genotype H, or a common surface protein sequence of HBV genotype H.

[0098] (b) Human papillomavirus (HPV) antigen

[0099] IL-21 can be combined with or in combination with human papillomavirus (HPV) antigens or fragments thereof or variants thereof. HPV antigens can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. HPV antigens can also be from HPV types 6 and 11, which cause genital warts and are known to be a cause of head and neck cancer.

[0100] HPV antigens can be from the HPV E6 or E7 domain of each HPV type. For example, for HPV type 16 (HPV16), the HPV16 antigen can include the HPV16E6 antigen, the HPV16E7 antigen, fragments thereof, variants thereof, or combinations thereof. Similarly, HPV antigens can be HPV 6E6 and / or E7, HPV 11E6 and / or E7, HPV 18E6 and / or E7, HPV 31E6 and / or E7, HPV 33E6 and / or E7, HPV 52E6 and / or E7, or HPV 58E6 and / or E7, fragments thereof, variants thereof, or combinations thereof.

[0101] (c) RSV antigen

[0102] IL-21 can also be combined with or in combination with RSV antigens, fragments thereof, or variants thereof. RSV antigens can be human RSV fusion proteins (also referred to herein as “RSV F,” “RSV F protein,” and “F protein”) or fragments thereof or variants thereof. Human RSV fusion proteins can be conserved between subtype A RSV and subtype B RSV. RSV antigens can be RSV F proteins, fragments thereof, or variants thereof derived from the RSV long chain (GenBank AAX23994.1). RSV antigens can be RSV F proteins, fragments thereof, or variants thereof derived from the RSV A2 chain (GenBank AAB59858.1). RSV antigens can be monomers, dimers, or trimers of RSV F proteins, fragments thereof, or variants thereof. RSV antigens can be shared RSV F amino acid sequences, fragments thereof, or variants thereof. RSV antigens can be optimized nucleic acids encoding RSV F amino acid sequences, fragments thereof, or variants thereof.

[0103] The post-fusion form of RSV F elicits high titers of neutralizing antibodies in immunized animals and protects them from RSV stimulation. This invention utilizes this immune response in the claimed vaccine. According to the invention, the RSV F protein can be in either a pre-fusion or post-fusion form.

[0104] RSV antigens can also be human RSV attachment glycoproteins (also referred to herein as “RSV G,” “RSV G protein,” and “G protein”) or fragments or variants thereof. Human RSV G proteins differ between subtype A and subtype B RSV. Antigens can be RSV G proteins or fragments or variants thereof derived from the RSV long chain (GenBank AAX23993). RSV antigens can be derived from the following RSVG proteins: subtype B RSV isolates H5601, H1068, H5598, and H1123, or fragments or variants thereof. RSV antigens can be shared RSV G amino acid sequences or fragments or variants thereof. RSV antigens can be optimized nucleic acids encoding RSV G amino acid sequences or fragments or variants thereof.

[0105] In other embodiments, the RSV antigen may be human RSV nonstructural protein 1 (“NS1 protein”) or a fragment or variant thereof. For example, the RSV antigen may be RSV NS1 protein or a fragment or variant thereof derived from RSV long chain (GenBank AAX23987.1). The RSV antigen may also be human RSV nonstructural protein 2 (“NS2 protein”) or a fragment or variant thereof. For example, the RSV antigen may be RSV NS2 protein or a fragment or variant thereof derived from RSV long chain (GenBank AAX23988.1). The RSV antigen may also be human RSV nucleocapsid (“N”) protein or a fragment or variant thereof. For example, the RSV antigen may be RSV N protein or a fragment or variant thereof derived from RSV long chain (GenBank AAX23989.1). The RSV antigen may be human RSV phosphoprotein (“P”) protein or a fragment or variant thereof. For example, the RSV antigen may be RSV P protein or a fragment or variant thereof derived from RSV long chain (GenBank AAX23990.1). RSV antigens can also be human RSV matrix protein (“M”) protein or fragments or variants thereof. RSV antigens can be RSV M protein or fragments or variants thereof derived from the RSV long chain (GenBank AAX23991.1).

[0106] In 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 or a fragment or variant thereof derived from the RSV long chain (GenBank AAX23992.1). 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 or a fragment or variant thereof derived from the RSV long chain (GenBank AAX23995.1). The RSV antigen may also 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 or a fragment or variant thereof derived from the RSV long chain (GenBank AAX23997.1). The RSV antigen may be the RSV polymerase L (“L”) protein or a fragment or variant thereof. For example, the RSV antigen can be the RSV L protein or a fragment or variant thereof derived from the RSV long chain (GenBank AAX23996.1).

[0107] In another embodiment, the RSV antigen may have a common 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 of the proteins encoded by the human RSV genome.

[0108] In other embodiments, the RSV antigen may be, but is not limited to: RSV F protein from the RSV long chain, RSV G protein from the RSV long chain, a shared RSV G amino acid sequence, optimized amino acids encoding the RSV G amino acid sequence, the human RSV genome from the RSV long chain, a shared RSV F amino acid sequence, optimized amino acids encoding the RSV F amino acid sequence, RSV NS1 protein from the RSV long chain, RSV NS2 protein from the RSV long chain, RSV N protein from the RSV long chain, RSV VP protein from the RSV long chain, RSV M protein from the RSV long chain, RSV SH protein from the RSV long chain, RSV M2-1 protein from the RSV long chain, RSV M2-2 protein from the RSV long chain, RSV L protein from the RSV long chain, RSV G protein from RSV isolate H5601 (subtype B), RSV G protein from RSV isolate H1068 (subtype B), and RSV G protein from RSV isolate H5598 (subtype B). G protein, RSV G protein from RSV isolator H1123 of subtype B, or fragments thereof or variants thereof.

[0109] (d) Influenza antigen

[0110] IL-21 can be combined with or in combination with influenza antigens, fragments thereof, or variants thereof. Influenza antigens are those that can elicit an immune response against one or more influenza serotypes in mammals. Antigens may contain the full-length translation product HA0, subunit HA1, subunit HA2, variants thereof, fragments thereof, or combinations thereof. Influenza hemagglutinin antigens may be derived from a common sequence of multiple influenza A serotype H1 strains, a common sequence of multiple influenza A serotype H2 strains, a hybrid sequence containing portions of two different common sequences of multiple influenza A serotype H1 strains from different groups, or a common sequence of multiple influenza B strains. Influenza hemagglutinin antigens may be derived from influenza B.

[0111] Influenza antigens may also contain at least one antigenic epitope that is effective against a specific influenza immunogen, against which an immune response can be induced. The antigen can provide the full lineage of immunogenic sites and epitopes present in the intact influenza virus. The antigen may be a common hemagglutinin antigen sequence derived from a hemagglutinin antigen sequence from multiple influenza A virus strains of a single serotype, such as multiple influenza A virus strains of serotype H1 or serotype H2. The antigen may be a hybrid common hemagglutinin antigen sequence obtained by combining two different common hemagglutinin antigen sequences or portions thereof. The two different common hemagglutinin antigen sequences may each be derived from multiple influenza A virus strains of a single serotype from different groups, such as multiple influenza A virus strains of serotype H1. The antigen may be a common hemagglutinin antigen sequence derived from a hemagglutinin antigen sequence from multiple influenza B virus strains.

[0112] In some embodiments, the influenza antigen may be an H1HA, H2HA, H3HA, H5HA, or BHA antigen. Alternatively, the influenza antigen may be a shared hemagglutinin antigen containing a shared H1 amino acid sequence or a shared H2 amino acid sequence. A shared hemagglutinin antigen may be a synthetic hybridized shared H1 sequence containing portions of two distinct shared H1 sequences, each derived from a different group of sequences from the other. An example of a shared HA antigen for synthesizing a hybridized shared H1 protein is a protein containing a U2 amino acid sequence. A shared hemagglutinin antigen may be a shared hemagglutinin protein derived from a hemagglutinin sequence from an influenza B strain, such as a protein containing a shared BHA amino acid sequence.

