Compositions Containing a Pathogenic Antigen and an Immune Stimulator

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/142,250, filed January 27, 2021, U.S. Provisional Application No. 63/142,229, filed January 27, 2021, U.S. Provisional Application No. 63/001,353, filed March 29, 2020, U.S. Provisional Application No. 62/994,375, filed March 25, 2020, and U.S. Provisional Application No. 62/985,192, filed March 4, 2020, each of which is incorporated herein by reference.

SEQUENCE LISTING

The Sequence Listing written in file 55179_SeqListing_ST25.txt is 96 kilobytes in size, was created February 16, 2021, and is hereby incorporated by reference.

INTRODUCTION

[1] Coronaviruses, which can infect and cause disease in both mammals and birds, typically lead to mild respiratory infections such as the common cold. However, some coronavirus strains can be lethal. In December 2019, a novel strain of coronavirus, initially coined 2019-nCoV by the World Health Organization (WHO) and later named SARS-CoV-2, was discovered after tracing the origins of a pneumonia outbreak in Wuhan, China. SARS- CoV-2 identifies as a novel strain of betacoronavirus and is capable of person-to-person spread (Zhu N et al. “Novel Coronavirus from Patients with Pneumonia in China, 2019” N Engl J Med. 2020 382(8):727-733). Previous international emergency betacoronavirus outbreaks include both the genetically similar Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) (Corman VM et al. “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.” Euro Surveill. 202025(3)). The World Health Organization determined the SARS-CoV-2 outbreak to be a global pandemic.

[2] Because new coronaviruses and other emerging pathogens represent a worldwide health threat, there is need for improved vaccines that can be readily adapted to newly identified infectious pathogens.

SUMMARY

[3] Described are vaccines directed against pathogens. The described vaccine compositions can be used to vaccinate against pathogen infection. Methods of vaccinating a subject against infection using the described vaccine compositions are also described. The pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoans, parasites, and fungi. A virus can be, but is not limited to: Hepatitis C virus, Hepatitis B virus, Hepatitis C virus, influenza virus, varicella virus, measles virus, mumps virus, poliovirus, rubella virus, rotavirus, human papillomavirus, enteroviruses, West Nile virus, Ebola virus, Zika virus, human immunodeficiency virus, lyssaviruses, rabies virus, yellow fever virus, Japanese encephalitis virus, hantavirus, and coronavirus. A bacterial pathogen can be, but is not limited to: Corynebacterium diphtherias, Haemophilus influenzae type b, Bordetella pertussis, Streptococcus pneumoniae, pneumococcus, Clostridium tetani, Neisseria meningitidis, Salmonella typhi, Vibrio cholerae, and Yersinia pestis. A parasitic pathogen can be, but is not limited to: Plasmodium spp., P. falciparum, P. vivax, P. ovale, P. malarias, Trypanosoma brucei , and Leishmania. The described vaccine compositions combine at least one antigenic polypeptide from the pathogen with at least one immune stimulator. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory cytokine may be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-a, IFN-b, IFN-γ, TGF- b, and C-X-C Motif Chemokine ligand 9 (CXCL9). The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1. In some embodiments, a nucleic acid encoding the genetic adjuvant further encodes a pathogenic antigen and the encoded product comprises a genetic adjuvant-pathogenic antigen fusion polypeptide. The pathogenic antigen can be an isolated polypeptide or a nucleic acid encoding the pathogenic antigen. The immune stimulator can be an isolated polypeptide or a nucleic acid encoding the immune stimulator.

[4] The described vaccines can be used to elicit an immune response against a pathogen, provide protective immunity against pathogen infection, prevent one or more symptoms associated with infection by a pathogen, prevent a disease caused by the pathogen, decrease severity or duration of disease associated with infection by a pathogen, or decrease severity or duration of one or more symptoms associated with infection by a pathogen.

[5] Described are nucleic acid-based vaccines. The described nucleic acid-based vaccine compositions can be used to vaccinate against infection. Methods of vaccinating a subject against infection using the described nucleic acid-based vaccine compositions are also described. [6] Described are vaccines directed against coronaviruses. The described coronavirus vaccine compositions can be used to vaccinate against coronavirus infection. In some embodiments, the described coronavirus vaccine compositions can be used to vaccinate against coronavirus infection or viral replication in a lung or a nasal cavity. Methods of vaccinating a subject against coronavirus infection using the described vaccine compositions are also described. The coronavirus can be, but is not limited to, a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS-CoV, a SARS-CoV-2, or a related betacoronavirus. The described coronavirus vaccine compositions combine at least one coronavirus antigenic polypeptide with at least one immune stimulator. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory cytokine may be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-a, IFN-b, IFN-γ, TGF- b, and C-X-C Motif Chemokine ligand 9 (CXCL9). The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1. In some embodiments, a nucleic acid encoding the genetic adjuvant further encodes a coronavirus antigenic polypeptide and the encoded product comprises a genetic adjuvant-coronavirus antigenic polypeptide fusion polypeptide. The coronavirus antigenic polypeptide can be an isolated polypeptide or a nucleic acid encoding the coronavirus antigenic polypeptide. The immune stimulator can be an isolated polypeptide or a nucleic acid encoding the immune stimulator.

[7] Described are hybrid protein/nucleic acid coronavirus vaccines. The described hybrid coronavirus vaccine compositions can be used to vaccinate against coronavirus infection. Methods of vaccinating a subject against coronavirus infection using the described vaccine compositions are also described. The coronavirus can be, but is not limited to, a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS- CoV, a SARS-CoV-2, or a related betacoronavirus. The described coronavirus vaccine compositions combine at least one coronavirus antigenic polypeptide with one or more nucleic acid sequences encoding at least one immune stimulator. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory cytokine may be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL- 15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-a, IFN-b, IFN-γ, TGF- b, and C-X-C Motif Chemokine ligand 9 (CXCL9). The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1. In some embodiments, the nucleic acid encoding the genetic adjuvant further encodes a coronavirus antigenic polypeptide and the encoded product comprises a genetic adjuvant-coronavirus antigenic polypeptide fusion polypeptide. The nucleic acid can be, but is not limited to, an RNA, an mRNA, a DNA, a plasmid, or an expression vector.

[8] Described are coronavirus nucleic acid-based vaccines. The described nucleic acid- based vaccine compositions can be used to vaccinate against coronavirus infection. Methods of vaccinating a subject against coronavirus infection using the described nucleic acid-based vaccine compositions are also described. The coronavirus can be, but is not limited to, a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS- CoV, a SARS-CoV-2, or a related betacoronavirus. The described coronavirus vaccine compositions combine at least one nucleic acid encoding a coronavirus antigenic polypeptide with one or more nucleic acid sequences encoding at least one immune stimulator. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half- BiTE. The immunostimulatory cytokine may be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-a, PTNG-b, IFN-γ, TGF-b, and C-X-C Motif Chemokine ligand 9 (CXCL9). The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1. In some embodiments, the nucleic acid encoding the genetic adjuvant further encodes a coronavirus antigenic polypeptide and the encoded product comprises a genetic adjuvant-coronavirus antigenic polypeptide fusion polypeptide. The nucleic acid can be, but is not limited to, an RNA, an mRNA, a DNA, a plasmid, or an expression vector.

[9] A coronavirus antigenic polypeptide can be, but is not limited to, betacoronavirus antigenic polypeptide, a lineage A betacoronavirus (subgenus Embecovirus ) antigenic polypeptide, a lineage B betacoronavirus (subgenus Sarbecovirus) antigenic polypeptide, a lineage C betacoronavirus (subgenus Merbecovirus ) antigenic polypeptide, a lineage D betacoronavirus (subgenus Nobecovirus) antigenic polypeptide, a SARS-CoV antigenic polypeptide, a MERS-CoV antigenic polypeptide, a SARS-CoV-2 antigenic polypeptide, or a related betacoronavirus antigenic polypeptide. A coronavirus antigenic polypeptide can be, but is not limited to, an S protein antigenic polypeptide. The S protein has been shown to induce long-term and potent neutralizing antibodies and/or protective immunity in various preclinical severe acute respiratory syndrome (SARS)-CoV studies and in both preclinical and clinical Middle East Respiratory Syndrome (MERS)-CoV studies. An S protein antigenic polypeptide comprises a coronavirus spike polypeptide (i.e., S glycoprotein or S protein) or an antigenic fragment thereof or a modified coronavirus spike polypeptide or an antigenic fragment thereof. A coronavirus spike protein or antigenic fragment thereof can be, but is not limited to, a SARS- CoV spike polypeptide or an antigenic fragment thereof, a MERS-CoV spike polypeptide or an antigenic fragment thereof, or a SARS-CoV-2 spike polypeptide or an antigenic fragment thereof. An S protein antigenic polypeptide can comprise an extracellular domain of an S protein or antigenic fragment thereof. In some embodiments, an S protein antigenic polypeptide can comprise a polypeptide corresponding to amino acids 1-1208, 14-1208, 21-1208, 14-305 (N-terminal domain), or 330-521 (receptor binding domain) of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33. An S protein antigenic polypeptide can comprise one or more additional components including, but not limited to, a secretion signal, a trimerization domain, or an Fc tag. A secretion signal (i.e., signal sequence or signal peptide) may be a native secretion signal (i.e., an S protein secretion signal) or a heterologous secretion signal. A trimerization domain can be a native trimerization domain or a heterologous trimerization domain. An S protein antigenic polypeptide can also contain one or more mutations that disrupt an internal furin- cleavage site and/or one or more mutations that stabilize the protein. A nucleic acid encoding the S protein antigenic polypeptide may be codon-optimized.

[10] In some embodiments, the immune stimulator comprises an IL-12. In some embodiments, the immune stimulator comprises a CXCL9. In some embodiments, the immune stimulator comprises a Flt3L. In some embodiments, the immune stimulator comprises an anti- CD3 half-BiTE. In some embodiments, the immune stimulator comprises an IL-12 and a CXCL9, a Flt3L, or an anti-CD3 half-BiTE. In some embodiments, the immune stimulator comprises an IL-12 and a CXCL9. In some embodiments, the immune stimulator comprises an IL-12 and a Flt3L. In some embodiments, the immune stimulator comprises an IL-12 and an anti-CD3 half-BiTE. In some embodiments, the immune stimulator comprises an IL-12, a CXCL9, and an anti-CD3 half-BiTE.

[11] In some embodiments, a nucleic acid encoding IL-12 comprises a nucleic acid encoding an IL-12 p35 subunit and an IL-12 p40 subunit separated by an internal ribosome entry site or 2 A peptide skipping motif. In some embodiments, a nucleic acid encoding IL-12 comprises SEQ ID NO: 3 or SEQ ID NO: 4, or a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, a nucleic acid encoding IL-12 comprises a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 5 or a nucleic acid encoding a polypeptide having at least 90% identify to the amino acid sequence of SEQ ID NO: 5.

[12] In some embodiments, a nucleic acid encoding IL-12 and a Flt3L comprises a nucleic acid sequence comprising SEQ ID NO: 23, a nucleic acid sequence having at least 90% identity to SEQ ID NO: 23, a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 24 (Flt3L), or nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 24.

[13] In some embodiments, a nucleic acid encoding IL-12 and CXCL9 comprises a nucleic acid sequence comprising SEQ ID NO: 21, a nucleic acid sequence having at least 90% identity to SEQ ID NO: 21, a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 22, a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 22, a nucleic acid sequence comprising SEQ ID NO: 6, a nucleic acid sequence having at least 90% identity to SEQ ID NO: 6, a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 7, or a nucleic acid sequence encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 7.

[14] In some embodiments, a nucleic acid encoding IL-12 and an anti-CD3 half-BiTE comprises a nucleic acid sequence comprising SEQ ID NO: 25, a nucleic acid sequence having at least 90% identity to SEQ ID NO: 25, a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 26, a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 26, a nucleic acid sequence comprising SEQ ID NO: 8, a nucleic acid sequence having at least 90% identity to SEQ ID NO: 8, a nucleic acid sequence encoding the amino acid sequence SEQ ID NO: 9, or a nucleic acid sequence encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 9.

[15] An isolated polypeptide(s) or a nucleic acid(s) encoding the polypeptide(s) can be administered by injection. The isolated polypeptide(s) and/or the nucleic acid(s) encoding the polypeptide(s) can be combined with one or more adjuvants, carriers, excipients, or a combination thereof. The isolated polypeptide(s) and/or the nucleic acid(s) encoding the polypeptide(s) can be formulated for injection and for inducing an immune response. The isolated polypeptide(s) or the nucleic acid(s) encoding the polypeptide(s) can be formulated for intramuscular injection, intradermal injection, intratumoral injection, or a combination thereof.

[16] A nucleic acid encoding a pathogenic antigen and/or an immune stimulator can be administered by electroporation or other non-viral means available in the art, including, but not limited to, direct injection (with or without electroporation), needless injection (with or without electroporation), microprojectile bombardment ( e.g ., gene gun), hydrodynamic injection, magneto-fection, sono-poration (e.g., ultrasound-mediated delivery), photo-poration, and hydro-poration. The nucleic acid sequence can be in a nanoparticle, a lipid nanoparticle, a liposome, a lipoplex, a polyplex, a lipopolyplex, or other non-viral particle or complex.

[17] A nucleic acid encoding a pathogenic antigen and/or an immune stimulator can be formulated for administration into the dermis (intradermal administration), skeletal muscle (intramuscular administration) and/or a tumor (intratumoral administration). In some embodiments, a nucleic acid encoding a pathogenic antigen and/or an immune stimulator can be formulated for administration into the dermis by intradermal electroporation (ID-EP), into muscle by intramuscular electroporation (IM-EP), into a tumor by intratumoral electroporation (IT-EP), or a combination thereof. In some embodiments, a nucleic acid encoding a pathogenic antigen and/or an immune stimulator can be formulated for administration into the dermis, muscle, and/or tumor by direct injection, needleless injection, microprojectile bombardment, hydrodynamic injection, magneto-fection, sono-poration, photo-poration, or hydro-poration. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[18] An isolated pathogenic antigen or immune stimulator peptide can be formulated for administration into the dermis (intradermal administration), skeletal muscle (intramuscular administration), and/or tumor (intratumoral administration). The isolated pathogenic antigen or immune stimulator peptide can be combined with an immune adjuvant prior to administration. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[19] Described are methods of eliciting an immune response against a coronavirus. The coronavirus can be, but is not limited to, a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS-CoV, a SARS-CoV-2, or a related betacoronavirus. In some embodiments, the methods comprise: administering to the subject an effective dose of a coronavirus antigenic polypeptide and an effective dose of an immune stimulator. In some embodiments, the methods comprise: (a) administering to the subject a first effective dose of a coronavirus antigenic polypeptide and (b) administering to the subject a first effective dose of an immune stimulator. In some embodiments, the method further comprises administering a second dose of the coronavirus antigenic polypeptide at a site distinct from administration of the first effective dose of the coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide and the immune stimulator are administered to the subject at the same time, within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 25 minutes, within about 30 minutes, within about 35 minutes, within about 40 minutes, within about 45 minutes, within about 50 minutes, within about 55 minutes, or within about 60 minutes of each other. The coronavirus antigenic polypeptide and the immune stimulator can be administered intradermally, intramuscularly, intratumorally, or a combination thereof.

[20] In some embodiments, the methods comprise: administering to the subject a first effective dose of a coronavirus antigenic polypeptide and a first effective dose of an immune stimulator by intradermal administration. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. In some embodiments, the first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator are administered by intradermal administration at the same site. In some embodiments, the first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator are administered by intradermal administration at the distinct sites. The coronavirus antigenic polypeptide and the immune stimulator can be separately administered to the dermis or they may be combined prior to intradermal administration. The intradermal administration site can be, but is not limited to, a shoulder, leg, or a buttocks of the subject.

[21] In some embodiments, the methods comprise: (a) administering to the subject a first effective dose of a coronavirus antigenic polypeptide and a first effective dose of an immune stimulator by intradermal administration, and (b) administering to the subject a second effective dose of the coronavirus antigenic polypeptide by intramuscular administration. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. In some embodiments, the first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator are administered by intradermal administration at the same site. The coronavirus antigenic polypeptide and the immune stimulator can be separately administered to the dermis or they may be combined prior to intradermal administration. The intradermal and intramuscular administration sites can be, but are not limited to, a shoulder, leg, or a buttocks of the subject. The intradermal and intramuscular administration sites can be in close proximity (<10 cm) or distant (>10 cm).

[22] In some embodiments, the methods comprise: administering to the subject a first effective dose of a coronavirus antigenic polypeptide and a first effective dose of an immune stimulator. In some embodiments, the methods further comprise administering a second effective dose of the coronavirus antigenic polypeptide and/or a second effective dose of the immune stimulator. The first effective dose of the coronavirus antigenic polypeptide can be administered to the dermis, muscle, or a tumor. The first effective dose of the immune stimulator can be administered to the dermis, muscle, or the tumor. The first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator can be administered to the same site or distinct sites. In some embodiments, the first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator are administered by intradermal, intramuscular, or intratumoral administration at the same site. Administration at different sites can be to the same tissue ( e.g ., distinct dermis sites, e.g, opposite limbs) or different tissues (e.g, dermis and muscle or tumor). In some embodiments, the second effective dose of the coronavirus antigenic polypeptide is administered with the first effective dose of the immune stimulator. In some embodiments, the second effective dose of the immune stimulator is administered with the first effective dose of the coronavirus antigenic polypeptide. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. For administration to the same site, the coronavirus antigenic polypeptide and the immune stimulator can be administered separately, or they may be combined prior to administration.

[23] Eliciting an immune response against a coronavirus can be used to:

(a) elicit a cellular immune response to the coronavirus;

(b) elicit a humoral immune response to the coronavirus;

(c) elicit both cellular and humoral immune responses to the coronavirus;

(d) enhance the proliferation of cytotoxic T lymphocytes;

(e) elicit the generation of anti-coronavirus antibodies;

(f) elicit the generation of neutralizing anti-coronavirus antibodies;

(g) elicit the generation of antibodies protective against coronavirus infection; (h) reduce the likelihood of coronavirus infection or lessen the severity of coronavirus infection;

(i) reduce the likelihood of developing COVID-19 or lessen the severity or duration of COVID-19;

(j) vaccinate a patient against coronavirus;

(k) elicit protective immunity against COVID-19 or severe COVID-19;

(l) prevent at least one symptom of COVID-19 disease, and/or

(m) prevent symptomatic COVID-19 disease.

[24] In some embodiments, methods of eliciting an immune response against a coronavirus comprise: administering to the subject a first effective dose of a nucleic acid encoding a coronavirus antigenic polypeptide and a first effective dose of a nucleic acid encoding an immune stimulator by intradermal administration. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. In some embodiments, the first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the nucleic acid encoding the immune stimulator are administered by intradermal administration at the same site. In some embodiments, the first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the nucleic acid encoding the immune stimulator are administered by intradermal administration at distinct sites. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immune stimulator can be separately administered to the dermis or they may be combined prior to intradermal administration. In some embodiments, intradermal administration comprises intradermal administration at a site in a shoulder of the subject. The intradermal administration site can be, but is not limited to, a shoulder, leg, or a buttocks of the subject.

[25] In some embodiments, methods of eliciting an immune response against a coronavirus comprise: (a) administering to the subject a first effective dose of a nucleic acid encoding a coronavirus antigenic polypeptide and a first effective dose of a nucleic acid encoding an immune stimulator by intradermal administration; (b) administering to the subject a second effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide by intramuscular administration. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. In some embodiments, the first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the nucleic acid encoding the immune stimulator are administered by intradermal administration at the same site. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immune stimulator can be separately administered to the dermis or they may be combined prior to intradermal administration. The intradermal and intramuscular administration sites can be, but are not limited to, a shoulder, leg, or a buttocks of the subject. The intradermal and intramuscular administration sites can be in close proximity (<10 cm) or distant (>10 cm). Intradermal administration can comprise intradermal electroporation (ID-EP). Intramuscular administration can comprise intramuscular electroporation (IM-EP). Intratumoral administration can comprise intratumoral electroporation (IM-EP).

[26] In some embodiments, the methods of eliciting an immune response against a coronavirus comprise: administering to the subject a first effective dose of a nucleic acid encoding a coronavirus antigenic polypeptide and a first effective dose of a nucleic acid encoding an immune stimulator. In some embodiments, the methods further comprise administering a second effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and/or a second effective dose of the nucleic acid encoding the immune stimulator. The first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide can be administered to the dermis, muscle, or a tumor. The first effective dose of the nucleic acid encoding the immune stimulator can be administered to the dermis, muscle, or the tumor. The first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the nucleic acid encoding the immune stimulator can be administered to the same site or distinct sites. In some embodiments, the first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the nucleic acid encoding the immune stimulator are administered by intradermal, intramuscular, or intratumoral administration at the same site. Administration at different sites can be to the same tissue ( e.g ., distinct dermis sites, e.g, opposite limbs) or different tissues (e.g, dermis and muscle or tumor). In some embodiments, the second effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide is administered with the first effective dose of the nucleic acid encoding the immune stimulator. In some embodiments, the second effective dose of the nucleic acid encoding the immune stimulator is administered with the first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immune stimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. For administration to the same site, the nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immune stimulator can be administered separately, or they may be combined prior to administration. Intradermal administration can comprise intradermal electroporation (ID-EP). Intramuscular administration can comprise intramuscular electroporation (IM-EP). Intratumoral administration can comprise intratumoral electroporation (IM-EP).

[27] Administration can comprise prime administration (single administration), or a prime administration and one or more boost administrations (two or more administrations). Prime and boost administrations can be performed at the same site or different sites in the subject, e.g ., in the same limb or difference limbs. In some embodiments, the methods comprise at least two rounds of administration of at least one coronavirus antigenic polypeptide and at least one immune stimulator. In some embodiments, administration of at least one coronavirus antigenic polypeptide and administration of at least one immune stimulator are both performed each round. The second round of administration (boost) can be performed 2 weeks to 12 months after the first round of administration (prime). In some embodiments, the first round (prime) is administered on day 1 and the second round (boost) is administered 14-63 days after the first round of administration. In some embodiments, the first and subsequence boost administrations can be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequence boost administrations can be performed at intervals of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. In some embodiments, an additional boost is administered about 1-5 years after the prime or boost administration.

[28] In some embodiments, the methods comprise a first (prime) administration of the combination of a coronavirus antigenic polypeptide and an immune stimulator. In some embodiments, the methods further comprise at least one boost administration of the combination of a coronavirus antigenic polypeptide and an immune stimulator, wherein the boost is administered after the prime administration (prime plus boost). The boost administration can be 2, 3, 4, 5, 6, 7, 8, or more weeks (each ±4 days) after the prime administration. The coronavirus antigenic polypeptide and/or an immune stimulator can be one or more nucleic acids encoding the coronavirus antigenic polypeptide and/or an immune stimulator.

[29] In some embodiments, the methods comprise a first (prime) intradermal administration of a coronavirus antigenic polypeptide and an immune stimulator. In some embodiments, the methods further comprise at least one boost intradermal administration of the coronavirus antigenic polypeptide and the immune stimulator, wherein the boost administration is administered after the prime administration. The boost administration can be

1, 2, 3, 4, 5, 6, 7, 8 (each ±4 days), or more weeks after the prime administration.

