Vaccines having antigen and interleukin-21 as adjuvant

Abstract

To provide a vaccine comprising an antigen and IL-21.SOLUTION: Disclosed herein is a vaccine comprising an antigen and IL-21. Also disclosed herein are methods for increasing an immune response in a subject. The methods may comprise administering the vaccine to the subject in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 058,304, filed October 1, 2014, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to vaccines comprising an antigen and IL-21, and methods of administering such vaccines. [Background technology]

[0003] Vaccines are used to stimulate an immune response in individuals and provide protection and / or treatment against specific diseases. Some vaccines contain antigens to induce an immune response. Some antigens induce strong immune responses, while others induce weak immune responses. Weak immune responses to antigens can be enhanced by including adjuvants in the vaccine. Adjuvants come in many different forms, such as aluminum salts, oil emulsions, bactericidal components of bacteria or other pathogens, cytokines, etc.

[0004] Cytokines are proteins produced by cells that affect the behavior of other cells and, unlike many adjuvants, can modulate specific immune responses. One such cytokine is interleukin-21 (IL-21), which acts on lymphoid and myeloid populations and 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 the proliferation and functional responses of CD8+ T cells, and play a role in the development of B cell immunoglobulin responses. IL-21 is produced by CD8+ and CD4+ T cell populations, including follicular helper T (TFH) cells, type 17 helper T (Th17) cells, and natural killer T (NKT) cells.

[0005] Additionally, vaccines are administered in many different ways (e.g., injection, oral, etc.) and to many different tissues (e.g., intramuscular, intradermal, etc.). However, not all delivery methods are equal. While some delivery methods allow for greater compliance within a population, others may affect the immunogenicity and / or safety of the vaccine. Thus, there remains a need in the art for the development of safe and more effective adjuvants that enhance antigen responses regardless of antigen identity and route of administration. Summary of the Invention

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

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

[0008] The antigen may be selected from the group consisting of human papillomavirus (HPV) antigens, human immunodeficiency virus (HIV) antigens, influenza antigens, Plasmodium falciparum antigens, Clostridium difficile antigens, and fragments thereof. The HPV antigen may be selected from the group consisting of HPV16E6 antigens, HPV16E7 antigens, and combinations thereof. The HIV antigen may be selected from the group consisting of EnvA, EnvB, EnvC, EnvD, B Nef-Rev, Gag, and any combinations thereof. The influenza antigen may be selected from the group consisting of H1 HA, H2 HA, H3 HA, H5 Ha, BHA antigens, and any combinations thereof. The Plasmodium falciparum antigen may include a circumsporozoite antigen. The Clostridium difficile antigen may be selected from the group consisting of toxin A, toxin B, and combinations thereof.

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

[0010] The present invention is also directed to a method for enhancing an immune response in a subject in need thereof. 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 a nucleotide sequence having at least about 95% identity to the nucleotide sequence set forth in SEQ ID NO:3 and the nucleotide sequence set forth in SEQ ID NO:3. IL-21 may be encoded by the nucleotide sequence set forth in SEQ ID NO:3.

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

[0012] The present invention is further directed to nucleic acid molecules comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 3 and a nucleotide sequence that is 95% or greater identical to SEQ ID NO: 3. The nucleic acid molecule may be a plasmid. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows a map of the plasmid pVAX-mIL-21 Opt, which contains an optimized nucleic acid encoding murine IL-21. [Figure 2] Figure 1 shows the expression of IL-21 in the supernatant of transfected HEK 293T cells. [Figure 3] A B cell ELISpot assay is used to demonstrate humoral immune responses in mice immunized via the intramuscular route with plasmids encoding toxin A and toxin B antigens from C. difficile. [Figure 4] Figure 1 shows the humoral immune response of mice immunized via the intramuscular route using the ELYSA assay. [Figure 5] Figure 1 shows the cellular immune response in mice immunized via the intramuscular route with plasmids encoding EnvA and EnvC antigens from HIV using an interferon gamma ELISpot assay. [Figure 6] The ELYSA assay is used to demonstrate the humoral immune response of mice immunized via the intramuscular route with plasmids encoding EnvA and EnvC antigens from HIV. DETAILED DESCRIPTION OF THE INVENTION

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

[0015] In some instances, IL-21 can function as a general-purpose adjuvant, eliciting a higher immune response in subjects regardless of the source of the antigen or route of administration compared to vaccines containing the antigen alone. IL-21 can further enhance the immune response to both viral and bacterial antigens, such as HIV antigens and Clostridium difficile antigens. IL-21 can also take the form of a DNA / peptide combination with an antigen, which is a nucleic acid / peptide combination to achieve a greater immune response than DNA or peptide alone with the antigen. In some instances, IL-21 can further enhance the immune response in both muscle and skin tissues, as indicated by increased interferon-γ (IFN-γ) production.