[0113] The co-hemagglutinin antigen may further include one or more additional amino acid sequence elements. The co-hemagglutinin antigen may further include an IgE or IgG leader amino acid sequence at its N-terminus. The co-hemagglutinin antigen may also include an immunogenic tag, which is a unique immunogenic epitope detectable by readily available antibodies. An example of such an immunogenic tag is a 9-amino acid influenza HA tag that can be attached to the C-terminus of the co-hemagglutinin. In some embodiments, the co-hemagglutinin antigen may further include an IgE or IgG leader amino acid sequence at its N-terminus and an HA tag at its C-terminus.

[0114] Common hemagglutinin antigens can be common hemagglutinin proteins composed of common influenza amino acid sequences or fragments and variants thereof. Common hemagglutinin antigens can also be common hemagglutinin proteins containing non-influenza protein sequences and influenza protein sequences or fragments and variants thereof.

[0115] Examples of shared H1 proteins include those that can be composed of a shared H1 amino acid sequence or those that also contain additional elements (such as an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag).

[0116] Examples of shared H2 proteins include those that can be composed of shared H2 amino acid sequences or those that also contain an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag.

[0117] Examples of hybrid shared H1 proteins include those hybrid shared H1 proteins that may consist of a shared U2 amino acid sequence or those hybrid shared H1 proteins that also contain an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag.

[0118] Examples of hybrid concordant influenza B hemagglutinin proteins include those that can consist of a concordant BHA amino acid sequence or that can contain an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag.

[0119] Concordant hemagglutinin proteins can be encoded by concordant hemagglutinin nucleic acids, their variants, or fragments thereof. Unlike concordant hemagglutinin proteins, which can be concordant sequences derived from multiple different hemagglutinin sequences from different strains and variants, concordant hemagglutinin nucleic acids refer to nucleic acid sequences encoding concordant protein sequences, and the coding sequences used can differ from those coding sequences used to encode specific amino acid sequences from multiple different hemagglutinin sequences from which the concordant hemagglutinin protein sequence originates. Concordant nucleic acid sequences can be codon-optimized and / or RNA-optimized. Concordant hemagglutinin nucleic acid sequences may contain a Kozak sequence located in the 5' untranslated region. Concordant hemagglutinin nucleic acid sequences may contain nucleic acid sequences encoding leader sequences. The coding sequence for the N-terminal leader sequence is the 5' of the hemagglutinin coding sequence. The N-terminal leader can promote secretion. The N-terminal leader can be an IgE leader or an IgG leader. Concordant hemagglutinin nucleic acid sequences may contain nucleic acid sequences encoding immunogenic tags. The immunogenic tag can be located at the C-terminus of the protein, and the sequence encoding it is the 3' of the concordant HA coding sequence. An immunogenic tag provides a unique epitope for which readily available antibodies are present, allowing the use of these antibodies in an assay to detect and confirm protein expression. An immunogenic tag may be an HA tag located at the C-terminus of a protein.

[0120] (e) Human Immunodeficiency Virus (HIV) Antigen

[0121] IL-21 can be conjugated or combined with HIV antigens, fragments thereof, or variants thereof. HIV antigens may include modified immunogenic common sequences. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly effective immunoglobulin leader sequences to increase the immunogenicity of the construct, may be included in the modified common sequences. Novel immunogens can be designed to elicit stronger and broader cellular immune responses than corresponding codon-optimized immunogens.

[0122] In some implementations, the HIV antigen may be a common envelope DNA sequence construct of subtype A, an IgE leader sequence linked to a common sequence of subtype A envelope protein, or a common envelope protein sequence of subtype A.

[0123] In other embodiments, the HIV antigen may be a construct of the subtype B common envelope DNA sequence, an IgE leader sequence linked to a common sequence of the subtype B envelope protein, or a subtype B common envelope protein sequence.

[0124] In other embodiments, the HIV antigen may be a C subtype common envelope DNA sequence construct, an IgE leader sequence linked to a common sequence of a C subtype envelope protein, or a C subtype common envelope protein sequence.

[0125] In another embodiment, the HIV antigen may be a D subtype common envelope DNA sequence construct, an IgE leader sequence linked to a common sequence of the D subtype envelope protein, or a D subtype common envelope protein sequence.

[0126] In some implementations, the HIV antigen may be a construct of the subtype B Nef-Rev common envelope DNA sequence, an IgE leader sequence linked to a common sequence of the subtype B Nef-Rev protein, or a subtype B Nef-Rev common protein sequence.

[0127] In other embodiments, the HIV antigen may be a Gag common DNA sequence of the A, B, C and D subtype DNA sequence construct, an IgE leader sequence linked to a common sequence of Gag common A, B, C and D subtype proteins, or a common Gag A, B, C and D subtype protein sequence.

[0128] In 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, and any combination thereof.

[0129] (2) Parasite antigen

[0130] Antigens can be parasitic antigens or fragments or variants thereof. Parasites can be protozoa, worms, or ectoparasites. Worms (i.e., intestinal worms) can be flatworms (e.g., flukes and tapeworms), acanthocephalans, or roundworms (e.g., pinworms). Ectoparasites can be lice, fleas, ticks, and mites.

[0131] Parasites can be any parasite that causes the following diseases: Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, giardiasis, raccoon ascariasis, Chagas disease, Clonorchiasis, Cochliomyiasis, Cryptosporidiosis, Sparganosis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobius vermicularis, Fasciolopsis buski, Fasciolopsis buski, Filariasis, Giardiasis, Gnathostoma spinigerum, Hymenolepis taeniasis, Isosporidiosis, Katayama fever, Leishmaniasis, Lyme disease. Diseases including malaria, metaclonal trematode disease, myiasis, onchocerciasis, lice, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm disease, toxocariasis, toxoplasmosis, trichinosis, and whipworm disease.

[0132] Parasites can include Acanthamoeba, Anisakis, Ascaris lumbricoides, Drosophila melanogaster, Balantidium coli, bedbugs, Tapeworms, Chiggers, Spiral fly larvae, Entamoeba histolytica, Fasciolopsis buski, Giardia lamblia, hookworms, Leishmania, Glossoptera serpentina, Liver flukes, Loaloa, Paragonimus westermani, Enterobius vermicularis, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0133] (a) Malaria antigen

[0134] IL-21 can be combined with or in combination with malaria antigens (i.e., PF antigens or PF immunogens) or fragments thereof or variants thereof. The antigens can originate from the parasite that causes malaria. The parasite causing malaria can be *Plasmodium falciparum*. *Plasmodium falciparum* antigens can include cyclosporine (CS) antigens.

[0135] In some implementations, 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. The immunogen may contain a shared sequence and / or modifications to improve expression.

[0136] In other embodiments, the malaria antigen may be a shared sequence of a TRAP (also known as SSP2) designed from all full-length Plasmodium falciparum TRAP / SSP2 sequences (a total of 28 sequences) in the GenBank database. The shared TRAP immunogen (i.e., ConTRAP immunogen) may contain a signal peptide (such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide), and in some embodiments, may contain an HA tag.

[0137] In other embodiments, the malaria antigen may be CelTOS, also known as Ag2, and is a highly conserved Plasmodium antigen. The common CelTOS antigen (i.e., ConCelTOS immunogen) may contain a signal peptide (such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide), and in some embodiments, may contain an HA tag.

[0138] In another embodiment, the malaria antigen may be Ama1, a highly conserved Plasmodium antigen. The malaria antigen may also be a conserved sequence of Ama1 (i.e., ConAmaI immunogen), which in some cases contains a signal peptide (such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide), and in some embodiments may contain an HA tag.

[0139] In some embodiments, the malaria antigen may be a common CS antigen (i.e., a common CS immunogen), which in some cases contains a signal peptide (such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide), and in some embodiments may contain an HA tag.

[0140] In other embodiments, the malaria antigen may be a fusion protein comprising a combination of two or more PF proteins described herein. For example, the fusion protein may comprise two or more of the following immunogens: ConCS, ConLSA1, ConTRAP, ConCelTOS, and ConAma1, linked directly adjacent to each other or with spacers between them having spacers or one or more amino acid linkages. 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.

[0141] Fusion proteins sharing two common PF immunogens may include: 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 sharing three common PF immunogens may include: 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 with four shared PF immunogens may include: 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 with five shared PF immunogens may include CS or CS-alt, LSA1, TRAP, CelTOS, and Ama1.

