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Title:
COMPOSITIONS AND METHOD FOR INDUCING AN IMMUNE RESPONSE
Document Type and Number:
WIPO Patent Application WO/2018/217897
Kind Code:
A1
Abstract:
Disclosed herein is an immunogenic comprising an antigen and optionally IL-12. Also disclosed herein is a method for administering the vaccine through intradermal electroporation.

Inventors:
WEINER DAVID (US)
YAN JIAN (US)
DEROSA STEPHEN (US)
Application Number:
PCT/US2018/034138
Publication Date:
November 29, 2018
Filing Date:
May 23, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
WEINER DAVID (US)
YAN JIAN (US)
DEROSA STEPHEN (US)
International Classes:
A61K38/00; A61K48/00; A61P33/00; A61P37/00; A61P41/00; C12N15/09
Foreign References:
CA2422863A12002-03-28
CA2821992A12012-04-05
US7744896B12010-06-29
JP2013052202A2013-03-21
US8710200B22014-04-29
Attorney, Agent or Firm:
NGUYEN, Quang, D. et al. (US)
Download PDF:
Claims:
Claims

What is claimed is:

1. An immunogenic formulation for intradermal (ID) administration comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an antigen.

2. The formulation of claim 1, wherein the formulation comprises a low dose of the nucleic acid molecule comprising a nucleic acid sequence encoding an antigen.

3. The formulation of claim 1 , wherein the formulation further

comprises a nucleic acid molecule comprising a nucleic acid sequence encoding IL-12.

4. The formulation of claim 3, wherein the formulation comprises a low dose of the nucleic acid molecule comprising a nucleic acid sequence encoding IL-12.

5. The formulation of claim 1, wherein the antigen is selected from the group consisting of a foreign antigen and a self-antigen.

6. The formulation of claim 5, wherein the foreign antigen is derived from a virus.

7. The formulation of claim 6, wherein the virus is selected from the group consisting of HIV and Ebolavirus.

8. The formulation of any of claims 1 wherein the nucleic acid

molecule is a plasmid.

9. The formulation of claim 1 , wherein the composition is formulated for delivery to an individual using electroporation.

10. A method of inducing an immune response against an antigen in a subject in need thereof, the method comprising intradermal (ID) administration of an immunogenic formulation of any of claims 1-9 to the subject.

11. The method of claim 10, wherein the immunogenic formulation is administered through intradermal electroporation.

12. A method of treating a disease in a subject, the method comprising intradermal (ID) administration of an immunogenic formulation of claims 1-9 to the subject.

13. The method of claim 12, wherein the immunogenic formulation is administered through intradermal electroporation.

14. The method of claim 12, wherein the ID electroporation is injected at a depth of about 0.2 to about 0.4 cm.

15. The method of claim 12, wherein the ID electroporation is injected between 1 and 10 sites.

Description:
COMPOSITIONS AND METHOD FOR INDUCING AN IMMUNE RESPONSE

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. provisional application number 62/510,116, filed May 23, 2017, the content of which is incorporated herein in its entirety.

TECHNICAL FIELD

The present invention relates to vaccines and methods of administering such vaccines. BACKGROUND

Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease. Some vaccines include an antigen to induce the immune response. Some antigens elicit a strong immune response while other antigens elicit a weak immune response. A weak immune response to an antigen can be strengthened by including an adjuvant in the vaccine

Vaccines are also administered in many different ways (e.g., injection, orally, etc.) into many different tissues (e.g., intramuscular, nasal, etc.). Not all delivery methods, however, are equal. Some delivery methods allow for greater compliance within a population of individuals while other delivery methods may affect immunogenicity and/or safety of the vaccine. Accordingly, a need remains in the art for the development of safe and more effective delivery methods for increased immune responses to the antigen.

SUMMARY OF THE INVENTION

The invention provides an immunogenic formulation for intradermal (ID)

administration comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an antigen.

In one embodiment, the formulation comprises a low dose of the nucleic acid molecule comprising a nucleic acid sequence encoding an antigen. In one embodiment, the formulation further comprises a nucleic acid molecule comprising a nucleic acid sequence encoding IL-12.

In one embodiment, the formulation comprises a low dose of the nucleic acid molecule comprising a nucleic acid sequence encoding IL-12.

In one embodiment, the antigen is selected from the group consisting of a foreign antigen and a self antigen.

embodiment, the foreign antigen is derived from a virus.

embodiment, the virus is selected from the group consisting of HIV and

Ebolavirus.

embodiment, the nucleic acid molecule is a plasmid.

In one embodiment, the formulation is formulated for delivery to an individual using electroporation.

The invention also provides a method of inducing an immune response against an antigen in a subject in need thereof. In one embodiment, the method comprises intradermal (ID) administration of an immunogenic formulation of the invention to the subject.

In one embodiment, the immunogenic formulation is administered through intradermal electroporation.

In one embodiment, the method comprises intradermal (ID) administration of an immunogenic formulation of the invention to the subject.

In one embodiment, the immunogenic formulation is administered through intradermal electroporation.

In one embodiment, the ID electroporation is injected at a depth of about 0.2 to about

0.4 cm.

embodiment, the ID electroporation is injected between 1 and 10 sites.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 depicts an illustration of exemplary PENNVAX ® -GP vaccine components utilized in the experiments. Components included Env A plasmid, Env C plasmid, hIL-12 plasmid, Pol plasmid, and Gag plasmid.

Figure 2 depicts an illustration of intramuscular electroporation (IM EP) compared to intradermal electroporation (ID EP). IM administration involves delivery of the vaccine at about 1.9 cm depth, while ID administration involves delivery of the vaccine at about 0.3 cm depth.

Figure 3 depicts a schematic utilized for the example experiment. Briefly, there were

4 groups. Group 1 received ID-delivered (0.1 mg env A + 0.1 mg env C + 0.2 mg gag + 0.2 mg pol = 0.6 mg total PENNVAX ® -GP) with 0.2 mg of IL-12 DNA, or placebo. Group 2 received ID-delivered (0.6 mg env A + 0.6 mg env C + 0.2 mg gag + 0.2 mg pol = 1.6 mg total PENNVAX ® -GP) with 0 mg of IL-12 DNA, or placebo. Group 3 received ID-delivered (0.6 mg env A + 0.6 mg env C + 0.2 mg gag + 0.2 mg pol = 1.6 mg total PENNVAX ® -GP) with 0.4 mg of IL-12 DNA, or placebo. Group 4 received IM-delivered (3 mg env A, 3 mg env C, 1 mg gag, 1 mg pol = 8 mg total PENNVAX ® -GP) with 1 mg of IL-12 DNA or placebo. All participants received injections at four timepoints: day 0, month 1, month 3, and month 6. Groups 2 and 3 received the doses indicated over 2 injection sites on the arm.

Figure 4 depicts results from example experiments demonstrating that ID and IM EP induce similar CD4 T-cell responses (to any HIV protein) at visit 9.

Figure 5 depicts results from example experiments demonstrating that IL-12 leads to increased CD4 T-cell response rates (to any HIV protein) at visit 9.

Figure 6 depicts results from example experiments demonstrating that there is a higher CD8 T-cell response rate after ID compared to IM EP (to any HIV protein) at visit 9.

Figure 7 depicts results from example experiments demonstrating that there sometimes may be a less pronounced benefit of IL-12 for CD8 T-cell responses (to any HIV protein) at visit 9.

Figure 8 depicts results from example experiments demonstrating that binding antibody to group M consensus envelope was detected in most ID-vaccinated individuals.

Figure 9 depicts results from example experiments demonstrating the trend for a higher magnitude of binding antibody to group M consensus envelope after 3 rd vaccination with IL-12. Figure 10 depicts results from example experiments demonstrating that binding antibody to gp41 was detected in most ID-vaccinated individuals (fewer for IM group).

Figure 11 depicts results from example experiments demonstrating that binding antibody to p24 was detected in about half of ID- and IM-vaccinated individuals with IL-12.

Figure 12 depicts results from example experiments demonstrating the level of discomfort with EP. Overall, ID was better tolerated than IM.

Figure 13 depicts results from example experiments demonstrating the appearance of the injection site. Visible lesions occurred with ID, but were not seen with IM. Overall, the appearance of the injection site was mostly acceptable.

Figure 14 depicts results from example experiments demonstrating IgG binding antibody to Con S gpl40 CFI, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 15 depicts results from example experiments demonstrating IgG binding antibody to Con 6 gpl20/B, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 16 depicts results from example experiments demonstrating IgG binding antibody to Al .con.env03 140 CF, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 17 depicts results from example experiments demonstrating IgG binding antibody to B.con.env03 140 CF, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 18 depicts results from example experiments demonstrating IgG binding antibody to C.con.env03 140 CF_avi, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 19 depicts results from example experiments demonstrating IgG binding antibody to gp41, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 20 depicts results from example experiments demonstrating IgG binding antibody to p24, net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination).

Figure 21 depicts results from example experiments demonstrating IgG binding antibody to p66 (RT), net response, at visit 7 (2 weeks after 3rd vaccination) and visit 9 (2 weeks after 4th vaccination). Figure 22 depicts binding antibody multiplex assay (BAMA) response rates for IgG for the antigens indicated under the treatments indicated, at day 98 and day 182 of the study.

Figure 23 depicts magnitude summary by isotype, antigen, and visit, among positive responders.

Figure 24 depicts results from example experiments demonstrating Neutralizing

Antibody (NAb) response for TZM-bl, Tier 1 at 2 weeks post 3rd vaccination, reverse cumulative distribution function (RCDF).

Figure 25 depicts results from example experiments demonstrating Neutralizing Antibody (NAb) response for TZM-bl, Tier 1 at 2 weeks post 4th vaccination, reverse cumulative distribution function (RCDF).

Figure 26 depicts results from example experiments demonstrating Tier 1

Neutralizing Antibody (NAb) response at visit 7 (2 weeks post 3rd vaccination).

Figure 27 depicts results from example experiments demonstrating Tier 1

Neutralizing Antibody (NAb) response at visit 9 (2 weeks post 4th vaccination).

Figure 28 depicts results from example experiments demonstrating neutralizing antibody titer response rates at visit 7 and visit 9 for all groups in the experiment.

Figure 29 depicts results from example experiments demonstrating descriptive statistics (Number, Minimum, Maximum, Mean, Median, and Standard Deviation) for neutralizing antibody assay among positive responders.

Figure 30 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 31 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any ENV PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 32 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any GAG PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 33 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any POL PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 34 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV- 1 -PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 35 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 36 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-3-PTEg, at visit 9 (2 weeks post 4th vaccination). Figure 37 depicts results from example experiments demonstrating ICS CD4+ T-cell response to GAG- 1 -PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 38 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-l-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 39 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 40 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 41 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any ENV PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 42 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any GAG PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 43 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any POL PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 44 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV- 1 -PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 45 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 46 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-3-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 47 depicts results from example experiments demonstrating ICS CD4+ T-cell response to GAG- 1 -PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 48 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-l-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 49 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 50 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 51 depicts depicts results from example experiments demonstrating HVTN 098 ICS CD4+ T-cell response to any ENV PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 52 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any GAG PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 53 depicts results from example experiments demonstrating ICS CD4+ T-cell response to any POL PTEg, at visit 9 (2 weeks post 4th vaccination). Figure 54 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-l-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 55 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 56 depicts results from example experiments demonstrating ICS CD4+ T-cell response to ENV-3-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 57 depicts results from example experiments demonstrating ICS CD4+ T-cell response to GAG-l-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 58 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-l-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 59 depicts results from example experiments demonstrating ICS CD4+ T-cell response to POL-2-PTEg, at visit 9 (2 weeks post 4th vaccination).

Figure 60 depicts results from example experiments demonstrating a ranking analysis for ICS response rates for IFN-γ and/or IL-2, and CD40L.

Figure 61 depicts results from example experiments demonstrating a ranking analysis for ICS response magnitudes among positive responders for IFN-γ and/or IL-2, and CD40L.

Figure 62 depicts results from example experiments demonstrating ICS response rates for IFN-γ and/or IL-2, and CD40L.

Figure 63 depicts results from example experiments demonstrating the percent of T- cells producing IFN-γ and/or IL-2, and CD40L among positive responders by T-cell subset, visit, and treatment arm.

Figure 64 shows EBOV-001 phase I clinical (NCT02464670) study strategy, cohort descriptions and demographics.

Figure 65 shows results from Example 3 demonstrating a comparison with other Ebola vaccine platforms currently in clinical trials.

Figure 66 shows results from Example 8 demonstrating ELISA Titers by Cohort and Time point for cohorts 1-3.

Figure 67 shows results from Example 8 demonstrating ELISA Titers by Cohort and Time point for cohorts 4 and 5.

Figure 68 shows results from Example 3 demonstrating EBOV-001 seroconversion in the phase I clinical trial.

Figure 69 shows results from Example 3 demonstrating induction of Ebola GP Specific T-Cell Responses (ELISpot) in Representative Patients. Figure 70 shows results from Example 3 demonstrating subject responses by peptide for cohort 1.

Figure 71 shows results from Example 3 demonstrating subject responses by peptide for cohort 2.

Figure 72 shows results from Example 3 demonstrating subject responses by peptide for cohort 3.

Figure 73 shows results from Example 3 demonstrating subject responses by peptide for cohort 4.

Figure 74 shows results from Example 3 demonstrating subject responses by peptide for cohort 5.

Figure 75 shows results from Example 3 demonstrating ELISpot summary by cohort.

Figure 76 shows results from Example 3 demonstrating ELISpot summary by cohort.

Figure 77 shows results from Example 3 demonstrating analysis of vaccine responders for all subjects.

Figure 78 shows results from Example 3 demonstrating analysis of vaccine responders for subjects with baseline outliers removed.

Figure 79 shows results from Example 3 demonstrating Wilcoxon Paired analysis based on cohort for ICS experiments.