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

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

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

[0019] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0020] As used herein, "complement" or "complementary" refers to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

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

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

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

[0024] As used herein, "genetic construct" or "construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that encodes a protein.The coding sequence comprises a start signal and a stop signal that are operably linked to regulatory elements that comprise a promoter and a polyadenylation signal that can direct 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 genetic construct or construct that contains the necessary regulatory elements operably linked to the coding sequence that encodes a protein, so that the coding sequence is expressed when present in the cells of an individual.

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

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

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

[0028] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, RNA, or hybrids, both genomic and cDNA, where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

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

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

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

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

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

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

[0035] As used herein, "substantially complementary" means that a first sequence is at least as complementary to the complement of a second sequence over 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. "Stringent hybridization" can mean that two sequences are 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each other, or that two sequences hybridize under stringent hybridization conditions.

[0036] As used herein, "substantially identical" means that the first and second amino acid sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10 ... "Identity" can mean 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% identity over a region of 00, 700, 800, 900, 1000, 1100, or more amino acids. In addition, being substantially identical means that the first nucleic acid sequence and the second nucleic acid sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, It can also mean at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 700, 800, 900, 1000, 1100 or more nucleotides.

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

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

[0039] A variant can be further defined as a peptide or polypeptide that differs in amino acid sequence due to amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. A variant also refers to a protein having an amino acid sequence substantially identical to a reference protein that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydrophilicity index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydrophilicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By considering the hydrophilicity of amino acids in a peptide, one can calculate the peptide's maximum local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids with hydrophilicity values within ±2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

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

[0041] As used herein, "vector" refers to a nucleic acid sequence that includes a replication origin.Vector can be a virus vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome.Vector can be a DNA or RNA vector.Vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid.Vector can contain or include one or more heterologous nucleic acid sequences.

[0042] When numerical ranges are recited herein, each intervening number to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 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 expressly contemplated.

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

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

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

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

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

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

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

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

[0051] Murine IL-21 may be an optimized nucleic acid sequence of SEQ ID NO: 1, which encodes SEQ ID NO: 2. In some embodiments, murine 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 entire length of the nucleic acid sequence set forth in SEQ ID NO: 1. In other embodiments, murine 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 entire length of the amino acid sequence set forth in SEQ ID NO: 2. Murine 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 entire length of the amino acid sequence set forth in SEQ ID NO: 2.

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

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

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

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

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

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

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

[0059] The antigen may be contained in a protein, nucleic acid, fragment thereof, variant thereof, or combination thereof derived from any number of organisms, such as a virus, parasite, bacterium, fungus, or mammal. The antigen may be associated with an autoimmune disease, allergy, or asthma. In other embodiments, the antigen may be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV), or human immunodeficiency virus (HIV). As described below, the antigen of the vaccine may be selected from the group consisting of HIV antigens, Clostridium difficile antigens, and fragments thereof. The HIV antigen may be selected from the group consisting of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof.

[0060] Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. Antigens can elicit a stronger immune response when combined with an adjuvant, as described above.