[0142] In some embodiments, the fusion protein includes a signal peptide linked to its N-terminus. In some embodiments, the fusion protein includes multiple signal peptides linked to the N-terminus of each common PF immunogen. In some embodiments, spacers may be included between the PF immunogens of the fusion protein. In some embodiments, the spacers between the PF immunogens of the fusion protein may be proteolytic cleavage sites. In some embodiments, the spacers may be proteolytic cleavage sites recognized by proteases found in cells intended to administer and / or ingest vaccines. In some embodiments, spacers may be included between the PF immunogens of the fusion protein, wherein the spacers are proteolytic cleavage sites recognized by proteases found in cells intended to administer and / or ingest vaccines, and the fusion protein includes multiple signal peptides linked to the N-terminus of each common PF immunogen such that, upon cleavage, the signal peptide of each common PF immunogen causes the corresponding common PF immunogen to translocate extracellularly.

[0143] (3) Bacterial antigens

[0144] Antigens can be bacterial antigens or fragments or variants thereof. Bacteria can originate from any of the following phyla: Acidobacteria, Actinobacteria, Aquagenic Bacteria, Bacteroidetes, Caldiserica, Chlamydia, Chlorobacteria, Chlorophytes, Chlorobacteria, Chlorophytes, Chlorobacteria, Chlorophytes, Agrobacteria, Anomalae, Anomalae, Elusimicrobia, Fibrobacteria, Firmicutes, Fusobacteria, Bacillus, Mucoidobacteria, Nitrifying Spirogyrae, Planctomyces, Proteobacteria, Spirogyrae, Altotrophic Bacteria, Amothermic Bacteria, Thermostodacteria, Thermostodacteria, and Verrucous Microbes.

[0145] Bacteria can be Gram-positive or Gram-negative. Bacteria can be aerobic or anaerobic. Bacteria can be autoaerobic or heteroaerobic. Bacteria can be thermophilic, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or hyperosmophilic.

[0146] The bacteria can be anthrax bacteria, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacteria, Staphylococcus bacteria, Streptococcus bacteria, or tetanus bacteria. The bacteria can also be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0147] (a) Mycobacterium tuberculosis antigen

[0148] IL-21 can be combined or conjugated with Mycobacterium tuberculosis antigens (i.e., TB antigens or TB immunogens) or fragments thereof or variants thereof. TB antigens can be derived from the Ag85 family of TB antigens, such as Ag85A and Ag85B. TB antigens can 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.

[0149] In some embodiments, the TB antigen may be a heterologous nucleic acid molecule (e.g., a plasmid) encoding one or more Mycobacterium tuberculosis immunogens from the Ag85 and Esx families. The immunogen may be a full-length protein or an immunogenic fragment. The immunogen may contain a common sequence and / or modifications to improve expression. The common immunogen may contain a signal peptide (e.g., an immunoglobulin signal peptide, such as an IgE or IgG signal peptide), and in some embodiments, may contain an HA tag.

[0150] (b) Clostridium difficile antigen

[0151] IL-21 can be conjugated or combined with Clostridium difficile antigen (i.e., CD antigen or CD immunogen) or fragments thereof or variants 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 can be a full-length protein or an immunogenic fragment of a full-length protein. The CD antigen may contain a common sequence and / or modifications to improve expression. The CD antigen may contain a signal peptide, such as an immunoglobulin signal peptide (e.g., IgE or IgG signal peptide), and in some embodiments, may contain an HA tag.

[0152] (4) Fungal antigens

[0153] The antigen can be a fungal antigen or a fragment or variant thereof. The fungi can be *Aspergillus* species, *Blastomyces dermatitidis*, *Candida* yeasts (e.g., *Candida albicans*), *Coccidioides*, *Cryptococcus neoformans*, *Cryptococcus gattii*, dermatophytes, *Fusarium* species, *Histoplasma capsulatum*, *Mucoromycotina*, *Pneumocystis jirovecii*, *Sporothrix schenckii*, *Exserohilum*, or *Cladosporium*.

[0154] c. Carrier

[0155] Vaccines may comprise one or more vectors containing one or more heterologous nucleic acids encoding antigens and adjuvants. One or more vectors may be capable of expressing both the antigen and the adjuvant. One or more vectors may be expression constructs, typically plasmids used to introduce specific genes into target cells. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the ribosome complex, the cellular transcription and translation machinery. Plasmids are often engineered to contain regulatory sequences that act as enhancer and promoter regions, leading to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA, and consequently, the proteins formed.

[0156] Vectors can contain expression signals (such as strong promoters and strong stop codons), regulate the distance between the promoter and the cloned gene, and insert transcription termination sequences and PTIS (mobile translation initiation sequences).

[0157] (1) Expression carrier

[0158] The vector can 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 appropriate recipient cells. The vector can have a promoter operatively linked to a nucleotide sequence encoding an antigen or an adjuvant, which can be operatively linked to a termination signal. The vector can also contain the sequence required for the correct translation of the nucleotide sequence. Vectors containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. Expression of the nucleotide sequence in the expression cassette can be controlled by a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to certain external stimuli. In the case of multicellular organisms, the promoter can also be tissue- or organ- or developmental stage-specific.

[0159] (2) Circular and linear carriers

[0160] The vector can be a circular plasmid, which can be used to transform target cells by integrating into the cell genome or exist outside the chromosome (e.g., an autonomously replicating plasmid with an origin of replication).

[0161] The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector that can express heterologous DNA encoding an antigen or adjuvant and enable cells to translate the sequence into an antigen or adjuvant that is recognized by the immune system.

[0162] This article also provides linear nucleic acid vaccines and / or linear expression cassettes (“LECs”) capable of being efficiently delivered to subjects via electroporation and expressing one or more desired antigens or one or more desired adjuvants. An LEC can be any linear DNA without a phosphate backbone. The DNA may encode one or more antigens and / or one or more adjuvants. An LEC may contain a promoter, introns, stop codons, and / or polyadenylation signals. Expression of the antigen or adjuvant may be controlled by the promoter. An LEC may not contain any antibiotic resistance genes and / or a phosphate backbone. An LEC may not contain any other nucleic acid sequences unrelated to the expression of the desired antigen gene or the desired adjuvant.

[0163] LECs can be derived from any plasmid capable of linearization. The plasmid can express antigens and / or adjuvants. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding an antigen or adjuvant and enabling the cell to translate the sequence into an antigen or adjuvant recognized by the immune system.

[0164] LEC can be pcrM2. LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (PuertoRico / 34) and pM2 (New Caledonia / 99), respectively.

[0165] (3) Promoter, intron, stop codon and polyadenylation signaling

[0166] The vector may have a promoter. The promoter can be any promoter capable of driving gene expression and regulating the expression of the isolated nucleic acid. The promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase, which transcribes the antigen or adjuvant sequence described herein. The choice of promoter for directing heterologous nucleic acid expression depends on the specific application. In the vector, the promoter may be located approximately at the same distance from the transcription start site as it is in its natural environment. However, variations in this distance can be tolerated without loss of promoter function.

[0167] The promoter can be operatively linked to a nucleic acid sequence encoding an antigen, as well as a signal required for efficient polyadenylation, ribosome binding, and translation termination of the transcript. The promoter can also be operatively linked to a nucleic acid sequence encoding an adjuvant, as well as a signal required for efficient polyadenylation, ribosome binding, and translation termination of the transcript.

[0168] The promoter can be the CMV promoter, the early SV40 promoter, the late SV40 promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrosis protein promoter, or another promoter that has shown effectiveness for expression in eukaryotic cells.

[0169] Vectors may contain enhancers and introns with functional splicing donor and acceptor sites. Vectors may contain transcription termination regions located downstream of structural genes to provide efficient termination. Termination regions may be obtained from genes with the same promoter sequence or from different genes.