Figure 80 shows results from Example 3 demonstrating ICS analysis for cohort 3 (INO4201 ID) Cytokines in CD4+ T cells.

Figure 81 shows results from Example 3 demonstrating ICS analysis for cohort 3 Cytokines in CD8+ T cells.

Figure 82 shows results from Example 3 demonstrating a detailed analysis of vaccine responders for each subject in cohorts 1-3.

Figure 83 shows results from Example 3 demonstrating a detailed analysis of vaccine responders for each subject in cohorts 4-5.

Figure 84 shows results from Example 3 demonstrating median responses by cohort and pool.

Figure 85 shows results from Example 3 demonstrating mean responses by cohort and pool.

Figure 86 depicts a comparison of binding antibodies in EBOV-001 to rVSV EBOV. All EBOV-001 cohorts had significant increases at both weeks 6 and 14 compared to week 0. Figure 87 depicts a summary of the Intradermal Delivery of EBOV GP DNA vaccine study. Cohorts were added to explore dosing, dose regimens and use of IL-12 DNA as an immuno-adj uvant.

Figure 88 depicts the results of ID cohorts at week 14. Sereoreactivity was observed in 125/127 (98.4%) subjects in all ID cohorts.

DETAILED DESCRIPTION

The present invention provides methods and compositions for inducing an immune response in a subject. In one embodiment, the invention provides DNA vaccines that are capable of generating in a mammal an immune response against a desired target (e.g. an antigen). The DNA plasmid vaccines are comprised of a DNA plasmid capable of expressing a consensus antigen in a mammal and a pharmaceutically acceptable excipient. The DNA plasmid is comprised of a promoter operably linked to a coding sequence that encodes the consensus antigen.

In one embodiment, composition comprises a nucleic acid encoding an antigen. The antigen can be a foreign antigen, such as a viral antigen, a parasitic antigen, or a bacterial antigen. The antigen can also be a self-antigen such as cytokines, antibodies against viruses, antigens affecting cancer progression or development, cell surface receptors or

transmembrane proteins.

The composition can also include a nucleic acid encoding an adjuvant wherein the adjuvant is IL-12. Compositions comprising both a nucleic acid encoding an antigen and a nucleic acid encoding an adjuvant may provide increased immune responses as the combination of an antigen and adjuvant is synergistic.

The composition can comprise a low dose of a nucleic acid encoding an antigen, a nucleic acid encoding an adjuvant, or both. In one embodiment, the composition comprises a low dose of a nucleic acid encoding an antigen.

Another aspect of the invention provides methods of generating an immune response in a subject. In some embodiments, the methods comprise administering to the subject a composition of the invention. For example, in one embodiment, the method comprises administering a composition comprising a low dose of a nucleic acid encoding an antigen to the subject. In some embodiments, the composition is administered through intradermal electroporation. 1. Definitions

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

The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

"Adjuvant" as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigens.

"Fragment" as used herein means a nucleic acid sequence or a portion thereof, that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below.

As used herein, the term "expressible form" refers to nucleic acid constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

The term "constant current" is used herein to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse delivered to same tissue. The electrical pulse is delivered from the electroporation devices described herein. This current remains at a constant amperage in said tissue over the life of an electrical pulse because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to alter its electrical energy output (e.g., increase voltage) so current in same tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller.

The term "feedback" or "current feedback" is used interchangeably and means the active response of the provided electroporation devices, which comprises measuring the current in tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment. Preferably, the feedback is accomplished by the electroporation component, e.g., controller, of the electroporation device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels. In some embodiments, the feedback loop is instantaneous as it is an analog closed-loop feedback.

The terms "electroporation," "electro-permeabilization," or "electro-kinetic enhancement" ("EP"), as used interchangeably herein, refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and/or water to pass from one side of the cellular membrane to the other.

The term "decentralized current" is used herein to define the pattern of electrical currents delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of electroporation related heat stress on any area of tissue being electroporated.

The term "feedback mechanism" as used herein refers to a process performed by either software or hardware (or firmware), which process receives and compares the impedance of the desired tissue (before, during, and/or after the delivery of pulse of energy) with a present value, preferably current, and adjusts the pulse of energy delivered to achieve the preset value. The term "impedance" is used herein when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current. In a preferred embodiment, the "feedback mechanism" is performed by an analog closed loop circuit.

"Immune response" as used herein means the activation of a host's immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both. The term "consensus" or "consensus sequence" is used herein to mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of an alignment of multiple strains of an antigen. The consensus sequence can be used to induce broad immunity against multiple subtypes or serotypes of the antigen.

"Nucleic acids" as used herein can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

The coding sequence, or "encoding nucleic acid sequence," can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.

"Operably linked" as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5'

(upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

A "peptide," "protein," or "polypeptide" as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

"Promoter" as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. 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 the CMV IE promoter.

"Treatment" or "treating," as used herein can mean protection of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after induction of the disease, but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to an animal after clinical appearance of the disease.

"Subject" as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

"Variant" as used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A

conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The

hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly, the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

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

"Vector" as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self- replicating extrachromosomal vector, and preferably, is a DNA plasmid.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. 2. Vaccine

Provided herein are immunogenic compositions, such as vaccines, comprising an antigen, or a fragment of an antigen. The immunogenic composition can increase antigen presentation and the overall immune response to the antigen in a subject. The immunogenic composition can further induce an immune response when administered to different tissues such as the muscle and the skin. This immune response provides increased efficacy in the treatment and/or prevention of any disease, pathogen, or virus, including cancer as described in more detail below.

In one embodiment, the immunogenic composition comprises an antigen and an adjuvant. The combination of antigen and adjuvant induces the immune system more efficiently than an immunogenic composition comprising the antigen alone.

The vaccine can be a DNA vaccine, a peptide vaccine, or a combination DNA and peptide vaccine. The DNA vaccine can include a nucleic acid sequence encoding the antigen. 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 additional sequences that encode linker, leader, or tag sequences that are linked to the antigen by a peptide bond. The peptide vaccine can include an antigenic peptide, an antigen, a variant thereof, a fragment thereof, or a combination thereof. The combination DNA and peptide vaccine can include the above described nucleic acid sequence encoding the antigen.

The immunogenic composition can comprise a low dose of a nucleic acid encoding an antigen, a nucleic acid encoding an adjuvant, or both. In one embodiment, the composition comprises a low dose of a nucleic acid encoding an antigen. For example, in one

embodiment, the composition comprises about 0.01 mg to about 10 mg, about 0.01 mg to about 9 mg, about 0.01 mg to about 8 mg, about 0.01 mg to about 7 mg, about 0.01 mg to about 6 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 4 mg, about 0.01 mg to about 3 mg, about 0.1 to about 2 mg, about 0.5 to about 1.6 mg or about 0.6 to about 1.6 mg of a nucleic acid encoding an antigen.

In some embodiments, the composition comprises about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, or 2.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, or 3.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, or 4.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, or 6.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, or 7.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, or 8.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, or 9.0 mg of a nucleic acid encoding an antigen. In some embodiments, the composition comprises about 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, or 10.0 mg of a nucleic acid encoding an antigen.

In some embodiments, the composition comprises multiple nucleic acids, each encoding one or more antigens. In some embodiments, each of the multiple nucleic acids in the composition are provided at a low dose. For example, in one embodiment, the composition comprises a low dose of a first nucleic acid encoding a first antigen and a low dose of a second nucleic acid encoding a second antigen. In one embodiment, the composition comprises a low dose of a first nucleic acid encoding a first antigen, a low dose of a second nucleic acid encoding a second antigen, and a low dose of a third nucleic acid encoding a third antigen. In one embodiment, the composition comprises a low dose of a first nucleic acid encoding a first antigen, a low dose of a second nucleic acid encoding a second antigen, a low dose of a third nucleic acid encoding a third antigen, and a low dose of a fourth nucleic acid encoding a fourth antigen. In one embodiment, the composition comprises a low dose of a first nucleic acid encoding a first antigen, a low dose of a second nucleic acid encoding a second antigen, a low dose of a third nucleic acid encoding a third antigen, a low dose of a fourth nucleic acid encoding a fourth antigen, and a low dose of a fifth nucleic acid encoding a fifth antigen. In some embodiments, the total nucleic acids in the composition are provided at a low dose.

In one embodiment, the composition comprises a low dose of a nucleic acid encoding an adjuvant. For example, in one embodiment, the composition comprises about 0.01 mg to about 1 mg, about 0.01 mg to about 0.5 mg, about 0.1 mg to about 0.5 mg, or about 0.2 mg to about 0.4 mg of a nucleic acid encoding an adjuvant.

In some embodiments, the composition comprises about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, or 2.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, or 3.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, or 4.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, or 6.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, or 7.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, or 8.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, or 9.0 mg of a nucleic acid encoding an adjuvant. In some embodiments, the composition comprises about 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, or 10.0 mg of a nucleic acid encoding an adjuvant.

The volume of the immunogenic composition and number of doses comprised within each composition can vary. Accordingly, the total amount of the nucleic acids described herein can vary with changes in volume and number of doses. For example, in one embodiment, the composition comprises a single dose, 2 or more doses, 3 or more doses, 4 or more doses, 5 or more doses, 10 or more doses, 15 or more doses, 20 or more doses, 30 or more doses, 40 or more doses, 50 or more doses, 100 or more doses, 200 or more doses, 300 or more doses, 500 or more doses, or 1000 or more doses. In one embodiment, the volume per dose can be between O.OlmL and 5mL. Thus, as a non-limiting example, the composition may comprise O.OlmL and a single or up to 5000 mL and 1000 or more doses at 0.01 mg to about 10 mg of a nucleic acid encoding an antigen per dose corresponding to lmg/mL to lOOOmg/mL. Similarly, the composition may comprise 5000 mL and 10 doses at 0.01 mg to about 10 mg of a nucleic acid encoding an antigen per dose corresponding to O^g/mL to 20 μg/mL.

The vaccine can induce a humoral immune response in the subject administered the vaccine. The induced humoral immune response can be specific for the antigen. The induced humoral immune response can be reactive with the antigen. The humoral immune response can be induced in the subject administered the vaccine by about 1.5 -fold to about 16-fold, about 2-fold to about 12-fold, or about 3 -fold to about 10-fold. The humoral immune response can be induced in the subject administered the vaccine by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5- fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5 -fold, or at least about 16.0-fold.

The vaccine can induce a cellular immune response in the subject administered the vaccine. The induced cellular immune response can be specific for the antigen. The induced cellular immune response can be reactive to the antigen. In some embodiments, the cellular response is cross-reactive against two or more strains of the antigen. The induced cellular immune response can include eliciting a CD8 + T cell response. The elicited CD8 + T cell response can be reactive with the antigen. The elicited CD8 + T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD8 + T cell response, in which the CD8 + T cells produce interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-a), interleukin-2 (IL-2), or a combination of IFN-γ and TNF-a.

The induced cellular immune response can include an increased CD8 + T cell response associated with the subject administered the vaccine as compared to the subject not administered the vaccine. The CD8 + T cell response associated with the subject administered the vaccine can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold as compared to the subject not administered the vaccine. The CD8 + T cell response associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0- fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0- fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0- fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0- fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD8 + T cells that produce IFN-γ. The frequency of CD3 + CD8 + IFN-y + T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15 -fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD8 + T cells that produce TNF-a. The frequency of CD3 + CD8 + TNF-a + T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14- fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD8 + T cells that produce IL-2. The frequency of CD3 + CD8 + IL-2 + T cells associated with the subject administered the vaccine can be increased by at least about 0.5 -fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD8 + T cells that produce both IFN-γ and TNF-a. The frequency of CD3 + CD8 + IFN- Y + TNF-a + T cells associated with the subject administered the vaccine can be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70- fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140- fold, 150-fold, 160-fold, 170-fold, or 180-fold as compared to the subject not administered the vaccine.

The cellular immune response induced by the vaccine can include eliciting a CD4 + T cell response. The elicited CD4 + T cell response can be reactive with the PEDV antigen. The elicited CD4 + T cell response can be poly functional. The induced cellular immune response can include eliciting a CD4 + T cell response, in which the CD4 + T cells produce IFN-γ, TNF- a, IL-2, or a combination of IFN-γ and TNF-a.

The induced cellular immune response can include an increased frequency of CD3+CD4+ T cells that produce IFN-γ. The frequency of CD3 + CD4 + IFN-y + T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15 -fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD4 + T cells that produce TNF-a. The frequency of CD3 + CD4 + TNF-a + T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,

15 - fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21 -fold, or 22-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD4 + T cells that produce IL-2. The frequency of CD3 + CD4 + IL-2 + T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,

16- fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26- fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3 + CD4 + T cells that produce both IFN-γ and TNF-a. The frequency of CD3 + CD4 + IFN- Y + TNF-a + associated with the subject administered the vaccine can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5- fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21 -fold, 22-fold, 23-fold 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31 -fold, 32-fold, 33-fold, 34-fold, or 35-fold as compared to the subject not administered the vaccine.

The vaccine of the present invention can have features required of effective vaccines such as being safe so the vaccine itself does not cause illness or death; is protective against illness resulting from exposure to live pathogens such as viruses or bacteria; induces neutralizing antibody to prevent invention of cells; induces protective T cells against intracellular pathogens; and provides ease of administration, few side effects, biological stability, and low cost per dose.

The vaccine can further induce an immune response when administered to different tissues such as the muscle or skin. The vaccine can further induce an immune response when administered via electroporation, or injection, or subcutaneously, intradermally, or intramuscularly.

The vaccine can comprise an adjuvant wherein the adjuvant is IL-12. 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 additional sequences that encode linker or tag sequences that are 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

3. Antigen

The vaccine can also comprise an antigen or fragment or variant thereof. The antigen can be anything that induces an immune response in a subject. The antigen 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 additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

The antigen can be contained in a protein, a nucleic acid, or a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal. The antigen can be associated with an autoimmune disease, allergy, or asthma. In other embodiments, the antigen can be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), or human immunodeficiency virus (HIV).