[0061] (1) Viral antigens The antigen may be a viral antigen, or a fragment or variant thereof. The viral antigen may be derived from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens include papillomaviruses, e.g., human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (variola major and variola minor), 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, haemophilus influenzae virus, leukemia virus (LEV-II), ... The virus may be derived from a virus such as a rabies virus, an Ebola virus, a Marburg virus, a measles virus, a mumps virus, a respiratory syncytial virus (RSV), a herpes simplex virus type 1 (oral herpes), a herpes simplex virus type 2 (genital herpes), a varicella zoster virus (varicella zoster, also known as chickenpox), a cytomegalovirus (CMV), such as human CMV, an Epstein-Barr virus (EBV), a flavivirus, a foot-and-mouth disease virus, a chikungunya virus, a Lassa fever virus, an arenavirus, or an oncogenic virus.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0086] The HPV antigen may be the HPV E6 or E7 domain of each HPV type. For example, in the case of HPV type 16 (HPV16), the HPV16 antigen may include the HPV16 E6 antigen, the HPV16 E7 antigen, a fragment, a variant, or a combination thereof. Similarly, the HPV antigen may be HPV6 E6 and / or E7, HPV11 E6 and / or E7, HPV18 E6 and / or E7, HPV31 E6 and / or E7, HPV33 E6 and / or E7, HPV52 E6 and / or E7, or HPV58 E6 and / or E7, a fragment, a variant, or a combination thereof.

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

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

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

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

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

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

[0093] In other embodiments, the RSV antigen is selected from the group consisting of, but not limited to, an RSV F protein from a RSV Long strain, an RSV G protein from a RSV Long strain, a consensus RSV G amino acid sequence, an optimized nucleic acid encoding the RSV G amino acid sequence, a human RSV genome of a RSV Long strain, a consensus RSV F amino acid sequence, an optimized nucleic acid encoding the RSV F amino acid sequence, an RSV NS1 protein from a RSV Long strain, an RSV NS2 protein from a RSV Long strain, an RSV N protein from a RSV Long strain, an RSV P protein from a RSV Long strain, an RSV M protein from a RSV Long strain, an RSV SH protein from a RSV Long strain, an RSV M2-1 protein from a RSV Long strain, an RSV M2-2 protein from a RSV Long strain, an RSV L protein from a RSV Long strain, an RSV G protein from a RSV subtype B isolate H5601, an RSV G protein from a RSV subtype B isolate H1068, an RSV G protein from a RSV subtype B isolate H559 ... Long strain, an RSV G protein from a RSV Long strain, an RSV G protein from a RSV subtype B isolate H5598, an RSV G protein from a RSV Long strain, an RSV G protein from a RSV Long strain, an RSV G protein from a RSV Long strain, an RSV G The G protein may be the RSV G protein from RSV subtype B isolate H1123, or a fragment or variant thereof.

[0094] (d) influenza antigen IL-21 can be bound or combined with an influenza antigen, or a fragment thereof, or a variant thereof. Influenza antigens are antigens that can induce an immune response against one or more influenza serotypes in mammals. The antigen can include the full-length translation product HA0, the subunit HA1, the subunit HA2, a variant thereof, a fragment thereof, or a combination thereof. The influenza hemagglutinin antigen can be a consensus sequence derived from multiple strains of influenza A serotype H1, a consensus sequence derived from multiple strains of influenza A serotype H2, a hybrid sequence containing portions of two different consensus sequences derived from different sets of multiple strains of influenza A serotype H1, or a consensus sequence derived from multiple strains of influenza B. The influenza hemagglutinin antigen can be derived from influenza B.

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

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

[0097] The consensus hemagglutinin antigen may further comprise one or more additional amino acid sequence elements. The consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence at its N-terminus. The consensus hemagglutinin antigen may further comprise an immunogenic tag, which is a unique immunogenic epitope that can be detected by a readily available antibody. One example of such an immunogenic tag is a 9-amino acid influenza HA tag that can be linked to the C-terminus of the consensus hemagglutinin. In some embodiments, the consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence at its N-terminus and an HA tag at its C-terminus.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0112] (2) Parasite antigens The antigen may be a parasitic antigen or a fragment or variant thereof. The parasite may be a protozoan, a protozoan, or an ectoparasite. The protozoan (i.e., helminth) may be a flatworm (e.g., a trematode and a tapeworm), an anchocyst, or a nematode (e.g., a pinworm). The ectoparasite may be a lice, a flea, a tick, or a mite.