[0170] d. Excipients and other components of the vaccine

[0171] The vaccine may also contain pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients can be functional molecules, such as mediators, adjuvants other than IL-21, carriers, or diluents. These pharmaceutically acceptable excipients can be transfection promoters, which may include surfactants such as immunostimulatory complexes (ISCOMS), Freunds incomplete adjuvants, LPS analogs (including monophosphoryl ester A), cell wall peptides, benzoquinone analogs, vesicles such as squalene and hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanionic, polycationic, or nanoparticles or other known transfection promoters.

[0172] Transfection promoters can be polyanionic, polycationic (including poly-L-glutamate (LGS)), or lipids. Transfection promoters can be poly-L-glutamate, and poly-L-glutamate can be present in the vaccine at a concentration of less than 6 mg / ml. Transfection promoters may also include surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including monophospholipid A), muramyl peptides, quinone analogs, and vesicles (such as squalene and squalene). Hyaluronic acid may also be used in conjunction with gene constructs for administration. DNA plasmid vaccines may also contain transfection promoters such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art, such as DNA-liposome mixtures (see, for example, WO9324640)), calcium ions, viral proteins, polyanionic, polycationic, 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.

[0173] Pharmaceutically acceptable excipients may be adjuvants other than IL-21. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in vaccines in combination with the plasmids described above. Adjuvants may 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), skin T-cell capture chemokine (CTACK), epithelial thymus expression chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with a deleted signal sequence and optionally including a signal peptide derived from IgE). The adjuvant may 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.

[0174] Other genes that can be used as adjuvants besides IL-21 include those encoding the following: MCP-1, MIP-1a, MIP-1p, 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, and CD40L. Angiogenic 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, p65Re1, MyD88, IRAK, TRAF6, IkB, inactivated 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 their functional fragments.

[0175] The vaccine may also contain a gene vaccine promoter as described in U.S. Serial No. 021,579, filed April 1, 1994, the entire patent document of which is incorporated herein by reference.

[0176] Vaccines can be formulated according to the intended method of administration. Injectable vaccine compositions can be sterile, pyrogen-free, and particulate-free. Isotonic formulations or solutions can be used. Additives for isotonicity may include sodium chloride, dextran, mannitol, sorbitol, and lactose. Vaccines may contain vasoconstrictors. Isotonic solutions may include phosphate-buffered saline. Vaccines may also contain stabilizers (including gelatin and albumin). Stabilizers allow the formulation to remain stable for an extended period at room temperature or ambient temperature; said stabilizers include LGS, polycationic, or polyanionic stabilizers.

[0177] 3. Methods of vaccination

[0178] The present invention also relates to methods for increasing the immune response in subjects through different routes of vaccine administration. Increased immune response can be used to treat and / or prevent diseases in subjects.

[0179] The method may include administering the vaccine disclosed herein to a subject. Subjects administered the vaccine have an increased or enhanced immune response compared to subjects administered only the antigen. In some embodiments, the immune response in subjects administered the vaccine may be increased by about 18% to about 650%. Alternatively, the immune response in subjects administered the vaccine may be increased by about 45% to about 260%. In other alternative embodiments, the immune response in subjects administered the vaccine may be increased by about 93% to about 130%.

[0180] In other implementations, the administered vaccine may increase or enhance the immune response in subjects by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0181] The vaccine dosage can be between 1 μg and 10 mg of the active component / kg body weight / time, and can be between 20 μg and 10 mg of the component / kg body weight / time. The vaccine can be administered 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 vaccine doses used for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0182] a. application

[0183] The vaccine can be formulated using standard techniques well known to those skilled in the art of pharmaceuticals. Such compositions can be administered using techniques well known to those skilled in the medical field, taking into account factors such as the age, sex, weight, condition, and route of administration of the specific subject. Subjects can be mammals, such as humans, horses, cattle, pigs, sheep, cats, dogs, rats, or mice.

[0184] Vaccines can be administered prophylactically or therapeutically. In prophylactic administration, a sufficient amount of vaccine may be administered to induce an immune response. In therapeutic administration, a sufficient amount of vaccine may be administered to the subject in need to achieve a therapeutic effect. The amount sufficient to achieve this goal is defined as the “therapeutic effective dose.” The effective amount for this purpose depends on, for example, the specific composition of the vaccine regimen, the route of administration, the stage and severity of the disease, the patient’s overall health condition, and the prescribing physician’s judgment.

[0185] Vaccines can be administered by methods well known in the art, as described in the following literature: Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (US Patent No. 5,580,859, issued December 3, 1996); Felgner (US Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (US Patent No. 5,679,647, issued October 21, 1997), all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be compounded with particles or beads that can be administered to an individual, for example, using a vaccine gun. Those skilled in the art will recognize that the choice of pharmaceutically acceptable carriers (including physiologically acceptable compounds) depends on, for example, the route of administration of the expression vector.

[0186] Vaccines can be delivered via a variety of routes. Typical routes of delivery include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral, intranasal, and intravaginal administration. Particularly for the DNA of the vaccine, it can be delivered into the interstitial space of an individual's tissues (Felgner et al., U.S. Patents 5,580,859 and 5,703,055, all of which are incorporated herein by reference in their entirety). Vaccines can also be administered intramuscularly, or via intradermal or subcutaneous injection, or percutaneously (e.g., through iontophoresis). Epidermal administration of the vaccine is also possible. Epidermal administration can involve mechanically or chemically stimulating the outermost layer of the epidermis to stimulate an immune response to the stimulus (Carson et al., U.S. Patent 5,679,647, the contents of which are incorporated herein by reference in their entirety).

[0187] Vaccines can also be formulated for administration via the nasal passage. Formulations suitable for nasal administration (where the carrier is solid) may include coarse powder having, for example, a particle size in the range of about 10 micrometers to about 500 micrometers, said coarse powder being administered via nasal inhalation, i.e., rapidly inhaled through the nasal passage from a container of powder near the nose. The formulation may be a nasal spray, nasal drops, or an aerosol administered via a nebulizer. The formulation may contain an aqueous or oily solution of the vaccine.

[0188] Vaccines can be liquid formulations, such as suspensions, syrups, or elixirs. Vaccines can also be formulations intended for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.

[0189] Vaccines can be incorporated into liposomes, microspheres, or other polymer matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vol. I–Vol. III (2nd ed., 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids and can be nontoxic, physiologically acceptable, and metabolizable carriers that are relatively simple to manufacture and administer.

[0190] Vaccines can be administered via electroporation, such as by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be performed by the methods and / or apparatus 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 contents of which are incorporated herein by reference in their entirety. Electroporation can be performed via minimally invasive devices.

[0191] Minimally invasive electroporation devices (“MIDs”) can be apparatuses for injecting the vaccines and related fluids described above into body tissues. The device may include a hollow needle, a DNA cartridge, and a fluid delivery component, wherein the device is adapted to actuate the fluid delivery component in use to simultaneously (e.g., automatically) inject DNA into the body tissue during needle insertion. This has the advantages of allowing for gradual injection of DNA and related fluids simultaneously with needle insertion, resulting in a more uniform distribution of fluid through the body tissue. Because the injected DNA is distributed over a larger area, pain experienced during injection is reduced.

[0192] Minimally invasive electroporation devices (MIDs) can inject vaccines into tissues without the use of needles. MIDs inject vaccines as small streams or jets, allowing the vaccine to penetrate the surface of the tissue and reach underlying tissues and / or muscles. The force following the small stream or jet can be provided by the expansion of a compressed gas (such as carbon dioxide passing through a tiny pore in a fraction of a second). Examples of minimally invasive electroporation devices and methods of use 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 contents of which are respectively incorporated herein by reference.

[0193] MIDs may include syringes that generate a high-speed jet of fluid that penetrates tissue painlessly. Such needle-free syringes are commercially available. Examples of needle-free syringes that may be used herein include those described in U.S. Patent Nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the contents of each of which are incorporated herein by reference.

[0194] A needle-free injector can be used to introduce (e.g., inject) a desired vaccine, in a form suitable for direct or indirect electrotransportation, into the tissue to be treated. This is typically achieved by contacting the tissue surface with the injector to actuate the delivery of the drug jet with a force sufficient to cause the vaccine to penetrate the tissue. For example, if the tissue to be treated is mucous membrane, skin, or muscle, the drug is sprayed toward the mucous membrane or skin surface with a force sufficient to cause the drug to penetrate through the stratum corneum and into the dermis, or into the underlying tissues and muscles, respectively.