In some embodiments, the antigen is foreign. In some embodiments, the antigen is a self-antigen. a. Foreign Antigens

In some embodiments, the antigen is foreign. A foreign antigen is any non-self substance (i.e., originates external to the subject) that, when introduced into the body, is capable of stimulating an immune response. (1) Viral Antigens

The foreign antigen can be a viral antigen, or fragment thereof, or variant thereof. The viral antigen can be from a virus from one of the following families: Adenoviridae,

Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,

Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen can be from human immunodeficiency virus (HIV), Chikungunya virus (CHIKV), dengue fever virus, papilloma viruses, for example, human papillomoa virus (HPV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, equine encephalitis viruses, 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, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, lassa virus, arenavirus, or cancer causing virus.

(a) Hepatitis Antigen

The viral antigen may include a hepatitis virus antigen (i.e., hepatitis antigen), or a fragment thereof, or a variant thereof. The hepatitis antigen can be an antigen or immunogen from one or more of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and/or hepatitis E virus (HEV).

The hepatitis antigen can be an antigen from HAV. The hepatitis antigen can be a

HAV capsid protein, a HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.

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

The hepatitis antigen can be an antigen from HDV. The hepatitis antigen can be a HDV delta antigen, fragment thereof, or variant thereof. The hepatitis antigen can be an antigen from HEV. The hepatitis antigen can be a HEV capsid protein, fragment thereof, or variant thereof.

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

In some embodiments, the hepatitis antigen can be an antigen from HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H.

The DNA vaccine may encode a hepatitis antigen. Examples of DNA vaccines encoding hepatitis antigens include those described in U.S. Patent Nos. 8,829,174, US 8,921,536, US 9,403,879, US 9,238,679, the contents of each are fully incorporated by reference.

(b) Human Papilloma Virus (HPV) Antigen

The viral antigen may comprise an antigen from HPV. The HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 which cause cervical cancer, rectal cancer, and/or other cancers. The HPV antigen can be from HPV types 6 and 11, which cause genital warts, and are known to be causes of head and neck cancer.

The HPV antigens can be the HPV E6 or E7 domains from each HPV type. For example, for HPV type 16 (HPV 16), the HPV 16 antigen can include the HPV 16 E6 antigen, the HPV 16 E7 antigen, fragments, variants, or combinations thereof. Similarly, the HPV antigen can be HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, fragments, variants, or combinations thereof.

The DNA vaccine may encode a HPV antigen. Examples of DNA vaccines encoding HPV antigens include those described in WO/2008/014521, published January 31, 2008; U.S. Patent Application Pub. No. 20160038584; U.S. Patent Nos. 8389706 and 9,050,287, the contents of each are fully incorporated by reference.

(c) RSV Antigen

The viral antigen may comprise a RSV antigen. The RSV antigen can be a human

RSV fusion protein (also referred to herein as "RSV F," "RSV F protein," and "F protein"), or fragment or variant thereof. The human RSV fusion protein can be conserved between RSV subtypes A and B. The RSV antigen can be a RSV F protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23994.1). The RSV antigen can be a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. The RSV antigen can be a monomer, a dimer, or trimer of the RSV F protein, or a fragment or variant thereof.

The RSV F protein can be in a prefusion form or a postfusion form. The postfusion form of RSV F elicits high titer neutralizing antibodies in immunized animals and protects the animals from RSV challenge.

The RSV antigen can also be human RSV attachment glycoprotein (also referred to herein as "RSV G," "RSV G protein," and "G protein"), or fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. The antigen can be RSV G protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23993). The RSV antigen can be RSV G protein from the RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSV subtype B isolate HI 123, or a fragment or variant thereof.

In other embodiments, the RSV antigen can be human RSV non-structural protein 1 ("NS1 protein"), or fragment or variant thereof. For example, the RSV antigen can be RSV NS 1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank

AAX23987.1). The RSV antigen human can also be RSV non-structural protein 2 ("NS2 protein"), or fragment or variant thereof. For example, the RSV antigen can be RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1). The RSV antigen can further be human RSV nucleocapsid ("N") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989.1). The RSV antigen can be human RSV Phosphoprotein ("P") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990.1). The RSV antigen also can be human RSV Matrix protein ("M") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).

In still other embodiments, the RSV antigen can be human RSV small hydrophobic ("SH") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). The RSV antigen can also be human RSV Matrix protein2-l ("M2-1") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M2-1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1). The RSV antigen can further be human RSV Matrix protein 2-2 ("M2-2") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV M2-2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1). The RSV antigen human can be RSV

Polymerase L ("L") protein, or fragment or variant thereof. For example, the RSV antigen can be RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996.1).

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

In other embodiments, the RSV antigen can be, but is not limited to, the RSV F protein from the RSV Long strain, the RSV G protein from the RSV Long strain, the optimized amino acid RSV G amino acid sequence, the human RSV genome of the RSV Long strain, the optimized amino acid RSV F amino acid sequence, the RSV NS1 protein from the RSV Long strain, the RSV NS2 protein from the RSV Long strain, the RSV N protein from the RSV Long strain, the RSV P protein from the RSV Long strain, the RSV M protein from the RSV Long strain, the RSV SH protein from the RSV Long strain, the RSV M2-1 protein from the RSV Long strain, the RSV M2-2 protein from the RSV Long strain, the RSV L protein from the RSV Long strain, the RSV G protein from the RSV subtype B isolate H5601, the RSV G protein from the RSV subtype B isolate H1068, the RSV G protein from the RSV subtype B isolate H5598, the RSV G protein from the RSV subtype B isolate HI 123, or fragment thereof, or variant thereof.

The DNA vaccine may encode a RSV antigen. Examples of DNA vaccines encoding RSV antigens include those described in U.S. Patent Application Pub. No. 20150079121, the content of which is incorporated by reference. (d) Influenza Antigen

The viral antigen may comprise an antigen from influenza virus. The influenza antigens are those capable of eliciting an immune response in a mammal against one or more influenza serotypes. The antigen can comprise the full length translation product HAO, subunit HA1, subunit HA2, a variant thereof, a fragment thereof or a combination thereof. The influenza hemagglutinin antigen can be derived from multiple strains of influenza A serotype HI, serotype H2, a hybrid sequence derived from different sets of multiple strains of influenza A serotype HI, or derived from multiple strains of influenza B. The influenza hemagglutinin antigen can be from influenza B.

The influenza antigen can also contain at least one antigenic epitope that can be effective against particular influenza immunogens against which an immune response can be induced. The antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus. The antigen may be derived from hemagglutinin antigen sequences from a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI or of serotype H2. The antigen may be a hybrid hemagglutinin antigen sequence derived from combining two different hemagglutinin antigen sequences or portions thereof. Each of two different hemagglutinin antigen sequences may be derived from a different set of a plurality of influenza A virus strains of one serotype such as a plurality of influenza A virus strains of serotype HI . The antigen may be a hemagglutinin antigen sequence derived from hemagglutinin antigen sequences from a plurality of influenza B virus strains.

In some embodiments, the influenza antigen can be HI HA, H2 HA, H3 HA, H5 HA, or a BHA antigen.

The DNA vaccine may encode a influenza antigen. Examples of DNA vaccines encoding influenza antigens include those described in WO/2008/014521, published January 31, 2008; U.S. Patent Nos. 9,592,285, US 8,298,820; U.S. Patent Application Pub. Nos. 20160022806, US 20160175427, the contents of each are fully incorporated by reference

(e) Human Immunodeficiency Virus (HIV) Antigen

The viral antigen may be from Human Immunodeficiency Virus (HIV) virus. In some embodiments, the HIV antigen can be a subtype A envelope protein, subtype B envelope protein, subtype C envelope protein, subtype D envelope protein, subtype B Nef-Rev protein, Gag subtype A, B, C, or D protein, MPol protein, a nucleic acid or amino acid sequences of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or any combination thereof. A DNA vaccine encoding an HIV antigen can include a vaccine encoding a subtype A envelope protein, subtype B envelope protein, subtype C envelope protein, subtype D envelope protein, subtype B Nef-Rev protein, Gag subtype A, B, C, or D protein, MPol protein, a nucleic acid or amino acid sequences of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or any combination thereof. Examples of DNA vaccines encoding HIV antigens include those described in U.S. Patent No. 8,168,769 and WO2015/073291, the contents of each are fully incorporated by reference.

(f) Lymphocytic Choriomeningitis Virus (LCMV) Antigen

The viral antigen may be from LCMV. The LCMV antigen can comprise consensus sequences 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 to increase the immunogenicity of constructs, can be included in the modified sequences. The LCMV antigen can comprise a signal peptide such as an immunoglobulin signal peptide (e.g., IgE or IgG signal peptide), and in some embodiments, may comprise an HA tag. The immunogens can be designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogen.

The LCMV antigen can be an antigen from LCMV Armstrong. The LCMV antigen can be an antigen from LCMV clone 13. The LCMV antigen can be a nucleoprotein (NP) from LCMV, a glycoprotein (GP; e.g., GP-1, GP-2, and GP-C) from LCMV, a L protein from LCMV, a Z polypeptide from LCMV, a fragment thereof, a variant thereof, or a combination thereof.

(g) Chikungunya Virus

The viral antigen may be from Chikungunya virus (CHIKV). Chikungunya virus belongs to the alphavirus genus of the Togaviridae family. Chikungunya virus is transmitted to humans by the bite of infected mosquitoes, such as the genus Aedes. In some

embodiments, the HIV antigen can be a CHIKV Capsid protein, El protein, E2 protein, or Env protein.

The DNA vaccine may encode a CHIKV antigen. Examples of DNA vaccines encoding CHIKV antigens include those described in U.S. Patent No. 8,852,609, the contents of which is fully incorporated by reference. (h) Dengue Virus

The viral antigen may be from Dengue virus. The Dengue virus antigen may be one of three proteins or polypeptides (C, prM, and E) that form the virus particle. The Dengue virus antigen may be one of seven other proteins or polypeptides (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) which are involved in replication of the virus. The Dengue virus may be one of five strains or serotypes of the virus, including DENV-1, DENV-2, DENV-3 and DENV-4. The antigen may be any combination of a plurality of Dengue virus antigens.

The DNA vaccine may encode a Dengue virus antigen. Examples of DNA vaccines encoding Dengue virus antigens include those described in U.S. Patent No. 8,835,620 and WO2014/144786, the contents of each are fully incorporated by reference.

(i) Ebola Virus

The viral antigen may be from Ebola virus. Ebola virus disease (EVD) or Ebola hemorrhagic fever (EHF) includes any of four of the five known Ebola viruses including Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Tai Forest virus (TAFV, also referred to as Cote d'lvoire Ebola virus (Ivory Coast Ebolavirus, CIEBOV).

The DNA vaccine may encode an Ebola antigen. Examples of DNA vaccines encoding Ebola antigens include those described in U.S. Patent Application Pub. No.

20150335726 and PCT Application No. PCT/US17/31215, the content of which is incorporated by reference.

(j) Zika Virus

The viral antigen may be from Zika virus. Zika disease is caused by infection with the Zika virus and can be transmitted to humans through the bite of infected mosquitoes or sexually transmitted between humans. The Zika antigen can include a Zika Virus Envelope protein, Zika Virus NS1 protein, or a Zika Virus Capsid protein.

Examples of DNA vaccines encoding Zika virus antigens include those described in PCT Application No. PCT/US17/19407, the content of which is incorporated by reference.

(k) Marburg Virus

The viral antigen may be from Marburg virus. Marburgvirus immunogens that can be used to induce broad immunity against multiple subtypes or serotypes of Marburgvirus. The antigen may be derived from a Marburg virus envelope glycoprotein. The DNA vaccine may encode a Marburg antigen. Examples of DNA vaccines encoding Marburg antigens include those described in U.S. Patent Nos. 9,597,388, the contents of which are fully incorporated by reference.

(2) Parasitic Antigens

The foreign antigen can be a parasite antigen or fragment or variant thereof. The parasite can be a protozoa, helminth, or ectoparasite. The helminth (i.e., worm) can be a fiatworm (e.g., flukes and tapeworms), a thorny-headed worm, or a round worm (e.g., pinworms). The ectoparasite can be lice, fleas, ticks, and mites.

The parasite can be any parasite causing any one of the following diseases:

Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis,

Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

The parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides , Botfly,

Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania,

Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti. (a) Lyme Antigen

The foreign antigen may be a Lyme disease antigen. The antigen may be an outer- surface protein A antigen (OspA antigen), or fragment thereof, or variant thereof. The antigen can be from a parasite causing malaria. The Lyme disease is caused by the bacterium Borrelia burgdorferi and is transmitted to humans through the bite of infected Ixodes scapularis (Blacklegged tick or Deer tick).

(b) Malaria Antigen

The foreign antigen may be a malaria antigen (i.e., PF antigen or PF immunogen), or fragment thereof, or variant thereof. The antigen can be from a parasite causing malaria. The malaria causing parasite can be Plasmodium falciparum. The Plasmodium falciparum antigen can include the circumsporozoite (CS) antigen.

In some embodiments, the malaria antigen can be one of P. falciparum immunogens CS; LSAl; TRAP; CelTOS; and Amal. The immunogens may be full length or immunogenic fragments of full length proteins.

In other embodiments, the malaria antigen can be TRAP, which is also referred to as SSP2. In still other embodiments, the malaria antigen can be CelTOS, which is also referred to as Ag2 and is a highly conserved Plasmodium antigen. In further embodiments, the malaria antigen can be Amal, which is a highly conserved Plasmodium antigen. In some

embodiments, the malaria antigen can be a CS antigen. In some embodiments, the malaria antigen can be a Plasmodium spp. liver stage (LS) immunogen. Exemplary LS immunogens include EXP1, EXP2, EXP23, ICP, TMP21, UIS3, UIS10, SPECT1, SPECT2 and RON2.