[0113] The parasite may be any parasite that causes the following diseases: acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, raccoon ascariasis, Chagas disease, clonorchiasis, spiral worm infection, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, tumefacilitatoriasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0114] The parasite may be Acanthamoeba, Anisakis, Ascaris, Horsefly, Balantidium coli, Bedbug, Tapeworm (Cestworm), Chigger, Screwworm, Entamoeba histolytica, Fasciola, Giardia lamblia, Hookworm, Leishmania, Rhino-glossum, Clonorchis, Loa loa, Paragonimus, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mites, Tapeworm, Toxoplasma gondii, Trypanosoma brucei, Trichuris trichiura, or Wuchereria bancrofti.

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

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

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

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

[0119] In a further embodiment, the malaria antigen may be the highly conserved malaria parasite antigen, Ama1. The malaria antigen may also be the consensus sequence of Ama1 (i.e., the ConAma1 immunogen), which in some instances may include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments may include an HA tag.

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

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

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

[0123] In some embodiments, the fusion protein comprises a signal peptide linked to the N-terminus. In some embodiments, the fusion protein comprises multiple signal peptides linked to the N-terminus of each consensus PF immunogen. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein. In some embodiments, the spacer between the PF immunogens of the fusion protein may be a proteolytic cleavage site. In some embodiments, the spacer may be a proteolytic cleavage site recognized by a protease found in cells into which the vaccine is intended to be administered and / or taken. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein, wherein the spacer is a proteolytic cleavage site recognized by a protease found in cells into which the vaccine is intended to be administered and / or taken, and the fusion protein comprises multiple signal peptides linked to the N-terminus of each consensus PF immunogen such that, upon cleavage, the signal peptide of each consensus PF immunogen translocates the respective consensus PF immunogen outside the cell.

[0124] (3) Bacterial antigen The antigen may be a bacterial antigen or a fragment or variant thereof. The bacterium may be from any one of the following phyla: Acidobacteria, Actinomycetes, Aquifex, Bacteroidetes, Caldicellicum, Chlamydia, Green Sulfur Bacteria, Green Non-Sulfur Bacteria, Chrysiogenes, Cyanobacteria, Dictyoglomers, Deinococcus Thermus, Fibrobacter, Firmicutes, Fusobacteria, Gemmatimonads, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, Thermodesulfobacteria, Thermotoga, and Verrucomicrobium.

[0125] The bacteria may be gram-positive or gram-negative. The bacteria may be aerobic or anaerobic. The bacteria may be autotrophic or heterotrophic. The bacteria may be mesophilic, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or densifloric.

[0126] The bacteria may be Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. The bacteria may be Mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, Methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

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

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

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

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

[0131] c. Vector A vaccine can include one or more vectors containing one or more heterologous nucleic acids encoding an antigen and an adjuvant. The one or more vectors can be capable of expressing the antigen and the adjuvant. The one or more vectors can be expression constructs, typically plasmids used to introduce specific genes into target cells. Once the expression vector enters the cell, the protein encoded by the gene is produced by the ribosomal complex, the cell's transcription and translation machinery. Plasmids are often genetically engineered to contain regulatory sequences that act as enhancers and promoter regions, resulting in efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and, therefore, proteins.

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

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

[0134] (2) Circular and linear vectors Vectors may be circular plasmids, which are capable of transforming target cells by integration into the cellular genome, or may exist extrachromosomally (eg, autonomous replication of plasmids having an origin of replication).

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

[0136] Also provided herein are linear nucleic acid vaccines or linear expression cassettes ("LECs") that can be efficiently delivered to a subject via electroporation and express one or more desired antigens and / or one or more desired adjuvants. The LEC may be any linear DNA that does not have any phosphate backbone. The DNA may encode one or more antigens and / or one or more adjuvants. The LEC may contain a promoter, introns, stop codons, and / or polyadenylation signals. Expression of the antigen or adjuvant may be controlled by a promoter. The LEC may be free of any antibiotic resistance genes and / or phosphate backbones. The LEC may be free of other nucleic acid sequences unrelated to desired antigen gene expression or desired adjuvant expression.

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

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

[0139] (3) Promoter, intron, stop codon, and polyadenylation signal The vector may be a promoter. The promoter may be any promoter capable of driving gene expression and regulating the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase, which transcribes the antigen sequence or adjuvant sequence described herein. The choice of promoter used to direct the expression of heterologous nucleic acid depends on the specific application. The promoter may be located approximately the same distance from the transcription start site in the vector as it is from the transcription start site in its natural environment. However, variations in this distance can be accommodated without losing promoter function.