[30] In some embodiments, the methods comprise a first (prime) intradermal administration of a nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immune stimulator. In some embodiments, the methods further comprise at least one boost intradermal administration of a nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immune stimulator, wherein the boost administration is administered after the prime administration. The boost administration can be

2, 3, 4, 5, 6, 7, 8, or more weeks (each ±4 days) after the prime administration.

[31] In some embodiments, the methods comprise a first (prime) intradermal administration of a coronavirus antigenic polypeptide and an immune stimulator and intramuscular administration of the coronavirus antigenic polypeptide. In some embodiments, the methods further comprise at least one boost intradermal administration of the coronavirus antigenic polypeptide and the immune stimulator and intramuscular administration of the coronavirus antigenic polypeptide, wherein the boost administration is administered after the prime administration. The boost administration can be 2, 3, 4, 5, 6, 7, 8, or more weeks (each ±4 days) after the prime administration.

[32] In some embodiments, the methods comprise a first (prime) intradermal administration of nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator and intramuscular administration of a nucleic acid encoding the coronavirus antigenic polypeptide. In some embodiments, the methods further comprise at least one boost intradermal administration of nucleic acids encoding the coronavirus antigenic polypeptide and the immune stimulator and intramuscular administration of the nucleic acid encoding the coronavirus antigenic polypeptide, wherein the boost administration is administered after the prime administration. The boost administration can be 2, 3, 4, 5, 6, 7, 8, or more weeks (each ±4 days) after the prime administration.

[33] In some embodiments, the methods comprise a first (prime) intratumoral administration of a nucleic acid encoding an immune stimulator and optionally a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the methods further comprise at least one boost intratumoral administration of the nucleic acid encoding the immune stimulator and optionally the nucleic acid encoding the coronavirus antigenic polypeptide, wherein the boost administration is administered after the prime administration. The boost administration can be 2, 3, 4, 5, 6, 7, 8, or more weeks (each ±4 days) after the prime administration. [34] For intradermal administration, a coronavirus antigenic polypeptide and an immune stimulator can be administered at the same site or at distinct sites. For administration at distinct sites, intradermal administration of the coronavirus antigenic polypeptide and intradermal administration of the immune stimulator can be near each other (<10 cm), or distant from each either other (>10 cm). Distant sites include, but are not limited to, contralateral sites (e.g, in opposite shoulders or thighs).

[35] The sites of intradermal administration and intramuscular administration can be near each other (<10 cm), or distant from each either other (>10 cm). Distant sites include, but are not limited to, contralateral sites. In some embodiments, the intradermal site is 1-5 cm or 2-3 cm from the intramuscular site. In some embodiments, the intradermal site is more than 10 cm from the intramuscular site. In some embodiments, the intradermal site is at a contralateral site from the intramuscular site.

[36] Intradermal administration can be performed before, concurrently with, or after intramuscular or intratumoral administration. In some embodiments, intradermal administration is performed within 60 minutes, 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes of intramuscular or intratumoral administration. In some embodiments, intradermal administration is performed prior to intramuscular or intratumoral administration. In some embodiments, intradermal administration is performed after intramuscular or intratumoral administration. In some embodiments, intradermal administration is performed within 5-15 minutes, before or after, of intramuscular or intratumoral administration.

[37] In some embodiments, the methods comprise as single round of ID-EP administration of nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator. In some embodiments, the methods comprise at least two rounds of ID-EP administration of nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator. The second round of ID-EP administration (boost) can be performed two weeks to 12 months after the first round of ID-EP administration (prime). In some embodiments, the second round of ID-EP administration is performed 14-63 days after the first round of ID-EP administration. In some embodiments, the first and subsequence boost administrations can be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequence boost administrations can be performed at intervals of about 2 about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. In some embodiments, an additional boost is administered about 1-5 years after the prime or boost administration. [38] The nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator can be administered by ID-EP at the same site or at distinct sites. For ID-EP at distinct sites, the sites can be near each other (<10 cm), or distant from each either other (>10 cm). Distant sites include, but are not limited to, contralateral sites ( e.g ., opposite shoulders or thighs). In some embodiments, the distinct sites are 1-5 cm or 2-3 cm apart.

[39] The ID-EP administration of a nucleic acid encoding a coronavirus antigenic polypeptide and ID-EP administration of a nucleic acid encoding an immune stimulator can occur concurrently or sequentially. For sequence administration, the ID-EP administrations can occur within 60 minutes, 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes of each other. In some embodiments, ID-EP administration of a nucleic acid encoding a coronavirus antigenic polypeptide is performed prior to ID-EP administration of a nucleic acid encoding an immune stimulator. In some embodiments, ID-EP administration of a nucleic acid encoding an immune stimulator is performed prior to ID-EP administration of a nucleic acid encoding a coronavirus antigenic polypeptide.

[40] In some embodiments, the methods comprise a single round of ID-EP administration of nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator and IM-ED administration of a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the methods comprise at least two rounds of ID-EP administration of nucleic acids encoding a coronavirus antigenic polypeptide and an immune stimulator and IM-ED administration of a nucleic acid encoding a coronavirus antigenic polypeptide. ID-EP and IM- EP administration are both performed each round. The second round of ID-EP and IM-EP administration (boost) can be performed two weeks to 12 months after the first round of ID-EP and IM-EP administration (prime). In some embodiments, the second round of ID-EP and IM- EP administration is performed 14-63 days after the first round of ID-EP and IM-EP administration. In some embodiments, the first and subsequence boost administrations can be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequence boost administrations can be performed at intervals of about 2 about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. In some embodiments, an additional boost is administered about 1-5 years after the prime or boost administration.

[41] The ID-EP and IM-EP sites can be near each other (<10 cm), or distant from each either other (>10 cm). Distant sites include, but are not limited to, contralateral sites. In some embodiments, the ID-EP site is 1-5 cm or 2-3 cm from the IM-EP site. In some embodiments, the ID-EP site is more than 10 cm from the IM-EP site. In some embodiments, the ID-EP site is at a contralateral site from the IM-EP. The IM-EP site can be, but is not limited to, the deltoid muscle. In some embodiments the ID-EP site is in one shoulder and the IM-EP site is in the deltoid muscle of the opposite shoulder.

[42] ID-EP administration can be performed before, concurrently with, or after the IM-EP administration. In some embodiments, ID-EP administration is performed within 60 minutes, 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes of IM-EP administration. In some embodiments, ID-EP administration is performed prior to IM-EP administration. In some embodiments, ID-EP administration is performed after IM-EP administration. In some embodiments, ID-EP administration is performed within 5-15 minutes, before or after, of IM-EP administration.

[43] In some embodiments, the methods comprise at least one round (cycle) of IT-EP administration of a nucleic acid encoding an immune stimulator (IT-EP IL-12 therapy) and IT- EP, ID-EP, or IM-EP administration of a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the methods comprise at least two rounds (cycles) of IT- EP administration of the nucleic acid encoding the immune stimulator and IT-EP, ID-EP, or IM-EP administration of the nucleic acid encoding the coronavirus antigenic polypeptide, wherein the second round is administered after the first round.

[44] IT-EP administration can be performed before, concurrently with, or after the ID-EP or IM-EP administration. In some embodiments, IT-EP administration is performed within 60 minutes, 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes of ID-EP or IM-EP administration. In some embodiments, IT-EP administration is performed prior to ID-EP or IM-EP administration. In some embodiments, IT-EP administration is performed after ID-EP or IM-EP administration. In some embodiments, IT-EP administration is performed within 5-15 minutes, before or after, of IT- EP or IM-EP administration.

[45] In some embodiments, a cycle of IT-EP IL-12 therapy comprises IT-EP administration of a nucleic acid encoding an immune stimulator on day 1 (±2 days); days 1 (±2 days) and 5 (±2 days); days 1 (±2 days) and 8 (±2 days); or days 1(±2 days), 5 (±2 days), and 8 (±2 days) of a 3 week cycle. In some embodiments, IL-12 is administered on days 1 (±2 days), 5 (±2 days), and 8 (±2 days) of each cycle and the coronavirus antigenic polypeptide is administered on day 1 (±2 days); day 5 (±2 days); day 8 (±2 days); days 1 (±2 days) and 5 (±2 days); days 1 (±2 days) and 8 (±2 days); days 1 (±2 days), 5 (±2 days), and 8 (±2 days); or days 5 (±2 days) and 8 (±2 days) of each cycle. In some embodiments, IL-12 is administered on days 1 (±2 days) and 8 (±2 days) of each cycle and the coronavirus antigenic polypeptide is administered on day 1 (±2 days), day 8 (±2 days), or days 1 (±2 days) and 8 (±2 days) of each cycle. In some embodiments, IL-12 is administered on days 1 (±2 days) and 5 (±2 days) of each cycle and the coronavirus antigenic polypeptide is administered on day 1 (±2 days), day 5 (±2 days), or days 1 (±2 days) and 5 (±2 days) of each cycle. In some embodiments, IL-12 is administered on days 1 (±2 days), 5 (±2 days), and 8 (±2 days) of each cycle and the coronavirus antigenic polypeptide is administered on day 1 (±2 days) or each cycle.

[46] In some embodiments, the coronavirus antigenic polypeptide and the immunostimulatory cytokine are injected into a tumor, such as a cancerous tumor, in a subject. In some embodiments, a nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immunostimulatory cytokine are injected into the tumor. In some embodiments, the nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulatory cytokine are administered to a tumor by intratumoral electroporation. The coronavirus antigenic polypeptide can be, but is not limited to, a SARS- CoV-2 S protein or antigenic fragment thereof. The immunostimulatory cytokine can be, but is not limited to, an interleukin, an IL-12, a CXCL9, an anti-CD3 half-BiTE, a genetic adjuvant, or a combination thereof.

[47] In some embodiments, the described vaccines are administered to a cancer patient. In some embodiments, coronavirus vaccination is combined with tumor treatment, by administering to a tumor in a subject, a nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immunostimulatory cytokine by intratumoral electroporation. The coronavirus antigenic polypeptide can be, but is not limited to, a SARS- CoV-2 S protein or antigenic fragment thereof. The immunostimulatory cytokine can be, but is not limited to, an interleukin, an IL-12, a CXCL9, an anti-CD3 half-BiTE, a genetic adjuvant, or a combination thereof. The described vaccines, which combine intratumoral electroporation (IT-EP) of a coronavirus antigenic polypeptide and IT-EP of an immunostimulatory cytokine, such as IL-12, provide for vaccination of these patients against coronavirus and anti-cancer therapy. Immune compromised patients, such as those with cancer, benefit from a vaccine that not only drives an anti-tumor response, but also creates lasting immunity to SARS-CoV-2 by boosting their immune systems to mount a defense against viral infection, such as COVID-19.

[48] In some embodiments, the electroporation is reversible electroporation and is administered using any suitable electroporation device. Such electroporation devices include, but are not limited to, Cliniporator (IGEA), Cliniporator Vitae (IGEA), Apollo (OncoSec), GenPulse (OncoSec), MedPulse (OncoSec), Cellectra (Inovio), and Agilepulse (BTX Harvard Apparatus). [49] Any of the methods disclosed herein for vaccinating against coronavirus can be modified to vaccinate against another pathogen by substituting an antigenic polypeptide from the pathogen of interest for the coronavirus polypeptide. Similarly, any of the coronavirus vaccine compositions described herein can be readily modified to prepare a vaccine composition against a different pathogen by substituting an antigenic polypeptide form the pathogen of interest for the coronavirus polypeptide.

[50] Described are expression vectors encoding immunostimulatory cytokines, pathogenic antigens, or an immunostimulatory cytokine and a pathogenic antigen for use in treatment of cancer. Methods of using the described expression vectors to treat tumors, including cancers and metastatic cancers, are also described. The described expression vectors, when delivered to a subject by intratumoral electroporation (IT-EP), optionally in combination with intradermal electroporation (ID-EP) or intramuscular electroporation (IM-EP), result in expression of the encoded proteins, leading to T cell recruitment and anti-tumor activity. The expression vectors and methods can be used to regress the tumor (i.e., decrease tumor volume), regress one or more tumors in the subject, soften the tumor, increase survival, increase tumor- free survival, and/or increase immune response to the tumor. In some embodiments, the described methods also result in abscopal effects, i.e., regression of one or more untreated tumors. In some embodiments, regression includes debulking of a solid tumor.

[51] Described are methods of treating a cancer comprising administering to a subject, by intratumoral electroporation (IT-EP), a composition comprising a therapeutically effective amount of an expression vector encoding an immunostimulatory cytokine and a therapeutically effective amount of an expression vector encoding a pathogenic antigen. The expression vector encoding the immunostimulatory cytokine and the expression vector encoding the pathogenic antigen can be present in the same plasmid of vector or different plasmids or vectors. For a plasmid or vector encoding both the immunostimulatory cytokine and the pathogenic antigen, the coding sequences for the immunostimulatory cytokine and the pathogenic antigen can be operably linked to a single promoter and linked by an IRES or 2A translation modification element, as in a multi cistronic plasmid or vector, or the coding sequences for the immunostimulatory cytokine and the pathogenic antigen can be operably linked to separate promoters. The composition is injected into a tumor, tumor microenvironment, and/or tumor margin tissue and electroporation therapy is applied to the tumor, tumor microenvironment, and/or tumor margin tissue. The electroporation therapy may be applied by any suitable electroporation system known in the art. In some embodiments, the electroporation is at a field strength of about 60 V/cm to about 1500 V/cm, and a duration of about 10 microseconds to about 20 milliseconds. In some embodiments, the electroporation incorporates Electrochemical Impedance Spectroscopy (EIS). The subject can be a mammal. The mammal can be, but is not limited to, a human, canine, feline, or equine.

[52] Described are methods of treating cancer comprising administering to a subject, by intratumoral electroporation (IT-EP), a composition comprising a therapeutically effective amount an expression vector encoding an immunostimulatory cytokine and administering to the subject, by intradermal electroporation (ID-EP) or intramuscular electroporation (IM-EP), an expression vector encoding a pathogenic antigen. A composition containing the expression vector encoding the immunostimulatory cytokine is injected into a tumor, tumor microenvironment, and/or tumor margin tissue and electroporation therapy is applied to the tumor, tumor microenvironment, and/or tumor margin tissue. A composition containing the expression vector encoding the pathogenic antigen is injected into the dermis or skeletal muscle and electroporation therapy is applied to the dermis or skeletal muscle at the site of the injection. The electroporation therapy may be applied by any suitable electroporation system known in the art. In some embodiments, the electroporation is at a field strength of about 60 V/cm to about 1500 V/cm, and a duration of about 10 microseconds to about 20 milliseconds. In some embodiments, the electroporation incorporates Electrochemical Impedance Spectroscopy (EIS). The subject can be a mammal. The mammal can be, but is not limited to, a human, canine, feline, or equine.

[53] The immunostimulatory cytokine can be, but is not limited to, IL-12 or IL-15. In some embodiments, the immunostimulatory cytokine comprises IL-12. IL-12 is a heterodimeric cytokine having both IL-12A (p35) and IL-12B (p40) subunits. The encoded IL-12 can comprise a fusion construct encoding an IL-12 p35-IL-12 p40 fusion protein (IL12 p70). In some embodiments, the IL-12 p35 and p40 coding sequences are expressed from a multi cistronic expression vector from a single promoter and linked by an IRES or 2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, a multi cistronic expression vector comprises an IL12 p35 coding sequence, and IL-12 p40 coding sequence, and pathogenic antigen coding sequence each separated by an IRES or 2A element. In some embodiments, the 2A element is a P2A element. An expression vector encoding the immunostimulatory cytokine can be delivered prior to, subsequent to, or concurrent with one or more of the pathogenic antigens.

[54] The pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoans, parasites, and fungi. A virus can be, but is not limited to: Hepatitis C virus, Hepatitis B virus, Hepatitis C virus, influenza virus, varicella virus, measles virus, mumps virus, poliovirus, rubella virus, rotavirus, human papillomavirus, enteroviruses, West Nile virus, Ebola virus, Zika virus, human immunodeficiency virus, lyssaviruses, rabies virus, yellow fever virus, Japanese encephalitis virus, hantavirus, and coronavirus. In some embodiments, the viral antigen is a coronavirus antigen. The coronavirus can be, but is not limited to, a betacoronavirus antigen, a lineage A betacoronavirus (subgenus Embecovirus ) antigen, a lineage B betacoronavirus (subgenus Sarbecovirus) antigen, a lineage C betacoronavirus (subgenus Merbecovirus ) antigen, a lineage D betacoronavirus (subgenus Nobecovirus) antigen, a SARS-CoV antigen, a MERS-CoV antigen, or a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen is a spike protein antigen. A bacterial pathogen can be, but is not limited to: Corynebacterium diphtheriae, Haemophilus influenzae type b, Bordetella pertussis, Streptococcus pneumoniae, pneumococcus, Clostridium tetani, Neisseria meningitidis, Salmonella typhi, Vibrio cholerae, and Yersinia pestis. A parasitic pathogen can be, but is not limited to: Plasmodium spp., P. falciparum, P. vivax, P. ovale, P. malariae , Trypanosoma brucei , and Leishmania. In some embodiments, the pathogenic antigen is a viral antigen. The pathogenic antigen can be any antigen that induces an immune response against the pathogen.

[55] In some embodiments, the pathogenic antigen is a SARS-CoV-2 spike protein antigen and the immunostimulatory cytokine is IL-12.

[56] In some embodiments, the methods further comprise administration of one or more additional therapies. The one or more additional therapies can be, but are not limited to, immune checkpoint therapy. Immune checkpoint therapy can be, but is not limited to, administration of one or more immune checkpoint inhibitors.

[57] In some embodiments, the methods comprise: administering to the subject a first effective dose of an expression vector encoding a pathogenic antigen and a first effective dose of an expression vector encoding an immunostimulatory cytokine by intratumoral electroporation. The pathogenic antigen can be a viral antigen. The viral antigen can be a coronavirus antigenic polypeptide. The coronavirus antigenic polypeptide can be, but is not limited to, a coronavirus spike protein or an antigenic fragment thereof. The immunostimulatory cytokine can be, but is not limited to, IL-12 or a combination of IL-12 and a second immune stimulator. In some embodiments, the immunostimulatory cytokine ( e.g. , IL- 12) and the pathogenic antigen (e.g, coronavirus S protein polypeptide) are encoded on the multi cistronic plasmid.

[58] Described are methods of treating a tumor in a subject comprising: administering at least one treatment cycle to the subject, the cycle comprising: administering to the tumor, by IT-EP, a composition comprising a therapeutically effective amount of one or more of the described expression vectors encoding pathogenic antigen and IL-12. In some embodiments, the cycle is a four, five, or six week cycle. In some embodiments, the cycle is a three week cycle. In some embodiments, the cycle is a four week cycle. In some embodiments, the cycle is a six week cycle. The composition can be administered by IT-EP on 1, 2, 3, 4, 5, or 6 days of a cycle. In some embodiments, the composition is administered by IT-EP on day 1 of each cycle. In some embodiments, the composition administered by IT-EP on days 1 and 5±2 of each cycle. In some embodiments, the composition is administered by IT-EP on days 1 and 8±2 of each cycle. In some embodiments, the composition is administered by IT-EP on days 1, 5±2, and 8±2 of each cycle. The cycles can be repeated as often as is necessary to treat the subject. In some embodiments, a cycle further comprises administration of an additional therapeutic. The additional therapeutic can be, but is not limited to, an immune checkpoint therapy. In some embodiments, the immune checkpoint therapy is administered to the subject on day 1, 2, or 3 of the cycle.

[59] Described are methods of treating a tumor in a subject comprising: administering at least one treatment cycle to the subject, the cycle comprising: administering to the tumor, by IT-EP, a composition comprising a therapeutically effective amount of an expression vector encoding IL-12 (IT-EP IL-12 treatment) and administering to the subject, by ID-EP or IM-EP, a composition comprising a therapeutically effective amount of an expression vector encoding a pathogenic antigen (ID-EP PA treatment or IM-EP PA treatment). In some embodiments, the cycle is a four, five, or six week cycle. In some embodiments, the cycle is a three week cycle. In some embodiments, the cycle is a four week cycle. In some embodiments, the cycle is a six week cycle. IT-EP IL-12 treatment can be administered on 1, 2, 3, 4, 5, or 6 days of a cycle. In some embodiments, IT-EP IL-12 treatment is administered on day 1 of each cycle. In some embodiments, IT-EP IL-12 treatment is administered on days 1 and 5±2 of each cycle. In some embodiments, IT-EP IL-12 treatment is administered on days 1 and 8±2 of each cycle. In some embodiments, IT-EP IL-12 treatment is administered on days 1 and 15±2 of each cycle. In some embodiments, IT-EP IL-12 treatment is administered on days 1, 5±2, and 8±2 of each cycle. ID-EP PA treatment or IM-EP PA treatment can be administered on 1, 2, 3, 4, 5, or 6 days of a cycle. In some embodiments, ID-EP PA treatment or IM-EP PA treatment is administered on day 1 of each cycle. In some embodiments, ID-EP PA treatment or IM-EP PA treatment is administered on days 1 and 5±2 of each cycle. In some embodiments, ID-EP PA treatment or IM-EP PA treatment is administered on days 1 and 8±2 of each cycle. In some embodiments, ID-EP PA treatment or IM-EP PA treatment is administered on days 1 and 15±2 of each cycle. In some embodiments, ID-EP PA treatment or IM-EP PA treatment is administered on days 1, 5±2, and 8±2 of each cycle. The cycles can be repeated as often as is necessary to treat the subject. In some embodiments, a cycle further comprises administration of an additional therapeutic. The additional therapeutic can be, but is not limited to, an immune checkpoint therapy. In some embodiments, the immune checkpoint therapy is administered to the subject on day 1, 2, or 3 of the cycle.

[60] In some embodiments, a subject is treated with one of more cycles of IT-EP IL-12 plus PA treatment, IT-EP IL12 treatment plus ID-EP PA treatment, or IT-EP IL-12 treatment plus IM-EP PA treatment. Any of the above cycles can be repeated in subsequent cycles. The subsequent cycles can be consecutive cycles or alternating cycles. Alternating cycles can have one or more intervening cycles of no therapy of alternative therapy ( e.g ., immune checkpoint therapy). For example, any of the described expression vectors can be administered on one or more of days 1, 5±2, 8±2 and 15±2 of alternating cycles (e.g., cycles 1, 3, 5, etc. as needed) and an alternative therapy can be administered, e.g, on day 1±2, 2±2, or 3±2, of consecutive cycles (e.g, cycles 1, 2, 3, 4, 5, etc. as needed).

[61] The cycles of immunostimulatory cytokine plus pathogenic antigen treatment can be administered with or without immune checkpoint inhibitor therapy. In other words, a subject can be administered a composition comprising a therapeutically effective amount of one or more of the described expression vectors encoding an immunostimulatory cytokine (e.g, IL- 12) and/or a pathogenic and administered immune checkpoint inhibitor therapy on odd numbered cycles (cycles 1, 3, etc.); and administered immune checkpoint inhibitor therapy on even numbered cycles (cycles 2, 4, etc.).