[0195] Needle-free injectors are ideally suited for delivering vaccines to all types of tissues, particularly to the skin and mucous membranes. In some embodiments, needle-free injectors can be used to push a vaccine-containing liquid onto a surface and into the skin or mucous membranes of a subject. Representative examples of various types of tissues that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, pharynx, lungs, heart, kidneys, muscles, breast, colon, prostate, thymus, testes, skin, mucous membranes, ovaries, blood vessels, or any combination thereof.

[0196] MIDs may have needle electrodes for electroporating tissue. Improved results, superior to those achieved by pulses between a single pair of electrodes, are provided by pulses between multiple pairs of electrodes in a multi-electrode array (e.g., arranged in a rectangular or square pattern). For example, U.S. Patent No. 5,702,359, entitled “Needle Electrodes for Mediated Delivery of Drugs and Genes,” discloses needle arrays in which pulses can be generated on multiple pairs of needles during therapeutic treatment. In this application (which is incorporated herein by reference as it is stated throughout), the needles are arranged in a circular array but have connectors and switching devices to achieve pulses between opposing pairs of needle electrodes. A pair of needle electrodes for delivering recombinant expression vectors to cells can be used. Such devices and systems are described in U.S. Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, a single-needle device may be used that allows DNA injection and single-needle electroporation similar to a normal injection needle, applying pulses at voltages lower than those delivered by currently used devices, thereby reducing the electrical sensation experienced by the patient.

[0197] MIDs may include one or more electrode arrays. An array may include two or more needles having the same or different diameters. The needles may be evenly or unevenly spaced. The needles may be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches, or between 0.015 inches and 0.020 inches. The diameter of the needles may be 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.

[0198] A MID may consist of a pulse generator and two or more needle-type vaccine injectors that deliver vaccine and electroporation pulses in a single step. The pulse generator allows for flexible programming of pulse and injection parameters, as well as comprehensive recording and storage of electroporation and patient data, via a personal computer operated via a flash memory card. The pulse generator can deliver multiple voltage pulses over a short period of time. For example, the pulse generator can deliver three 15-volt pulses lasting 100 ms. An example of such a MID is the Elgen 1000 system from Inovio Biomedical Corporation, described in U.S. Patent No. 7,328,064, the contents of which are incorporated herein by reference.

[0199] MIDs can be CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) devices and systems, which are modular electrode systems that facilitate the introduction of macromolecules, such as DNA, into cells of selected tissues in the body or plant. A modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing a conductive link from a programmable constant-current pulse controller to the multiple needle electrodes, and a power supply. An operator can grasp the multiple needle electrodes, which are fixed to a support structure, and firmly insert them into the selected tissue in the body or plant. The macromolecule is then delivered into the selected tissue via the hypodermic needle. The programmable constant-current pulse controller is activated, and constant-current electrical pulses are applied to the multiple needle electrodes. The applied constant-current electrical pulses facilitate the introduction of the macromolecule into cells between the multiple electrodes. Cell death due to overheating of the cells is minimized by limiting power consumption in the tissue by means of the constant-current pulses. The Cellectra device and system are described in U.S. Patent No. 7,245,963, the contents of which are incorporated herein by reference.

[0200] The MID can be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device providing a hollow needle; and a fluid delivery component, wherein the device is adapted to actuate the fluid delivery component in use to simultaneously (e.g., automatically) inject fluid (the vaccine described herein) into said body tissue during needle insertion. An advantage is that the ability to gradually inject fluid while the needle is inserted results in a more uniform distribution of fluid through the body tissue. It is also believed that pain experienced during injection is reduced because the volumetric distribution of the injected fluid over a larger area.

[0201] Furthermore, automated fluid injection facilitates the automatic monitoring and registration of the actual dose of the injected fluid. This data can be stored via a control unit as needed for documentation purposes.

[0202] It should be understood that the injection rate can be linear or non-linear, and the injection can be performed after the needle has been inserted through the skin of the subject to be treated and while the needle is being further inserted into body tissue.

[0203] The fluid can be injected into suitable tissues, including tumor tissue, skin, or liver tissue, but also muscle tissue, via the device of the present invention.

[0204] The device also includes a needle insertion component for guiding the needle into body tissue. The rate of fluid injection is controlled by the rate of needle insertion. This has the advantages of controlling both needle insertion and fluid injection, allowing the insertion rate to be matched to the injection rate as needed. It also makes the device easier for the user to operate. Components for automatically inserting the needle into body tissue can be provided if needed.

[0205] The user can choose when to begin fluid injection. However, ideally, injection should begin when the tip of the needle reaches the muscle tissue, and the device may include components for sensing when the needle has been inserted to a depth sufficient to begin fluid injection. This means that when the needle reaches the desired depth (which will typically be the depth at the beginning of the muscle tissue), a prompt can be made to automatically begin fluid injection. The depth at the beginning of the muscle tissue can, for example, be taken as a preset needle insertion depth (such as a value of 4 mm), which would be considered sufficient for the needle to penetrate the skin layer.

[0206] The sensing component may include an ultrasound probe. The sensing component may include a component for sensing changes in impedance or resistance. In this case, the component may not record the depth of the needle in body tissue, but will instead be adapted to sense changes in impedance or resistance as the needle moves from different types of body tissue into muscle. Any of these alternatives provides a sensing component that can be initiated with relatively accurate and simple operation. The insertion depth of the needle can be further recorded as needed, and said depth can be used to control the injection of fluid so that the volume of fluid to be injected is determined based on the needle insertion depth being recorded.

[0207] The device may also include: a base for supporting the needle and a housing for receiving the base therein, wherein 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 into the housing, and when the base is in a second forward position within the housing, the needle extends out of the housing. This is advantageous to the user because the housing can be aligned on the patient's skin, and the needle can then be inserted into the patient's skin by moving the housing relative to the base.

[0208] As described above, it is desirable to achieve a controlled rate of fluid injection so that the fluid is uniformly distributed along the length of the needle when it is inserted into the skin. The fluid delivery component may include a piston drive adapted to inject fluid at a controlled rate. The piston drive may be activated, for example, by a servo motor. However, the piston drive may be actuated by a base that moves relative to the housing in the axial direction. It should be understood that alternative devices for fluid delivery can be provided. Therefore, for example, a closed container that can be squeezed for fluid delivery at a controlled or uncontrolled rate can be provided instead of the injection tube and piston system.

[0209] The device described above can be used for any type of injection. However, it is envisioned to be particularly useful in the field of electroporation, and therefore it may also include components for applying voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during the electroporation process. This is particularly advantageous because it means that an electric field is applied to the same area as the injected fluid. Traditionally, electroporation suffers from the problem of accurately aligning the electrode with the previously injected fluid, and therefore users tend to inject a fluid volume larger than required over a larger area and apply an electric field over a higher area in an attempt to ensure overlap between the injected material and the electric field. Using this invention, the volume of the injected fluid and the magnitude of the applied electric field can be reduced while achieving good alignment between the electric field and the fluid.

[0210] The present invention has several aspects, which are illustrated by the following non-limiting embodiments.

[0211] 3. Example

[0212] Example 1

[0213] IL-21 expression

[0214] Construct a plasmid encoding the IL-21 gene (i.e., pVAX-mIL-21Opt) to express IL-21. Figure 1 Before insertion into the plasmid, the DNA sequence of IL-21 was optimized for codons and RNA.

[0215] The plasmid was transfected into HEK 293T cells to confirm IL-21 expression. Cell supernatant was analyzed by ELISA. Results showed that IL-21 was expressed in HEK 293T cells. Figure 2 ).

[0216] Example 2

[0217] IL-21 increases serum titers of IgG and IgA.

[0218] When the vaccine is administered intramuscularly, mice are used as a model system to determine whether IL-21 can act as an adjuvant. The vaccine contains toxin A and toxin B antigens from Clostridium difficile encoded by their respective plasmids, as well as IL-21.