In other embodiments, the malaria antigen can be a fusion protein comprising a combination of two or more of the PF proteins set forth herein. For example, fusion proteins may comprise two or more of CS immunogen, ConLSAl immunogen, ConTRAP immunogen, ConCelTOS immunogen, and ConAmal immunogen linked directly adjacent to each other or linked with a spacer or one or more amino acids in between. In some embodiments, the fusion protein comprises two PF immunogens; in some embodiments the fusion protein comprises three PF immunogens, in some embodiments the fusion protein comprises four PF immunogens, and in some embodiments the fusion protein comprises five PF immunogens. Fusion proteins with two PF immunogens may comprise: CS and LSAl; CS and TRAP; CS and CelTOS; CS and Amal; LSAl and TRAP; LSAl and CelTOS; LSAl and Amal; TRAP and CelTOS; TRAP and Amal; or CelTOS and Amal. Fusion proteins with three PF immunogens may comprise: CS, LSAl and TRAP; CS, LSAl and CelTOS; CS, LSAl and Amal; LSAl, TRAP and CelTOS; LSAl, TRAP and Amal; or TRAP, CelTOS and Amal. Fusion proteins with four PF immunogens may comprise: CS, LSAl, TRAP and CelTOS; CS, LSAl, TRAP and Amal; CS, LSAl, CelTOS and Amal; CS, TRAP, CelTOS and Amal; or LSAl, TRAP, CelTOS and Amal. Fusion proteins with five PF immunogens may comprise CS or CS-alt, LSAl, TRAP, CelTOS and Amal.

The DNA vaccine may encode a malaria antigen. Examples of DNA vaccines encoding malaria antigens include those described in U.S. Patent Application Pub. No.

20130273112 and PCT Application No. PCT/US17/33617, the content of which is incorporated by reference. (3) Bacterial Antigens

The foreign antigen can be a bacterial antigen or fragment or variant thereof. The bacterium can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes,

Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

The bacterium can be a gram positive bacterium or a gram negative bacterium. The bacterium can be an aerobic bacterium or an anerobic bacterium. The bacterium can be an autotrophic bacterium or a heterotrophic bacterium. The bacterium can be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, an halophile, or an osmophile.

The bacterium can be an anthrax bacterium, an antibiotic resistant bacterium, a disease causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. The bacterium can be a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. The bacterium can be Mycobacterium tuberculosis.

Examples of DNA vaccines encoding Clostridium difficile antigens include those described in U.S. Patent Application Pub. No. 20140341936, the content of which is incorporated by reference.

Examples of DNA vaccines encoding MRSA antigens include those described in U.S. Patent Application Pub. No. 20140341944, the content of which is incorporated by reference.

(a) Pseudomonas Antigens

The bacterial antigen may be a Pseudomonas antigen. For example, in one embodiment, the antigen may be a Pseudomonas aeruginosa antigen, or fragment thereof, or variant thereof. The Pseudomonas aeruginosa antigen can be from a virulence factor.

Virulence factors associated with Pseudomonas aeruginosa include, but are not limited to structural components, enzymes and toxins. A Pseudomonas aeruginosa virulence factor can be one of exopolysaccharide, Adhesin, lipopolysaccharide, Pyocyanin, Exotoxin A, Exotoxin S, Cytotoxin, Elastase, Alkaline protease, Phospholipase C, Rhamnolipid, and components of a bacterial secretion system. In one embodiment, a Pseudomonas antigen is an extracellular polysaccharide (e.g. Alginate, Pel and Psl). In one embodiment, an antigen is one of polysaccharide synthesis locus (psl), a gene contained therein (e.g. psl A, pslB, pslC, pslD, pslE, pslF, pslG, pslH, psll, pslJ, pslK, pslL, pslM, pslN and pslO), a protein or enzyme encoded therein (e.g. a glycosyl transferase, phosphomannose isomerase/GDP-D-mannose pyrophosphorylase, a transporter, a hydrolase, a polymerase, an acet lase, a dehydrogenase and a topoisomerase) or a product produced therefrom (e.g. Psl exopolysaccharide, referred to as "Psl").

In one embodiment, a Pseudomonas antigen is a component of a bacterial secretion system. Six different classes of secretion systems (types I through VI) have been described in bacteria, five of which (types I, II, II, V and VI) are found in gram negative bacteria, including Pseudomonas aeruginosa. In one embodiment, an antigen is one of a gene (e.g. an apr or has gene) or protein (e.g. AprD, AprE, AprF, HasD, HasE, HasF and HasR) or a secreted protein (e.g. AprA, AprX and HasAp) of a type I secretion system. In one embodiment, an antigen is one of a gene (e.g. xcpA/pilD, xphA, xqhA, xcpP to Q and xcpR to Z) or protein (e.g. GspC to M, GspAB, GspN, GspO, GspS, XcpT to XcpX, FppA, ) or a secreted protein (e.g. LasB, LasA, PlcH, PlcN, PlcB, CbpD, ToxA, PmpA, PrpL, LipA, LipC, PhoA, PsAP, LapA) of a type II secretion system. In one embodiment, an antigen is one of a gene (e.g. a psc, per, pop or exs gene) or protein (e.g. PscC, PscE to PscF, PscJ, PscN, PscP, PscW, PopB, PopD, PcrH and PcrV ) or a secreted protein (e.g. ExoS, ExoT, ExoU and ExoY) of a type III secretion system. In one embodiment, an antigen is a regulator of a type III secretion system (e.g. ExsA and ExsC). In one embodiment, an antigen is one of a gene (e.g. est A) or protein (e.g. EstA, CupB3, CupB5 and LepB) or a secreted protein (e.g. EstA, LepA, and CupB5) of a type V secretion system. In one embodiment, an antigen is one of a gene (e.g. a HSI-I, HSI-II and HSI-III gene) or protein (e.g. Fhal, ClpVl, a VgrG protein or a Hep protein) or a secreted protein (e.g. Hcpl) of a type VI secretion system.

Examples of DNA vaccines encoding Pseudomonas antigens include those described in PCT Application No. PCT/US 17/31449, the content of which is incorporated by reference,

(b) Borrelia Antigens

The bacterial antigen may be a Borrelia spp antigen, or fragment thereof, or variant thereof. The Borrelia spp antigen can be from any one of Borrelia burgdorferi, Borrelia lusitaniae, Borrelia afzelii, Borrelia bissettii, Borreliella bavariensis, Borrelia chilensis, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, and Borrelia finlandensis.

The bacterial antigen may be a Borrelia spp antigen, or fragment thereof, or variant thereof. The Borrelia spp antigen can be from a bacterial product that allows a Borrelia spp to replicate or survive. Bacterial products that allow a Borrelia spp to replicate or survive include, but are not limited to structural components, enzymes and toxins. Such a product can be one of a lipoprotein, an outer surface protein, a product required for infectivity or persistence within vertebrate hosts, and a product involved in motility and chemotaxis.

In one embodiment, an antigen is a lipoprotein (e.g. BptA). In one embodiment, an antigen is an outer surface protein (e.g. OspA, OspB, and OspC). In one embodiment, an antigen is a product required for infectivity or persistence within vertebrate hosts (e.g. PncA, DbpA, DbpB, Bgp, P66 and VlsE). (c) Mycobacterium tuberculosis Antigens

The bacterial antigen may be a Mycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen), or fragment thereof, or variant thereof. The TB antigen can be from the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigen can be from the Esx family of TB antigens, for example, EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.

The DNA vaccine may encode a Mycobacterium tuberculosis antigen. Examples of DNA vaccines encoding Mycobacterium tuberculosis antigens include those described in U.S. Patent Application Pub. No. 20160022796, the content of which is incorporated by reference.

(4) Fungal Antigens

The foreign antigen can be a fungal antigen or fragment or variant thereof. The fungus can be Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium. b. Self Antigens

In some embodiments, the antigen is a self antigen. A self antigen may be a constituent of the subject's own body that is capable of stimulating an immune response. In some embodiments, a self antigen does not provoke an immune response unless the subject i in a disease state, e.g., an autoimmune disease. Self antigens may include, but are not limited to, cytokines, antibodies against viruses such as those listed above including HIV and Dengue, antigens affecting cancer progression or development, and cell surface receptors or transmembrane proteins.

(1) WT-1

The self-antigen antigen can be Wilm's tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor containing at the N-terminus, a proline/glutamine-rich DNA-binding domain and at the C-terminus, four zinc finger motifs. WT1 plays a role in the normal development of the urogenital system and interacts with numerous factors, for example, p53, a known tumor suppressor and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with a cytotoxic drug. Mutation of WT1 can lead to tumor or cancer formation, for example, Wilm's tumor or tumors expressing WT1.

The DNA vaccine may encode a WT-1 antigen. Examples of DNA vaccines encoding WT-1 antigens include those described in U. S. Patent Application Pub. Nos. 20150328298 and 20160030536, the contents each are incorporated by reference.

(2) EGFR

The self-antigen may include an epidermal growth factor receptor (EGFR) or a fragment or variation thereof. EGFR (also referred to as ErbB-1 and HER1) is the cell- surface receptor for members of the epidermal growth factor family (EGF -family) of extracellular protein ligands. EGFR is a member of the ErbB family of receptors, which includes four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB- 2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Mutations affecting EGFR expression or activity could result in cancer.

The antigen may include an ErbB-2 antigen. Erb-2 (human epidermal growth factor receptor 2) is also known as Neu, HER2, CD340 (cluster of differentiation 340), or pi 85 and is encoded by the ERBB2 gene. Amplification or over-expression of this gene has been shown to play a role in the development and progression of certain aggressive types of breast cancer. In approximately 25-30% of women with breast cancer, a genetic alteration occurs in the ERBB2 gene, resulting in the production of an increased amount of HER2 on the surface of tumor cells. This overexpression of HER2 promotes rapid cell division and thus, HER2 marks tumor cells. (3) Cocaine

The self-antigen may be a cocaine receptor antigen. Cocaine receptors include dopamine transporters. (4) PD-1

The self-antigen may include programmed death 1 (PD-1). Programmed death 1 (PD- 1) and its ligands, PD-L1 and PD-L2, deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 is a 288 amino acid cell surface protein molecule including an extracellular IgV domain followed by a transmembrane region and an intracellular tail.

The DNA vaccine may encode a PD-1 antigen. Examples of DNA vaccines encoding PD-1 antigens include those described in U.S. Patent Application Pub. No. 20170007693, the content of which is incorporated by reference. (5) 4-lBB

The self-antigen may include 4- IBB ligand. 4- IBB ligand is a type 2 transmembrane glycoprotein belonging to the TNF superfamily. 4-lBB ligand may be expressed on activated T Lymphocytes. 4-lBB is an activation-induced T-cell costimulatory molecule. Signaling via 4-lBB upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells.

(6) CTLA4

The self-antigen may include CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD 152 (Cluster of differentiation 152). CTLA-4 is a protein receptor found on the surface of T cells, which lead the cellular immune attack on antigens. The antigen may be a fragment of CTLA-4, such as an extracellular V domain, a transmembrane domain, and a cytoplasmic tail, or combination thereof.

(7) IL-6

The self-antigen may include interleukin 6 (IL-6). IL-6 stimulates the inflammatory and auto-immune processes in many diseases including, but not limited to, diabetes, atherosclerosis, depression, Alzheimer's Disease, systemic lupus erythematosus, multiple myeloma, cancer, Behcet's disease, and rheumatoid arthritis. (8) MCP-1

The self-antigen may include monocyte chemotactic protein-1 (MCP-1). MCP-1 is also referred to as chemokine (C-C motif) ligand 2 (CCL2) or small inducible cytokine A2. MCP-1 is a cytokine that belongs to the CC chemokine family. MCP-1 recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation produced by either tissue injury or infection.

(9) Amyloid beta

The self-antigen may include amyloid beta (Αβ) or a fragment or a variant thereof. The Αβ antigen can comprise an Αβ(Χ-Υ) peptide, wherein the amino acid sequence from amino acid position X to amino acid Y of the human sequence Αβ protein including both X and Y, in particular to the amino acid sequence from amino acid position X to amino acid position Y of the amino acid sequence that corresponds to amino acid positions 1 to 47. (10) IP- 10

The self-antigen may include interferon (IFN)-gamma-induced protein 10 (IP-10). IP- 10 is also known as small-inducible cytokine B10 or C-X-C motif chemokine 10 (CXCLIO). CXCLIO is secreted by several cell types, such as monocytes, endothelial cells and fibroblasts, in response to IFN-γ.

(11) Prostate Antigen

The self-antigen may include prostate antigens such as prostate-specific membrane antigen (PSMA), PSA antigen, STEAP antigen, PSCA antigen, Prostatic acid phosphatase (PAP) antigen, and other known prostate tumor antigensPSMA is also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), NAAG peptidase, or folate hydrolase (FOLH). PMSA is an integral membrane protein highly expressed by prostate cancer cells.

The DNA vaccine may encode a PSMA antigen. Examples of DNA vaccines encoding PSMA antigens include those described in U.S. Patent Application Pub. No.

20130302361, the content of which is incorporated by reference.

(12) TERT

The self-antigen may include TERT (telomerase reverse transcriptase). TERT is a telomerase reverse transcriptase that synthesizes a TTAGGG tag on the end of telomeres to prevent cell death due to chromosomal shortening. Hyperproliferative cells with abnormally high expression of TERT may be targeted by immunotherapy. Recent studies demonstrate that TERT expression in dendritic cells transfected with TERT genes can induce CD8+ cytotoxic T cells and elicit a CD4+ T cells in an antigen-specific fashion.