[62] The expression vectors and methods can be used to treat a subject having an advanced, metastatic, treatment refractory tumor. A treatment refractory tumor can be, but is not limited to, an immune checkpoint inhibitor refractory tumor, a hormone refractory tumor, a radiation refractory tumor, or a chemotherapy refractory tumor. In some embodiments, the subject has failed to respond to at least one course of immune checkpoint inhibitor therapy. In some embodiments, the subject is progressing on or has progressed on one or more anti-cancer therapies, such as, but not limited to, checkpoint inhibitor therapy.

[63] The expression vectors and methods can be used to treat subjects having tumors predicted to be refractory to or not respond to one or more anti-cancer therapies. In some embodiments, the subject has low tumor infiltrating lymphocytes, low partially cytotoxic lymphocytes, or exhausted T cells. In some embodiments, the subject has advanced on one or more prior cancer therapies. [64] The expression vectors and methods can be used to treat a subject that has previously been infected with the pathogen, a subject that has previously had a disease or condition associated with infection by the pathogen, a subject that has previously been exposed to the pathogen, or a subject that has previously been vaccinated against the pathogen. In some embodiments, the pathogen is SARS-CoV-2 and the disease associated with infection by the pathogen is COVID-19.

BRIEF DESCRIPTION OF THE FIGURES

[65] FIG. 1A-B. (A) Model of coronavirus, showing membrane display of coronavirus spike glycoprotein. (B) Illustration of the coronavirus spike glycoprotein domains (SS = signal sequence (secretion signal), Extracellular domain, TM = transmembrane domain, ICD - intracellular domain).

[66] FIG. 2A-C. (A) Schematic representation of nucleic acids encoding IL-12 and a fusion protein of Flt3L and an antigen. (B) Flow cytometry graphs illustrating the percentage of OVA-specific CD8+ T cells following administration of PIIM-OVA (IL-12 p35-P2A-IL- 12 p40-Flt3L-OVA ^{241 270aa} (SIINFEKL)). (C) Graph illustrating percent CD8+ cells (n=5) following administration of PIIM-OVA.

[67] FIG. 3. Graph illustrating differential systemic expression of a secreted human model protein 7 days post-EP.

[68] FIG. 4. Plasmid map of SARS-CoV-2 S-2P_defurin_F3CH2S

[69] FIG. 5. Plasmid map of pUMVC3.SARS-CoV-2-2_S-2P.foldon.tagoff.

[70] FIG. 6. Schematic representation of nucleic acids constructs encoding coronavirus antigenic polypeptides ( e.g ., CoV antigen), IL-12, CXCL9, anti-CD3 half-BiTE, Flt3L and combinations thereof (Tl, T2 = translation modification elements, P2A = P2A element, IRES = internal ribosome entry site, ECD = extracellular domain, IL-12 p35 = 35 kDa subunit of IL- 12, IL-12 p40 = 40 kDa subunit of IL-12).

[71] FIG. 7. Schematic representation of nucleic acids constructs encoding coronavirus antigenic polypeptides (trimer = trimerization domain, SS = secretion signal, · = cleavage site mutation, * = stabilizing mutation).

[72] FIG. 8. Schematic representation of nucleic acids constructs encoding coronavirus antigenic polypeptides (ECD = extracellular domain, trimer = trimerization domain, SS = secretion signal). [73] FIG. 9. Graph illustrating anti-S protein IgGl antibody levels in peripheral blood in Balb/C (A) or C57bl/6 (B) mice following IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 vs. control mice.

[74] FIG. 10. Graph illustrating anti-S protein IgG2a antibody levels in peripheral blood in Balb/C (A) or C57bl/6 (B) mice following IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 vs. control mice.

[75] FIG. 11. Graph illustrating presence of anti-S protein neutralization antibody levels in peripheral blood in Balb/C (A) or C57bl/6 (B) mice following IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 vs. control mice.

[76] FIG. 12A. Graph illustrating anti-S protein IgGl (upper panel) and IgG2a (lower panel) serial dilution analyses at day 31 in mice vaccinated with various combinations of spike plasmid and IL-12 (key shown in FIG. 12B).

[77] FIG. 12B. Graph illustrating anti-S protein neutralizing antibody analyses at day 31 in mice vaccinated with various combinations of spike plasmid and IL-12.

[78] FIG. 13 A. Graph illustrating anti-S protein IgGl (upper panel) and IgG2a (lower panel) serial dilution analyses at day 42 in mice vaccinated with various combinations of spike plasmid and IL-12 (key shown in FIG. 13B).

[79] FIG. 13B. Graph illustrating anti-S protein neutralizing antibody analyses at day 42 in mice vaccinated with various combinations of spike plasmid and IL-12.

[80] FIG. 14A. Graph illustrating presence of anti-S protein IgGl levels in serum at day 92 in mice vaccinated with various combinations of spike plasmid and IL-12 (key shown in FIG. 14B).

[81] FIG. 14B. Graph illustrating anti-S protein neutralizing antibody analyses at day 92 in mice vaccinated with various combinations of spike plasmid and IL-12.

[82] FIG. 15 A. Graph illustrating anti-S protein IgG2a (upper panel) and IgGl (lower panel) serial dilution analyses at day 31 in mice vaccinated with various combinations of spike plasmid and IL-12 (key shown in FIG. 15B).

[83] FIG. 15B. Graph illustrating anti-S protein neutralizing antibody analyses at day 31 in mice vaccinated with various combinations of spike plasmid and IL-12.

[84] FIG. 16A. Graph illustrating anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody levels in serum at day 31 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[85] FIG. 16B. Graph illustrating anti-S protein neutralizing antibody analyses at day 31 in mice vaccinated with various combinations of spike plasmid and IL-12. [86] FIG. 17A. Graph illustrating anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody levels in serum at day 42 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[87] FIG. 17B. Graph illustrating neutralizing antibody analyses at day 42 in mice vaccinated with various combinations of spike plasmid and IL-12.

[88] FIG. 18 A. Graph illustrating anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody levels in serum at day 61 in mice vaccinated with the indicated combinations of plasmid protein and IL-12.

[89] FIG. 18B. Graph illustrating anti-S protein neutralizing antibody analyses at day 61 in mice vaccinated with various combinations of spike plasmid and IL-12.

[90] FIG. 19A. Graph illustrating anti-S protein IgGl antibody levels in serum at day 31 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[91] FIG. 19B. Graph illustrating anti-S protein IgG2a antibody levels in serum at day 31 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[92] FIG. 19C. Graph illustrating anti-S protein neutralizing antibody analyses at day 32 in mice vaccinated with various combinations of spike plasmid and IL-12

[93] FIG. 20. Graph illustrating anti-S protein IgGl (upper panel) and IgG2a (middle panel) serial dilution analyses or anti-S protein neutralizing antibody analyses (bottom panel) at day 42 in mice vaccinated with various combinations of spike plasmid and IL-12.

[94] FIG. 21 A. Graph illustrating (CD19 ⁺ ) B cell levels at day 90 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[95] FIG. 2 IB. Graph illustrating (CD3 ⁺ CD4 ⁺ ) T cell levels at day 90 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[96] FIG. 21C. Graph illustrating (CD3 ⁺ CD8 ⁺ ) T cell levels at day 90 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[97] FIG. 2 ID. Graph illustrating memory B cell levels at day 90 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[98] FIG. 22A. Graph illustrating anti-S protein IgGl (upper panel) and IgG2a (lower panel) antibody levels at day 31 in mice vaccinated with various amounts of spike plasmid and IL-12.

[99] FIG. 22B. Graph illustrating anti-S protein neutralizing antibody analyses at day 31 in mice vaccinated with various amounts of spike plasmid and IL-12. [100] FIG. 23 A. Graph illustrating anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody levels in serum at day 31 in mice vaccinated with various amounts of spike plasmid and IL-12.

[101] FIG. 23B. Graph illustrating anti-S protein neutralizing antibody analyses at day 31 in mice vaccinated with various amounts of spike plasmid and IL-12

[102] FIG. 24. Graph illustrating B cell levels in peripheral blood at day 15 (upper panel) ad day 24 (lower panel) in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[103] FIG. 25. Graph illustrating memory B cell levels in peripheral blood at day 15 (upper panel) and day 24 (lower panel) in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[104] FIG. 26. Graph illustrating germinal center B cell levels in tumor draining lymph node in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[105] FIG. 27 Graphs illustrating IgGl or IgG2a (upper panel) or class-switched B cell levels (lower panel) in tumor draining lymph node in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[106] FIG. 28. Graphs illustrating CD4 ⁺ T cell levels (upper panel) or T-bet ⁺ CD4 ⁺ cells (lower panel) in tumor draining lymph node in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[107] FIG. 29. Graphs illustrating TEH cell levels (upper panel) or CD8 ⁺ T cells (lower panel) in tumor draining lymph node in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[108] FIG. 30. Graphs illustrating anti-spike IgGl (upper panel) and IgG2a (lower panel) levels in serum at days 15 and 25 in mice vaccinated with the indicated combinations of spike plasmid and IL-12.

[109] FIG. 31. Graphs illustrating tumor growth the mice treating with IT-EP IL-12 and TG- EP or ID-EP SARS-CoV-2 spike protein or control mice.

[110] FIG. 32. Graphs illustrating tumor growth the mice treating with IT-EP IL-12 and IT- EP or ID-EP SARS-CoV-2 spike protein or control mice.

[111] FIG. 33. Graph illustrating percent tumor-free mice in mice treating with IT-EP IL- 12 and IT-EP or ID-EP SARS-CoV-2 spike protein or control mice. DESCRIPTION

1. Definitions:

[112] A "vaccine" is a substance(s) or composition(s) used to stimulate an immune response, such as the production of antibodies, and provide immunity against a disease without inducing the disease. Vaccines are often prepared from a causative agent of a disease or a product of the causative agent, such as a polypeptide or nucleic acid encoding the polypeptide. When administered to a subject, a vaccine induces or stimulates an immune response. A vaccine can render a subject resistant or immune to a particular disease or infection. A vaccine can also reduce severity or duration of infection.

[113] A "pathogen" is a bacterium, virus, or other microorganism that can infect a host, such as a human, and/or cause disease.

[114] A "nucleic acid" includes both RNA and DNA. RNA and DNA include, but are not limited to, cDNA, genomic DNA, plasmid DNA, condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides or cationic polymers, RNA, and mRNA. Nucleic acid also includes modified RNA or DNA. Nucleic acids, including plasmids, can be manufactured in large scale quantities and/or in high yield. Nucleic acids can further be manufacture using cGMP manufacturing. Nucleic acids can be formulated to be safe and effective for injection into a mammalian subject.

[115] A "homologous" sequence (e.g, nucleic acid sequence or amino acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (/%., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (/%., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences. [116] An "expression vector" refers to a nucleic acid ( e.g ., RNA or DNA) encoding one or more expression products (e.g., peptide (i.e., polypeptide or protein)). An expression vector may be, but is not limited to, a virus, an attenuated virus, a plasmid, a linear DNA molecule, or an mRNA. An expression vector is capable of expressing one or more polypeptides in a cell, such a mammalian cell. The expression vector may comprise one or more sequences necessary for expression of the encoded expression product. A variety of sequences can be incorporated into an expression vector to alter expression of the coding sequence. The expression vector may comprise one or more of: a 5' untranslated region (5' UTR), an enhancer, a promoter, an intron, a 3' untranslated region (3' UTR), a terminator, and a polyA signal operably linked to the DNA coding sequence. Any of the described nucleic acids encoding a coronavirus antigenic polypeptide and/or immune stimulator can be part of an expression vector designed to express the coronavirus antigenic polypeptide or an immune stimulator in a cell.

[117] The term "plasmid" refers to a nucleic acid that includes at least one sequence encoding a polypeptide (e.g, an expression vector) that is capable of being expressed in a mammalian cell. A plasmid can be a closed circular DNA molecule. A variety of sequences can be incorporated into a plasmid to alter expression of the coding sequence or to facilitate replication of the plasmid in a cell. Sequences can be used that influence transcription, stability of a messenger RNA (mRNA), RNA processing, or efficiency of translation. Such sequences include, but are not limited to, 5' untranslated region (5' UTR), promoter, introns, and 3' untranslated region (3 ' UTR). In some embodiments, plasmids can be transformed into bacteria, such as E. coli.

[118] A "promoter" is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g, directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter may comprise one or more additional regions or elements that influence transcription initiation rate, including, but not limited to, enhancers. A promoter can be, but is not limited to, a constitutively active promoter, a conditional promoter, an inducible promoter, or a cell-type specific promoter. Examples of promoters can be found, for example, in WO 2013/176772. The promoter can be, but is not limited to, CMV promoter, IgK promoter, mPGK, SV40 promoter, b-actin promoter (such as, but not limited to a human or chicken b-actin promoter), a-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, and EFla promoter. The CMV promoter can be, but is not limited to, CMV immediate early promoter, human CMV promoter, mouse CNV promoter, and simian CMV promoter. The promoter can also be a hybrid promoter. Hybrid promoters include, but are not limited to, C AG promoter.

[119] A "translation modification element" enables translation of two or more genes from a single transcript. Translation modification elements include Internal Ribosome Entry Sites (IRES), which allow for initiation of translation from an internal region of an mRNA, and 2A peptides, derived from picornavirus, which cause the ribosome to skip the synthesis of a peptide bond at the C-terminus of the element. Incorporation of a translation modulating element results in co-expression of two or more polypeptide from a single multi cistronic mRNA. 2A modulators include, but are not limited to, P2A, T2A, E2A or F2A. 2A modulators contain a PG/P cleavage site.

[120] "Operable linkage" or being "operably linked" refers to the juxtaposition of two or more components ( e.g ., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[121] A "secretion signal" (also termed signal sequence or signal peptide) is an amino acid sequence, typically 10-35 amino acids in length, that directs newly synthesized proteins to the secretory pathway. Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases. A secretion signal may be a native secretion signal or a heterologous secretion signal. An encoded signal peptide can be operably linked to the 5' end of a nucleic acid sequence encoding a polypeptide.

[122] A "trimerization domain" is an amino acid sequence that promotes, facilitates, or stabilizes assembly of a polypeptide into trimers. The assembly can occur through association with other trimerization domains. When linked to a polypeptide, the trimerization domain can be used to form artificial trimers. Trimerization domains may use coiled-coil motifs. The term is also used to refer to a nucleic acid sequence that encodes such an amino acid sequence. Trimerization domains include, but are not limited to, basic leucine zipper domains, GCN4pII trimerization heptad repeat, modifications of the GCN4 domain thereof that allow for formation of trimeric coiled coils, and the C-terminal domain of T4 fibritin. A T4 Fibritin trimerization domain (also termed “foldon”) comprises an amino acid sequence that naturally trimerizes. In some embodiments, a T4 Fibritin trimerization domain is linked to a coronavirus S protein antigenic polypeptide. T4 Fibritin trimerization domains include, but are not limited to, peptides comprising: GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 29), GSGYIPEAPRDGQ AYVRKDGEWVLLSTFL (SEQ ID NO: 30), or MQALQEAGYIPEAP- RDGQAYVRKDGEWVLLSTFLSPA (SEQ ID NO: 31). In some embodiments, a minifibritin trimerization domain comprising: ADIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQ- Y GSEGSMNRPT V S VLRNVEVLDKNIGILKTSLETAN SDIKTIQEAGYIPEAPRDGQ AY - VRKDGEWVLLSTFLSPALVPRGSHHHHHHS AW SHPQFEK (SEQ ID NO: 46) is linked to a coronavirus S protein antigenic polypeptide.

[123] An "Fc domain" refers to the "crystallizable fragment" region of a heavy chain of an immunoglobin. The fragment crystallizable region (Fc domain) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. An Fc domain ca be, but is not limited to, a human Fc domain. An Fc domain can be, but is not limited to, an IgG Fc domain, IgGl Fc domain, IgG4 Fc domain. The Fc domain can contain one of more modifications (such as amino acid substitutions, deletions, of insertions) that reduce binding affinity to an Fc receptor and/or reduce effector function, as compared to a native IgGl Fc domain.

[124] A "heterologous" sequence is a sequence which is not normally present in a cell, genome, or gene in the genetic context in which the sequence is currently found. A heterologous sequence can be a sequence derived from a different gene or species than a reference gene or species. A heterologous sequence can be from a homologous gene from a different species, from a different gene in the same species, or from a different gene from a different species. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.

[125] "Immunostimulatory cytokine" includes cytokines that mediate or enhance immune response to a foreign antigen, including viral, bacterial, or tumor antigens. Immunostimulatory cytokines can include, but are not limited to: TNFα, IL-1, IL-7, IL-10, IL-12, IL-12 p35, IL-12 p40, IL-15, IL-15R α IL-21IL-23, IL-27, IFNα, IFNβ, IFNγ , IL-2, IL-4, IL-5, IL-7, IL-9, IL- 21, TGFβ, and CXCL9.

[126] The term "electroporation" refers to the use of an electroporative pulse to facilitate entry of biomolecules such as a plasmid, nucleic acid, or drug, into a cell. [127] "Reversible electroporation" is the reversible, or temporary, permeabilization of cell membranes to molecules that are normally impermeable to the cell membranes using an electric pulse that is below the electric field threshold of the target cells. Because the electric pulse is below the cells' electric threshold, the cells can repair and are not killed by the electric pulse. Reversible electroporation can be used to delivery macromolecules, such as nucleic acid, into a cell without killing the cell. Reversible electroporation is a method that applies electric pulses to facilitate uptake of macromolecules, such as nucleic acids, into cells. Unless indicated otherwise, reference herein to electroporation includes reversible electroporation.

[128] A "draining lymph node" is a lymph node that filters lymph from a particular region or organ. In context of nucleic acid-based vaccines, a draining lymph node lies downstream of the treated area ( e.g ., the site of intradermal or intramuscular administration).

[129] An "immune checkpoint" molecule is any one of a group of immune cell surface receptor/ligands which induce T cell dysfunction or apoptosis. These immune inhibitory targets attenuate excessive immune reactions and ensure self-tolerance. Tumor cells harness the suppressive effects of these checkpoint molecules. Immune checkpoint target molecules include, but are not limited to, Cytotoxic T Lymphocyte Antigen-4 (CTLA-4), Programmed Death 1 (PD-1), Programmed Death Ligand 1 (PD-L1), Lymphocyte Activation Gene-3 (LAG- 3), T cell Immunoglobulin Mucin-3 (TIM3), Killer Cell Immunoglobulin-like Receptor (MR), B- and T- Lymphocyte Attenuator (BTLA), Adenosine A2a Receptor (A2aR), and Herpes Virus Entry Mediator (HVEM). "Immune checkpoint inhibitors" include molecules that prevent immune suppression by blocking the effects of immune checkpoint molecules. Checkpoint inhibitors include, but are not limited to, antibodies and antibody fragments, nanobodies, diabodies, soluble binding partners of checkpoint molecules, small molecule therapeutics, and peptide antagonists. An immune checkpoint inhibitor can be, but is not limited to, a PD-1 and/or PD-L1 antagonist. A PD-1 and/or PD-L1 antagonist can be, but is not limited to, an anti-PD-1 or anti-PD-Ll antibody. Anti-PD-l/PD-Ll antibodies include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

[130] The term "cancer" includes a myriad of diseases generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. Examples of cancer include, but are not limited to, breast cancer, triple negative breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, ovarian cancer, kidney cancer, brain cancer, or sarcomas.

[131] A “treatment-refractory cancer” is a cancer that does not respond, or has not responded, to at least one prior medical treatment. In some embodiments, a treatment- refractory, with respect to a treatment, indicates an inadequate response to a treatment or the lack of a partial or complete response to the treatment. For example, patients may be considered refractory to a treatment, ( e.g ., checkpoint inhibitor therapy such as a PD-1 or PD-L1 inhibitor therapy) if they do not show at least a partial response after receiving at least 2 doses of the treatment.

[132] The “tumor microenvironment” refers to the environment around a tumor and includes the non-malignant vascular and stromal tissue that aid in growth and/or survival of a tumor, such as by providing the tumor with oxygen, growth factors, and nutrients, or inhibiting immune response to the tumor. A tumor microenvironment includes the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix.

[133] The “tumor margin” or “margin tissue” is the visually normal tissue immediately near or surrounding a tumor. Typically, the margin tissue is the visually normal tissue within 0.1-2 cm of the tissue. Tumor margin tissue is often removed when a tumor is surgically resected.

II. Vaccine Compositions

[134] Described are vaccine compositions suitable for inducing an immune response against a pathogen in a subject, the composition comprising a pathogenic antigen in combination with at least one immune stimulator. The pathogenic antigen can be an isolated polypeptide or a nucleic acid encoding the pathogenic antigen. The immune stimulator can be an isolated polypeptide or a nucleic acid encoding the immune stimulator. The nucleic acid can be, but is not limited to, an RNA, an mRNA, a DNA, a plasmid, or an expression vector.

[135] In some embodiments, the pathogen is a coronavirus. The described coronavirus vaccine compositions can be used to vaccinate against coronavirus infection. The coronavirus can be, but is not limited to, a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS-CoV, a SARS-CoV-2, or a related betacoronavirus. The described coronavirus vaccine compositions combine at least one coronavirus antigenic polypeptide with one or more immune stimulators. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory cytokine may be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TGF- b, and C-X-C Motif Chemokine ligand 9 (CXCL9). The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1.

[136] In some embodiments, the described vaccine compositions comprise nucleic acid- based vaccines. Nucleic acid-based vaccine compositions are advantageous for several reasons. Nucleic acid ( e.g ., plasmids or expression vectors) can be can quickly adapted (in a matter of weeks, or even days) to new mutations arising in a virus. The described nucleic acid-based vaccines can induce humoral immunity while also stimulating a Thl-biased T cell response. Most vaccine approaches rely on neutralizing antibodies (humoral immunity). However, analysis of SARS-CoV, the nearest known relative to SARS-CoV-2, has shown that CD4 effector cytokines are a significant vaccine component and an over-abundance of neutrophils associated with a Th2 -biased response may limit vaccine efficacy. Coordination of a humoral response with a Thl-biased T cell response may provide an improved immune protective response.

[137] The pathogenic antigen and/or the immune stimulator can be administered to a subj ect by administering one or more nucleic acids encoding the pathogenic antigen and/or the immune stimulator. The one or more nucleic acids can be administered by electroporation or other non- viral means available in the art, including, but not limited to, direct injection (with or without reversible electroporation), needless injection (with or without reversible electroporation), microprojectile bombardment (e.g., gene gun), hydrodynamic injection, magneto-fection, sono-poration (e.g, ultrasound-mediated delivery), photo-poration, and hydro-poration. The nucleic acid sequence can be in a nanoparticle, liposome, lipoplex, polyplex, lipopolyplex, or other non-viral particle or complex. In some embodiments, the immunogenic compositions further comprise one or more electroporation applicators.

[138] Nucleic acid-based vaccines represent an affordable, safe, and effective alternative to conventional vaccines. Nucleic acid-based vaccines are considerably faster and easier to manufacture than most other vaccine platforms. Additionally, nucleic acids are considerably safer to handle than live/attenuated viral vaccines (Ruprecht R. “Live attenuated AIDS viruses as vaccines: promise or peril?” Immunol. Rev., 1999 170:135-149; and Sardesai NY et al. “Electroporation delivery of DNA vaccines: prospects for success.” Curr Opin Immunol, 2011 23(3):421-429). Plasmid DNA vaccine are also stable at room temperature with long shelf life.