[0219] Specifically, using plasmid pVAX-mIL-21Opt( Figure 1 As described in Example 1 above, and using plasmids encoding toxin A and toxin B antigens from Clostridium difficile as described above, one group of mice was immunized. A second group of mice was immunized using only plasmids encoding toxin A and toxin B antigens. A third group of mice was immunized using only the empty control plasmid pVAX. Mice were immunized via an intramuscular route using electroporation. Circulating antigen-specific IgG and IgA antibody-secreting cells in the blood of the immunized animals were then analyzed to determine the effect of the IL-21 adjuvant.

[0220] like Figure 4 As shown in the figure above, compared with immunization using the antigen alone, IL-21 adjuvant increased the total serum antitoxin A IgG, with a strong titer observed at a 1:2000 dilution in the IL-21 group, while none was observed in the antigen-only group. When analyzing serum IgA, it was noted that IL-21 adjuvant produced detectable levels of antigen-specific IgA titrated at a 1:2000 dilution, while immunization using the antigen alone did not show any strong signal in the novice animals. Figure 4 (See image below).

[0221] The data above show that IL-21 has the ability to act as an adjuvant when administered intramuscularly, as it enhances the humoral immune response to toxin A and toxin B antigens from Clostridium difficile. The data also indicate that IL-21 can act as an adjuvant for bacterial antigens.

[0222] Example 3

[0223] IL-21 increases cellular and humoral immune responses to HIV antigens.

[0224] The IL-21 adjuvant is also administered in combination with plasmids encoding the EnvA and EnvC antigens from HIV. Including IL-21 in the vaccine enhances both cellular and humoral immune responses to EnvC. The vaccine contains the EnvA and EnvC antigens from HIV, as well as IL-21. The EnvA antigen, EnvC antigen, and IL-21 are encoded by separate plasmids.

[0225] Specifically, the EnvA antigen is a common protein (SEQ ID NO:6) encoded by the nucleotide sequence listed in SEQ ID NO:5. This nucleotide sequence listed in SEQ ID NO:5 is incorporated into the plasmid.

[0226] The EnvC antigen is a common protein (SEQ ID NO:8) encoded by the nucleotide sequence listed in SEQ ID NO:7. This nucleotide sequence listed in SEQ ID NO:7 is incorporated into the plasmid.

[0227] Specifically, using plasmid pVAX-mIL-21Opt( Figure 1 As described in Example 1 above, and using plasmids encoding EnvA and EnvC antigens as described above, one group of mice was immunized. A second group of mice was immunized using only plasmids encoding EnvA and EnvC antigens. A third group of mice was immunized using only the empty control plasmid pVAX. Mice were immunized via an intramuscular route using electroporation. Cellular immune responses in the immunized mice were assessed using an interferon-γ ELISpot assay.

[0228] like Figure 5 As shown, immunization with IL-21 increased the cellular immune response to the EnvC antigen by more than 2-fold compared to the antigen alone. Therefore, these data suggest that IL-21 can be used as an adjuvant in muscle tissue because it enhances the cellular immune response to both EnvA and EnvC antigens.

[0229] Antibody response was measured in the serum of immunized animals after a third immunization via ELISA targeting the EnvA protein. Figure 6 At a 1:400 dilution, the mean OD for HIV EnvA / C was approximately 0.6, while the mean OD reading for the IL-21 adjuvant group was 1.0.

[0230] In summary, these data suggest that when included in DNA vaccines, IL-21 exhibits novel adjuvant activity in the following forms: increased frequency of antibody-secreting cells, increased amount and class switching of IgG produced, increased IgA production, and increased IFN-γ secretion.

[0231] It should be understood that the preceding detailed description and accompanying embodiments are illustrative only and should not be construed as limiting the scope of the invention, which is defined only by the appended claims and their equivalents.