(13) Tyrosinase

The self-antigen may include tyrosinase (Tyr). Tyr is an important target for immune mediated clearance by inducing (1) humoral immunity via B cell responses to generate antibodies that block monocyte chemoattractant protein- 1 (MCP-1) production, thereby retarding myeloid derived suppressor cells (MDSCs) and suppressing tumor growth; (2) increase cytotoxic T lymphocyte such as CD8+ (CTL) to attack and kill tumor cells; (3) increase T helper cell responses; (4) and increase inflammatory responses via IFN-γ and TFN-a or all of the aforementioned.

Tyrosinase is a copper-containing enzyme that can be found in plant and animal tissues. Tyrosinase catalyzes the production of melanin and other pigments by the oxidation of phenols such as tyrosine. In melanoma, tyrosinase can become unregulated, resulting in increased melanin synthesis. Tyrosinase is also a target of cytotoxic T cell recognition in subjects suffering from melanoma. Accordingly, tyrosinase can be an antigen associated with melanoma.

The antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-Tyr immune responses can be induced. The Tyr antigen can comprise the full-length translation product, a variant thereof, a fragment thereof or a combination thereof.

The Tyr antigen can comprise a consensus protein. The Tyr antigen induces antigen- specific T-cell and high titer antibody responses both systemically against all cancer and tumor related cells. As such, a protective immune response is provided against tumor formation by vaccines comprising the Tyr consensus antigen. Accordingly, any user can design an immunogenic composition of the present invention to include a Tyr antigen to provide broad immunity against tumor formation, metastasis of tumors, and tumor growth. Proteins may comprise sequences homologous to the Tyr antigens, fragments of the Tyr antigens and proteins with sequences homologous to fragments of the Tyr antigens.

(14) NY-ESO-1

The self-antigen may include NY-ESO-1. NY-ESO-1 is a cancer-testis antigen expressed in various cancers where it can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the gene for NY-ESO-1, CTAGIB, in myxoid and round cell liposarcomas.

(15) MAGE

The self-antigen may include MAGE (Melanoma-associated Antigen). The MAGE antigen may include MAGE-A4 (melanoma associated antigen 4). NY-ESO-1 is a cancer- testis antigen expressed in various cancers where it can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the gene for NY-ESO-1, CTAGIB, in myxoid and round cell liposarcomas.

MAGE-A4 is expressed in male germ cells and tumor cells of various histological types such as gastrointestinal, esophageal and pulmonary carcinomas. MAGE-A4 binds the oncoprotein, Gankyrin. This MAGE-A4 specific binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partly inhibit the adhesion-independent growth of Gankyrin-overexpressing cells in vitro and suppress the formation of migrated tumors from these cells in nude mice. This inhibition is dependent upon binding between MAGE-A4 and Gankyrin, suggesting that interactions between Gankyrin and MAGE-A4 inhibit Gankyrin-mediated carcinogenesis. It is likely that MAGE expression in tumor tissue is not a cause, but a result of tumorgenesis, and MAGE genes take part in the immune process by targeting early tumor cells for destruction.

Melanoma-associated antigen 4 protein (MAGEA4) can be involved in embryonic development and tumor transformation and/or progression. MAGEA4 is normally expressed in testes and placenta. MAGEA4, however, can be expressed in many different types of tumors, for example, melanoma, head and neck squamous cell carcinoma, lung carcinoma, and breast carcinoma. Accordingly, MAGEA4 can be antigen associated with a variety of tumors.

The MAGEA4 antigen can induce antigen-specific T cell and/or high titer antibody responses, thereby inducing or eliciting an immune response that is directed to or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-a). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immune suppression factors that promote growth of the tumor or cancer expressing the antigen, for example, but not limited to, factors that down regulate MHC presentation, factors that up regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor associated macrophages, tumor associated fibroblasts.

The MAGEA4 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-MAGEA4 immune responses can be induced. The MAGEA4 antigen can comprise the full length translation product, a variant thereof, a fragment thereof or a combination thereof.

(16) FSHR

The self-antigen may include FSHR (Follicle stimulating hormone receptor). FSHR is an antigen that is selectively expressed in women in the ovarian granulosa cells (Simoni et al., Endocr Rev. 1997, 18:739-773) and at low levels in the ovarian endothelium (Vannier et al., Biochemistry, 1996, 35: 1358-1366). Most importantly, this surface antigen is expressed in 50-70% of ovarian carcinomas.

(17) Tumor Microenvironment Antigens

The self-antigen may include Tumor microenvironment antigen. Several proteins are overexpressed in the tumor microenvironment including, but not limited to, Fibroblast Activation Protein (FAP), Platelet Derived Growth Factor Receptor Beta (PDGFR-β), and Glypican-1 (GPC1). FAP is a membrane-bound enzyme with gelatinase and peptidase activity that is up-regulated in cancer-associated fibroblasts in over 90% of human carcinomas. PDGFR-β is a cell surface tyrosine kinase receptor that has roles in the regulation of many biological processes including embryonic development, angiogenesis, cell proliferation and differentiation. GPC1 is a cell surface proteoglycan that is enriched in cancer cells.

(18) PRAM I

The self-antigen may include PRAME (Melanoma antigen preferentially expressed in tumors). PRAME is a protein that in humans is encoded by the PRAME gene. This gene encodes an antigen that is predominantly expressed in human melanomas and that is recognized by cytolytic T lymphocytes. It is not expressed in normal tissues, except testis. The gene is also expressed in acute leukemias. Five alternatively spliced transcript variants encoding the same protein have been observed for this gene. Proteins may comprise sequences homologous to the PRAME antigens, fragments of the PRAME antigens and proteins with sequences homologous to fragments of the PRAME antigens.

(19) Tumor Antigen

The self-antigen may include a tumor antigen. In the context of the present invention,

"tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder," refers to antigens that are common to specific hyperproliferative disorders such as cancer. The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding moiety of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA,

Her2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success. The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:

Differentiation antigens such as MART- 1/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C- associated protein, TAAL6, TAG72, TLP, and TPS.

c. Other Antigens

In some embodiments, the antigen is an antigen other than the foreign antigen and/or the self-antigen. (a) HIV-1 VRCOl

The other antigen can be HIV-1 VRCOl. HIV-1 VCR01 is a neutralizing CD4- binding site-antibody for HIV. HIV-1 VCR01 contacts portions of HIV-1 including within the gpl20 loop D, the CD4 binding loop, and the V5 region of HIV-1.

(b) HIV-1 PG9

The other antigen can be HIV-1 PG9. HIV-1 PG9 is the founder member of an expanding family of gly can-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus.

(c) HIV-1 4E10

The other antigen can be HIV-1 4E10. HIV-1 4E10 is a neutralizing anti-HIV antibody. HIV-1 4E10 is directed against linear epitopes mapped to the membrane-proximal external region (MPER) of HIV-1, which is located at the C terminus of the gp41 ectodomain.

(d) DV-SF1

The other antigen can be DV-SF1. DV-SF1 is a neutralizing antibody that binds the envelope protein of the four Dengue virus serotypes.

(e) DV-SF2

The other antigen can be DV-SF2. DV-SF2 is a neutralizing antibody that binds an epitope of the Dengue virus. DV-SF2 can be specific for the DENV4 serotype.

(f) DV-SF3

The other antigen can be DV-SF3. DV-SF3 is a neutralizing antibody that binds the EDIII A strand of the Dengue virus envelope protein.

4. Vector

The vaccine can comprise one or more vectors that include a nucleic acid encoding the antigen. The one or more vectors can be capable of expressing the antigen. The vector can have a nucleic acid sequence containing an origin of replication. Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self- replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U. S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

The one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.

The vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

(1) Expression Vectors

The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The vector can have a promoter operably linked to the antigen- encoding nucleotide sequence, or the adjuvant-encoding nucleotide sequence, which may be operably linked to termination signals. The vector can also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

A plasmid may comprise a nucleic acid sequence that encodes one or more of the various immunogens disclosed above including coding sequences that encode the antigen capable of eliciting an immune response.

A single plasmid may contain coding sequence for an antigen, coding sequence for two antigens, coding sequence for three antigens, coding sequence for four antigens, coding sequence for antigens, or coding sequence for six antigens. A single plasmid may contain a coding sequence for a single antigen which can be formulated together. In some

embodiments, a plasmid may comprise coding sequence that encodes IL-12.

The plasmid may further comprise an initiation codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The initiation and termination codon may be in frame with the coding sequence.

The plasmid may also comprise a promoter that is operably linked to the coding sequence The promoter operably linked to the coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US patent application publication no. US20040175727, the contents of which are incorporated herein in its entirety.

The plasmid may also comprise a polyadenylation signal, which may be downstream of the coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA). The plasmid may also comprise an enhancer upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV, RSV or EBV. Polynucleotide function enhances are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94/016737, the contents of each are fully incorporated by reference.

The plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell. The plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered. The coding sequence may comprise a codon that may allow more efficient transcription of the coding sequence in the host cell.

The coding sequence may also comprise an Ig leader sequence. The leader sequence may be 5' of the coding sequence. The consensus antigens encoded by this sequence may comprise an N-terminal Ig leader followed by a consensus antigen protein. The N-terminal Ig leader may be IgE or IgG.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif), which may be used for protein production in Escherichia coli (E.coli). The plasmid may also be pYES2 (Invitrogen, San Diego, Calif), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif), which may be used for protein production in insect cells. The plasmid may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. (2) RNA vectors

In one embodiment, the nucleic acid is an RNA molecule. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of the antigens. The RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription. A RNA molecule useful with the invention may have a 5' cap (e.g. a 7- methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of a RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. A RNA molecule useful with the invention may be single- stranded.

(3) Circular and Linear Vectors

The vector may be a circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen, or the adjuvant and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.

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

The LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the antigen, or the adjuvant. The plasmid may be capable of expressing the adjuvant ISG15. The plasmid can 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 the antigen, or encoding the adjuvant, and enabling a cell to translate the sequence to an antigen that is recognized by the immune system, or the adjuvant.

The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively. (4) Promoter, Intron, Stop Codon, and Polyadenylation Signal

The vector may have a promoter. A promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the antigen sequence, or the adjuvant sequence described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

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

The promoter may be a CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.

The vector may include an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. (5) Viral Vectors

In one embodiment, viral vectors are provided herein which are capable of delivering a nucleic acid of the invention to a cell. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

5. Excipients and other components of the Vaccine

The vaccine of the invention may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.

The transfection facilitating agent is a poly anion, poly cation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the vaccine at a concentration less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. The DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, poly cation, including poly-L-glutamate (LGS), or lipid. 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.

The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the plasmid above in the vaccine.

The vaccine may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference. The vaccine can be formulated according to the mode of administration to be used. An 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 comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions. 6. Method of Treatment

Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by administering the immunogenic composition, such as a vaccine, to the subject. Administration of the vaccine to the subject can induce or elicit an immune response in the subject. In one embodiment, the composition is administered to the subject through intradermal electroporation (ID EP) or intramuscular EP (IM EP). As described below, ID EP and IM EP allow for a low dose of the nucleic acid encoding the antigen and/or adjuvant to be administered to the subject while maintaining the generation of an immune response.

The present invention is also directed to a method of generating an immune response in a subject. Generating the immune response can be used to treat and/or prevent disease associated with the antigen in the subject, as described in more detail below. The method can include administering the herein disclosed composition or vaccine to the subject.

The vaccine dose can be between 1 μg to 10 mg active component/kg body weight/time, and can be 20 μg to 10 mg component/kg body weight/time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. a. Treatment and Prevention of Cancer

The subject administered the composition can generate an immune response. The immune response can be used to treat and/or prevent disease in the subject. The disease can be cancer, for example, an HPV-associated cancer, HBV-associated cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, head and neck cancer, throat cancer, lung cancer, liver cancer, cancer of the pancreas, kidney cancer, bone cancer, melanoma, metastatic cancer, hTERT-associated cancer, FAP-antigen associated cancer, non-small cell lung cancer, blood cancer, esophageal squamous cell carcinoma, cervical cancer, bladder cancer, colorectal cancer, gastric cancer, anal cancer, synovial carcinoma, testicular cancer, recurrent respiratory papillomatosis, skin cancer, glioblastoma, hepatocarcinoma, stomach cancer, acute myeloid leukemia, triple-negative breast cancer, and primary cutaneous T cell lymphoma.

The method can further include reducing the size of an established tumor or lesion in the subject. The tumor can be reduced in size by about 5% to about 100%, 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 90% to about 95%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 80% to about 90%, compared to not administering the composition. The tumor can be reduced in size by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 80%, by about 81%, by about 82%, by about 83%, by about 84%, by about 85%, by about 86%, by about 87%, by about 88%, by about 89%, by about 90%, by about 91 %, by about 92%, by about 93%, by about 94%, by about 95%, by about 96%, by about 97%, by about 98%, by about 99%, or by about 100%, compared to not administering the composition.

In some embodiments, administration of the vaccine can tumor can reduce tumor size by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, or by at least 90%, compared to not administering the composition.

The method can further include increasing tumor regression in the subject as compared to the subject administered the antigen alone. Administration of the vaccine can increase tumor regression by about 40% to about 60%, about 45% to about 55%, or about 50%, compared to not administering the composition. Administration of the vaccine can also increase the rate of tumor regression. Administration of the vaccine can further achieve tumor regression in the subject of about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 80% to about 90%, or about 85% to about 90%, compared to not administering the composition. Tumor regression can be about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% in the subject administered the vaccine, compared to not administering the composition. Tumor regression in the subject administered the vaccine can further be about 90% or about 100%.

In some embodiments, administration of the vaccine can increase tumor regression by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, or by at least 90%, compared to not administering the composition.