A) Pathogenic antigen [139] The pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoans, parasites, and fungi. A virus can be, but is not limited to: Hepatitis C virus, Hepatitis B virus, Hepatitis C virus, influenza virus, varicella virus, measles virus, mumps virus, poliovirus, rubella virus, rotavirus, human papillomavirus, enteroviruses, West Nile virus, Ebola virus, Zika virus, human immunodeficiency virus, lyssaviruses, rabies virus, yellow fever virus, Japanese encephalitis virus, hantavirus, and coronavirus. A bacterial pathogen can be, but is not limited to: Corynebacterium diphtherias, Haemophilus influenzae type b, Bordetella pertussis, Streptococcus pneumoniae, pneumococcus, Clostridium tetani, Neisseria meningitidis, Salmonella typhi, Vibrio cholerae, and Yersinia pestis. A parasitic pathogen can be, but is not limited to: Plasmodium spp., P. falciparum, P. vivax, P. ovale, P. malarias, Trypanosoma brucei , and Leishmania.

[140] A pathogenic antigen is a polypeptide derived from the pathogen that is capable of eliciting an immune response (i.e., is immunogenic). In some embodiments, the pathogenic antigen comprises a polypeptide expressed on the surface of the pathogen or an antigenic fragment thereof. In some embodiments, the pathogen is a coronavirus. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[141] The coronavirus antigenic polypeptide can be, but is not limited to, a betacoronavirus antigenic polypeptide, a lineage A betacoronavirus (subgenus Embecovirus ) antigenic polypeptide, a lineage B betacoronavirus (subgenus Sarbecovirus ) antigenic polypeptide, a lineage C betacoronavirus (subgenus Merbecovirus ) antigenic polypeptide, a lineage D betacoronavirus (subgenus Nobecovirus ) antigenic polypeptide, a SARS-CoV antigenic polypeptide, a MERS-CoV antigenic polypeptide, a SARS-CoV-2 antigenic polypeptide, or a related betacoronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide comprises a coronavirus spike glycoprotein {also termed S glycoprotein or S protein) or an antigenic fragment thereof. A coronavirus spike polypeptide or an antigenic fragment thereof can be, but is not limited to, a betacoronavirus spike protein or an antigenic fragment thereof, a lineage A betacoronavirus (subgenus Embecovirus) spike protein or an antigenic fragment thereof, a lineage B betacoronavirus (subgenus Sarbecovirus ) spike protein or an antigenic fragment thereof, a lineage C betacoronavirus (subgenus Merbecovirus ) spike protein or an antigenic fragment thereof, a lineage D betacoronavirus (subgenus Nobecovirus ) spike protein or an antigenic fragment thereof, a SARS-CoV spike protein or an antigenic fragment thereof, a MERS-CoV spike protein or an antigenic fragment thereof, a SARS-CoV- 2 spike protein or an antigenic fragment thereof, or a related betacoronavirus spike protein or an antigenic fragment thereof.

[142] Coronavirus spike glycoproteins (S protein) facilitate interaction with host cells. SARS-CoV and SARS-CoV-2 spike glycoproteins (S protein) facilitate interaction with the host cell through binding to the ACE2 receptor (FIG. 1 A). The spike glycoprotein contains a large extracellular domain followed by a transmembrane sequence and a short intracellular tail, with a predicted molecular weight of ~141kDa (FIG. IB). The S glycoprotein SI subunit contains the receptor-binding domain (RBD) which recognizes the host-cell receptor, angiotensin-converting enzyme 2 (ACE2) for SARS-CoV-2. The S2 subunit contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate fusion of the viral and host-cell membranes by undergoing a large conformational rearrangement. The SI and S2 subunits trimerize to form a large prefusion spike.

[143] Phylogenetic analyses have revealed that of all the viral strains from the family Coronaviridae, SARS-CoV-2 share the greatest similarity with the severe acute respiratory syndrome (SARS) strain (Lu R et al. “Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.” The Lancet 2020; and Wu F et al. “A new coronavirus associated with human respiratory disease in China.” Nature. 2020). Three strains of SARS-CoV-2 together with two bat-derived SARS-like strains, form a distinct clade of coronaviruses. As such, relevant immune mechanisms and associated antigens from SARS-CoV have been applied to a SARS-CoV-2 coronavirus vaccine. The coronavirus S glycoprotein has demonstrated immunogenicity, eliciting both cellular and humoral immune responses (Li CK et al. “T Cell Responses to Whole SARS Coronavirus in Humans.” Journal of Immunology , 2008 5490-5499; Muthumani K et al. “A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates.” Science Translational Medicine, 2015 7(301):301; and Kong W et al. “Modulation of the Immune Response to the Severe Acute Respiratory Syndrome Spike Glycoprotein by Gene-Based and Inactivated Virus Immunization.” Journal of Virology, 2005 13915-13923).

[144] A conserved genetic sequence that codes for the S glycoprotein was identified by comparing multiple isolates of SARS-CoV-2 (Lu et al. 2020). The S glycoprotein genetic sequence has limited synonymous mutations between strains, which may help to limit immune escape variants.

[145] The S protein antigenic polypeptide can contain any combination of one or more of: one or more mutations to disrupt an internal furin-cleavage site, one or more mutations to stabilize the protein, a native or heterologous secretion signal, a native or heterologous transmembrane domain, a native or heterologous trimerization domain, an Fc tag, and a reading frame codon-optimized for human expression (FIG. 7-8). In addition, S protein antigenic polypeptide may optionally comprise an HRV3C protease cleavage site and/or one or more affinity (epitope) tags.

[146] A mutation to disrupt an internal furin-cleavage site in the S-protein can be, but is not limited to, substitution of GSAS for RRAR corresponding to amino acid positions 664-667 of SEQ ID NO: 1. In some embodiments, the S protein antigenic polypeptide contains the native furin cleavage site. In some embodiments, the S protein antigenic polypeptide contains a transmembrane domain and the native furin cleavage site.

[147] In some embodiments, the S protein antigenic polypeptide contains one or more mutations to stabilize the protein. Mutations to stabilize the S-protein can comprise mutations that stabilize the spike protein in prefusion conformations. Stabilizing the prefusion conformation can increase immunogenicity of the spike protein. Stabilizing the prefusion conformation can also increase expression of the spike protein from a nucleic acid expressing the spike protein. A mutation that stabilizes the spike protein in a prefusion conformation can be, but is not limited to, a substitution of prolines (2P) for the amino acids at the apex of the central helix and heptad repeat 1 (corresponding to amino acids K ₉₈₆ and V ₉₈₇ of SEQ ID NO: 1). The 2P mutation has been shown to stabilize the spike protein in a prefusion conformation in a variety of betacoronaviruses, including MERS-CoV, SARS-CoV and HCoV-HKUl.

[148] In some embodiments, the S protein antigenic polypeptide contains a secretion signal. The secretion signal may be a native secretion signal or a heterologous secretion signal. A heterologous secretion signal can be, but is not limited to, a tyrosine protein phosphatase alpha secretion signal or an Ig-kappa secretion signal. In some embodiments, the native secretion signal is used. In some embodiments, the SARS-CoV-2 S protein antigenic polypeptide comprises a native SARS-CoV-2 S protein secretion signal. In some embodiments, the S protein antigenic polypeptide does not contain a secretion signal.

[149] An S protein antigenic polypeptide may or may not contain the native S protein transmembrane and/or intracellular domain tail. An S protein antigenic polypeptide may contain a heterologous transmembrane domain.

[150] A trimerization domain facilitates trimerization of the spike protein extracellular domain. A heterologous trimerization domain can be, but is not limited to, a C-terminal bacteriophage T4 fibritin trimerization motif (foldon domain). In some embodiments, the S protein antigenic polypeptide contains a native or a heterologous trimerization domain. In some embodiments, the S protein antigenic polypeptide does not contain a native a heterologous trimerization domain.

[151] In some embodiments, a nucleic acid encoding a coronavirus antigenic polypeptide comprises a mammalian codon-optimized nucleic acid encoding minifibritin foldon sequence (amino acids 1-112 of SEQ ID NO: 46). The foldon sequence may further contain a C-terminal thrombin cleavage site, 6x His-tag, and Strep-Tagll (ADIVLNDLPFVDGPPAEGQSRISWI- KNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQE- AGYIPEAPRDGQAYVRKDGEWVLLSTFLSPALVPRGSHHHHHHSAWSHPQFEK (SEQ D NO: 46).

[152] An affinity tag can be, but is not limited to, STREP-TAG®II , TWIN-STREP-TAG®, or a His Tag (such as, but not limited to, a 6x His tag or an 8x His tag).

[153] In some embodiments, the SARS-CoV-2 spike protein is encoded on a nucleic acid as described in Wrapp et al. (“Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation.” Science 2020367(6483): 1260-1263). This SARS-CoV-2 antigenic polypeptide contains ectodomain (i.e., extracellular domain) residues 1 to 1208 of SARS-CoV-2 S protein and has two stabilizing proline mutations (e.g., K ₉₈₆ P and V ₉₈₇ P of SEQ ID NO: 1 and corresponding to residues V1060 and L1061 of the MERS S protein) in the C-terminal S2 fusion machinery using stabilization strategy that proved effective for other betacoronavirus S proteins (Pallesen et al. “ Immunogenicity and structures of a rationally designed prefusion MERS-CGV spike antigen.” Proc. Natl. Acad. Sci. U.S.A. 2017 114:E7348 E7357 (2017); Kirchdoerfer et al. “Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis. Sci. Rep. 2018 8, 15701). The proline mutations (KV _986-987 PP, i.e., 2P) help stabilize prototypic prefusion morphology In the polypeptide (i.e., profusion-stabilizing mutations). In some embodiments, a nucleic acid encoding a S protein antigenic polypeptide comprises a secretion signal that results in secretion of the expressed polypeptide.

[154] In some embodiments, a nucleic acid encoding a coronavirus antigenic polypeptide comprises DNA. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled. The DNA can be an expression vector or a plasmid. In some embodiments, a nucleic acid encoding a coronavirus antigenic polypeptide comprises RNA. The RNA can be an mRNA.

[155] Nucleic acid-based vaccines can be quickly adapted to new mutations in the targeted virus, such as SARS-CoV-2. The nucleic acid-based vaccines can be adapted to new mutations in the viral genome in a matter of weeks, or even days. For a new coronavirus strain or mutation of an existing strain, the nucleic acid sequence encoding the S protein is sequenced using methods known in the art. A nucleic acid encoding the new S protein antigenic polypeptide, corresponding to the nucleic acids encoding S protein antigenic polypeptides described herein, are then prepared, and combined with nucleic acids encoding an immune stimulator for form a vaccine composition for eliciting an immune response against the new coronavirus strain or mutation.

B) Immune stimulator

[156] The coronavirus antigenic polypeptide is co-administered with one or more immune stimulators. The immune stimulator can be an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE. The immune stimulator(s) supports innate effector cells, presentation of the coronavirus antigenic polypeptide, and priming of a cellular anti-viral T cell response. The immune stimulator can be an isolated polypeptide or a nucleic acid encoding the immune stimulator. In some embodiments, the nucleic acid encoding an immune stimulator comprises DNA. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled. The DNA can be an expression vector or a plasmid. In some embodiments, the nucleic acid encoding an immune stimulator comprises RNA. The RNA can be an mRNA.

[157] In some embodiments, the coronavirus antigenic polypeptide comprises an isolated polypeptide and the immune stimulator comprises a nucleic acid encoding the immune stimulator. In some embodiments, the coronavirus antigenic polypeptide comprises a nucleic acid encoding the coronavirus antigenic polypeptide and the immune stimulator comprises an isolated polypeptide. In some embodiments, the coronavirus antigenic polypeptide and the immune stimulator comprise isolated polypeptides. In some embodiments, the coronavirus antigenic polypeptide comprises a nucleic acid encoding the coronavirus antigenic polypeptide and the immune stimulator comprises a nucleic acid encoding the immune stimulator.

[158] In some embodiments, the immune stimulator comprises an immunostimulatory cytokine. Immunostimulatory cytokines include, but are not limited to, IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/Receptor a, IL-21, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TGF-β and CXCL9.

C) IL-12

[159] In some embodiments, the immunostimulatory cytokine comprises an IL-12. IL-12 is a proinflammatory cytokine known to efficiently support NK (natural killer) cells and macrophage function as well as polarize Thl -biased T cell responses. IL-12 has been reported to increase in both cellular and humoral immune responses. Interleukin 12 (IL-12) plays a role in the induction of a Thl -biased response while coordinating both the innate and adaptive immune systems. IL-12 can also augment antigenicity via activation of antigen presenting cells and assist the humoral response by encouraging isotype switching towards a more functional antibody.

[160] IL-12 is a heterodimeric cytokine having both IL-12A (p35) and IL-12B (p40) subunits. A nucleic acid encoding IL-12 can comprise a nucleic acid sequence encoding an IL- 12 p40-IL-12 p35 fusion protein (an IL-12 p70), a nucleic acid sequence encoding an IL-12 p35-IL-12 p40 fusion protein (an IL-12 p70), or an IL-12 p35 subunit and an IL-12 p40 subunit. The nucleic acid sequences encoding the IL-12 p35 and IL-12 p40 subunits can be on a contiguous nucleic acid sequence separated by a translation modification element, allowing both subunits to be expressed from a single promoter or a single mRNA. The translation modification element can be an internal ribosome entry site (IRES) element or a ribosome skipping modulator. A ribosome skipping modulator can be, but is not limited to, a 2A element (also termed 2A peptide or 2A self-cleaving peptide). The 2A element can be, but is not limited to, a P2A, T2A, E2A or F2A element. The IL-12 p35 and p40 coding sequences can be expressed from a multi cistronic expression vector from a single promoter and separated by an IRES or 2A element.

D) CXCL9

[161] In some embodiments, the immunostimulatory cytokine comprises a CXCL9. C-X-C Motif Chemokine ligand 9 (CXCL9) is a small cytokine belonging to the CXC chemokine family. CXCL9 is also known as Monokine Induced by Gamma interferon (MIG). CXCL9 is a T-cell chemoattractant, and facilitates chemotactic recruitment of tumor infiltrating lymphocytes (TIL).

E) Flt3L

[162] In some embodiments, the immunostimulatory cytokine comprises a genetic adjuvant. A “genetic adjuvant” is a polypeptide that can be encoded by a nucleic acid and that acts as adjuvant, enhancing an antigen-specific immune response compared with the immune response generated in the absence of the genetic adjuvant. The genetic adjuvant can be selected from the group consisting of: Fms-like tyrosine kinase 3 ligand (also termed Flt3-Ligand or Flt3L), LAMP-1, calreticulin, human heat shock protein 96, GM-CSF, and CSF Receptor 1. In some embodiments, the genetic adjuvant comprises Flt3L.

[163] Flt3L is an immunomodulatory growth factor that supports dendritic cell differentiation and maturation, both of which are involved in T cell priming. Clinical data suggests Flt3L can augment humoral and T cell responses to antigens (Bhardwaj, 2016). Plasmid encoding IL-12 cytokine with the Flt3L immune modulator has been shown to enhance immune response induced by a DNA-vaccine against the model antigen OVA (SIINFEKL epitope) (FIG. 2A-C).

[164] In some embodiments, the genetic adjuvant is linked to the pathogenic antigen, thereby forming a genetic adjuvant-pathogenic antigen fusion polypeptide. In some embodiments, the genetic adjuvant-pathogenic antigen fusion polypeptide comprises a genetic adjuvant-SARS-CoV-2 antigen fusion polypeptide. In some embodiments, the genetic adjuvant-SARS-CoV-2 antigen fusion polypeptide comprises a Flt3L-SARS-CoV-2 antigen fusion polypeptide. The SARS-CoV-2 antigenic polypeptide can be, but is not limited to, any of the SARS-CoV-2 antigenic polypeptides described herein.

[165] In some embodiments, immunogenicity of a pathogenic antigen is enhanced by co- delivery with IL-12 and/or Flt3 ligand. The pathogenic antigen can be expressed from the same plasmid as the IL-12 and/or Flt3 ligand or a different plasmid than the IL-12 and/or Flt3 ligand.

F) Anti-CD3 half-BiTE

[166] In some embodiments, the immunostimulatory cytokine comprises an anti-CD3 half- BiTE. An anti-CD3 half-BiTE comprises an anti-CD3 single-chain variable fragment (scFv) fused to a transmembrane domain (TM). An scFv comprises a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. An anti-CD3 scFv can be identified from phage display. An anti-CD3 scFv can also be generated by subcloning the VH and VL from a known anti-CD3 antibody, such as from a hybridoma. Known anti-CD3 antibodies have been described, for instance in US20180117152, US20140193399, US20100183554, and US20060177896. Known anti-CD3 antibodies also include, but are not limited to, OKT3 (Muromonab-CD3), 145-2C11, 17A2, SP7, and UCHT1. In some embodiments, the VH and or VL domains of an anti-CD3 scFv can be humanized. A humanized antibody (or antibody fragment or domain) is an antibody from a non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. In some embodiments, humanized antibodies can be made by inserting the relevant complementarity-determining regions (CDRs, also termed hypervariable regions (HVRs)) of an anti-CD3 antibody into human VH and VL domain scaffolds.

[167] An anti-CD3 scFv can be formed by linking the C terminus of the VH chain with the N terminus of the VL. Alternatively, the C terminus of the VL can be linked to the N-terminus of the VH. The peptide linker can be about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is rich in glycine. An scFv peptide linker can be, but is not limited to, (G4S) _x where x is an integer from 2 to 5 (inclusive). In some embodiments, the scFv peptide linker comprises Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 34); also termed [(Gly) ₄ Ser] , (G ₄ S) ₃ or G4S (x3)). In some embodiments, the scFv peptide linker consists of G4S (x3).

[168] A transmembrane domain (TM) comprises a polypeptide capable of being inserted into a biological lipid bilayer (membrane) and anchoring the anti-CD3 half-BiTE to the membrane. TMs are known in the art and typically consist predominantly of nonpolar amino acids. The transmembrane domain can be, but is not limited to, a PDGFRβ transmembrane domain or a PDGFRα transmembrane domain (PDGFR is Platelet-derived growth factor receptor). In some embodiments, a spacer is included between the anti-CD3 scFv and the transmembrane domain. In some embodiments, the TM domain comprises an amino acid sequence selected from the group comprising: VGQDTQEVIVVPHSLPFKVVVISAILALV- VLTIISLIILIMLW QKKPR (SEQ ID NO: 35), AVGQDTQEVIVVPHSLPFKVVVISAILA- L VVLTIISLIILIMLW QKKPR (SEQ ID NO: 36), VVISAILALVVLTVISLIILI (PDGFRβ) (SEQ ID NO: 37), VVISAILALVVLTIISLIILI (PDGFRβ) (SEQ ID NO: 38), AAVLVLLVI- VIISLIVLVVIW (PDGFRa) (SEQ ID NO: 39), and AAVLVLLVIVIVSLIVLVVIW (PDGFRa) (SEQ ID NO: 40). In some embodiments, the TM domain is encoded by a nucleic acid sequence selected from the group comprising: gtgggccaggacacgcaggaggtcatcgtggtgc- cacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggt-gctc accatcatctcccttatcatcctcatcatgct- ttggcagaagaagccacgt (SEQ ID NO: 41), gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgc- cctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctccc ttatcatcctcatcatgctttggcagaagaa- gccacgt (SEQ ID NO: 42), tggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctca tc (PDGFRβ) (SEQ ID NO: 43), gtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctc atc (PDGFRβ) (SEQ ID NO: 44), gctgcagtcctggtgctgttggtgattgtgatcatctcacttattgtcctggttgtcatt - tggaa (PDGFRa) (SEQ ID NO: 45).

[169] In some embodiments, the encoded anti-CD3 half-BiTE polypeptide includes a signal peptide such as an IgK signal peptide.

[170] In some embodiments, the one or more immune stimulator comprises an IL-12 and either a CXCL9, a Flt3L, or an anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulator comprises an IL-12, a CXCL9, and an anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulators comprises a CXCL9 and an anti-CD3 half- BiTE. In some embodiments, the one or more immune stimulator comprises an IL-12 and a CXCL9. In some embodiments, the one or more immune stimulator comprises an IL-12 and an anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulator comprises an IL-12 and a Flt3L. The Flt3L may be linked to a pathogenic antigen. The pathogenic antigen linked to the Flt3L may be any of the SARS-CoV-2 antigenic polypeptides described herein.

G) Multi -cistronic expression vectors

[171] The nucleic acids encoding the at least one immune stimulator may be present on a single plasmid or on multiple plasmids. Similarly, the nucleic acid encoding the pathogenic antigen may be present on a plasmid containing one or more nucleic acid sequences the encode at least one immune stimulator or the nucleic acid encoding the pathogenic antigen may be present on a separate nucleic acid. Nucleic acids containing nucleic acid sequences coding for more than one polypeptide may contain multi cistronic expression vectors.

[172] In some embodiments, a nucleic acid encoding an IL-12 also encodes a Flt3 ligand, a Flt3L-pathogenic antigen fusion polypeptide, a CXCL9, an anti-CD3 half-BiTE, or a pathogenic polypeptide. In some embodiments, the IL-12 and the Flt3L, the Flt3L-pathogenic antigen fusion polypeptide, the CXCL9, the anti-CD3 half-BiTE or the pathogenic antigen are expressed from a multi cistronic expression vector from a single promoter. In some embodiments, a nucleic acid encoding a CXCL9 also encodes an anti-CD3 half-BiTE. The CXCL9 and the anti-CD3 half-BiTE may be expressed from a multi cistronic expression vector from a single promoter. The coding sequence in a multi cistronic expression vector may be separated by an IRES or 2A translation modification element. In some embodiments, the translation modification element comprises an IRES element. In some embodiments, the translation modification element comprises an 2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[173] An expression vector or plasmid may contain a multi cistronic expression vector. Multi cistronic expression vectors express two or more separate proteins from the same mRNA and contain one or more translation modification elements. In some embodiments, an expression vector encoding IL-12 expresses two or three polypeptides expressed from a single promoter, with one or more translation modification elements to allow the two or three polypeptides to be expressed from a single mRNA. In some embodiments, the expression vector comprises:

(a) P-A-T ¹ -B,

(b) P-B-T ¹ -A, (c) R-B-T ¹ -B'

(d) R-A-T ¹ -B-T ¹ -B' or

(e) R-B-T ¹ -B'-T ¹ -A wherein P is a promoter, A encodes a pathogenic antigen, a Flt3L, a Flt3L-pathogenic antigen fusion polypeptide, a CXCL9, or an anti-CD3 half-BiTE, B and B' encode IL-12 or IL-12 subunits, and T ¹ and T ² are both translation modification elements. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[174] In some embodiments, T ¹ and T ² are independently internal ribosome entry site (IRES) elements or ribosomal skipping modulators. A ribosome skipping modulator can be, but is not limited to, a 2A element (also termed 2A peptide or 2A self-cleaving peptide). The 2A element can be, but is not limited to, a P2A (SEQ ID NO: 28), T2A, E2A or F2A element.