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

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

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

[0143] d. Vaccine excipients and other components The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule such as a vehicle, an adjuvant other than IL-21, a carrier, or a diluent. The pharmaceutically acceptable excipient may be a transfection-enhancing agent, including surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, endoplasmic reticulum such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-enhancing agents.

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

[0145] In addition to IL-21, the pharmaceutically acceptable excipient may be an adjuvant. Additional adjuvants may be other genes expressed on separate plasmids or delivered as proteins in combination with the above-mentioned plasmids in the vaccine. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 containing IL-15 with a deleted signal sequence, and optionally containing a signal peptide derived from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0146] In addition to IL-21, other genes that may be useful as adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M- CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, F lt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase These include genes encoding ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, 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 functional fragments thereof.

[0147] The vaccine may further comprise a genetic vaccine facilitator as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0148] The vaccine can be formulated according to the mode of administration used. The injectable vaccine pharmaceutical composition can be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can also include a vasoconstrictor. An isotonic solution can include phosphate-buffered saline. The vaccine can further include stabilizers, including gelatin and albumin. Stabilizers, such as LGS or polycations or polyanions, can stabilize the formulation for long periods of time at room or ambient temperature.

[0149] 3. Vaccination Methods The present invention is also directed to methods for enhancing an immune response in a subject by different routes of administration of a vaccine. Enhanced immune responses can be used to treat and / or prevent disease in a subject.

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

[0151] In other embodiments, the administered vaccine is capable of enhancing or boosting the immune response in a subject by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0152] The vaccine dose may be 1 μg to 10 mg of active ingredient per kg of body weight per hour, or 20 μg to 10 mg of ingredient per kg of body weight per hour. The vaccine may 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 doses of vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

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

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

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

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

[0157] Vaccines can also be formulated for administration via the nasal cavity. When the carrier is a solid, formulations suitable for nasal administration can include, for example, a coarse powder having a particle size ranging from about 10 to about 500 microns, administered in a sniffing manner, i.e., by rapid inhalation through the nasal passages from a container of the powder held close to the nose. Formulations can also be administered by nasal spray, nasal drops, or aerosol administration via a nebulizer. Formulations can include aqueous or oily solutions of the vaccine.

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

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

[0160] The vaccine can be administered by electroporation, such as using the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation may be 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 entireties. Electroporation may also be performed by a minimally invasive device.

[0161] A minimally invasive electroporation device ("MID") may be an apparatus for injecting the aforementioned vaccines and associated fluids into body tissue. The device may include a hollow needle, a DNA cassette, and a fluid delivery means, and the device is configured to activate and use the fluid delivery means to inject DNA into the body tissue simultaneously (e.g., automatically) while inserting the needle into the body tissue. This has the advantage of providing a more uniform distribution of fluids throughout the body tissue due to the ability to gradually inject the DNA and associated fluids while the needle is being inserted. Pain experienced during injection may be reduced because the injected DNA is distributed over a wider area.

[0162] MIDs can inject vaccines into tissue without the use of needles. MIDs can inject vaccines as a small stream or jet, with such force that the vaccine penetrates the tissue surface and enters the underlying tissue and / or muscle. The motive force for the small stream or jet may be provided by the expansion of compressed gas, such as carbon dioxide, through a minute opening in a short burst. Examples of minimally invasive electroporation devices and their 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 each of which are incorporated herein by reference).

[0163] MID can be equipped with a syringe that produces a high-speed jet of liquid without causing pain.Such needleless syringes are commercially available.Examples of needleless syringes that can be used herein include those described in U.S. Patent No. 3,805,783, U.S. Patent No. 4,447,223, U.S. Patent No. 5,505,697 and U.S. Patent No. 4,342,310 (each of which is incorporated herein by reference).

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

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

[0166] The MID may have needle electrodes for electroporating tissue. For example, generating pulses between multiple pairs of electrodes in a multi-electrode array configured in a rectangular or square pattern provides improved results over pulsing between a pair of electrodes. For example, U.S. Patent No. 5,702,359, entitled "Needle Electrodes for Mediated Delivery of Drugs and Genes," discloses a needle array capable of applying pulses to multiple pairs of needles during therapeutic treatment. In this application, which is incorporated herein by reference as fully described, the needles are arranged in a circular array but have connectors and switching devices that allow pulsing between opposing needle electrodes. A pair of needle electrodes may also be used to deliver recombinant expression vectors to cells. Such a device and system is 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 electroporation using a single needle similar to a regular syringe needle, applying pulses of lower voltage than those delivered by currently used devices, thereby reducing the electrical stimulation experienced by patients.