The method can further include preventing cancer or tumor growth in the subject administered the vaccine. This prevention can allow the subject administered the vaccine to survive a future cancer. In other words, the vaccine affords protection against cancer to the subject administered the vaccine. The subject administered the vaccine can have about 90% to about 100% survival of cancer, compared to not administering the composition. The subject administered the vaccine can have about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% survival of cancer, compared to not administering the composition. b. Treatment and Prevention of Infectious Disease

The subject administered the vaccine can have an increased or boosted immune response as compared to the subject administered the antigen alone. The increased immune response can be used to treat and/or prevent disease in the subject. The disease can be infectious disease, for example, viral and bacterial infections. The bacterial infection can be an anthrax bacterium, an antibiotic resistant bacterium, a disease causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. The bacterium can be a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. The bacterium can be

Mycobacterium tuberculosis . listeria monocytogenes, lymphocytic choriomeningitis virus.

The viral infection can be a papilloma virus, for example, human papillomoa virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis B virus, hepatitis C virus, smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhino viruses, dengue fever virus, equine encephalitis viruses, 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, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella- zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, chikungunya virus, lassa virus, Ebolavirus, Zika virus, arenavirus, lymphocytic choriomeningitis virus (LCMV), or cancer causing virus.

The method can further include preventing the deleterious effects of an infectious disease in the subject administered the vaccine. This prevention can allow the subject administered the vaccine to survive the infectious disease. In other words, the vaccine affords protection against infectious disease to the subject administered the vaccine. The subject administered the vaccine can have about 50% to about 100% survival of infectious disease, compared to not administering the composition. The subject administered the vaccine can have about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% survival of infectious disease, compared to not administering the composition. c. Administration

The vaccine can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the vaccines are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No.

5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21 , 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

The vaccine can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U. S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of 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 its entirety).

The vaccine can be administered via electroporation, such as by a method described in U. S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. The electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874;

5,676,646; 6,241 ,701 ; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6, 181,964; 6, 150, 148;

6, 120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety. The electroporation may be carried out via a minimally invasive device.

In some embodiments, the vaccine is administered via intradermal electroporation (ID

EP). In one embodiment, vaccine administered via ID EP comprises a low dose of a nucleic acid encoding the antigen, a nucleic acid encoding the adjuvant, or both. ID EP allows for injection of the compositions described herein from smaller needles. IM EP devices usually comprise about 5 needles which are typically about 1.8-2.0 cm in length while ID EP devices usually comprise about 3 needles which are typically about 0.2-0.4 cm in length. ID delivery is better tolerated in patients than IM.

In some aspects, the ID EP injects the vaccine into the intradermal layer of the tissue and does not reach the subdermis. Accordingly, in some embodiments the ID EP inj ects the vaccine 0.20 cm, 0.21 cm, 0.22 cm, 0.23 cm, 0.24 cm, 0.25 cm, 0.26 cm, 0.27 cm, 0.28 cm, 0.29 cm, 0.30 cm, 0.31 cm, 0.32 cm, 0.33 cm, 0.34 cm, 0.35 cm, 0.36 cm, 0.37 cm, 0.38 cm, 0.39 cm, 0.40 cm, 0.41 cm, 0.42 cm, 0.43 cm, 0.44 cm, 0.45 cm, 0.46 cm, 0.47 cm, 0.48 cm, 0.49 cm, or 0.50 cm below the surface of the skin. The ID EP can be injected at one or more sites. For example, in some embodiments the ID EP is injected at 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites or 10 sites.

The minimally invasive electroporation device ("MID") may be an apparatus for injecting the vaccine described above and associated fluid into body tissue. The device may comprise a hollow needle, DNA cassette, and fluid delivery means, wherein the device is adapted to actuate the fluid delivery means in use so as to concurrently (for example, automatically) inject DNA into body tissue during insertion of the needle into the said body tissue. This has the advantage that the ability to inject the DNA and associated fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. The pain experienced during injection may be reduced due to the distribution of the DNA being inj ected over a larger area.

The MID may inject the vaccine into tissue without the use of a needle. The MID may inject the vaccine as a small stream or jet with such force that the vaccine pierces the surface of the tissue and enters the underlying tissue and/or muscle. The force behind the small stream or jet may be provided by expansion of a compressed gas, such as carbon dioxide through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655; U.S. Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent NO. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; and U.S. Patent No. 6,763,264, the contents of each of which are herein incorporated by reference.

The MID may comprise an injector that creates a high-speed jet of liquid that painlessly pierces the tissue. Such needle-free injectors are commercially available. Examples of needle-free injectors that can be utilized herein include those described in U.S. Patent Nos. 3,805,783; 4,447,223; 5,505,697; and 4,342,310, the contents of each of which are herein incorporated by reference.

A desired vaccine in a form suitable for direct or indirect electrotransport may be introduced (e.g., injected) using a needle-free injector into the tissue to be treated, usually by contacting the tissue surface with the injector so as to actuate delivery of a jet of the agent, with sufficient force to cause penetration of the vaccine into the tissue. For example, if the tissue to be treated is mucosa, skin or muscle, the agent is projected towards the mucosal or skin surface with sufficient force to cause the agent to penetrate through the stratum comeum and into dermal layers, or into underlying tissue and muscle, respectively.

Needle-free injectors are well suited to deliver vaccines to all types of tissues, particularly to skin and mucosa. In some embodiments, a needle-free inj ector may be used to propel a liquid that contains the vaccine to the surface and into the subject's skin or mucosa. Representative examples of the various types of tissues that can be treated using the invention methods include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

The MID may have needle electrodes that electroporate the tissue. By pulsing between multiple pairs of electrodes in a multiple electrode array, for example set up in rectangular or square patterns, provides improved results over that of pulsing between a pair of electrodes. Disclosed, for example, in U. S. Patent No. 5,702,359 entitled "Needle Electrodes for Mediated Delivery of Drugs and Genes" is an array of needles wherein a plurality of pairs of needles may be pulsed during the therapeutic treatment. In that application, which is incorporated herein by reference as fully set forth, needles were disposed in a circular array, but have connectors and switching apparatus enabling a pulsing between opposing pairs of needle electrodes. A pair of needle electrodes for delivering recombinant expression vectors to cells may be used. Such a device and system is described in U. S. Patent No. 6,763,264, the contents of which are herein incorporated by reference. Alternatively, a single needle device may be used that allows injection of the DNA and electroporation with a single needle resembling a normal injection needle and applies pulses of lower voltage than those delivered by presently used devices, thus reducing the electrical sensation experienced by the patient.

The MID may comprise one or more electrode arrays. The arrays may comprise two or more needles of the same diameter or different diameters. The needles may be evenly or unevenly spaced apart. The needles may be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches; or between 0.015 inches and 0.020 inches. The needle may be 0.0175 inches in diameter. The needles may be 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 spaced apart.

The MID may consist of a pulse generator and a two or more-needle vaccine injectors that deliver the vaccine and electroporation pulses in a single step. The pulse generator may allow for 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 may deliver a variety of volt pulses during short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms in duration. An example of such a 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 herein

incorporated by reference.

The MID may be a CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting, PA) device and system, which is a modular electrode system, that facilitates the introduction of a macromolecule, such as a DNA, into cells of a selected tissue in a body or plant. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The macromolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to overheating of cells is minimized by limiting the power dissipation in the tissue by virtue of constant- current pulses. The CELLECTRA device and system is described in U.S. Patent No.

7,245,963, the contents of which are herein incorporated by reference.

The MID may be an Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may comprise device that provides a hollow needle; and fluid delivery means, wherein the apparatus is adapted to actuate the fluid delivery means in use so as to concurrently (for example automatically) inject fluid, the described vaccine herein, into body tissue during insertion of the needle into the said body tissue. The advantage is the ability to inject the fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. It is also believed that the pain experienced during injection is reduced due to the distribution of the volume of fluid being injected over a larger area.

In addition, the automatic injection of fluid facilitates automatic monitoring and registration of an actual dose of fluid injected. This data can be stored by a control unit for documentation purposes if desired. It will be appreciated that the rate of injection could be either linear or non-linear and that the injection may be carried out after the needles have been inserted through the skin of the subject to be treated and while they are inserted further into the body tissue.

Suitable tissues into which fluid may be injected by the apparatus of the present invention include tumor tissue, skin or liver tissue but may be muscle tissue.

The apparatus further comprises needle insertion means for guiding 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 the needle insertion and injection of fluid can be controlled such that the rate of insertion can be matched to the rate of injection as desired. It also makes the apparatus easier for a user to operate. If desired means for automatically inserting the needle into body tissue could be provided.

A user could choose when to commence injection of fluid. Ideally however, injection is commenced when the tip of the needle has reached muscle tissue and the apparatus may include means for sensing when the needle has been inserted to a sufficient depth for injection of the fluid to commence. This means that injection of fluid can be prompted to commence automatically when the needle has reached a desired depth (which will normally be the depth at which muscle tissue begins). The depth at which muscle tissue begins could for example be taken to be a preset needle insertion depth such as a value of 4 mm which would be deemed sufficient for the needle to get through the skin layer.

The sensing means may comprise an ultrasound probe. The sensing means may comprise a means for sensing a change in impedance or resistance. In this case, the means may not as such record the depth of the needle in the body tissue but will rather be adapted to sense a change in impedance or resistance as the needle moves from a different type of body tissue into muscle. Either of these alternatives provides a relatively accurate and simple to operate means of sensing that injection may commence. The depth of insertion of the needle can further be recorded if desired and could be used to control injection of fluid such that the volume of fluid to be injected is determined as the depth of needle insertion is being recorded.

The apparatus may further comprise: a base for supporting the needle; and a housing for receiving the base therein, wherein the base is moveable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends out of the housing when the base is in a second forward position within the housing. This is advantageous for a user as the housing can be lined up on the skin of a patient, and the needles can then be inserted into the patient's skin by moving the housing relative to the base.

As stated above, it is desirable to achieve a controlled rate of fluid injection such that the fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may comprise piston driving means adapted to inject fluid at a controlled rate. The piston driving means could for example be activated by a servo motor. However, the piston driving means may be actuated by the base being moved in the axial direction relative to the housing. It will be appreciated that alternative means for fluid delivery could be provided. Thus, for example, a closed container which can be squeezed for fluid delivery at a controlled or non-controlled rate could be provided in the place of a syringe and piston system.

The apparatus described above could be used for any type of inj ection. It is however envisaged to be particularly useful in the field of electroporation and so it may further comprise 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 as it means that the electric field is applied to the same area as the injected fluid. There has traditionally been a problem with electroporation in that it is very difficult to accurately align an electrode with previously injected fluid and so users have tended to inject a larger volume of fluid than is required over a larger area and to apply an electric field over a higher area to attempt to guarantee an overlap between the injected substance and the electric field. Using the present invention, both the volume of fluid inj ected and the size of electric field applied may be reduced while achieving a good fit between the electric field and the fluid.

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

7. Examples

Example 1: PENNVAX®-GP delivered by electroporation (EP) in HVTN 098 induces a strong immune response

First-generation vaccines were improved by changing nucleic acid constructs and pharmaceutical formulation, co-administration with a molecular adjuvant, and improved delivery by in vivo electroporation (EP). It was previously demonstrated that the addition of EP and IL-12 as an adjuvant induced higher CD4+ and CD8+ immune responses. To date, vaccines have been delivered intramuscularly (IM).

ID administration of the vaccine, compared to IM administration, has potential for better quality of immune response based on ID environment, may allow for dose sparing, and may be better tolerated than IM (shallower penetration: ID = 0.3 cm vs IM = 1.9 cm).

Because of poor immune responses to prior env DNA vaccines (env B) (Kalams et al., 2013, J Infect Dis., 208(5):818-29), env A and env C were used for the present experiments at higher IM doses.

The present studies were designed to obtain safety and tolerability information, and to test the efficacy of ID compared with that of IM delivery of PENNV AX ® -GP vaccine, using only a fraction (20%) of the antigen in ID compared to IM. Another aim was to determine whether there is a difference in the T-cell and antibody immune responses in the presence or absence of IL-12 in ID administration. The materials and methods employed in these experiments are now described.

The vaccine comprised nucleotide sequences encoding several HIV-associated antigens (env A, env C, gag, pol), in the presence or absence of an adjuvant (e.g., hIL-12) (Figure 1). The vaccine was delivered by electroporation either intramuscularly (IM; 1.9 cm depth) or intradermally (ID; 0.3 cm depth) (Figure 2). The schema for the study is provided (Figure 3). Briefly, there were 4 groups. Group 1 received ID-delivered (0.1 mg env A + 0.1 mg env C + 0.2 mg gag + 0.2 mg pol = 0.6 mg total PENNV AX ® -GP) with 0.2 mg of IL-12 DNA, or placebo. Group 2 received ID-delivered (0.6 mg env A + 0.6 mg env C + 0.2 mg gag + 0.2 mg pol = 1.6 mg total PENNV AX ® -GP) with 0 mg of IL-12 DNA, or placebo. Group 3 received ID-delivered (0.6 mg env A + 0.6 mg env C + 0.2 mg gag + 0.2 mg pol = 1.6 mg total PENNV AX ® -GP) with 0.4 mg of IL-12 DNA, or placebo. Group 4 received IM- delivered (3 mg env A, 3 mg env C, 1 mg gag, 1 mg pol = 8 mg total PENNV AX ® -GP) with 1 mg of IL-12 DNA or placebo. All participants received injections at four timepoints: day 0, month 1, month 3, and month 6. Groups 2 and 3 received the doses indicated over 2 injection sites on the arm.

Intracellular cytokine staining (ICS) was performed following 6-hour ex vivo stimulation with global potential T cell epitope (PTE) peptide pools for Env, Gag, and Pol.

Binding antibody multiplex assay (BAMA) was performed for the following gpl40 Envelope proteins: Group M consensus (ConS), Clade A consensus (A con), Clade B consensus (B con), and Clade C consensus (C con), as well as gp41 , p24 Gag, and p66 RT. Two (2) weeks following the 3 rd vaccination, BAMA was performed, and 2 weeks following the 4 th vaccination, ICS and BAMA were performed.

To date, the ICS analysis was completed for the 2-week time point following the 4 th vaccination, and BAMA analysis was completed for the 2-week time points following the 3 rd and 4 th vaccination.