H) Nucl ei c aci d F ormul ati on

[175] Nucleic acids encoding any of the described pathogenic antigens and/or immune stimulators can be formulated for in vivo administration. In some embodiments, the nucleic acids are formulated for in vivo administration by electroporation. The nucleic acids can be made using methods known in the art, including, but not limited to, forming overlapping geneblock fragments. The fragments can be stitched together using PCR or Gibson assembly and cloned into a vector such as, but not limited to, pUMVC3. Protein expression and secretion can be analyzed in model cell lines using epitope-tagged variants.

[176] An mRNA can be in vitro transcribed mRNA. The in vitro transcribed mRNA can be produced from a DNA template using an RNA polymerase. The polymerase can be, but is not limited to, a T7, a T3, or a Sp6 phage RNA polymerase. The DNA template can be a supereoiled plasmid, a relaxed circular DNA, or a linear DNA template. The transcribed mRNA may contain one or more of: 5' UTR, 3' UTR, 5' cap, polyA tail, pseudouridine modified nucleotides, and 1-metliylpseudouridine modified nucleotides. In some embodiments, the RNA is a self- amplifying mRNA. A self-amplifying RNA encodes both the antigen or immune stimulator of interest and an RNA dependent RNA polymerase that enables RNA replication.

[177] In some embodiments, the DNA template encoding the coronavirus antigenic polypeptide is flanked with 5' and 3' untranslated regions and a poly-A tail. The 5' UTR can be, but is not limited to, 5'-GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUA- UAAGAGCC ACC-3' (SEQ ID NO: 47). The 3' UTR can be, but is not limited to, 5'- UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC- CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA- GUCUGA-3' (SEQ ID NO: 48).

[178] In some embodiments, a donor methyl group S-adenosylmethionine (SAM) is added to the methylated capped RNA.

I) Polypeptide formulation

[179] In some embodiments, a pathogenic antigen or immune stimulator can be prepared by biosynthesis of the polypeptide in a host cell. For biosynthesis in a host cell, a nucleic acid encoding the pathogenic antigen or immune stimulator is created and transformed or transfected into the host cell. In some embodiments, the host cell is an industrially scalable microorganism or eukaryotic culture cell. In some embodiments, a pathogenic antigen or immune stimulator is isolated and/or purified. The polypeptides can be isolated and purified from the synthesis reaction mixture by means of peptide purification known to in the art. For example, the polypeptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution.

III. Formulation

[180] The described vaccine compositions can be formulated for intradermal, intramuscular, and/or intratumoral administration to a subject. Intradermal administration comprises injection into or delivery to the dermis. Intramuscular administration comprises injection into or delivery to skeletal muscle. Intratumoral administration comprises injection into or delivery to a tumor. Intradermal administration, intramuscular administration, and intratumoral administration can be performed using devices know in the art for administration to the dermis, skeletal muscle, or tumor, including, but not limited to, conventional syringes and needles.

[181] The nucleic acids encoding pathogenic antigens and immune stimulators can be formulated for administration into the dermis (intradermal administration), skeletal muscle (intramuscular administration), or a tumor (intratumoral administration).

[182] The nucleic acid can be administered as naked nucleic acid or the nucleic acid can be combined with protamine, cationic nanoemulsion, dendrimer nanoparticle, protamine liposome, liposome, cationic polymer, polysaccharide particle, polyliposome, cationic lipid nanoparticle, cationic lipid cholesterol particle, or cationic lipid cholesterol PEG nanoparticle. In some embodiments, electroporation is used to enhance delivery of the nucleic acid to cells.

[183] In some embodiments, nucleic acids encoding pathogenic antigens and/or immune stimulators are encapsulated in liposomes, such as lipid nanoparticles (LNP). Formation of LNPs can be performed using methods available in the art, including, but not limited to, ethanol drop nanoprecipitation. In some embodiments, the LNP contains l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, and PEG-lipid; or ionizable cationic lipid, phospholipid, cholesterol, and PEG-lipid. In some embodiments, the LNP contains lipid, DSPC, cholesterol, and PEG-lipid at molar ratio of 50: 10:38.5: 1.5 (ionizable lipid:DSPC:cholesterol:PEG-lipid, or ionizable cationic lipid, phospholipid, cholesterol, and PEG-lipid). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the LNP are between 80-100 nm in size.

[184] In some embodiments, the nucleic acids encoding pathogens antigens and immune stimulators can be formulated for administration into the dermis by intradermal electroporation (ID-EP), intramuscular electroporation (IM-EP), and/or intratumoral electroporation (IT-EP). Detection in serum of secreted protein following electroporation of a nucleic acid encoding a model protein is shown in FIG. 3. In some embodiments, the nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators can be formulated for administration into the dermis, muscle, or tumor by direct injection, needleless injection, microprojectile bombardment, hydrodynamic injection, magneto-fection, sono-poration, photo-poration, or hydro-poration. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[185] Intradermal electroporation (ID-EP) comprises inj ecting one or more nucleic acids into the dermis and administering at least one electroporative pulse to the dermis at the site of the injection. The one or more nucleic acids can be injected prior to administering the electroporative pulse or substantially simultaneously with administering the electroporative pulse. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[186] Intramuscular electroporation (IM-EP) comprises injecting one or more nucleic acids into a skeletal muscle and administering at least one electroporative pulse to the skeletal muscle at the site of the injection. The one or more nucleic acids can be injected prior to administering the electroporative pulse or substantially simultaneously with administering the electroporative pulse. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[187] Intratumoral electroporation (IT-EP) comprises injecting one or more nucleic acids into a tumor and administering at least one electroporative pulse to the tumor. The one or more nucleic acids can be injected prior to administering the electroporative pulse or substantially simultaneously with administering the electroporative pulse. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject. [188] For administration of isolated pathogenic antigen polypeptide or isolated immune stimulator polypeptide, the isolated polypeptide can be combined with a vaccine adjuvant. In some embodiments, the isolated pathogenic antigens or isolated immune stimulator polypeptide is administered with a vaccine adjuvant. In some embodiments, the polypeptide is combined with adjuvant prior to injection. The adjuvant can be, but is not limited to, alum, Sigma Adjuvant System, or Freund’s adjuvant. Alum can be, but is not limited to, alum hydrogel. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[189] Any of the described vaccine compositions may comprise one or more pharmaceutically acceptable excipients. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to one or more of the nucleic acids encoding a pathogenic antigen or immune stimulator. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

[190] Pharmaceutically acceptable excipients ("excipients") are substances other than the Active Pharmaceutical ingredient (API, therapeutic product; e.g., nucleic acid encoding a coronavirus antigenic polypeptide or immune stimulator) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[191] Excipients include, but are not limited to: agents that enhance transfection, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. Agents that enhance transfection include, but are not limited to, lipids, cationic lipids, lipids, polycations, cell-penetrating peptides, and combinations thereof.

[192] The vaccine compositions can contain other additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to, anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g, antihistamine, diphenhydramine, etc.). As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective dose" refers to that amount of a described nucleic acid to produce the intended pharmacological, therapeutic, or preventive result.

[193] Vaccine compositions suitable for injectable use include sterile aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions. For intradermal, intramuscular, or intratumoral injection, including ID-EP, IM-EP and IT-EP, suitable carriers include, but are not limited to, physiological saline, and phosphate buffered saline (PBS).

IV. Kits

[194] Any of the described isolated polypeptides, nucleic acids and/or vaccine compositions comprising the isolated polypeptides and/or nucleic acids disclosed herein may be packaged or included in a kit, container, pack, or dispenser. Any of the described isolated polypeptides, nucleic acids and/or vaccine compositions comprising the isolated polypeptides, nucleic acids may be packaged in pre-filled syringes or vials. The isolated polypeptides, nucleic acids and/or vaccine compositions may be provided as a lyophilized powder or they may be provided in a solution. A kit can comprise a reagent utilized in performing a method disclosed herein. A kit can also comprise a composition, tool, or instrument disclosed herein, such as, but not limited to, an electroporation applicator. In some embodiments, the kit comprises one or more of the described nucleic acids and/or vaccine compositions comprising nucleic acids and an electroporation device or applicator. In some embodiments, the kit comprises one or more or the described nucleic acids, one or more electroporation applicators, syringes, and injection needles. A nucleic acid encoding a coronavirus antigenic polypeptide (such as the SARS-CoV- 2 spike protein and an antigenic fragment thereof) and a nucleic acid encoding an immune stimulator (such as IL-12) may be provided in separate containers (vials, etc.) or they may be combined in a single container. An isolated coronavirus antigenic polypeptide and an isolated immune stimulator may be provided in separate containers (vials, etc.) or they may be combined in a single container.

[195] In some embodiments, a vaccine composition comprises a first container containing a nucleic acid encoding a coronavirus antigenic polypeptide and a second container containing a nucleic acid encoding an immune stimulator. In some embodiments, a vaccine composition comprises a first container containing a coronavirus antigenic polypeptide and a second container containing a nucleic acid encoding an immune stimulator. In some embodiments, a vaccine composition comprises a first container containing a coronavirus antigenic polypeptide and a second container containing an immune stimulator polypeptide. In some embodiments, a vaccine composition comprises a first container containing a nucleic acid encoding a coronavirus antigenic polypeptide and a second container containing an immune stimulator polypeptide.

[196] In some embodiments, a vaccine composition comprises a first container containing a nucleic acid encoding a coronavirus antigenic polypeptide, a second container containing a nucleic acid encoding an immune stimulator, and a third container containing a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, a vaccine composition comprises a first container containing a coronavirus antigenic polypeptide, a second container containing an immune stimulator polypeptide, and a third container containing a coronavirus antigenic polypeptide. In some embodiments, a vaccine composition comprises a first container containing a coronavirus antigenic polypeptide, a second container containing a nucleic acid encoding an immune stimulator, and a third container containing a coronavirus antigenic polypeptide. In some embodiments, a vaccine composition comprises a first container containing a nucleic acid encoding coronavirus antigenic polypeptide, a second container containing an immune stimulator polypeptide, and a third container containing a nucleic acid encoding a coronavirus antigenic polypeptide.

[197] In some embodiments, a vaccine composition comprises a first container containing a nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immune stimulator and a second container containing a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, a vaccine composition comprises a first container containing coronavirus antigenic polypeptide and immune stimulator polypeptide and a second container containing coronavirus antigenic polypeptide.

[198] The containers can contain a sufficient amount of polypeptide or nucleic acid to provide a single effective dose or multiple effective doses. The polypeptides or nucleic acids encoding the coronavirus antigenic polypeptide and the immune stimulator(s) can be any of the described coronavirus antigenic polypeptides, immune stimulators, or combinations thereof.

[199] In some embodiments, a kit further contains one or more of: instructions for use, or a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of the products.

V. Vaccination

[200] Prophylactic anti-viral vaccines frequently rely on neutralizing antibodies by triggering a humoral immune response. This antibody-directed immunity is commonly generated by an attenuated or subunit vaccine in a Th2 -polarized environment. Thl-type cytokines tend to produce the proinflammatory responses responsible for killing intracellular pathogens. In excess, Th2 responses can inhibit or counteract the Thl-type response. By co- administering an immune stimulator, a Thl-type immune response is elicited. A Thl-type immune response may limit clinical pathology sometimes associated with Th2-specific cytokines and associated immune subsets. Eliciting a Thl-polarized immune response may help drive a cellular T cell response, which can be important for isotype switching and for induction of anti-viral effector cytokines. In some embodiments, the described vaccines compositions and methods can elicit both a Th2 -biased response (humoral immunity) and a Thl -biased T cell response

[201] IL-12 plays a role in the induction of a Thl-biased response while coordinating both the innate and adaptive immune systems. Besides triggering cellular immunity, IL-12 can augment antigenicity via activation of antigen presenting cells. IL-12 can also assist the humoral response by encouraging isotype switching towards a more functional antibody. With the inclusion of immune stimulators, such as IL-12, the described vaccine compositions drive a coordinated immune response, capable of drawing upon the innate, adaptive humoral, and adaptive cellular arms.

[202] The location and cellular architecture of the tissue used for delivery of nucleic acid- base vaccines can modulate the effectiveness and/or type of immune response that the vaccine generates. Intramuscular administration of non-viral nucleic acid vaccines has been shown in some cases to be effective at generating antibody-mediated (humoral) immunity (Brown PA et al. “Delivery of DNA into skeletal muscle in large animals.” Methods Mol Bio, 2008 423:215- 224.). Properties such as accessibility, large area, and extensive vascularization of muscle tissue enables durable expression and efficient circulation of expressed proteins (Brown PA et al. 2008). Electroporation-mediated delivery targeting the skin has been described (Hirao LA et al. “Multivalent Smallpox DNA Vaccine Delivered by Intradermal Electroporation Drives Protective Immunity in Nonhuman Primates Against Lethal Monkeypox Challenge. J Infect Dis, 2011 203(1):95-102). Skin contains antigen presenting cells (APC), such as Langerhans cells and skin-resident dendritic cells, that present antigens and prime cellular (T cell-mediated) anti-viral responses (Hirao LA et al. 2011; and Tobin D “Biochemistry of human skin— our brain on the outside. Chem Soc Rev, 2006 35(l):52-67).

[203] Described are methods of co-administering a coronavirus antigenic polypeptide and one or more immune stimulators, optionally at spatially distinct sites. Also described are methods of co-administering nucleic acids encoding a coronavirus antigenic polypeptide and one or more immune stimulators via electroporation. Intramuscular delivery of nucleic acid encoding a coronavirus antigenic polypeptide results in expression of diffusible coronavirus antigenic polypeptide. Intradermal and/or intratumoral delivery of nucleic acids encoding the coronavirus antigenic polypeptide and/or at least one of the described immune stimulators results in expression of these polypeptide in these tissues.

[204] Local administration or expression of the coronavirus antigenic polypeptide and one or more immune stimulators can induce a local Thl -biased response and a cellular anti -viral T cell response. Since local cellular priming does not preclude the priming of humoral immunity in secondary lymphoid tissues, such as draining lymph nodes, the described vaccination methods are capable of producing both cellular and humoral anti-coronavirus immunity.

[205] In some embodiments, the described vaccines compositions and methods are able to take advantage of delivery of the coronavirus antigenic polypeptide and immune stimulator to spatially distinct sites ( e.g ., dermis and skeletal muscle). The discrete modular deployment of antigen and immune stimulator(s) creates an immunogenic cellular microenvironment in the dermis with IL-12 readily supporting innate effector cells, as well as presentation of the antigen, such as a SARS-CoV-2 S antigenic polypeptide, and subsequent priming of a cellular anti-viral T cell response.

[206] In some embodiments, the described vaccines induce coordinated immune responses that involve humoral and cellular immune response pathways. In some embodiments, the described vaccines induce a humoral immune response and a cellular immune response.

[207] In some embodiments, the described vaccines induce a humoral immune response. In some embodiments, the described vaccines mediate production of neutralizing antibodies. In some embodiments, the neutralizing antibodies protect against pulmonary viral replication.

[208] In some embodiments, the described vaccines induce a cellular immune response. The cellular immune response can include innate (NK) and adaptive (CD4 ⁺ T cells) arms and result in production of Thl -directed cytokines such as IFN-γ and TNF-a (Chen J et al. “The Immunobiology of SARS*” Annu Rev Immunol, 200725:443-472; and Li CK et al. 2008).

[209] In some embodiments, the nucleic acid encoding the coronavirus antigenic polypeptide and the one or more nucleic acid sequences encoding the at least one immune stimulator are delivered to the subject by ID-EP, IM-EP and/or IT-EP.

[210] In some embodiments, the nucleic acid encoding the coronavirus antigenic polypeptide and the one or more nucleic acid sequences encoding the at least one immune stimulator are delivered to the subject having at least one tumor by IT-EP, ID-EP, IM-EP, or combination thereof. [211] In some embodiments, intramuscular electroporation (IM-EP) administration of nucleic acid comprises injecting into skeletal muscle tissue a solution containing the nucleic acid and administering at least one electroporative pulse at the injection site. The muscle can be, but is not limited to, a deltoid muscle, a leg muscle, or a muscle in the buttocks.

[212] In some embodiments, 0.1-3 mg of nucleic acid is injected into skeletal muscle for IM-EP. In some embodiments, 0.1±0.05 mg of the nucleic acid is injected. In some embodiments, 0.25±0.15 mg of the nucleic acid is injected. In some embodiments, 0.5±0.4 mg of the nucleic acid is injected. In some embodiments, 0.8±0.5 mg of the nucleic acid is injected. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non-viral vector.

[213] In some embodiments, the nucleic acid is injected into skeletal muscle in a volume of 20-1000 μL. In some embodiments, the nucleic acid is injected in a volume of, 250±10 μL, 50±30 μL, 250±50 μL, 500±100 μL, or 750±250 μL. The injection(s) can be in a single location or in two or more locations. For injection in two or more locations, the injections can be adjacent to each other or in separate locations. For example, a subject can receive 500 μL in two adjacent injections of 250 μL each. Similarly, a subject can receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be sufficiently close such that the electroporation applicator electrodes are able to span both injection sites.

[214] In some embodiments, the nucleic acid is injected into skeletal muscle at a depth of 0.5 to 1.5 cm or more. In some embodiments, the nucleic acid is injected at a depth of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 cm, or greater than 1.5 cm. In some embodiments, the nucleic acid is injected at a depth of 1.0±0.5 cm or 1.5±0.5 cm.

[215] In some embodiments, intradermal electroporation (ID-EP) administration of nucleic acid comprises injecting into dermis a solution containing the nucleic acid and electroporation of the injection site. The site of injection can be, but is not limited to, a shoulder, a leg, or buttocks.

[216] In some embodiments, 0.1-3 mg of the nucleic acid polypeptide is injected into the dermis. In some embodiments, 0.25±0.01 mg, 0.25±0.05 mg, 0.25±0.1 mg, or 0.25±0.15 mg of the nucleic acid is injected. In some embodiments, 0.1±0.05 mg, 0.25±0.15 mg, 0.5±0.4 mg, or 0.8±0.5 mg of the nucleic acid is injected. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non- viral vector.

[217] In some embodiments, the nucleic acid is injected into the dermis in a volume of 20- 1000 μL. In some embodiments, the nucleic acid is injected in a volume of 50±30 μL, 100±50 μL, 250±500 μL, 500±100 μL, or 750±250 mL. The injection(s) can be in a single location or in two or more locations. For injection in two or more locations, the injections can be adjacent to each other or in separate locations. For example, a subject can receive 500 μL in two adjacent injections of 250 μL each. Similarly, a subject can receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be sufficiently close such that the electroporation applicator electrodes are able to span both injection sites.

[218] In some embodiments, the nucleic acid is injected into the dermis at a depth of 0.01 to 0.25 cm. In some embodiments, the nucleic acid is injected at a depth of about 0.1 cm. In some embodiments, the nucleic acid is injected at a depth of less than 0.1 cm. In some embodiments, the nucleic acid is injected into the outermost living tissue layer, the stratum granulosum.

[219] In some embodiments, intratumoral electroporation (ID-EP) administration of nucleic acid comprises injecting into a tumor a solution containing the nucleic acid and electroporation of the injection site. The tumor can be, but is not limited to, a solid tumor, a cutaneous tumor, a subcutaneous tumor, or a visceral tumor.

[220] In some embodiments, 0.01-3 mg of the nucleic acid polypeptide is injected into the tumor. In some embodiments, 0.1±0.05 mg, 0.25±0.15 mg, 0.5±0.4 mg, 0.8±0.5 mg, 1±0.5 mg, or 2±0.5 mg of the nucleic acid is injected. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non- viral vector.

[221] In some embodiments, the nucleic acid is injected into the tumor in a volume of 20- 1000 μL. In some embodiments, the nucleic acid is injected in a volume of, 50±30 μL, 250±50 μL, 500±100 μL, 750±250 μL. In some embodiments, the nucleic acid is injected into the tumor in a volume corresponding to 25±10% of the calculated volume of the tumor. In some embodiments, the nucleic acid, at a concentration of 0.5-1.0 mg/ml is injected into the tumor in a volume corresponding to 25±10% of the calculated volume of the tumor. Injection into a tumor can include injection into margin tissue around the tumor. Injection into a tumor can include injecting the tumor, and optionally the margin tissue around the tumor, as uniformly as possible (i.e., dispersing the injection throughout the tumor and optionally the margin tissue around the tumor).

[222] For each of ID-EP, IM-EP, and IT-EP, the electrodes of the EP applicator are inserted such that the electrodes to span the injection site(s). The electrodes are typically set at about the same depth as the injection. In some embodiments, the electrodes of the EP applicator are set to the same depth ±0.3 cm as the injection. In some embodiments, the electrodes of the EP applicator are set to the same depth ±0.2 cm as the injection. In some embodiments, the electrodes of the EP applicator are set to the same depth ±0.1 cm as the inj ection. The electrodes of the EP applicator are inserted such that the electroporative pulse is administered to cells at the injection site.

[223] In some embodiments, 1-10 pulses at a field strength (E±) of 100-1500 V/cm and pulse width (duration) of about 0.1-20 ms are administered. In some embodiments, 1-10 pulses at a field strength (E±) of 400±100 V/cm and pulse width of about 1-20 ms are administered. In some embodiments, six to eight pulses at a field strength (E±) of about 400 V/cm and pulse width of about 10 ms at 0.3-1 -second intervals are administered. In some embodiments, 1-10 pulses at a field strength (E±) of 1300-1500 V/cm and pulse width of about 0.1 ms are administered

[224] In some embodiments, the pulse(s) is(are) administered at a field strength of about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, or about 600 V/cm. In some embodiments, the pulse(s) is(are) administered at a field strength of 400 ±250 V/cm, 400 ±100 V/cm, 400 ±75 V/cm, or 400 ±50 V/cm, or. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pulses are administered at a field strength of 100- 1500 V/cm. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pulses are administered at a field strength of about 400 V/cm. In some embodiments, pulse width is about

0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 msec. In some embodiments, pulse width is about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 msec and the filed strength is about 400 V/cm.

[225] A nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immune stimulator can be present in the same solution or different solutions. If present in separate solutions, the nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding an immune stimulator can be injected concurrently or sequentially. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding immune stimulator can be combined prior to injection.

[226] An isolated coronavirus antigenic polypeptide and an isolated immune stimulator polypeptide can be present in the same solution or different solutions. If present in separate solutions, the isolated coronavirus antigenic polypeptide and the isolated immune stimulator polypeptide can be injected concurrently or sequentially. The isolated coronavirus antigenic polypeptide and the isolate immune stimulator polypeptide can be combined prior to injection.

[227] A nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immune stimulator can be co-injected into the dermis, muscle, and/or tumor. A nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immune stimulator can combined prior to injecting into the dermis, muscle, and/or tumor. A nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immune stimulator can be present on the same vector ( e.g ., plasmid or RNA). When present on the same vector, the nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding the immune stimulator can be expressed from different promotors or from a single promoter, as in a multi cistronic vector.

[228] Intradermal administration includes, but is not limited to, injection into the dermis (e.g., the dermis of a shoulder, thigh, or buttocks. In some embodiments, intradermal administration comprises ID-EP.