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

[0168] The MID may consist of a pulse generator and a vaccine syringe with two or more needles that delivers the vaccine and electroporation pulse in a single step. The pulse generator may allow flexible programming of pulse and injection parameters via a flash card-operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver various voltage pulses over a short period of time. For example, the pulse generator can deliver three 15-voltage pulses over a 100-ms period. One example of such an MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064 (the contents of which are incorporated herein by reference).

[0169] The MID may be the CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) device and system, a modular electrode system that facilitates the introduction of macromolecules, such as DNA, into cells of selected tissues within a body or plant. The modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector that provides conductive connections from a programmable constant electrode pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissues within a body or plant. The macromolecule is then delivered through the hypodermic needle into the selected tissue. A programmable constant current controller is activated to apply constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses facilitate the introduction of the macromolecule into cells between the multiple electrodes. The constant current pulses limit power dissipation within the tissue, thereby minimizing cell death due to overheating. The Cellectra device and system are described in U.S. Patent No. 7,245,963, the contents of which are incorporated herein by reference.

[0170] The MID may be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may comprise a device providing a hollow needle and a fluid delivery means, which is configured to activate and use the fluid delivery means to inject the desired vaccine described herein into the body tissue simultaneously (e.g., automatically) while inserting the needle into the body tissue. The ability to gradually inject the fluid while the needle is being inserted is advantageous in that it results in a more uniform distribution of the fluid throughout the body tissue. It is also believed that the pain experienced during injection is reduced because the volume of the injected fluid is distributed over a wider area.

[0171] Furthermore, automatic injection of fluids facilitates automatic monitoring and registration of the actual amount of fluid injected, and this data may be stored by the control device for documentation purposes, if desired.

[0172] It should be understood that the rate of injection may be either linear or non-linear, and that injection may be performed after the needles are inserted through the skin of the subject to be treated and while they are further inserted into the body tissue.

[0173] Suitable tissues that can be infused with fluid by the device of the present invention include tumor tissue, skin or liver tissue, but may also be muscle tissue.

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

[0175] The user can select when to begin fluid injection. Ideally, however, injection begins when the tip of the needle reaches muscle tissue, and the device may include means for sensing when the needle has been inserted deep enough to begin fluid injection. This means that fluid injection can be prompted to begin automatically when the needle reaches a desired depth (usually the depth where muscle tissue begins). The depth where muscle tissue begins may be interpreted as the insertion depth of the needle of the present invention, such as a value of 4 mm, which is considered sufficient for the needle to pass through the skin layer.

[0176] The sensing means may comprise an ultrasound probe. The sensing means may comprise means for sensing changes in impedance or resistance. In this case, the means may not be able to record the depth of the needle within the body tissue by itself, but rather is configured to sense changes in impedance or resistance as the needle moves through a different type of body tissue and into muscle. Either of these alternative means provides a relatively accurate and simple to operate means of sensing that injection may begin. The depth of needle insertion may further be recorded if desired and used to control the injection of fluid such that the volume of fluid to be injected is determined once the depth of needle insertion is recorded.

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

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

[0179] The aforementioned device can be used for any type of injection. However, it is envisioned that it will be particularly useful in the field of electroporation, and may further include a means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during electroporation. This is particularly advantageous because it means that the electric field is applied to the same area as the fluid being injected. Previously, electroporation has been problematic in that it is very difficult to accurately align the electrode with the previously injected fluid, leading users to inject a larger volume of fluid than necessary over a wider area and apply the electric field over a higher area to ensure overlap between the injected material and the electric field. Using the present invention, both the volume of the injected fluid and the magnitude of the applied electric field can be reduced while achieving good matching between the electric field and the fluid.

[0180] The present invention has multiple aspects, illustrated by the following non-limiting examples.