To measure the level of acceptability with EP administration, the tolerability of ID compared to IM delivery was determined by asking participants to fill out an acceptability questionnaire 2 weeks after each vaccination and 6 months after the last vaccination. ID low dose, ID (no IL-12), ID (+IL-12), and IM (+IL-12) were compared for level of discomfort and overall acceptability.

The results of the experiments are now described. Compared to IM, ID can lead to increased CD4 T-cell response rates

Compared to IM, ID delivery of vaccine can lead to similar (96% compared to 96%; Figure 4) or elevated (64% compared to 44%; Figure 6) T cell response rates, in the presence of IL-12 DNA adjuvant at 2 weeks following the 4 th vaccination. ID inclusion of IL-12 can lead to increased CD4 T-cell response rates

Compared to vaccine lacking IL-12, inclusion of IL-12 in ID-delivered vaccines can lead to elevated (96% compared to 56%; Figure 5) or similar (56% compared to 64%; Figure 7) T cell response rates at 2 weeks following the 4 th vaccination. Immunization correlates with production of binding antibodies

Compared to IM administration, ID administration resulted in elevated group M consensus envelope binding antibody (Figure 8). In addition, there was a trend for a higher magnitude of binding antibody to group M consensus envelope after the 3 rd vaccination with IL-12 (visit 7) compared to ID administration without IL-12 (96% compared to 92%; Figure 9). In addition, binding antibody to gp41 was elevated in the majority of ID-vaccinated individuals, compared to IM-vaccinated individuals (75% compared to 29% at visit 9; Figure 10). Finally, the amount of binding antibody to p24 detected was about half of ID- and IM- vaccinated individuals with inclusion of IL-12 (57% and 52% at visit 9; Figure 1 1). Compared to IM, ID can be better tolerated among patients

According to results from the acceptability questionnaire, ID administration of the vaccine is better tolerated than IM administration of the vaccine (Figure 12). Upon inspection of the injection site, visible lesions were sometimes observed with ID, though these faded with time and were generally acceptable (Figure 13).

Reactogenicity and adverse events

Overall, pain and tenderness were mild or moderate; there was no severe local reactogenicity. Systemic symptoms were mostly mild or moderate. One third of patients had none. Three of ninety -four (3/94) reported severe systemic symptoms such as malaise/fatigue or headache. Adverse events which were assessed as vaccine-related involved 34 (36.2%) participants (may include placebos).

Summary

PENNVAX ® -GP DNA alone (Env A, Env C) delivered by EP demonstrated Env- specific CD4+ T cell and binding antibody response rates close to 100% for either ID or IM (with IL-12). Regarding the cellular response, ID electroporation results in similar CD4 and higher CD8 T-cell response rates, using only l/5th of the dose. In addition, IL-12 leads to increased response rates, especially for CD4 T cells, but magnitudes are similar across arms. Regarding the humoral response, ID electroporation results in similar or higher response rates using only l/5th of the dose for Env-, gp41-, and p24-specific antibodies. In addition, IL-12 may lead to a higher magnitude Env-specific antibody response after only 3 vaccinations.

Example 2: Clinical parameters and results

The frequency and magnitude of IgG binding antibody responses were measured by the HIV-1 binding antibody multiplex assay (BAMA) from serum specimens obtained at visit 7 and 9, corresponding to 2 weeks after the 3rd vaccination (day 98) and 4th vaccination (day 182), respectively.

Neutralization ID50 titers were measured by HIV neutralizing antibody assay from specimens obtained at visit 7 (visit day 98) and visit 9 (visit day 182), corresponding to 2 weeks after the 3rd and the 4th vaccination, respectively. The ICS endpoints include CD4+ and CD8+ T-cell response rates and magnitudes measured by intracellular cytokine staining (ICS) on PBMC samples obtained at visit 9 (day 182), corresponding to 2 weeks after the 4th (last) vaccination. Binding Antibody Multiplex Assay (BAMA)

Serum HIV- 1 -specific IgG responses (1:50 dilution) against antigens listed below were measured on a Bio-Plex instrument (Bio-Rad) using a standardized custom HIV-1 Luminex assay (Tomaras and Yates et al, J Virology 2008). The readout was background- subtracted mean fluorescence intensity (MFI), where background referred to a plate level control (i.e., a blank well run on each plate). The positive control was purified polyclonal IgG from HIV subjects (HIVIG) using a 10-point standard curve (4PL fit). The negative controls were NHS (HIV-1 sero-negative human sera) and blank beads.

Several criteria were used to determine if data from an assay were acceptable and could be statistically analyzed. First, the blood draw date must have been within the allowable visit window as determined by the protocol. Second, if the blank bead negative control exceeded 5,000 MFI, the sample was repeated. If the repeat value exceeded 5,000 MFI, the sample was excluded from analysis due to high background.

There were 7 HIV proteins used as antigens in these binding assays to examine cross- clade envelope responses (A, B, C) and non-envelope proteins contained in the vaccine (p24 Gag, p66 Reverse Transcriptase):

Samples from post-enrollment visits were declared to have positive responses if they met three conditions: (1) the MFI minus Blank values were > antigen-specific cutoff at the 1 :50 dilution level (based on the 95th percentile of the baseline visit serum samples and at least 100 MFI), (2) the MFI minus Blank values were greater than 3 times the baseline (day 0) MFI minus Blank values, and (3) the MFI values were greater than 3 times the baseline MFI values.

Tables show the response rates and corresponding 95% confidence intervals calculated by the Wilson score method (Agresti A and Coull BA., 1998, The American Statistician, 52(2): 1 19-126).

The MFI minus blank responses (MFI*) at the 1 :50 dilution level were used to summarize the magnitude at a given time-point. These distributions are displayed graphically by treatment arm. Plots include data from responders in red and non-responders in blue with box plots based on data from responders superimposed on the distribution. The mid-line of the box denotes the median and the ends of the box denote the 25th and 75th percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range (i.e., height of the box) or if no value meets this criterion, to the data extremes.

Vaccine regimens T2, T3 and T4 were ranked by antigen and time point based on response rates and response magnitudes among responders, separately. Response magnitudes were ranked based on the mean response magnitude among responders divided by the standard error of each arm using log-transformed MFI* values. Immune variables with fewer than 3 positive responders in at least 2 arms were excluded in the ranking analysis of response magnitudes. The ranking analyses were applied to 7 antigens excluding p66 (RT) (due to low response). Two types of ranking p-values based on permutation tests were provided: one for the overall ranking of T2, T3 and T4; and another for the highest ranked arm among T2, T3 and T4. The two permutation p-values were computed as the proportion of permuted datasets (m=5000) that render the same overall ranking or the same highest ranked arm as observed, respectively. The false-discovery-rate (FDR) adjusted p-values (Benjamini and Hochberg, 1995) were calculated to account for the total number of ranking analyses across antigens and analysis endpoints (response rate and response magnitude) at each time point for each type of permutation tests.

As an exploratory analysis, responses between visits 7 and 9 were also compared by treatment arm and antigen using the Lachenbruch test of response rate (unpaired) and response magnitude (among positive responders paired at both visits) for T2, T3 and T4, and for all 7 antigens excluding p66 (RT). The FDR-adjusted p-values (Benjamini and Hochberg, 1995) were calculated to account for multiple treatment arms and antigens tested.

Figure 22 is a table showing response rates by antigen, visit, day, and treatment arm. Figure 23 is a table providing the summary for the magnitudes of response by antigen and visit among positive responders.

The graphs (Figure 14 through Figure 21) display the distribution of MFI* "Net Response (MFI - Blank)" for each treatment group by antigen and individual responses over time.

At visit 7, positive IgG responses were observed in all treatment groups, response rates ranging from 25%-96.2% to gpl40 antigens, 0%-42.3% to gpl20 antigens, 16%-73.1 % to gp41 , and 0%-36% to other antigens. No positive responses were observed to any proteins in placebo groups.

At visit 9 (peak time point), higher IgG response rates were observed in all treatment arms, ranging from 66.7%-100% to gpl40 antigens, 0%-57.1 % to gpl20 antigens, 28.6%- 92.3% to gp41, and 0%-57.1% to other antigens. No positive responses were observed to any proteins in placebo groups.

Overall, at both visits 7 and 9, the ID regimens (T3 or T2) generated higher or comparable response rates and response magnitudes than the IM group (T4). However, no ranking of response rates or response magnitudes of the 3 vaccine regimens was statistically significant.

In general, responses seemed to be boosted after the 4th vaccination, most obviously for gp41 responses in T2.

Neutralizing Antibody (NAb) Titer Response Rates

Neutralizing antibodies against HIV-1 were measured as a function of reductions in

Tat-regulated luciferase (Luc) reporter gene expression in TZM-bl cells. The assay measured neutralization titers against a heterologous clade C Env-pseudotyped virus that exhibits a tier 1A neutralization phenotype (MW965.26). Neutralization assays may also be performed against a global virus panel of heterologous Env-pseudotyped viruses (246F3, Cell 76, CNE55, XI 632, Ce0217, BJOX2000, 25710, TROl 1, CHI 19, X2278, CNE8 and 398F1). Additional assays may be performed in TZM-bl cells to measure neutralization titers against other tier 1 and tier 2 viruses. Data from blood draw dates outside the allowable visit window and assay results deemed unreliable for analysis by the lab are excluded from the analysis. Response to a virus/isolate in the TZM-bl assay is considered positive if the neutralization titer is above a pre-specified cutoff (one-half the lowest dilution tested). A titer is defined as the serum dilution that reduces relative luminescence units (RLUs) by 50% relative to the RLUs in virus control wells (cells + virus only) after subtraction of background RLU (cells only). The prespecified cutoff is 10 for TZM-bl cells. Figure 28 shows the response rates and corresponding 95% confidence intervals calculated by the Wilson score method (Agresti and Coull, 1998, The American Statistician, 52(2): 119-126).

The distribution of the neutralizing antibody titers at each visit is displayed graphically, with data from responders in red and non-responders in blue. Box plots are superimposed on the distributions. The mid-line of a box plot denotes the median and the ends of the box denote the 25th and 75th percentiles. Whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Titers are also displayed longitudinally by cell and isolate, with one line for each participant. The reverse cumulative distribution of the magnitude for TZM-bl is also displayed by visit with one line per treatment group

Figure 28 is a table showing the neutralizing antibody titer response rates by cell type, assay type, isolate, visit, and treatment arm. Figure 29 is a table showing summary statistics for NAb titers among positive responders. Figures show boxplots (Figure 26 and Figure 27) and reverse CDF (Figure 24 and Figure 25) of NAb titers by isolate, visit and treatment arm.

At visit 7, 2 weeks post the 3rd vaccination, the response rates for the tier 1 isolate MW965.26 were 0% in Tl, 27.8% in T2, 53.8% in T3, and 31 % in T4. The neutralization titers for responders range from 20.00 to 31.00 with mean 26.60 in T2, 22.00 to 93.00 with mean 46.14 in T3, and 21.00 to 276.00 with mean 73.56 in T4. No positive response was observed in placebo groups.

At the peak time point, 2 weeks after the 4th vaccination, the response rates for the tier 1 isolate MW965.26 were 0% in Tl, 56.3% in T2, 75% in T3, 50% in T4. The neutralization titers for responders range from 24.00 to 86.00 with mean 44.67 in T2, 25.00 to 103.00 with mean 58.57 in T3, and 21.00 to 164.00 with mean 61.46 in T4. No positive response was observed in the placebo groups.

Intracellular cytokine staining (ICS) responses

Flow cytometry is used to examine HIV- 1 -specific CD4+ and CD8+ T-cell responses using a validated 17-color panel assay. In this report, the responses evaluated are to the HIV global potential T cell epitope (PTEg) peptide pools as follows: 1) Env-l-PTEg; 2) Env-2-PTEg; 3) Env-3-PTEg; 4) Gag-l-PTEg; 5) Pol-l-PTEg; 6) Pol-2-PTEg.

Previously cryopreserved PBMC are stimulated with the synthetic peptide pools. As a negative control, cells are incubated with DMSO, the diluent for the peptide pools. As a positive control, cells are stimulated with a polyclonal stimulant, staphylococcal enterotoxin B (SEB). All stimulations are run in singlicate with the exception of the negative control, which is run in duplicate.

Several criteria are used to determine if data from an assay are acceptable and can be statistically analyzed. The blood draw date must be within the allowable visit window as determined by the protocol. Post-infection samples from HIV-infected participants are excluded. After sample thawing and overnight incubation, the viability of the PBMC must be 66% or greater for testing to proceed. If the viability is not 66% or greater, a new specimen for that participant at that time point must be thawed for testing. If the PBMC viability of the second thawed aliquot is below this threshold, the ICS assay is not performed and no data are reported to the statistical center for that time point. For the negative control acceptance criteria, if the average cytokine response for the negative control wells is above 0.1% for either the CD4+ or CD8+ T cells, then the sample must be retested. If the retested results are above 0.1% then the data are excluded from analysis; otherwise the retest data are used.

The total number of CD4+ and CD8+ T cells must exceed certain thresholds. If the number of CD4+ or CD8+ T cells is less than 5,000 for any of the peptide pools or for one of the negative control replicates for a particular sample, data for that stimulation will be filtered and data from the single negative control will be kept. If both negative control replicates are <5,000 cells, the sample will be retested and the same criteria applied as before. If both negative control replicates from the retest for a T-cell subset are <5,000, then data for the T- cell subset are not included in the analysis.