[229] Intramuscular administration includes, but is not limited to, injection into muscle (e.g., a shoulder muscle (e.g, a deltoid muscle), a leg muscle (e.g, anterolateral thigh muscle, vastus lateralis, or gastrocnemius), or a muscle of the buttocks (e.g, dorsogluteal muscle). In some embodiments, intramuscular administration comprises IM-EP.

[230] Intratumoral administration includes, but is not limited to, injection into a tumor. In some embodiments, intratumoral administration comprises IT-EP.

[231] In some embodiments, the vaccination occurs in a single step comprising: intradermal administration, intramuscular administration, or intratumoral administration.

[232] In some embodiments, the vaccination occurs in two steps comprising: (a) administering a first dose by intradermal administration and administering a second dose by intramuscular administration; (b) administering a first dose by intradermal administration and administering a second dose by intratumoral administration; or (c) administering a first dose by intramuscular administration and administering a second dose by intratumoral administration; wherein the first dose comprises a coronavirus antigenic polypeptide, an immune stimulator, or a combination of a coronavirus antigenic polypeptide and an immune stimulator, and the second dose comprises a coronavirus antigenic polypeptide, an immune stimulator, or a combination of a coronavirus antigenic polypeptide and an immune stimulator. Intradermal administration can be performed prior to, subsequently to, or concurrently with intramuscular administration. Intradermal administration can be performed prior to, subsequent to, or concurrently with intratumoral administration. Intramuscular administration can be performed prior to, subsequently to, or concurrently with intratumoral administration. In some embodiments, the first dose comprises a coronavirus antigenic polypeptide and the second dose comprises an immune stimulator. In some embodiments, the first dose comprises a coronavirus antigenic polypeptide and the second dose comprises a coronavirus antigenic polypeptide and an immune stimulator. In some embodiments, both the first dose and the second dose comprise a coronavirus antigenic polypeptide and an immune stimulator. The two steps can occur within 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes of each other.

[233] In some embodiments, the first dose comprises 0.25-1 mg of a nucleic acid encoding a coronavirus antigenic polypeptide and 0.25-1 mg of a nucleic acid encoding an immune stimulator and the second dose comprises 0.25-1 mg of a nucleic acid encoding coronavirus antigenic polypeptide. In some embodiments, the first dose comprises 0.25-1 mg of a nucleic acid encoding a coronavirus antigenic polypeptide and the second dose comprises 0.25-1 mg of a nucleic acid encoding coronavirus antigenic polypeptide.

[234] The coronavirus antigenic polypeptide can be any of the coronavirus antigenic polypeptides described herein, including a nucleic acid encoding the coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide comprises a SARS- CoV-2 Spike polypeptide or antigenic fragment thereof. The immune stimulator can be any of the immune stimulators described herein, including a nucleic acid encoding the immune stimulator. In some embodiments, the second dose comprises a nucleic acid encoding IL-12. In some embodiments, the second dose comprises a nucleic acid encoding IL-12 and a Flt3L, a CXCL9, or an anti-CD3 half-BiTE.

[235] A subject can be given a single vaccination or a subject can be given a prime vaccination and one or more boost vaccinations. In some embodiments, prime and boost vaccinations may be administered to the subject in the same location on the subject ( e.g ., in the same limb) or in different locations (e.g., different limbs).

[236] In some embodiments, the methods comprise at least two rounds of administration (vaccinations) of at least one coronavirus antigenic polypeptide and at least one immune stimulator, a prime vaccination, and a boost vaccination. Administration of at least one coronavirus antigenic polypeptide and administration of at least one immune stimulator are both performed each round. The second round of administration (boost) can be performed 2 weeks to 12 months after the first round of administration (prime). In some embodiments, the second round of administration is performed 2 weeks to 6 months after, 14-63 days after, or 14-45 days months after the first round of administration. In some embodiments, the second round of administration is performed about 14 days, about 21 days, about 28 days, about 30 days, about 35 days, about 42 days, about 49 days, about 56 days, or about 63 days after the first round of administration. In some embodiments, the first round (prime) is administered on day 1 and the second round (boost) is administered on day 63 ±35 days, day 42 ±21 days, day 28 ±14 days, day 28 ±7 days, or day 21 ±7 days. In some embodiments, an additional round is administered about 5 years, about 4 years, about 3 years, about 2 years, about 1 year, about 9 months, 6 months, 63±35 days, 42±21 days, 28 ±14 days, or day 28 ±7 days, or 21 days ±7 days after the previous administration.

[237] In some embodiments, the first and subsequence boost administrations can be performed at intervals of 2-6 weeks. In some embodiments, the interval is 2 weeks ±5 days. In some embodiments, the interval is 3 weeks ±5 days. In some embodiments, the interval is 4 weeks ±5 days. In some embodiments, the interval is 5 weeks ±5 days. In some embodiments, the interval is 6 weeks ±5 days.

VI. Immune Response

[238] In some embodiments, the described vaccines induce one or more of: neutralizing antibody production, increased CD8± T cell proliferation and/or response, increased CD4± T cell proliferation and/or response, increased memory T cell proliferation and/or response, balanced Thl/Th2 antibody isotype responses, S-specific IgG2a and IgGl responses, protection against upper airway coronavirus infection, protection against lower airway coronavirus infection, protective immunity against coronavirus infection, prevention of symptomatic COVID-19 disease, prevention at least one symptom of COVID-19 disease, a decrease in severity or duration of one or more symptoms of COVID-19 disease, and prevention of severe COVID-19 disease.

[239] In some embodiments, the described methods can be used to: induce neutralizing antibody production, increase CD8± T cell proliferation and/or response, increase CD4± T cell proliferation and/or response, increase memory T cell proliferation and/or response, induce a balanced Thl/Th2 antibody isotype response, induce S-specific IgG2a and IgGl responses, protect against upper airway coronavirus infection, protect against lower airway coronavirus infection, induce protective immunity against coronavirus infection, prevent symptomatic COVID-19 disease, prevent at least one symptom of COVID-19 disease, decrease severity or duration of one or more symptoms of COVID-19 disease, or prevent severe COVID-19 disease after administration of a single dose.

[240] In some embodiments, the described methods can be used to: induce neutralizing antibody production, increase CD8± T cell proliferation and/or response, increase CD4± T cell proliferation and/or response, increase memory T cell proliferation and/or response, induce a balanced Thl/Th2 antibody isotype response, induce S-specific IgG2a and IgGl responses, protect against upper airway coronavirus infection, protect against lower airway coronavirus infection, induce protective immunity against coronavirus infection, prevent symptomatic COVID-19 disease, prevent at least one symptom of COVID-19 disease, decrease severity or duration of one or more symptoms of COVID-19 disease, or prevent severe COVID-19 disease after administration of a prime administration and at least one boost administration.

[241] In some embodiments, administration of the described vaccines protects against coronavirus infection in lung and/or nose. In some embodiments, administration of the described vaccines results in decreased viral replication in lungs and nose after challenge. In some embodiments, administration of the described vaccines results in decreased viral replication in nasal turbinates after challenge.

VII. Methods of Treating Cancer

[242] The pathogenic antigen and the immune stimulator can be administered to a subject by administering one or more nucleic acids encoding the pathogenic antigen and the immune stimulator. In some embodiments, the immune stimulator is an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is IL-12. In some embodiments, the nucleic acid encoding the immune stimulator is administered to the tumor and the nucleic acid encoding the pathogenic antigen is administered to the tumor, dermis, or a skeletal muscle.

[243] Described are methods for treatment of a tumor in a subj ect comprising, administering to the subject an effective dose of an expression vector encoding an immunostimulatory cytokine ( e.g ., IL-12) and an effective dose of an expression vector encoding a pathogenic antigen. The expression vector encoding the immunostimulatory cytokine is administered to the subject by injecting the expression vector into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue (IT-EP treatment). The expression vector encoding the pathogenic antigen is administered to the subject by (a) injecting the expression vector into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue (IT-EP treatment), (b) injecting the expression vector into the dermis and administering electroporation therapy to the dermis at the site of the injection (ID-EP treatment), or (c) injecting the expression vector into skeletal muscle and administering electroporation therapy to the muscle at the site of the injection (IM-EP treatment). The expression vector encoding the immunostimulatory cytokine can be administered before, concurrently with, or after administering the expression vector encoding the pathogenic antigen. For administering both the expression vector encoding the immunostimulatory cytokine and the expression vector encoding the pathogenic antigen a tumor, the expression vectors can be combined prior to injecting, injected separately, or the expression vectors can be present on a single plasmid, RNA, or viral vector.

[244] The treated tumor can be a cutaneous tumor, a subcutaneous tumor, or a visceral tumor. The tumor can be cancerous or non-cancerous. The tumor can be, but is not limited to, a solid tumor, a surface lesion, a non-surface lesion, a lesion within 15 cm of body surface, or a visceral lesion. In some embodiments, the described methods and expression vectors can be used to treat primary tumors as well as distant (i.e., untreated) tumors and metastases. In some embodiments, the described compositions and methods provide for reducing the size of or inhibiting the growth of a tumor, inhibiting the growth of cancer cells, inhibiting or reducing metastases, reducing or inhibiting the development of metastatic cancer, and/or reducing recurrence of cancer in a subject suffering from cancer. The tumor is not limited to a specific type of tumor or cancer.

[245] IT-EP pathogenic antigen (PA) therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective dose of a described expression vector encoding a pathogenic antigen and administering at least one electroporative pulse to the tumor. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[246] IT-EP S protein antigen therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective dose of a described expression vector encoding a SARS-CoV-2 S protein antigen and administering at least one electroporative pulse to the tumor. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[247] ID-EP pathogenic antigen (PA) therapy or treatment comprises injecting the dermis with an effective dose of a described expression vector encoding a pathogenic antigen and administering at least one electroporative pulse to dermis at the site of the injection. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[248] ID-EP S protein antigen therapy or treatment comprises injecting the dermis with an effective dose of a described expression vector encoding the S protein antigen and administering at least one electroporative pulse to dermis at the site of the injection. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[249] IM-EP pathogenic antigen (PA) therapy or treatment comprises injecting a skeletal muscle with an effective dose of a described expression vector encoding a pathogenic antigen and administering at least one electroporative pulse to skeletal muscle at the site of the injection. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[250] ID-EP S protein antigen therapy or treatment comprises injecting a skeletal muscle with an effective dose of a described expression vector encoding the S protein antigen and administering at least one electroporative pulse to skeletal muscle at the site of the injection. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[251] IT-EP immunostimulatory cytokine therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective dose of an expression vector encoding an immunostimulatory cytokine and administering at least one electroporative pulse to the tumor. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[252] IT-EP IL-12 therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective dose of an expression vector encoding IL-12 and administering at least one electroporative pulse to the tumor. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[253] IT-EP immunostimulatory cytokine-pathogenic antigen therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective dose of an expression vector encoding an immunostimulatory cytokine and an effective dose of an expression vector encoding a pathogenic antigen and administering at least one electroporative pulse to the tumor. The electroporative pulse can be performed using any known electroporation device suitable for use in a mammalian subject.

[254] The described expression vectors and methods are contemplated for use in subjects afflicted with cancer. Tumors treated with the methods of the present embodiment may be any of noninvasive, invasive, superficial, papillary, flat, metastatic, localized, unicentric, multicentric, low grade, and high grade tumors. These growths may manifest themselves as any of a lesion, polyp, neoplasm (e.g. papillary urothelial neoplasm), papilloma, malignancy, tumor (e.g. Klatskin tumor, hilar tumor, noninvasive papillary urothelial tumor, germ cell tumor, Ewing's tumor, Askin' s tumor, primitive neuroectodermal tumor, Ley dig cell tumor, Wilms' tumor, Sertoli cell tumor), sarcoma, carcinoma (e.g. squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma, hepatocellular carcinoma, invasive papillary urothelial carcinoma, flat urothelial carcinoma), lump, or any other type of cancerous or non-cancerous growth. The expression vectors and methods can be used to treat advanced, metastatic, or treatment refractory cancer.

[255] The term "cancer" includes a myriad of diseases generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. The cancer can be, but is not limited to, solid cancer, sarcoma, carcinoma, and lymphoma. The cancer can also be, but is not limited to, pancreas, skin, brain, liver, gall bladder, stomach, lymph node, breast, lung, head and neck, larynx, pharynx, lip, throat, heart, kidney, muscle, colon, prostate, thymus, testis, uterine, ovary, cutaneous, and subcutaneous cancers. Skin cancer can be, but is not limited to, melanoma and basal cell carcinoma. Breast cancer can be, but is not limited to, ER positive breast cancer, ER negative breast cancer, and triple negative breast cancer. In some embodiments, the described methods can be used to treat cell proliferative disorders. The term "cell proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. In some embodiments, the described methods can be used to treat a human. In some embodiments, the described methods can be used to treat non-human animals or mammals. A non-human mammal can be, but is not limited to, mouse, rat, rabbit, dog, cat, pig, cow, sheep, and horse.

[256] The expression vectors and methods described herein are contemplated for use in, e.g ., adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer) bladder cancer, benign and cancerous bone cancer (e.g. osteoma, osteoid osteoma, osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astrocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell) esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer, both melanoma and non-melanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

[257] The described methods can be used to cause one or more of the following: inflame a tumor, induce T cell infiltration to the tumor or tumor microenvironment (increase the number of tumor infiltrating lymphocytes (TILs)), enhance systemic T cell response, induce activation of T cells, induce activation of tumor-specific T cells, increase T cell response, increase antigen-specific T cell response, increase proliferation of T cells, increase proliferation of antigen-specific T cells, increase polyclonal T cells response, increased NK and/or NKT cells in a tumor or tumor microenvironment, enhance an immune response against treated and/or untreated tumors, decrease T cell exhaustion, increase lymphocyte and monocyte cell surface markers in one or more treated or untreated tumors, increase intratumoral levels of INFγ regulated genes in one or more treated or untreated tumors, increase proliferating effector memory T cells in the subject’s blood, increase short-lived effector cells in the subject’s blood, increase expression of genes present in activated natural killer cells in a cancerous tumor, increase expression of genes that function in antigen presentation in a cancerous tumor, increase expression of genes that function in T cell survival and T cell mediated cytotoxicity in a cancerous tumor, induce regression of treated and/or untreated tumors, induce debulking of a treated and/or untreated tumor, and improve response to a second therapy, such as, but not limited to, immune checkpoint inhibitor therapy. In some embodiments, treatment of cancer includes one or more of: enhancement of immune reaction to the tumor, tumor regression, metastatic tumor regression, increased survival of the subject, and increase tumor-free survival.

[258] In some embodiments, ID-EP or IT-EP pathogenic antigen therapy enhances an IL- 12 effect resulting in increased effective trafficking of tumor specific lymphocytes, increased tumor regression, decreased tumor volume, and/or increase survival or tumor-free survival.

A) Treatment Regimens/Cycles [259] The described cancer therapies can be administered at various intervals, depending upon such factors, for example, as the nature of the tumor, the condition of the subject, the size and chemical characteristics of the molecule and half-life of the molecule.

[260] In some embodiments, and IT-EP IL-12-pathogenic antigen therapy is given to the tumor on day 1 (±2 days), day 5 (±2 days), and day 8 (±2 days) of a cycle. In some embodiments, and IT-EP IL-12-pathogenic antigen therapy is given to the tumor on day 1 (±2 days) and day 5 (±2 days) of a cycle. In some embodiments, and IT-EP IL-12-pathogenic antigen therapy is given to the tumor on day 1 (±2 days) and day 8 (±2 days) of a cycle.

[261] A treatment cycle can comprise 1-6 IT-EP treatments. In some embodiments, a treatment cycle comprises 1, 2, or 3 IT-EP treatments. A cycle can be from about 1 week to about 6 weeks. In some embodiments, a cycle is 2 weeks. In some embodiments, a cycle is 3 weeks. In some embodiments, a cycle is 4 weeks. In some embodiments, a cycle is 5 weeks. In some embodiments, a cycle is 6 weeks.

[262] In some embodiments, a cycle comprises 1-3 IT-EP treatments. The treatments can occur on days 1 (± 2 days), 5 (± 2 days) and/or day 8 (± 2 days) (i.e., days 0 (± 2 days), 4 (± 2 days) and/or day 7 (± 2 days)). Each treatment can comprise one or more of IT-EP IL-12 therapy, IT-EP IL-12-pathogenic antigen therapy, IT-EP IL-12 therapy plus IT-EP pathogenic antigen therapy, and IT-EP IL-12 therapy plus ID-EP or IM-EP pathogenic antigen therapy.

[263] In some embodiments, a treatment can be administered every cycle or every other cycle. A cycle may be repeated such that 2 or more cycles are administered to a subject. Repeated cycles may be administered consecutively, alternated with one or more different cycles of treatment, or run concurrently with one or more difference cycles of treatment. Any of the above described treatments can be combined with other cancer therapies. For example, an IT-EP cycle can be combined with checkpoint inhibitor therapy.

B) Combination Therapy

[264] In some embodiments, a therapeutic method includes a combination therapy. A combination therapy comprises a combination of therapeutic molecules or treatments. Therapeutic treatments include, but are not limited to, electric pulse (i.e., electroporation), radiation, antibody therapy, checkpoint inhibitor therapy, and chemotherapy. Therapeutic electroporation can be combined with, or administered with, one or more additional therapeutic treatments. The one or more additional therapeutics can be delivered by systemic delivery, intratumoral injection, intratumoral injection with electroporation, and/or radiation. The one or more additional therapeutics can be administered prior to, concurrent with, or subsequent to the IT-EP IL12-pathogenic antigen therapy. VIII. Electroporation (EP):

[265] The described nucleic acids encoding pathogenic antigens and/or immune stimulators, can be delivered by electroporation. Nucleic acid-based vaccines can be delivered with viral and bacterial vectors but are often limited by poor transfection rates and/or rapidly cleared by neutralizing antibodies (MacGregor RR et al. “First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response.” J Infect Dis, 1998 178(1):91-100; and Lin F et al. “Optimization of electroporation- enhanced intradermal delivery of DNA vaccine using a minimally invasive surface device.” Hum Gene Ther Methods, 201223(3), 157-68). Electroporation (EP) is atechnique that applies electric pulses to transiently permeabilize a cell membrane, promoting uptake of macromolecules such as nucleic acids into the cell. In vivo EP has been used in several clinical trials to deliver DNA vaccines and drugs to various tissues (Draghia-Akli R et al. “ Gene and cell therapy: Therapeutic mechanisms and strategies. ” 2009). Electroporation has been shown to dramatically improve gene delivery (100- 1000-fold; Sardesai et al. 2011 and Livingston BD et al. “Comparative performance of a licensed anthrax vaccine versus electroporation based delivery of a PA encoding DNA vaccine in rhesus macaques.” Vaccine, 201028(4): 1056-61).

[266] The Apollo C-System (ACS), a medical electroporation device system, consists of three main components: an Electroporation Generator that generates electric pulses, a Sterile Applicator containing a 2 needle array, and an Applicator cable assembly that connects to the Electroporation Generator at the proximal end and connects to the Applicator at the distal end. The APOLLO-C Generator delivers electrical pulses of specific voltage and duration. The Generator delivers controlled electroporation (EP) pulses in a square wave pulse pattern, resulting in a transmembrane potential for electroporation to occur. The applicator electroporation parameters are pre-programmed into the applicator cable assembly via a memory chip which determines the applied pulse width, pulse interval, number of pulses, and applied potential for each electroporation sequence. The ACS Applicator is a sterile (gamma irradiated), single-use disposable device consisting of a plastic molded assembly with two stainless steel Trocar needles and a reusable cable assembly with a proximal connector that connects to the APOLLO-C Generator. The reusable cable assembly contains a memory chip which stores the specific pulsing parameters: applied pulse width, pulse interval, number of pulses, and applied potential for each electroporation therapy. The stainless steel Trocar needles are space 0.5 cm apart. The needle length is 1.5 cm and the needle insertion depth is adjustable by the user to between 0 and 1.5 cm. [267] Additional electroporation devices suitable for use with the described compounds, compositions, and methods include, but are not limited to, those described in U. S. Patent Nos. 7245963, 5439440, 6055453, 6009347, 9020605, and 9037230, and U.S. Patent Publication Nos. 2005/0052630, 2019/0117964, and patent applications PCT/US2019/030437 and US. Patent Applications Serial No. 16/269,022.

[268] In some embodiments, electroporation therapy comprises the administration of one or more voltage pulses. The nature of the electric field to be generated is determined by the nature of the tissue, the size of the selected tissue and its location. The voltage pulse that can be delivered to the tumor may be about 100 V/cm to about 1500V/cm. In some embodiments, the voltage pulse is about 700 V/cm to 1500 V/cm. In some embodiments, the voltage pulse may be about 600 V/cm, 650 V/cm, 700 V/cm, 750 V/cm, 800 V/cm, 850 V/cm, 900 V/cm, 950 V/cm, 1000 V/cm, 1050 V/cm, 1100 V/cm, 1150 V/cm, 1200 V/cm, 1250 V/cm, 1300 V/cm, 1350 V/cm, 1400 V/cm, 1450 V/cm, or 1500 V/cm. In some embodiments, the voltage pulse is about 10 V/cm to 700 V/cm. In some embodiments, the electric is about 100 V/cm, 150 V/cm, 200 V/cm, 250 V/cm, 300 V/cm, 350 V/cm, or 400 V/cm, 450 V/cm, 500 V/cm, 550 V/cm, 600 V/cm 650 V/cm, or 700 V/cm.

[269] The pulse duration of the electroporative pulse may be from 10 psec to 1 second. In some embodiments, the pulse duration is from about 10 psec to about 100 milliseconds (ms). In some embodiments, the pulse duration is 100 psec, 1 ms, 10 ms, or 100 ms. The interval between pulses sets can be any desired time, such as one second. The waveform, electric field strength and pulse duration may also depend upon the type of cells and the type of molecules that are to enter the cells via electroporation.

[270] The waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train, a bipolar oscillating pulse train, or a combination of any of these forms. Square wave electroporation systems deliver controlled electric pulses that rise quickly to a set voltage, stay at that level for a set length of time (pulse length), and then quickly drop to zero.

[271] 1 to 100 pulses may be administered. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pulses are administered. In some embodiments, 6 pulses are administered. In some embodiments, 6x0.1 msec pulses are administered. In some embodiments, 6 pulses are administered. In some embodiments, 6x0.1 msec pulses are administered at 1300-1500 V/cm. In some embodiments 8 pulses are administered. In some embodiments 8x10 msec pulses are administered. In some embodiments 8x10 msec pulses are administered at 300-500 V/cm. [272] In some embodiments, the EP applicator is inserted such that the electrodes span the nucleic acid injection site.

[273] The electroporation device can comprise a single needle electrode, a pair of needle electrode, or a plurality or array of needle electrodes. In some embodiments, the electroporation device an comprise a hypodermic needle or equivalent. In some embodiments, the electroporation device can comprise an electro-kinetic device ("EKD device") able to produce a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters.

IX. Listing of embodiments

[274] 1. A method of eliciting an immune response against a pathogen in a subject comprising: administering to the subject an effective dose of a pathogenic antigen and an effective dose of an immune stimulator, wherein the immune stimulator comprises an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE.

[275] 2. The method of embodiment 1, wherein the pathogen is a coronavirus is selected from the group consisting of: a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS-CoV, and a SARS-CoV-2.