[0232] Various modifications and improvements to the disclosed embodiments will be apparent to those skilled in the art. Such modifications and improvements may be made relating to, but not limited to, the chemical structures, substituents, derivatives, intermediates, synthesis, compositions, formulations, and / or methods of use of the invention, without departing from its nature and scope. sequence list <110> University of Pennsylvania Board of Trustees Aino Pharmaceutical Co., Ltd. David B. Weiner Matthew P. Morrow <120> Vaccines with interleukin-21 as an adjuvant <130> 206108‑0032‑00‑WO.604750 / UPVG0052 <150> US 62 / 058,304 <151> 2014-10-01 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 495 <212> DNA <213> Artificial sequence <220> <223> Chemically synthesized <400> 1 atggactgga cctggatcct gttcctggtc gccgcagcca caagggtgca cagcgagagg 60 accctggtct gcctggtggt gatcttcctg ggcaccgtgg cccacaagag cagcccccag 120 ggccccgaca ggctgctgat caggctgagg cacctgatcg acatcgtgga gcagctgaag 180 atctacgaga acgacctgga ccccgagctg ctctccgccc ctcaagacgt gaagggccac 240 tgcgagcacg ccgccttcgc ctgcttccag aaggccaagc tgaagcccag caaccccggc 300 aacaacaaga ccttcatcat cgacctggtg gcccagctga gaagaaggct gcccgccaga 360 aggggcggca agaagcagaa acacatcgcc aagtgcccca gctgcgacag ctacgagaag 420 aggaccccca aggaatttct ggagaggctg aagtggctgc tgcagaagat gattcaccag 480 cacctgagct gatga 495 <210> 2 <211> 163 <212> PRT <213> Artificial Sequence <220> <223> Chemically Synthesized <400> 2 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr 20 25 30 Val Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg 35 40 45 Leu Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn 50 55 60 Asp Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His 65 70 75 80 Cys Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro 85 90 95 Ser Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln 100 105 110 Leu Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His 115 120 125 Ile Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys 130 135 140 Glu Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln 145 150 155 160 His Leu Ser <210> 3 <211> 537 <212> DNA <213> Artificial sequence <220> <223> Chemically synthesized <400> 3 atggactgga cttggattct gtttctggtc gcagcagcaa ctagagtgca ttcacgcagc 60 agccctggga acatggagag gattgtcatc tgcctgatgg tgattttcct gggcacactg 120 agccctggga acatggagag gattgtcatc tgcctgatgg tgattttcct gggcacactg 120 gtccacaaga gctcctctca gggacaggac aggcatatga tcaggatgcg acagctgatc 180 gtccacaaga gctcctctca gggacaggac aggcatatga tcaggatgcg acagctgatc 180 gacatcgtgg atcagctgaa gaactacgtg aacgacctgg tcccagagtt tctgcctgca 240 gacatcgtgg atcagctgaa gaactacgtg aacgacctgg tcccagagtt tctgcctgca 240 ccagaggatg tcgaaactaa ctgcgaatgg agtgccttct catgtttcca gaaggcacag 300 ccagaggatg tcgaaactaa ctgcgaatgg agtgccttct catgtttcca gaaggcacag 300 ctgaagtccg ctaacaccgg aaacaatgag cgaatcatca acgtgagcat taagaaactg 360 ctgaagtccg ctaacaccgg aaacaatgag cgaatcatca acgtgagcat taagaaactg 360 aagcgaaagc cccctagtac caatgctggc cggagacaga agcacagact gacatgccct 420 aagcgaaagc cccctagtac caatgctggc cggagacaga agcacagact gacatgccct 420 agctgtgatt cctacgaaaa gaaaccaccc aaggagttcc tggaacgctt taaaagcctg 480 agctgtgatt cctacgaaaa gaaaccaccc aaggagttcc tggaacgctt taaaagcctg 480 ctgcagaaaa tgattcacca gcacctgtct tccagaaccc acggctcaga ggattca 537 ctgcagaaaa tgattcacca gcacctgtct tccagaaccc acggctcaga ggattca 537 <210> 4<210> 4 <211> 179<211> 179 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220> <220> <223> 化学合成的 <223> Chemically synthesized <400> 4 <400> 4 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 1 5 10 15 His Ser Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu His Ser Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu 20 25 30 20 25 30 Met Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly 35 40 45 Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp 50 55 60 Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala 65 70 75 80 Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe 85 90 95 Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile 100 105 110 Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn 115 120 125 Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser 130 135 140 Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu 145 150 155 160 Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser 165 170 175 Glu Asp Ser <210> 5 <211> 2142 <212> DNA <213> Artificial Sequence <220> <223> Chemically synthesized <400> 5 ggatccatgg actggacctg gattctgttc ctggtggccg ccgccaccag agtgcacagc 60 agagtgatgg gcatccagcg gaattgccag cacctgtgga gatggggcac catgatcctg 120 ggcatgatca tcatctgctc tgccgccgag aacctgtggg tgaccgtgta ctacggcgtg 180 cctgtgtgga aggacgccga gaccaccctg ttctgcgcca gcgacgccaa ggcctacgat 240 accgaagtgc acaatgtgtg ggccacccac gcctgcgtgc ctaccgatcc caacccccag 300 gagatcaacc tggagaacgt gaccgaggag ttcaacatgt ggaagaacaa catggtggag 360 cagatgcaca ccgacatcat cagcctgtgg gaccagagcc tgaagccttg cgtgaagctg 420 acccctctgt gcgtgaccct gaactgcagc aacgtgaacg tgaccaccaa catcatgaag 480 ggcgagatca agaactgcag cttcaacatg accaccgagc tgcgggacaa gaagcagaaa 540 gtgtacagcc tgttctacaa gctggacgtg gtgcagatca acaagagcaa cagcagcagc 600 cagtaccggc tgatcaactg caacaccagc gccatcaccc aggcctgccc caaagtgagc 660 ttcgagccca tccccatcca ctactgcgcc cctgccggct tcgccatcct gaagtgcaag 720 gacaaggagt ttaacggcacggccctgc aagaatgtga gcaccgtgca gtgcacccac 780 ggcatcaagc ccgtggtgtc cacccagctg ctgctgaacg gcagcctggc cgaggagaa 840 gtgatgatcc ggagcgagaa catcaccaac aacgccaaga acatcatcgt gcagctgacc 900 aagcccgtga agatcaattg cacccggccc aacaacaaca cccggaagag catcagaatc 960 ggccctggcc aggccttcta cgccaccggc gacatcatcg gcgatatcag gcaggcccac 1020 tgcaatgtga gccggaccga gtggaacgag accctgcaga aagtggccaa gcagctgcgg 1080 aagtacttca acaacaagac catcatcttc accaacagca gcggcggcag actgagaatc 1140 accacccaca gcttcaattg tggcggcgag ttcttctact gcaatacctc cggcctgttc 1200 aacagcacct ggaacggcaa cggcaccaag aagaagaaca gcaccgagag caacgacacc 1260 atcaccctgc cctgccggat caagcagatc atcaatatgt ggcagagggt gggccaggcc 1320 atgtacgcccc ctcccatcca gggcgtgatc agatgcgaga gcaacatcac cggcctgctg 1380 ctgaccagag atggcggcga caacaacagc aagaacgaga ccttcagacc tggcggcgga 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 ctgggcgtgg cccccaccaa ggccaagaga agagtggtgg agcgggaga gagagctgtg ggcatcggcg ccgtgttcct gggcttcctg ggagccgccg gaagcaccat gggagccgcc 1620 agcatcacc tgaccgtgca ggccagacag ctgctgagcg gcattgtgca gcagcagagc 1680. aacctgctga gagccatcga ggcccagcag cacctgctga agctgacagt gtggggcatc 1740. aaacagctgc aggcccgcgt gctggccgtg gagagatacc tgaaggacca gcagctgctg ggcatctggg gctgcagcgg caagctgatc tgcaccacca acgtgccctg gaatagcagc tggagcaaca agagccagag cgagatctgg gacaacatga cctggctgca gtgggacaag gagatcagca actacaccga tatcatctac aacctgatcg aggagagcca gaaccagcag gagaagaacg agcaggatct gctggccctg gacaagtggg ccaacctgtg gaactggttc 2100. 2100. 2100. 2100. 2100. 2100. 2100. 2100 ggcctgagaa tcgtgttcgc cgtgctgtct gtgtgactcg ag 2142 <210> 6 <211> 709 <212> PRT <213> Synthetic Sequence <220> <223> Chemically Synthesized <400> 6 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val[[ID=P17]] 1 5 10 15 His Ser Arg Val Met Gly Ile Gln Arg Asn Cys Gln His Leu Trp Arg 20 25 30 Trp Gly Thr Met Ile Leu Gly Met Ile Ile Ile Cys Ser Ala Ala Glu 35 40 45 Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Asp Ala 50 55 60 Glu Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 65 70 75 80 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 85 90 95 Pro Gln Glu Ile Asn Leu Glu Asn Val Thr Glu Glu Phe Asn Met Trp 100 105 110 Lys Asn Asn Met Val Glu Gln Met His Thr Asp Ile Ile Ser Leu Trp 115 120 125 Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 130 135 140 Leu Asn Cys Ser Asn Val Asn Val Thr Thr Asn Ile Met Lys Gly Glu 145 150 155 160 Ile Lys Asn Cys Ser Phe Asn Met Thr Thr Glu Leu Arg Asp Lys Lys 165 170 175 Gln Lys Val Tyr Ser Leu Phe Tyr Lys Leu Asp Val Val Gln Ile Asn 180 185 190 Lys Ser Asn Ser Ser Ser Gln Tyr Arg Leu Ile Asn Cys Asn Thr Ser 195 200 205 Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile 210 215 220 His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys 225 230 235 240 Glu Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val Gln Cys 245 250 255 Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 260 265 270 Ser Leu Ala Glu Glu Glu Val Met Ile Arg Ser Glu Asn Ile Thr Asn 275 280 285 Asn Ala Lys Asn Ile Ile Val Gln Leu Thr Lys Pro Val Lys Ile Asn 290 295 300 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro 305 310 315 320 Gly Gln Ala Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln 325 330 335 Ala His Cys Asn Val Ser Arg Thr Glu Trp Asn Glu Thr Leu Gln Lys 340 345 350 Val Ala Lys Gln Leu Arg Lys Tyr Phe Asn Asn Lys Thr Ile Ile Phe 355 360 365 Thr Asn Ser Ser Gly Gly Arg Leu Arg Ile Thr Thr His Ser Phe Asn 370 375 380 Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Ser Gly Leu Phe Asn Ser 385 390 395 400 Thr Trp Asn Gly Asn Gly Thr Lys Lys Lys Asn Ser Thr Glu Ser Asn 405 410 415 Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp 420 425 430 Gln Arg Val Gly Gln Ala Met Tyr Ala Pro Pro Ile Gln Gly Val Ile 435 440 445 Arg Cys Glu Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 450 455 460 Asp Asn Asn Ser Lys Asn Glu Thr Phe Arg Pro Gly Gly Gly Asp Met 465 470 475 480 Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 485 490 495 Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Glu 500 505 510 Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu 515 520 525 Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val 530 535 540 Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu 545 550 555 560 Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Lys Leu Thr Val Trp 565 570 575 Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu 580 585 590 Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile 595 600 605 Cys Thr Thr Asn Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln 610 615 620 Ser Glu Ile Trp Asp Asn Met Thr Trp Leu Gln Trp Asp Lys Glu Ile 625 630 635 640 Ser Asn Tyr Thr Asp Ile Ile Tyr Asn Leu Ile Glu Glu Ser Gln Asn 645 650 655 Gln Gln Glu Lys Asn Glu Gln Asp Leu Leu Ala Leu Asp Lys Trp Ala 660 665 670 Asn Leu Trp Asn Trp Phe Asp Ile Ser Asn Trp Leu Trp Tyr Ile Lys 675 680 685 Ile Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Val Phe 690 695 700 Ala Val Leu Ser Val 705 <210> 7 <211> 2140 <212> DNA <213> Artificial sequence <220> <223> Chemically synthesized <400> 7 ggatccgcca ccatggattg gacctggatt ctgttcctgg tggccgccgc cacaagagtg 60 cacagcagag tgcggggcat cctgagaaat tgccagcagt ggtggatctg gggcattctg 120 gggttctgga tgctgatgat ctgcaacgtg atgggcaacc tgtgggtgac cgtgtactac 180 ggcgtgcctg tgtggagga ggccaagacc accctgttct gtgccagcga tgccaaggcc 240 tacgagaccg aggtgcacaa tgtgtgggcc acccacgcct gtgtgcccac cgatcccaac 300 cctcaggaga tggtgctgga gaacgtgacc gagaacttca acatgtgaa gaacgacatg 360 gtggaccaga tgcacgagga catcatcagc ctgtgggacc agagcctgaa gccttgcgtg 420 aagctgaccc ctctgtgcgt gaccctgaac tgccggaaca acgtgaacaa caacaacacc 480 540 cagaaggtgt acgccctgtt ctaccggctg gacatcgtgc ccctgaacga gaaacaac 600 agcaacgact accggctgat caactgcaac accagcgcca tcacccaggc ctgtcccaag 660 gtgtccttcg accccatccc catccactat tgtgcccctg ccggctacgc catcctgaag 720 tgcaacaa agaccttcaa cggcaccggc ccctgcaata atgtgagcac cgtgcagtgt 780 acccacggca tcaagcctgt ggtgtccacc cagctgctgc tgaatggcag cctggccgag 840 gaggagatta tcatccggag cgagaacctg accaacaacg ccaagaccat cattgtgcac 900 ctgaatgaga gcgtggagat cgtgtgtacc cggcccaaca acaatacccg gaagagcatc 960 agaatcggcc ctggccagac cttttacgcc accggcgaca tcatcggcga tatcaggcag 1020 gcccactgca atatcagcga ggagaagtgg aacaagaccc tgcagcgggt gtccgagaag 1080 ctgaaggagc acttccccaa taagaccatc aagttcgccc ctagcagcgg cggcagactg 1140 gagatcacca cccacagctt caactgcagg ggcgagttct tctactgcaa taccagcaag 1200 ctgttcaaca gcacctacat gcccaacagc accaacaata ccaacaccac catcaccctg 1260 ccctgccgga tcaagcagat catcaatatg tggcaggaag tgggcagagc catgtacgcc 1320 cctcccatcg agggcaacat cacctgcaag tccaacatca ccggcctgct gctgacaaga 1380 gatggcggca agaacgacac caatgacacc gagaccttca gacctggcgg cggagacatg 1440 agggacaact ggcggagcga gctgtacaag tacaaggtgg tggagatcaa gcctctgggc 1500 gtggccccta ccaaggccaa gaggagagtg gtggagaggg agaagagagc cgtgggcatc 1560 ggcgccgtgt ttctgggctt tctgggagcc gccggatcta caatgggagc cgccagcatc 1620 acactgaccg tgcaggccag acagctgctg agcggcatcg tgcagcagca gagcaatctg 1680 ctgagagcca tcgaggccca gcagcacatg ctgcagctga cagtgtgggg catcaagcag 1740 ctgcagacca gagtgctggc catcgagcgc tacctgaagg atcagcagct gctgggcatc 1800 tggggctgta gcggcaagct gatctgtacc accgccgtgc cttggaatag cagctggagc 1860 aacaagagcc aggaggacat ctgggacaac atgacctgga tgcagtggga ccgggagatc 1920 agcaactaca ccgacaccat ctacaggctg ctggaggaca gccagaacca gcaggagaag 1980 aacgagaagg acctgctggc cctggacagc tggaagaacc tgtggaactg gttcgacatc 2040 accaactggc tgtggtacat caagatcttc atcatgattg tgggcggcct gatcggcctg 2100 agaatcatct tcgccgtgct gagcatctga tagcggccgc 2140 <210> 8 <211> 705 <212> PRT <213> Artificial Sequence <220> <223> Chemically Synthesized <400> 8 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Arg Val Arg Gly Ile Leu Arg Asn Cys Gln Gln Trp Trp Ile 20 25 30 Trp Gly Ile Leu Gly Phe Trp Met Leu Met Ile Cys Asn Val Met Gly 35 40 45 Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 50 55 60 Lys Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Thr Glu 65 70 75 80 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 85 90 95 Pro Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp 100 105 110 Lys Asn Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp 115 120 125 Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 130 135 140 Leu Asn Cys Arg Asn Asn Val Asn Asn Asn Asn Thr Met Lys Glu Glu 145 150 155 160 Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Glu Leu Arg Asp Lys Lys 165 170 175 Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Leu Asn 180 185 190 Glu Lys Asn Asn Ser Asn Asp Tyr Arg Leu Ile Asn Cys Asn Thr Ser 195 200 205 Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Ile 210 215 220 His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys Asn Asn Lys 225 230 235 240 Thr Phe Asn Gly Thr Gly Pro Cys Asn Asn Val Ser Thr Val Gln Cys 245 250 255 Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 260 265 270 Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn 275 280 285 Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu Ser Val Glu Ile Val 290 295 300 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro 305 310 315 320 Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln 325 330 335 Ala His Cys Asn Ile Ser Glu Glu Lys Trp Asn Lys Thr Leu Gln Arg 340 345 350 Val Ser Glu Lys Leu Lys Glu His Phe Pro Asn Lys Thr Ile Lys Phe 355 360 365 Ala Pro Ser Ser Gly Gly Arg Leu Glu Ile Thr Thr His Ser Phe Asn 370 375 380 Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Lys Leu Phe Asn Ser 385 390 395 400 Thr Tyr Met Pro Asn Ser Thr Asn Asn Thr Asn Thr Thr Ile Thr Leu 405 410 415 Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Arg 420 425 430 Ala Met Tyr Ala Pro Pro Ile Glu Gly Asn Ile Thr Cys Lys Ser Asn 435 440 445 Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Lys Asn Asp Thr Asn 450 455 460 Asp Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp 465 470 475 480 Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly 485 490 495 Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg 500 505 510 Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly 515 520 525 Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln 530 535 540 Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile 545 550 555 560 Glu Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln 565 570 575 Leu Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln 580 585 590 Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 595 600 605 Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Glu Asp Ile Trp 610 615 620 Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr Thr 625 630 635 640 Asp Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu Lys 645 650 655 Asn Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp Asn 660 665 670 Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 675 680 685 Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu Ser 690 695 700 authorities 705 <210> 9 <211> 9 <212> PRT <213> artificial sequence <220> <223> Chemical composition <400> 9 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5