To assess positivity for an antigen within a T-cell subset, a two-by-two contingency table is constructed comparing the peptide stimulated and negative control data. The four entries in each table are the number of cells positive for IL-2 and/or IFN-γ and the number of cells negative for IL-2 and/or IFN-γ, for both the stimulated and the negative control data. A one-sided Fisher's exact test is applied to the table, testing whether the number of cytokine- producing cells for the stimulated data is equal to that for the negative control data. Since multiple individual tests are conducted simultaneously, a multiplicity adjustment is made to the HIV antigen p-values using the discrete Bonferroni adjustment method. If the adjusted p- value for an antigen is <0.00001, the response to the antigen for the T-cell subset is considered positive. Because the sample sizes (i.e., total cell counts for the T-cell subset) are large (e.g., 100,000 cells), the Fisher's exact test has high power to reject the null hypothesis for very small differences. Therefore, the adjusted p-value significance threshold was chosen stringently (<0.00001). If any peptide pool is positive for a T-cell subset, then the overall response for that T-cell subset is considered positive. Tables show the response rates and corresponding 95% confidence intervals calculated by Wilson's score method (Agresti and Coull, 1998, The American Statistician, 52(2): 119-126), as well as the ranking of arms at a specific time-point based on the observed response rates of T2, T3 and T4.

The distribution of the magnitude of IL-2 and/or IFN-γ T-cell response is displayed graphically by T-cell subset, peptide pool, cytokine subset, visit, and treatment arm.

Magnitudes are background-adjusted values. Figure 30 through Figure 59 include data from responders in red and non-responders in blue. Box plots based upon data from responders only are superimposed on the distributions. The mid-line of the box denotes the median and the ends of the box denote the 25th and 75th percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range (i.e., height of the box) or if no value meets this criterion, to the data extremes. The ranking of T2, T3 and T4 at a specific time-point was also done based on response magnitudes using the mean response magnitude divided by the standard error of the mean of each arm among positive responders. Immune variables with fewer than 3 positive responders in at least 2 arms were excluded in the ranking analysis of response magnitude.

To limit the number of multiple tests, the ranking analyses of response rates and response magnitudes were only applied to the overall peptide pool categories: ANY PTEG, ANY Env, ANY Gag and ANY Pol. Two types of ranking p-values based on permutation tests were provided: one for the overall ranking of T2, T3 and T4; and another for the highest ranked arm among T2, T3 and T4. The two permutation p-values were computed as the proportion of permuted datasets (m=5000) that render the same overall ranking or the same highest ranked arm as observed, respectively. The false-discovery-rate adjusted p-values (Benjamini and Hochberg, 1995) were calculated to account for the total number of ranking analyses. The two types of permutation p-values were adjusted separately.

The analysis above is also applied to CD4+ T cells expressing CD40L (CD 154). Figure 62 is a table reporting ICS response rates by cytokine, T-cell subset, peptide pool, visit and treatment group for cells expressing IL-2 and/or IFN-γ and CD40L. Figure 63 is a table providing descriptive statistics for magnitude of response by cytokine, T-cell subset, peptide pool, visit and treatment group. Figure 30 through Figure 59 show response rates and magnitudes by cytokine, T-cell subset, peptide pool, and treatment group. Additional figures (Figure 60, Figure 61) depict a table providing the ranking results for response rates and magnitude.

CD4+ and CD8+ T-cells expressing IL-2 and/or IFN-γ

In summary, in terms of responses across treatment arms, T4 had the highest response rate for CD4+ and CD8+ T cells to any protein. In terms of responses across T-cell subsets, positive CD4+ T-cell responses expressing IL-2 and/or IFN-γ were observed in all treatment groups and fewer positive CD8+ T-cell responses were observed in all arms. In terms of responses across HIV proteins, CD4+ T-cell response rates and magnitudes to Env were the highest, and the response rates and magnitudes to Pol were the lowest. No positive response to any HIV protein was observed among the placebo recipients.

Specifically, the CD4+ T-cell response rate to any HIV protein was 60% in Tl, 56.3% in T2, 96.4% in T3, and 96.3% in T4; to Env, 20% in Tl, 50% in T2, 85.7% in T3, and 92.6% in T4; to Gag, 40% in Tl, 31.3% in T2, 50% in T3, and 55.6% in T4; and to Pol, 0% in Tl, 0% in T2, 0% in T3, and 7.4% in T4. The CD8+ T-cell response rate to any HIV protein was 20% in Tl, 56.3% in T2, 64.3% in T3 and 44.4% in T4; to Env, 20% in Tl, 50% in T2, 57.1% in T3, 44.4% in T4; to Gag, 0% in Tl, 0% in T2, 7.1% in T3, and 0% in T4; and to Pol, 0% in Tl, 6.3% in T2, 14.3% in T3, and 7.4% in T4.

CD4+ T-cells expressing CD40L

In summary, positive CD4+ T-cell responses expressing CD40L were observed in all treatment groups, and in terms of responses across treatment arms, T4 had the highest positive CD4+ T-cell response rate to any protein. In terms of responses across HIV proteins, CD4+ T-cell response rates and magnitudes to Env were also the highest, and the response rates and magnitudes to Pol were the lowest. No positive response was observed in CD4+ T cells among the 9 placebo recipients.

Specifically, the CD4+ T-cell response rate to any HIV protein was 60% in Tl, 81.3%% in T2, 92.9% in T3, and 96.3% in T4; to Env, 40% in Tl, 81.3% in T2, 85.7% in T3, and 92.6% in T4; to Gag, 60% in Tl, 18.8% in T2, 53.6% in T3, and 59.3% in T4; and to Pol, 0% in Tl, 0% in T2, 3.6% in T3, and 11.1% in T4.

No ranking of response rates or response magnitudes of the 3 vaccine regimens by assay endpoints and peptide pools was statistically significant. Example 3:EP delivery of Ebolavirus DNA vaccine

An Open-Label study of INO-4212 (with or without INO-9012 was conducted. INO- 4212 was administered IM or ID followed by electroporation in healthy volunteers. Safety and immunological assessments were monitored. Intradermal delivery and intramuscular delivery were compared. There were 69 total subjects. ELISA analysis was performed before immunization (baseline), and at weeks 2, 6, and 14. Seropositive is defined as a positive IgG antibody response to Ebola Zaire glycoprotein.

INO-4201 is a DNA vaccine formulated with the consensus envelope glycoprotein of Zaire Ebolavirus (ConEBOVGP#l) generated by using the envelope glycoprotein sequences of the 1976, 1994, 1995, 1996, 2003, 2005, 2007 and 2008 outbreak strains, driven by a human CMV promoter (hCMV promoter) with the bovine growth hormone 3 'end poly- adenylation signal (bGH poly A). pGX4201 was made by cloning the synthetic consensus envelope glycoprotein gene of Zaire Ebolavirus into pGXOOOl at the BamHI and Xhol sites. The ConEBOVGP#l (ConGPl) sequence was constructed by generating a consensus envelope glycoprotein sequence of Zaire Ebolavirus using the envelope glycoprotein sequences of the 1976, 1994, 1995, 1996, 2003, 2005, 2007 and 2008 outbreak strains.

Briefly, a consensus GP sequence was first generated based on six envelope sequences of the 1976, 1994, 1995, 1996, 2003 and 2005 outbreak strains. Then three non-consensus residues at the positions 377, 430 and 440 were weighted towards the 2003, 2005, 2007 and 2008 strains since they were the most recent and lethal outbreaks with published sequence data. The GenBank accession numbers for selected outbreak strain GP sequences are: Q05320, P87671, AAC57989, AEK25495, ABW34743, P87666, AER59718, AER59712,

ABW34742, AAL25818. Once the consensus GP1 sequence was obtained, an upstream Kozak sequence was added to the N-terminal. Furthermore, in order to have a higher level of expression, the codon usage of this gene was adapted to the codon bias of Homo sapiens genes. In addition, RNA optimization was also performed: regions of very high (>80%) or very low (<30%) GC content and the cis-acting sequence motifs such as internal TATA boxes, chi-sites and ribosomal entry sites were avoided. The synthesized ConGPl was digested with BamHI and Xhol, and cloned into the expression vector.

INO-4202 is a DNA vaccine formulated with a DNA plasmid expressing the envelope glycoprotein of Zaire Ebolavirus isolated from the 2014 outbreak in Guinea (GuineaGP), driven by a human CMV promoter (hCMV promoter) with the bovine growth hormone 3 'end poly-adenylation signal (bGH poly A). INO-4212 is a bivalent vaccine of INO-4201 and INO-4202.

Figure 64 depicts the vaccine formulation schedule, route and dose for each cohort. After the first injection, 15% or less of the patients was seriopositive. After the second injection, 50-100% of the patients were seriopositive. After the third injection 79-100% of the patients were seriopositive (Figure 68). Two representative patients with a moderate IFNy ELISpot, or a high IFNy ELISpot showed specific T-cell responses (Figure 69).

While other Ebola vaccines platforms, including NIAID VRC/GSK and

rVSV/ZEBOVGP, are currently in clinical trials the present bivalent and trivalent vaccines described herein have advantages not observed in the other vaccine platforms. For example, the bi- or tri-valent vaccines can be administered IM or ID, while the other vaccine platforms are only administered IM. Importantly, NIAID VRC/GSK and rVSV/ZEBOVGP show side effects including fever, fatigue, arthralgia, and lymphopenia while the bi- and tri-valent vaccines do not show any side effects. It should be noted however, that some of the side effects of rVSV/ZEBOVGP and ChAd3/MVAGP overlap with symptoms of Ebola. Further the bi- and tri-valent vaccines give antibody titers one to two orders of magnitude larger than rVSV/ZEBOVGP and ChAd3/MVAGP (Figure 65).

Subjects (n=15) were assigned to receive INO-4201 at a 2 mg DNA/dose given as two separate 1 mg (0.1 mL) ID (Mantoux) injections followed by EP with the CELLECTRA®-3P device. Subjects received a 3-dose series with immunizations at 0, 4 weeks, and 12 weeks (0- 4-12-week schedule). Antibodies specific for EBOV glycoprotein (GP) were measured from the sera of vaccinated subjects with a binding ELISA. Reciprocal endpoint titers above Day 0 are shown two weeks post each immunization (Figure 66). 100% of subjects vaccinated with INO-4201 seroconverted after 2 immunizations (Figure 66, Cohort 3 and Figure 68). Described herein is immune response data for the three EBOV DNA constructs described above: a consensus sequence of ZEBOV (1976-1996) (INO-4201; pGX4201), ZEBOV (2002-2008) (INO-9201 ; pGX6001), and a matched ZEBOV sequence from the 2014 Guinea outbreak. (INO-4202; pGX4202). Five vaccines were developed, monovalent vaccines which comprise only a single DNA construct, a bivalent vaccine formulation, INO- 4212, which comprises pGX4201 and pGX4202; and a trivalent formulation which comprises all three of pGX4201, pGX4202 and pGX6001. 69 Subjects from cohorts 1-5 were included in analysis of immune response by ELISA and 75 subjects from cohorts 1-5 were included in analysis of immune response by ELISpot (Figure 64). ELISA Titers by Cohort and Timepoint

Titers of anti-EBOVR were determined for each cohort at weeks 2, 6 and 14. By week 6, each cohort saw an increase in antibody titer above Day 0 (Figures 66-67). There was little to no reactivity for the first dose in each cohort. Dose 2 begins to drive seroconversion, with Cohorts 3 and 5 seeing the largest frequency. Dose three drives >90% serconversion in 4/5 cohorts. Cohorts 3 and 5 show 100% seroconversion at this time point (Figure 64).

Subject responses by peptide pool

Cohort responses were analyzed by peptide pool (Figures 69-77). To analyze ELISpot outliers, day 0 values for each pool and total EBOV responses were used to create an outlier threshold (Mean day 0 values +( 3x STDEV of day 0 values)). This threshold should encompass 99% of a normally distributed population. Any subject that displayed baseline values greater than the outlier threshold was removed and a responder criteria was generated with the remaining subjects.

ICS Analysis

47 subjects from all cohorts were included in the analysis, however cohort 5 is underrepresented (Figure 64). ICS analysis performed at baseline and week 14. A single EBOV peptide pool composed of Pools 1-4 is used for stimulation. Analysis of T cell activity in the form of IFNg or TNFa production from both CD4 and CD8 compartments suggests significant elevation of TNFa in both CD4 and CD8 compartments as well as elevation of TNFa and/or IFNg in Cohort 3 only (Wilcoxon matched paired analysis, two-tailed)

Immunology Summary

100% of Cohort 3 (ID) patients seroconverted after 2 doses. 92% of Cohort 5 (IM+IL12) patients seroconverted after 2 doses and 100% after 3 doses. Other cohorts showed 67% at best after 2 doses and ranged as high as 93% after 3 doses.

When analyzing all patients: the best response frequency were Cohorts 2 and 4 with

53% and 57% respectively. Cohort 3 showed 40% responders. Addition of IL-12 in Cohort 5 did not seem to influence response rates (47%). When analyzing patients with 8 outliers removed the response frequency were Cohorts 2 and 4 with 84.6% and 76.9% respectively. Cohort 3 showed 64.3% responders. Addition of IL-12 in Cohort 5 did not seem to influence response rates (53.3%).

Both CD4 and CD8 T cells showed high expression of TNFa and TNFa and/or IFNg in Cohort 3 (statistically significant to baseline, Wilcoxon matched pairs test, 2 tailed).

Immunization with INO-4201 was well tolerated in healthy volunteers with no Grade 3 or Grade 4 SAEs noted. INO-4201 induced robust Ebola GP-specific antibody (GMT 46,968) and resulted in 100% seroconversion, as gauged by binding ELISA, after only two doses of INO-4201. Administration of INO-4201 generated EBOV GP specific T cell responses as assessed by Interferon gamma (IFNy) ELISpot (295.3 SFU per 10 6 PBMCs) and significant increases in in the production of IFNy or TNFa in both the CD8+ T and CD4+ T cell compartments. Intradermal administration of INO-4201 using the Cellectra device is both well tolerated and immunogenic as assessed by both humoral and cellular EBOV GP-specific immunoassays. These results indicate that INO-4201 is a strong candidate for further clinical development of a prophylactic Ebola vaccine.

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.