[276] 3. The method of embodiment 2, wherein the pathogenic antigen is a coronavirus antigenic polypeptide comprising a coronavirus spike protein or an antigenic fragment thereof.

[277] 4. The method of embodiment 3, wherein the coronavirus spike protein or the antigenic fragment thereof comprises an extracellular domain of the coronavirus spike protein or an antigenic fragment thereof.

[278] 5. The method of embodiment 3 or 4, wherein the coronavirus spike protein or the antigenic fragment thereof comprises a SARS-CoV-2 spike protein or an antigenic fragment thereof.

[279] 6. The method of embodiment 5, wherein the SARS-CoV-2 spike protein or an antigenic fragment thereof comprises amino acids 1-1208, 14-1208, or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 or a polypeptide having at least 90% identity to amino acids 1 to 1208, 14-1208, or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 33.

[280] 7. The method of any one of embodiments 3-6, wherein the coronavirus spike protein or the antigenic fragment thereof comprises a modified coronavirus spike protein or an antigenic fragment thereof. [281] 8. The method of embodiment 7, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises one or more of: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain, and an affinity tag.

[282] 9. The method of embodiments 7 or 8, wherein modified coronavirus spike protein or the antigenic fragment thereof comprises one or more mutations that disrupt an internal peptidase cleavage site and/or one or more mutations that stabilize the protein in a prefusion conformation.

[283] 10. The method of embodiment 9, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises proline substitutions at amino acids corresponding to amino acid positions 986 and 987 of SEQ ID NO: 1.

[284] 11. The method of any one of embodiments 1-10, wherein administering to the subject the effective dose of the pathogenic antigen comprises administering to the subject an isolated pathogenic antigen, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

[285] 12. The method of any one of embodiments 1-10, wherein administering to the subject the effective dose of the pathogenic antigen comprises administering to the subject a nucleic acid encoding the pathogenic antigen and/or administering to the subject the effective dose of the immune stimulator comprises administering to the subject a nucleic acid encoding the immune stimulator, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide

[286] 13. The method of any one of embodiments 1-12, wherein the immune stimulator comprises interleukin- 12 (IL-12).

[287] 14. The method of embodiment 13, wherein administering to the subject the effective dose of the immune stimulator comprises administering to the subject an isolated IL- 12 polypeptide.

[288] 15. The method of embodiment 13, wherein administering to the subject the effective dose of the immune stimulator comprises administering to the subject a nucleic acid encoding IL-12.

[289] 16. The method of embodiment 12 or 15, wherein the nucleic acid comprises a plasmid or an mRNA.

[290] 17. The method of embodiment 15 or embodiment 16, wherein the nucleic acid encoding IL-12 comprises: a nucleic acid sequence encoding an IL-12 p40-IL-12 p35 fusion protein, a nucleic acid sequence encoding an IL-12 p35-IL-12 p40 fusion protein, or a nucleic acid sequence encoding an IL-12 p35 subunit and an IL-12 p40 subunit separated by an internal ribosome entry site (IRES) element or a 2A peptide skipping motif.

[291] 18. The method of 17, wherein the nucleic acid encoding IL-12 comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[292] 19. The method embodiment 17, wherein the nucleic acid encoding IL-12 further encodes a Fms-like tyrosine kinase 3 ligand (Flt3L), a C-X-C Motif Chemokine ligand 9 (CXCL9), or an anti-CD3 half-BiTE.

[293] 20. The method embodiment 19, wherein the nucleic acid encoding IL-12 comprises:

(a) a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 22;

(b) a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 22;

(c) a nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 21; or

(d) a nucleic acid sequence having at least 90% identify to the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 21.

[294] 21. The method embodiment 20, wherein the nucleic acid encoding IL-12 comprises:

(a) a nucleic acid sequence encoding a polypeptide having the amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 9;

(b) a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9

(c) a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8; or

(d) a nucleic acid sequence having at least 90% identify to the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.

[295] 22. The method of any one of embodiments 15-21, wherein administering to the subject the effective dose of the coronavirus antigenic polypeptide and the effective dose of the immune stimulator comprises:

(a) administering to the subject a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and a first effective dose of the nucleic acid encoding IL-12 by intradermal administration; or

(b) administering to the subject a first effective dose of the nucleic acid encoding IL- 12 and optionally a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof by intradermal administration; and administering to the subject a second effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof by intramuscular administration.

[296] 23. The method of embodiment 22, wherein the first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and the first effective dose of the nucleic acid encoding IL-12 are injected into the same site.

[297] 24. The method of embodiment 23, wherein first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and the first effective dose of the nucleic acid encoding IL-12 are combined prior to intradermal administration.

[298] 25. The method of any one of embodiments 15-21, wherein administering to the subject the effective dose of the coronavirus antigenic polypeptide and the effective dose of the immune stimulator comprises:

(a) administering to the subject a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and a first effective dose of the nucleic acid encoding IL-12 by intratumoral administration;

(b) administering to the subject a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof by intradermal or intramuscular administration and a first effective dose of the nucleic acid encoding IL-12 by intratumoral administration; or

(c) administering to the subject a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and a first effective dose of the nucleic acid encoding IL-12 by intratumoral administration and a second first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof by intradermal or intramuscular administration;

[299] wherein the subject has a cancerous tumor.

[300] 26. The method of any one of embodiments 22-25, wherein the coronavirus spike protein or the antigenic fragment thereof comprises the SARS-CoV-2 spike protein or the antigenic fragment thereof

[301] 27. The method of any one of embodiments 22-26, wherein the intradermal administration comprises intradermal electroporation (ID-EP), the intramuscular administration comprises intramuscular electroporation (IM-EP), and the intratumoral administration comprises intratumoral electroporation (IT-EP).

[302] 28. The method of embodiment 27, wherein ID-EP, IM-EP, and IT-EP comprise administration of at least one voltage pulse having a field strength of about 100-1500 V/cm. [303] 29. The method of embodiment 28, wherein ID-EP, IM-EP, and IT-EP comprise administration of at least one voltage pulse having a field strength of about 400 V/cm and a duration of about 10 milliseconds.

[304] 30. The method of any one of embodiments 1-29 further comprising administering to the subject a second effective dose of the pathogenic antigen and a second effective dose of the immune stimulator about 14 days to 6 months after administering the effective dose of the pathogenic antigen and the effective dose of the immune stimulator, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

[305] 31. The method of any one of embodiments 1-30, wherein the immune response comprises a cellular immune response, a humoral immune response, or both a cellular and humoral immune response.

[306] 32. The method of any one of embodiments 1-31, wherein the immune response comprises one or more of: neutralizing antibody production, increased CD8+ T cell proliferation and/or response, increased CD4+ T cell proliferation and/or response, increased memory T cell proliferation and/or response, balanced Thl/Th2 antibody isotype responses, a S-specific IgG2a and IgGl response, protection against upper airway coronavirus infection, protection against lower airway coronavirus infection, protective immunity against coronavirus infection, prevention of symptomatic COVID-19 disease, prevention at least one symptom of COVID-19 disease, a decrease in severity or duration of one or more symptoms of COVID-19 disease, and prevention of severe COVID-19 disease.

[307] 33. A method of eliciting an immune response against a SARS-CoV-2 virus in a subject comprising:

(a) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12 by ID-EP;

(b) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof by IM-EP and a first effective dose of a nucleic acid encoding IL-12 by ID-EP; or

(c) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or the antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12 by ID-EP; and administering to the subject a second effective dose of the nucleic acid encoding the SARS-CoV-2 spike protein or the antigenic fragment thereof by IM-EP. [308] 34. A method of eliciting an immune response against a SARS-CoV-2 virus in a subject having cancer comprising:

(a) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12 by IT-EP;

(b) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof by ID-EP or IM-EP and a first effective dose of a nucleic acid encoding IL-12 by IT-EP; or

(c) administering to the subject a first effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12 by IT-EP and a second effective dose of the nucleic acid encoding the SARS-CoV-2 spike protein or the antigenic fragment thereof by ID-EP or IM-EP.

[309] 35. A vaccine for generating an immune response against a pathogen in a subject comprising: an effective dose of a pathogenic antigen or a nucleic acid encoding the pathogenic antigen and an effective dose of an immune stimulator or a nucleic acid encoding the immune stimulator, wherein the immune stimulator comprises an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE or a combination thereof.

[310] 36. The vaccine of embodiment 35, wherein the pathogen is a coronavirus is selected from the group consisting of: a betacoronavirus, a lineage A betacoronavirus (subgenus Embecovirus ), a lineage B betacoronavirus (subgenus Sarbecovirus ), a lineage C betacoronavirus (subgenus Merbecovirus ), a lineage D betacoronavirus (subgenus Nobecovirus ), a SARS-CoV, a MERS-CoV, a SARS-CoV-2.

[311] 37. The vaccine of embodiment 35 or embodiment 36, wherein the pathogenic antigen is a coronavirus antigenic polypeptide comprising a coronavirus spike protein or an antigenic fragment thereof.

[312] 38. The vaccine of embodiment 37, wherein the coronavirus spike protein or the antigenic fragment thereof comprises an extracellular domain of the coronavirus spike protein or an antigenic fragment thereof.

[313] 39. The vaccine of embodiment 37 or 38, wherein the coronavirus spike protein or the antigenic fragment thereof comprises a SARS-CoV-2 spike protein or an antigenic fragment thereof.

[314] 40. The vaccine of embodiment 39, wherein the SARS-CoV-2 spike protein or an antigenic fragment thereof comprises amino acids 1-1208, 14-1208, or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 or a polypeptide having at least 90% identity to amino acids 1 to 1208, 14-1208, or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 33.

[315] 41. The vaccine of any one of embodiments 37-40, wherein the coronavirus spike protein or the antigenic fragment thereof comprises a modified coronavirus spike protein or an antigenic fragment thereof.

[316] 42. The vaccine of embodiment 41, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises one or more of: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain, and an affinity tag.

[317] 43. The vaccine of embodiment 41 or embodiment 42, wherein modified coronavirus spike protein or the antigenic fragment thereof comprises one or more mutations that disrupt an internal peptidase cleavage site and/or one or more mutations that stabilize the protein in a prefusion conformation.

[318] 44. The vaccine of embodiment 43, wherein modified coronavirus spike protein or the antigenic fragment thereof comprises proline substitutions at amino acids corresponding to amino acid positions 986 and 987 of SEQ ID NO: 1.

[319] 45. The vaccine of any one of embodiments 35-43, the coronavirus antigenic polypeptide comprises an isolated coronavirus antigenic polypeptide.

[320] 46. The vaccine of any one of embodiments 35-43, the coronavirus antigenic polypeptide comprises the nucleic acid encoding the coronavirus antigenic polypeptide.

[321] 47. The vaccine of any one of embodiments 35-46, the immune stimulator comprises an isolated immune stimulator polypeptide.

[322] 48. The vaccine of any one of embodiments 35-47, wherein the immune stimulator comprises interleukin- 12 (IL-12).

[323] 49. The vaccine of embodiment 48, wherein the IL-12 comprises a nucleic acid encoding IL-12.

[324] 50. The vaccine of embodiment 46 or 49, wherein the nucleic acid comprises a plasmid or an mRNA.

[325] 51. The vaccine embodiment 49, wherein the nucleic acid encoding IL-12 comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[326] 52. The vaccine of embodiment 49, wherein the nucleic acid encoding IL-12 comprises: a nucleic acid sequence encoding an IL-12 p40-IL-12 p35 fusion protein, a nucleic acid sequence encoding an IL-12 p35-IL-12 p40 fusion protein, or a nucleic acid sequence encoding an IL-12 p35 subunit and an IL-12 p40 subunit separated by an internal ribosome entry site (IRES) element or a 2A peptide skipping motif. [327] 53. The vaccine embodiment 52, wherein the nucleic acid encoding IL-12 further encodes a Fms-like tyrosine kinase 3 ligand (Flt3L), a C-X-C Motif Chemokine ligand 9 (CXCL9), or an anti-CD3 half-BiTE.

[328] 54. The vaccine embodiment 53, wherein the nucleic acid encoding IL-12 comprises:

(a) a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 22;

(b) a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 22;

(c) a nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 21; or

(d) a nucleic acid sequence having at least 90% identify to the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 21.

[329] 55. The vaccine embodiment 54, wherein the nucleic acid encoding IL-12 comprises:

(a) a nucleic acid sequence encoding a polypeptide having the amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 9;

(b) a nucleic acid sequence encoding a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9

(c) a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8; or

(d) a nucleic acid sequence having at least 90% identify to the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.

[330] 56. The vaccine of any one of embodiments 35-54, wherein the pathogenic antigen or the nucleic acid encoding the pathogenic antigen and the immune stimulator or the nucleic acid encoding the immune stimulator are formulated for intradermal, intramuscular, and/or intratumoral administration, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

[331] 57. The vaccine of any one of embodiments 35-56, wherein the pathogenic antigen or the nucleic acid encoding the pathogenic antigen and the immune stimulator or the nucleic acid encoding the immune stimulator are combined, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

[332] 58. The vaccine of any one of embodiments 35-57, wherein the vaccine comprises the nucleic acid encoding the pathogenic antigen and the nucleic acid encoding the immune stimulator, wherein the nucleic acid encoding the pathogenic antigen and the nucleic acid encoding the immune stimulator are formulated for ID-EP, IM-EP, and/or IT-EP, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

[333] 59. The vaccine of embodiment 58, wherein the nucleic acid encoding the pathogenic antigen is encoded on a first expression vector and the nucleic acid encoding the immune stimulator is encoded on a second expression vector.

[334] 60. The vaccine of embodiment 59, wherein the nucleic acid encoding the pathogenic antigen and the nucleic acid encoding immune stimulator are encoded on a single expression vector.

[335] 61. The vaccine of any one of embodiments 35-60, wherein the vaccine comprises

(a) a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by ID-EP;

(b) a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by IT-EP;

(c) a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by ID-EP and a second effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide formulated for administration by IM-EP;

(d) a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by ID-EP and a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide formulated for administration by IM-EP;

(e) a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide formulated for administration by ID-EP or IM-EP and a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by IT-EP;

(f) a first effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide and a first effective dose of the nucleic acid encoding the immune stimulator formulated for administration by IT-EP and a second effective dose of the coronavirus antigenic polypeptide formulated for administration by ID-EP or IM-EP.

[336] 62. The vaccine of any one of embodiments 35-61, wherein

(a) the effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide and the effective dose of the immune stimulator or the nucleic acid encoding the immune stimulator are provided in separate containers in solutions or as lyophilized powders; (b) the effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide and the effective dose of the immune stimulator or the nucleic acid encoding the immune stimulator are provided are provided together in a container in a solution or as a lyophilized powder; or

(c) the first effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator or the nucleic acid encoding the immune stimulator are provided are provided together in a first container in a solution or as a lyophilized powder, and the second effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide is provided in a second container in a solution or as a lyophilized powder.

[337] 63. The vaccine of any one of embodiments 35-62, wherein the effective dose of the pathogenic antigen or the nucleic acid encoding the pathogenic antigen and the effective dose of the immune stimulator or the nucleic acid encoding the immune stimulator, are independently provided with one or more pharmaceutically acceptable carriers and/or excipients.

[338] 64. The vaccine of any one of embodiments 35-63, wherein the nucleic acid encoding the pathogenic antigen comprises a nucleic acid encoding the SARS-CoV-2 spike protein or the antigenic fragment thereof and the immune stimulator comprises the nucleic acid encoding IL-12.

[339] 65. A method of reducing the likelihood or severity of pathogen infection in a patient, the method comprising administering to the patient an immunogenic composition comprising the vaccine of any one of embodiments 35-64.

[340] 66. A method of reducing the likelihood or severity of pathogen infection in a patient comprising the steps of: (a) administering to the patient a first immunogenic composition comprising the vaccine of any one of embodiments 35-64; (b) waiting for a predetermined amount of time to pass after step (a); and (c) administering to the patient a second immunogenic composition comprising the vaccine of any one of embodiments 35-63.

[341] 67. A method of eliciting an immune response against a pathogen in a subject diagnosed with cancer comprising: administering to the subject by intratumoral electroporation an effective dose of a nucleic acid encoding a pathogenic antigen and an effective dose of a nucleic acid encoding an immune stimulator, wherein the immune stimulator comprises an immunostimulatory cytokine, a genetic adjuvant, or an anti-CD3 half-BiTE, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide. [342] 68. The method of embodiment 67, wherein the coronavirus antigenic polypeptide comprises a SARS-CoV-2 spike protein or the antigenic fragment thereof and the immune stimulator comprises IL-12.

[343] 69. An expression vector comprising: a first nucleotide sequence encoding an immunostimulatory cytokine and a second nucleotide sequence encoding a pathogenic antigen, wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to a translation modification element.

[344] 70. The expression vector of embodiment 69, wherein the first nucleotide sequence and the second nucleotide sequence are operatively linked to a promoter.

[345] 71. The expression vector of embodiment 70, wherein the promoter is selected from the group consisting of: CMV promoter, mPGK, SV40 promoter, b-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, and EFla promoter.

[346] 72. The expression vector any one of embodiments 69-71, wherein the immunostimulatory cytokine comprises an interleukin.

[347] 73. The expression vector of embodiment 72, wherein the interleukin comprises IL- 12

[348] 74. The expression vector of embodiment 73, wherein the nucleotide sequence encoding IL-12 comprises an IL-12 p35 coding sequence and an IL-12 p40 coding sequence operably linked to a translation modification element.

[349] 75. The expression vector of any one of embodiments 69-74, wherein the pathogenic antigen comprises a viral antigen.

[350] 76. The expression vector of embodiment 75, wherein the viral antigen comprises a coronavirus spike protein antigen.

[351] 77. The expression vector of embodiment 76, wherein the coronavirus spike protein antigen comprises a SARS-CoV-2 spike protein or antigenic fragment thereof.

[352] 78. The expression vector of any one of embodiments 69-77, wherein the expression vector comprises the formula represented by:

P-A-T ¹ -B-T ² -B' or P-T ¹ -B-T ² -B'-A

[353] wherein P is the promoter, A encodes the pathogenic antigen, T ¹ and T ² are translation modification elements, B encodes IL-12 p35, and B' encodes IL-12 p40.

[354] 79. The expression vector of embodiment 78, wherein T ¹ and T ² encode 2A peptides selected from the group consisting of: a P2 A peptide, a T2A peptide, a E2 A peptide, and a F2A peptide.

[355] 80. A plasmid for expressing an immunostimulatory cytokine and a pathogenic antigen comprising the expression vector of any one of embodiments 69-79.

[356] 81. The expression vector of any one of embodiments 69-79 or the plasmid of embodiment 80, for use in treating cancer in a subject.

[357] 82. The expression vector of any one of embodiments 69-79 or the plasmid of embodiment 80, wherein the expression vector or the plasmid is formulated for intratumoral electroporation therapy.

[358] 83. A method of treating a subj ect having a tumor comprising inj ecting an effective dose of an expression vector encoding an immunostimulatory cytokine into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue and

(a) injecting an effective dose of an expression vector encoding a pathogenic antigen into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue;

(b) injecting an effective dose of the expression vector encoding the pathogenic antigen into the dermis and administering electroporation therapy to the dermis at the site of the injection; or

(c) injecting an effective dose of the expression vector encoding the pathogenic antigen into skeletal muscle and administering electroporation therapy to the muscle at the site of the injection, thereby reducing the size of or inhibiting the growth of a tumor, inhibiting the growth of cancer cells, inhibiting or reducing metastases, reducing or inhibiting the development of metastatic cancer, and/or reducing recurrence of cancer in the subject.

[359] 84. The method of embodiment 83, wherein the expression vector encoding an immunostimulatory cytokine and the expression vector encoding the pathogenic antigen are operably linked to a translation modification element.

[360] 85. The method of embodiment 84, wherein the expression vector encoding an immunostimulatory cytokine and the expression vector encoding the pathogenic antigen are operatively linked to a promoter.

[361] 86. The method of embodiment 85, wherein the promoter is selected from the group consisting of: CMV promoter, mPGK, SV40 promoter, b-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, and EFla promoter.

[362] 87. The method any one of embodiments 83-86, wherein the immunostimulatory cytokine comprises an interleukin.

[363] 88. The method of embodiment 87, wherein the interleukin comprises IL-12.

[364] 89. The method of embodiment 88, wherein the nucleotide sequence encoding IL-12 comprises an IL-12 p35 coding sequence and an IL-12 p40 coding sequence operably linked to a translation modification element.

[365] 90. The method of any one of embodiments 83-89, wherein the pathogenic antigen comprises a viral antigen.

[366] 91. The method of embodiment 90, wherein the viral antigen comprises a coronavirus spike protein antigen.

[367] 92. The method of embodiment 91, wherein the coronavirus spike protein antigen comprises a SARS-CoV-2 spike protein or antigenic fragment thereof.

[368] 93. The method of any one of embodiments 83-92, wherein the expression vector comprises the formula represented by:

P-A-T ¹ -B-T ² -B' or P-T ¹ -B-T ² -B'-A

[369] wherein P is the promoter, A encodes the pathogenic antigen, T ¹ and T ² are translation modification elements, B encodes IL-12 p35, and B' encodes IL-12 p40.

[370] 94. The method of embodiment 93, wherein T ¹ and T ² encode 2A peptides selected from the group consisting of: a P2A peptide, a T2A peptide, a E2A peptide, and a F2A peptide

[371] 95. The method of any one of embodiments 83-94, wherein the electroporation therapy comprises administration of at least one voltage pulse over a duration of about 100 microseconds to about 1 millisecond.

[372] 96. The method of embodiment 95, wherein the least one voltage pulse comprises 1-10 voltage pulses.

[373] 97. The method of embodiment 96, wherein the least one voltage pulse comprises 6-8 voltage pulses.

[374] 98. The method of any one of embodiments 95-97, wherein the at least one voltage pulse has a field strength of about 200 V/cm to about 1500 V/cm.

[375] 99. The method of any one of embodiments 83-98, wherein the first and second nucleic acids are injected to the tumor into the tumor and the electroporation therapy is administered on day 1±2 days, day 5±2 days, and/or day 8±2 days of a cycle.

[376] 100. The method of embodiment 99, wherein the cycle is 3-6 weeks.

[377] 101. The method of any one of embodiments 83-100, further comprising administering to the subject at least one additional therapeutic.

[378] 102. The method of any one of embodiments 83-101, wherein the method results in one or more or: increased tumor infiltrating lymphocytes, increased activation and/or proliferation of T cells, regression of the treated tumor, and regression of one or more untreated tumors.

[379] 103. The method of any one of embodiments 83-102, wherein the method comprises injecting the expression vector encoding the immunostimulatory cytokine and the expression vector encoding pathogenic antigen into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue.

[380] 104. The method of any one of embodiments 83-102, wherein the method comprises injecting the expression vector encoding the immunostimulatory cytokine into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue and injecting the expression vector encoding the pathogenic antigen into the dermis and administering electroporation therapy to the dermis at the site of the injection.