Claims

1. A vaccine comprising a) Selected from at least one of the following groups: antigen and nucleotide molecule encoding the antigen; and b) A nucleotide molecule consisting of a nucleotide sequence encoding IL-21, wherein the nucleotide sequence is SEQ ID NO:

3.

2. The vaccine of claim 1, wherein the antigen is encoded by a first nucleic acid and IL-21 is encoded by a second nucleic acid.

3. The vaccine of claim 2 further comprises an antigenic peptide and an IL-21 peptide, wherein the antigenic peptide is encoded by the same nucleic acid sequence as the antigen of claim 2, and wherein the IL-21 peptide is encoded by the same nucleic acid sequence as the IL-21 of claim 2.

4. The vaccine of claim 2, wherein the second nucleic acid further comprises an expression vector.

5. The vaccine of claim 1, wherein the antigen is 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.

6. The vaccine of claim 5, wherein the HPV antigen is selected from the group consisting of HPV16 E6 antigen, HPV16 E7 antigen, and combinations thereof.

7. The vaccine of claim 5, 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.

8. The vaccine of claim 5, 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.

9. The vaccine of claim 5, wherein the Plasmodium falciparum antigen comprises cyclosporine (CS) antigen.

10. The vaccine of claim 5, wherein the Clostridium difficile antigen is selected from the group consisting of toxin A, toxin B, and combinations thereof.

11. The vaccine of claim 1, further comprising a pharmaceutically acceptable excipient.

12. Use of IL-21 in the preparation of vaccines to enhance immune responses in subjects in need. IL-21 is encoded by the following nucleotide sequence: the nucleotide sequence listed in SEQ ID NO:3, The vaccine was administered to the subject.

13. The use as claimed in claim 12, wherein administering the vaccine includes electroporation.

14. The use as claimed in claim 12, wherein increasing the immune response in the subject comprises increasing the cellular immune response, humoral immune response, or both cellular and humoral immune responses in the subject.

15. A nucleic acid molecule for expressing IL-21, comprising a coding sequence listed in SEQ ID NO:

3.

16. The nucleic acid molecule of claim 15, wherein the nucleic acid molecule is a plasmid.

Images