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

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

[0183] Example 2 IL-21-enhanced IgG and IgA serum titers Mice were used as a model system to determine whether IL-21 could function as an adjuvant when administered intramuscularly in a vaccine containing Clostridium difficile toxin A and toxin B antigens and IL-21 encoded by the respective plasmids.

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

[0185] As shown in the top panel of Figure 4, the IL-21 adjuvant increased total serum anti-toxin A IgG compared to immunization with antigen alone, with robust titers at a 1:2000 dilution in the IL-21 group but not in the group receiving antigen alone. When serum IgA was analyzed, it was noted that the inclusion of the IL-21 adjuvant resulted in detectable levels of antigen-specific IgA, titering at a 1:2000 dilution, whereas immunization with antigen alone did not produce a robust signal compared to naive animals (Figure 4, bottom panel).

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

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

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

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

[0190] Specifically, one group of mice was immunized with the plasmid pVAX-mIL-21 Opt (FIG. 1 and described above in Example 1) and the aforementioned plasmids encoding EnvA and EnvC antigens. A second group of mice was immunized with only the plasmid encoding EnvA and EnvC antigens. A third group of mice was immunized with the empty control plasmid pVAX. Mice were immunized intramuscularly using electroporation. An interferon-γ ELISpot assay was used to examine the cellular immune responses in the immunized mice.

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

[0192] Antibody responses were measured in the blood of immunized animals after the third immunization by ELISA against the EnvA protein (Figure 6). At a dilution of 1:400, the mean OD of HIV EnvA / C was approximately 0.6, while the IL-21 adjuvant group had a mean OD reading of 1.0.

[0193] Taken together, these data suggest that IL-21 exerts novel adjuvant activity in the form of increased frequency of antibody-secreting cells, increased amount and class switching of IgG produced, increased IgA production, and increased IFN-γ secretion when included in a DNA vaccine.

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

[0195] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

Claims

1. a) at least one selected from the group consisting of an antigen and a nucleic acid encoding the antigen; and b) A nucleic acid molecule comprising a nucleotide sequence encoding IL-21, wherein the nucleotide sequence comprises a nucleotide sequence consisting of SEQ ID NO: 3 and a nucleotide sequence having 90% or more identity to SEQ ID NO:

3. Including vaccines.

2. The vaccine of claim 1 , wherein the nucleotide sequence comprises SEQ ID NO:

3.

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

4. An antigenic peptide having the same encoded nucleic acid sequence as the antigen of claim 3; The vaccine of claim 3, further comprising an IL-21 peptide having the same encoded nucleic acid sequence as the IL-21 of claim 3.

5. The vaccine of claim 3 , wherein the second nucleic acid further comprises an expression vector.

6. 2. The vaccine of claim 1, wherein the antigen is selected from the group consisting of a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, an influenza antigen, a Plasmodium falciparum antigen, a Clostridium difficile antigen, and fragments thereof.

7. 7. The vaccine of claim 6, wherein the HPV antigen is selected from the group consisting of an HPV16 E6 antigen, an HPV16 E7 antigen, and a combination thereof.

8. 7. The vaccine of claim 6, wherein the HIV antigen is selected from the group consisting of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, and any combination thereof.

9. 7. The vaccine of claim 6, wherein the influenza antigen is selected from the group consisting of an H1 HA, an H2 HA, an H3 HA, an H5 HA, a BHA antigen, and any combination thereof.

10. 7. The vaccine of claim 6, wherein the Plasmodium falciparum antigen comprises a circumsporozoite (CS) antigen.

11. 7. The vaccine of claim 6, wherein the Clostridium difficile antigen is selected from the group consisting of toxin A, toxin B, and combinations thereof.

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

13. 10. A method for enhancing an immune response in a subject in need thereof, comprising administering to said subject a vaccine according to claim 1 or 2.

14. 14. The method of claim 13, wherein administering the vaccine comprises electroporation.

15. 14. The vaccine of claim 13, wherein the enhanced immune response in the subject comprises a cellular immune response, a humoral immune response, or both a cellular and humoral immune response in the subject.

16. A nucleic acid molecule comprising one or more optimized nucleotide sequences selected from the group consisting of SEQ ID NO:3 and a nucleotide sequence having 90% or greater identity to SEQ ID NO:

3.

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

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