[381] 105. The method of any one of embodiments 83-102, wherein the method comprises injecting the expression vector encoding the immunostimulatory cytokine into the tumor, tumor microenvironment, and/or tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or the tumor margin tissue and injecting the expression vector encoding the pathogenic antigen into the skeletal muscle and administering electroporation therapy to the skeletal muscle at the site of the injection.

[382] 106. The method of any one of embodiment 83-105, wherein the method further comprises administering an immune checkpoint inhibitor to the subject.

EXAMPLES

Example 1. Sequence alignment

[383] Approximately one hundred DNA sequences of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) cases in the USA (Global Initiative on Sharing All Influenza Data (GISAID)) were evaluated. All sequences of the ‘S’ gene, coding for spike glycoprotein, were aligned to reference gene ID 43740568 (NC_045512.2 bases 21563- 25384). Multi-sequence alignment of these -100 gene sequences resulted in no alignment gaps ( i.e . , no indels or significant structural alterations). The aligned DNA sequences were translated to amino acids and evaluated for percent identity to the reference protein sequence. All positions of spike glycoprotein had 100% identity to the reference sequence with the exception of amino acids H49 (99%), F157 (99%), G181 (99%), V483 (96%), D614 (97%), and H655 (99%). Notably, V483 is in the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). V483 is not a contact residue, and occurs as an alanine substitution, and is of low conservation among homologous coronavirus strains.

Example 2. Phase I trial of SARS-CoV-2 spike protein (S protein) with pIL-12 nucleic acid vaccine in human subjects

[384] Described is a Phase 1, study to evaluate the safety and profile of S protein in combination with IL-12 nucleic acid vaccine administered by electroporation. Nucleic acids encoding S protein and IL-12 are administered by ID-EP and/or IM-EP. Subjects are given a prime vaccination, and a boost vaccination 3-4 weeks after the prime vaccination. Subjects are divided into age groups of 18-50 and >50 years old.

Table 1. Vaccines combining SARS-CoV-2 S protein with IL-12 and healthy subjects aged 18-

50 and 50 years. [385] pIL-12 (TAVO; pIL-12): Plasmid encoding interleukin- 12 (IL-12) can be GLP grade and formulated in phosphate buffered saline (PBS) for direct intradermal injection followed by in vivo electroporation (EP). The plasmid is supplied at a concentration of 3.3 mg/mL in a fill volume of 0.225 mL.

[386] SARS-CoV-2 spike protein (S protein): Plasmid encoding S protein can be GLP grade and formulated in phosphate buffered saline (PBS) for direct intradermal and/or intramuscular injection followed by in vivo electroporation (EP). The plasmid is supplied at a concentration of 1.67 mg/mL in a fill volume of 0.7 mL.

[387] Electroporation can be performed using, for example, the Apollo C-System (ACS) electroporation apparatus or the IGEA Cliniporator electroporation system. [388] Electroporation (EP) is co-localized at the site of each injection. Electroporation can be administered concurrently with plasmid injection, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes. In some embodiments, electroporation (EP) comprises 6-8 10 millisecond pulses at a field strength of 400 V/cm.

[389] Administration: Prior to plasmid injection, local anesthetics such as 1% lidocaine may be injected around the planned injection site to obtain local anesthesia. Alternative local anesthetics may be used as clinically indicated and deemed clinically suitable. In addition, the patient may be given analgesics, anxiolytics, or conscious sedation. Icepacks to cool and numb an area are also acceptable.

[390] In some embodiments, a suitable site which readily allows for intramuscular (IM) delivery at 0.5 to 1.5 cm depth (such as, but limited to, the shoulder) and adjacent intradermal (ID) delivery 2-3 cm away from the IM injection site are selected. In some embodiments, the ID-EP site can be more than 10 cm from the IM-EP site In some embodiments, the ID-EP site is at a contralateral site from the IM-EP. In some embodiments, the vaccination occurs in two steps: intradermal injection + EP ( e.g ., right shoulder); followed by intramuscular injection + EP to the matched contralateral site (e.g. left shoulder deltoid muscle). In some embodiments, ID-EP is administered prior to IM-EP.

[391] Intramuscular administration (e.g, IM-EP) can be performed concurrently with intradermal administration (e.g, ID-EP), prior to intradermal administration, or subsequent to intradermal administration. Intramuscular administration can be performed within 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, or 30 minutes of intradermal administration. In some embodiments, intradermal administration is performed about 5 minutes after intramuscular administration.

[392] Intradermal administration can be performed at a site that is within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm of the site of intramuscular administration. In some embodiments, intradermal administration is performed at a site that is within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm of the site of intramuscular administration.

[393] The vaccine may be administered to a subject in the outpatient setting.

Example 3. Intramuscular, intradermal, and intratumoral ejection and electroporation of nucleic acids encoding a coronavirus S protein antigenic polypeptide and IL-12.

A. Intramuscular electroporation (IM-EP) administration of nucleic acid encoding coronavirus S protein antigenic polypeptide, IL12, or a combination of coronavirus S protein antigenic polypeptide and IL12. [394] IM-EP administration of nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 comprises injecting into skeletal muscle tissue a solution containing the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 and electroporation of the injection site.

[395] In some embodiments, 0.1-3 mg of nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL12 is injected. In some embodiments, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.1 mg, 0.1±0.01 mg, or 0.1±0.05 mg of the nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.25 mg, 0.25±0.01 mg, 0.25±0.05 mg, 0.25±0.1 mg, or 0.25±0.15 mg of the nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.5 mg, 0.5±0.1 mg, 0.5±0.2 mg, 0.5±0.3 mg, or 0.5±0.4 mg of the nucleic acid a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.8 mg, 0.8±0.1 mg, 0.8±0.2 mg, 0.8±0.3 mg, or 0.8±0.4 mg, or 0.8±0.5 mg of the nucleic acid a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. The coronavirus S protein antigenic polypeptide can be, but is not limited to, a SARS-CoV-2 spike protein or an antigenic fragment thereof. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non-viral vector. The above indicated amounts can be for each of a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL- 12, or for both a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12 together (whether on separate vectors or the same vector).

[396] In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is injected in a volume of 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 900, or 1000 μL. In some embodiments, the nucleic acid is injected in a volume of 50 μL, 50±10 μL, 100 μL, 100±10 μL, 100±25 μL, or 100±50 μL. In some embodiments, the nucleic acid is injected in a volume of 250 μL, 250±10 μL, 250±25 μL, or 250±50 μL. In some embodiments, the nucleic acid is injected in a volume of 500 μL, 500±10 μL, 500±25 μL, or 500±50 μL. In some embodiments, the nucleic acid is injected in a volume of 750 μL, 750±10 μL, 750±20 μL, 750±30 μL, 750±40 μL, 750±50 μL. [397] In some embodiments, the nucleic acid is injected at a depth of 0.5 to 1.5 cm. In some embodiments, the nucleic acid is injected at a depth of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 cm, or greater than 1.5 cm. In some embodiments, the nucleic acid is injected at a depth of 1.0 cm, 1.0±0.1 cm, 1.0±0.2 cm, 1.0±0.3 cm, 1.0±0.4 cm, 1.0±0.5 cm, 1.5 cm, 1.5±0.1 cm, 1.5±0.2 cm, 1.5±0.3 cm, 1.5±0.4 cm, or 1.5±0.5 cm. The EP applicator is set to the same depth ±0.1 cm as the injection. The EP applicator is inserted such that the electrodes span the nucleic acid injection site. In some embodiments, 1-10 pulses at a field strength (E±) of 400±100 V/cm and pulse width of about 1-20 ms at about 1-second intervals are administered. In some embodiments, eight pulses at a field strength (E±) of 400 V/cm and pulse width of about 10 ms at 1 -second intervals are administered. In some embodiments, the IM-EP site is the deltoid muscle.

B. Intradermal electroporation (ID-EP) administration of nucleic acid encoding a coronavirus S protein antigenic polypeptide IL12, or a combination of coronavirus S protein antigenic polypeptide and IL12.

[398] ID-EP administration of nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 comprises injecting into dermis a solution containing the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 and electroporation of the injection site.

[399] In some embodiments, 0.1-3 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.1 mg, 0.1±0.01 mg, or 0.1±0.05 mg of the nucleic acid encoding coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.25 mg, 0.25±0.01 mg, 0.25±0.05 mg, 0.25±0.1 mg, or 0.25±0.15 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.5 mg, 0.5±0.1 mg, 0.5±0.2 mg, 0.5±0.3 mg, or 0.5±0.4 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.8 mg, 0.8±0.1 mg, 0.8±0.2 mg, 0.8±0.3 mg, or 0.8±0.4 mg, or 0.8±0.5 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. The coronavirus S protein antigenic polypeptide can be, but is not limited to, a SARS-CoV-2 spike protein or an antigenic fragment thereof. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non- viral vector. The above indicated amounts can be for each of a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12, or for both a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12 together (whether on separate vectors or the same vector).

[400] In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is injected in a volume of 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 600 μL, 650, 700, 750, 800, 900, or 1000. In some embodiments, the nucleic acid is injected in a volume of 50 μL, 50±10 μL, 100 μL, 100±10 μL, 100±25 μL, or 100±50 μL. In some embodiments, the nucleic acid is injected in a volume of 250 μL, 250±10 μL, 250±25 μL, or 250±50 μL. In some embodiments, the nucleic acid is injected in a volume of 500 μL, 500±10 μL, 500±25 μL, or 500±50 μL. In some embodiments, the nucleic acid is injected in a volume of 750 μL, 750±10 μL, 750±20 μL, 750±30 μL, 750±40 μL, 750±50 μL.

[401] In some embodiments, the nucleic acid is injected at a depth of 0.1 to 0.25 cm. In some embodiments, the nucleic acid is injected at a depth of 0.1, 0.15, 0.2, or 0.25 cm. In some embodiments, the nucleic acid is injected at a depth of 0.1 cm. The EP applicator is set to the same depth ±0.1 cm as the inj ection. The EP applicator is inserted such that the electrodes span the nucleic acid injection site. In some embodiments, 1-10 pulses at a field strength (E±) of 400±100 V/cm and pulse width of about 1-20 ms at about 1-second intervals are administered. In some embodiments, eight pulses at a field strength (E±) of 400 V/cm and pulse width of about 10 ms at 1-second intervals are administered.

C. Intratumoral electroporation (ID-EP) administration of nucleic acids encoding coronavirus S protein antigenic polypeptide, IL-12, or a combination of coronavirus S protein antigenic polypeptide and IL12.

[402] IT-EP administration of nucleic acids encoding coronavirus S protein antigenic polypeptide and/or IL-12 comprises injecting into a tumor a solution(s) containing the nucleic acids encoding coronavirus S protein antigenic polypeptide and/or IL-12 and electroporation of the injection site.

[403] In some embodiments, 0.1 - 3 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3 mg of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and/or IL12 is injected. In some embodiments, 0.1 mg, 0.1±0.01 mg, or 0.1±0.05 mg of the nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.25 mg, 0.25±0.01 mg, 0.25±0.05 mg, 0.25±0.1 mg, or 0.25±0.15 mg of the nucleic acid encoding a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.5 mg, 0.5±0.1 mg, 0.5±0.2 mg, 0.5±0.3 mg, or 0.5±0.4 mg of the nucleic acid a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. In some embodiments, 0.8 mg, 0.8±0.1 mg, 0.8±0.2 mg, 0.8±0.3 mg, or 0.8±0.4 mg, or 0.8±0.5 mg of the nucleic acid a coronavirus S protein antigenic polypeptide and/or IL-12 is injected. The coronavirus S protein antigenic polypeptide can be, but is not limited to, a SARS-CoV-2 spike protein or an antigenic fragment thereof. The nucleic acid can be DNA or RNA. The nucleic acid can be an expression vector or a plasmid. In some embodiments, the nucleic acid is a non-viral vector. The above indicated amounts can be for each of a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12, or for both a nucleic acid encoding a coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12 together (whether on separate vectors or the same vector).

[404] In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is injected in a volume of 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 900, or 1000 μL. In some embodiments, the nucleic acid is injected in a volume of 50 μL, 50±10 μL, 100 μL, 100±10 μL, 100±25 μL, or 100±50 μL. In some embodiments, the nucleic acid is injected in a volume of 250 μL, 250±10 μL, 250±25 μL, or 250±50 μL. In some embodiments, the nucleic acid is injected in a volume of 500 μL, 500±10 μL, 500±25 μL, or 500±50 μL. In some embodiments, the nucleic acid is injected in a volume of 750 μL, 750±10 μL, 750±20 μL, 750±30 μL, 750±40 μL, 750±50 μL. In some embodiments, the nucleic acid is injected in a volume corresponding to about 1/4 the volume of the tumor. In some embodiments, the nucleic acid is injected into the tumor in a volume corresponding to 25±10%, 25±5%, 25±2.5% of the calculated volume of the tumor. In some embodiments, the nucleic acid, at a concentration of 0.5-1.0 mg/ml is injected into the tumor in a volume corresponding to 25±10%, 25±5%, 25±2.5% of the calculated volume of the tumor. Injection into a tumor can include injection into margin tissue around the tumor. Injection into a tumor can include injecting the tumor, and optionally the margin tissue around the tumor, as uniformly as possible (i.e., dispersing the injection throughout the tumor and optionally the margin tissue around the tumor).

[405] A nucleic acid encoding the coronavirus S protein antigenic polypeptide and a nucleic acid encoding IL-12 can be present in the same solution or different solutions. If present in separate solutions, the nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12 can be injected concurrently or sequentially. The nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12 can be combined prior to injection. A nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding IL-12 can be present on the same vector ( e.g ., plasmid or RNA). When present on the same vector, the nucleic acid encoding the coronavirus antigenic polypeptide and a nucleic acid encoding IL-12 can be expressed from different promotors or from a single promoter, as in a multi cistronic vector

[406] In some embodiments, an ID-EP site can be 0.5 to 10 cm from an IM-EP site. In some embodiments, the ID-EP site is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,

1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,

3.9, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 cm from the IM-EP site. In some embodiments, the ID-EP site is 1-5 cm or 2-3 cm from the IM-EP site. In some embodiments, the ID-EP site is 2.5±0.1, 2.5±0.2, 2.5±0.3, 2.5±0.4, 2.5±0.5, 2.5±0.6, 2.5±0.7, 2.5±0.8, 2.5±0.9, 2.5±1 cm from the IM-EP site. In some embodiments, the ID-EP site is 2-3 cm from the IM-EP site.

Example 4. Analysis of immune response.

[407] Immunologic Monitoring. Blood sampling for plasmid expression levels and immune response measurements can be performed on one or more of days 1, 2, 3, 15, 30, 31, 32, 45, 60 and 90 of the study. The sample are analyzed for antibody and cellular immune response to the vaccine. Flow cytometry is performed with a panel of markers including CD4, CD8, NK, B cell and monocyte/DC panels. These include CD4 T conventional, Treg, T memory, Ki-67, CD38, HLA-DR, CD40, ICOS, granzyme B, PD-1, TIM-3, LAG-3, 4-1BB, CTLA-4, OX40R, NKG2A, CD94, NKp46, NKG2D, NKp30, CD158el, KIRc, CD 158b, CDl lb, CD15, CD33, CD54, CD80, CD83, CD86, PD-L1, PD-L2. Sorted immune cell subsets are processed for RNA/DNA extraction and genome-wide CpG methylation by target-capture DNA library preparation and Illumina sequencing. [408] Immune response gene expression analyses', one or more of Ki-67, CD38, HLA-

DR, CD40, ICOS, granzyme B, PD-1, TIM-3, LAG-3, 4-1BB, CTLA-4, OX4QR, NKG2A, CD94, NKp46, NKG2D, NKp30, CD158el, KERc, CD158b, CDl lb, CD15, CD33, CD54, CD80, CD83, CD86, PD-L1, and PD-L2 are analyze by gene or protein expression analyses.

[409] Neutralization assay. Antibody neutralization of SARS-CoV-2 is assessed using a pseudotyped lentivirus expressing the SARS-CoV-2 S protein and luciferase. The ability of sera from vaccinated subject to prevent infection of susceptible target cells indicated induction of an anti-SARS-CoV-2 immune response in the subject. Readout is the ability of sera from vaccinated subjects to prevent infection of susceptible target cells. Alternatively, the ability of sera to block hACE2R binding to spike is performed.

[410] Cytokine analyses. Serum cytokine profiling is evaluated using the Quanterix Simoa platform. Systemic IL-12 (p70) and up to 35 additional cytokines, including IFN- gamma, TNF-alpha, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A and TGF-beta, are assayed.

[411] T cell response. SARS-CoV-2 S protein-specific cellular immune responses is measured by cytokine-release assay for type I, type 2, and IL-17 cytokine responses using cytokine bead arrays. IFN-γ positive responses are assessed by IFN-γ ELISPOT assay using unfractionated PBMC and peptide pools of epitopes considered to be dominant epitopes for the HLA expressed by the specific subject. Peptide pools containing 15-mer peptides overlapping by 11 amino acids are also explored. ELISPOT response to whole protein is predicted to be dominated by CD4 ⁺ T-cell response. The response to overlapping peptides is further expected to comprise both CD4 ⁺ and CD8 ⁺ T-cell responses. Alternatively, fractionated PBMC is tested by intracellular cytokine staining (ICS) for CD4 ⁺ T-cell and CD8 ⁺ T-cell responses.

[412] B cell response. Antibody response to SARS-CoV-2 spike S glycoprotein is evaluated using Microaffinity proteomic (MAP) bead technology. Bead arrays containing SARS-CoV-2 proteins are used to assess IgM and the isotype of IgG responses that develop following vaccination. Humoral response is monitored by B-Cell ELISPOT, evaluating PBMCs for IgA, IgM, and IgG response to the COVID-19 S spike to determine the B cell response.

[413] Coordinated humoral/cellular response. Single-cell RNA sequencing is performed at one or more of days 0, 15, 30, 45, 60 and 90. Paired ab TCRseq and tandem BCRseq using the Archer Immunoverse platform allows tracking of T and B cell clones. Recognition of epitopes of the SARS-CoV-2 spike protein by dominant clones is assessed. It is predicted that persisting neutralizing Ab responses will result from a coordinated CD4, CD8 and B cell response to the SARS-CoV-2 spike (S) glycoprotein.

[414] Whole genome sequencing. Whole genome sequencing is performed to assess polymorphisms in immune-response related genes and to provide HLA haplotypes. This information is evaluated for relationships to the magnitude of the immune response to vaccination. Correlations with the depth and magnitude of response to vaccination are also analyzed.

[415] Immune response to variants. In addition to analyzing immune response to a virus having the particular antigenic polypeptide used in the vaccine, the above assays can be used to analyze immune response to variants of the pathogen. For example, with respect to SARS- CoV-2, immune response to the United Kingdom (UK) variant (known as 20I/501Y.V1, VOC 202012/01, or B.l.1.7), South Africa variant (known as 20H/501Y.V2 or B.1.351), Brazil variant (known as P.l or B 11248), and the Californian variant (known as CAL.20C, or 2oC/S:452R;/Bi429) can also be analyzed. Induction of an immune response against a variant indicated the vaccine is effective against the variant.

Example 5. Phase I study of immune response in health subjects vaccinated with nucleic acids encoding SARS-CoV-2 spike glycoprotein (S protein) and IL-12.

[416] This study will evaluate the safety and anti-viral immune stimulating efficacy of combining electroporation of plasmid encoding both the coronavirus spike glycoprotein (S protein) and IL-12. The effect of vaccinating with IL-12 in addition to S protein on production of neutralizing antibodies, induction of cellular immunity, induction of Thl response, and protection against SARS-CoV-2 infection in analyzed.

[417] Recent reports suggest that patients who have succumbed to COVID-19 had exhausted T cells. Since IL-12 can support T cell responses and reduce T cell exhaustion, it is anticipated that subjects receiving S protein in combination with IL-12 will develop more sustained T cell responses to SARS-CoV-2. Sustained T cell responses will support higher titers of neutralizing antibody against the spike protein. Vaccination with is expected to provide protection from exposure to SARS-CoV-2 and may provide cross-protection against other coronaviruses.

[418] The phenomenon of antibody-dependent enhancement (ADE, wherein non- neutralizing antiviral proteins facilitate virus entry into host cells) has been reported for dengue (Boonnak, K. et al. Role of dendritic cells in antibody-dependent enhancement of dengue virus infection. J. Virol. 82, 3939-3951 (2008)), HIV-1 (Willey, S. et al. Extensive complement- dependent enhancement of HIV- 1 by autologous non-neutralising antibodies at early stages of infection. Retrovirology 8, 16 (2011)) and influenza (Gotoff, R. et al. Primary influenza A virus infection induces cross-reactive antibodies that enhance uptake of virus into Fc receptor- bearing cells. J. Infect. Dis. 169, 200-203 (1994)). Wang et al, demonstrated experimentally that monoclonal antibodies to SARS-CoV spike protein resulted in ADE (Wang, S. F. et al. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochemical and biophysical research communications 451, 208-214 (2014)), whereas polyclonal, undiluted anti-sera, neutralized SARS-CoV infection. Inclusion of IL-12 in the vaccine platform under study may safeguard against ADE, acting as the adjuvant to trigger both the effector CD8 ⁺ T cell response as well as the complimentary CD4 ⁺ T cell response which can deepen the humoral response.

[419] All subjects in this protocol will receive the plasmid encoding the S protein via ID- EP and/or IM-EP. Half of the subjects will also receive plasmid encoding IL-12 via ID-EP. Subjects are administered an initial round of S protein and IL-12 on day 1 (prime vaccination) and second round of S protein and IL-12 (boost) about 3-4 weeks later. Various doses of both the nucleic acid encoding the S protein and the nucleic acid encoding IL-12 to optimize the immune response.

[420] The plasmid encoding the S protein encodes a spike protein from SARS-CoV-2 and is driven by a cytomegalovirus (CMV) promoter or chicken b actin promoter. The S protein has been stabilized by addition of two prolines and was reported by). The S protein is the prefusion spike ectodomain, a gene encoding residues 1-1208 of SARS-CoV-2 S (GenBank: MN908947) with proline substitutions at residues 986 and 987 (Wrapp D et al. "Cryo-EM Structure of the SARS-CoV-2 Spike in the Prefusion Conformation." Science. 2020 367(6483): 1260-1263), a “GSAS” substitution at the furin cleavage site (residues 682-685), a C -terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8><HisTag was synthesized and cloned into the mammalian expression vector paH. The SARS-CoV-2 S -2P_defuri n_F 3 CH2 S plasmid map is in FIG. 4. The SARS-CoV-2 S- 2P_defurin_F3CH2S plasmid encoding SARS-CoV-2 S protein was received from the Vaccine Research Center, NT ATP (21MAR2020). The His8 and StrepII tag were removed from SARS- CoV-2 S-2P_defurin_F3CH2S plasmid by restriction enzyme digestion with BamHI and Xhol and replacement with 122 bp PCR product that encodes the fold-on only domain. SARS-CoV- 2 S-2P_defurin_F3CH2S plasmid was used as the template for PCR. The resulting plasmid was named as SARS-CoV-2 S-2P_dfurin_foldon. The CMV promoter, chimeric intron, and S-2P foldon fragment that encodes the prefusion mixed S-2P ECD and foldon fusion protein was excised by restriction digestion with Spel and Xhol and cloned into Spel and Notl digested IL- 12 p35 _p40 pUMVC3 clinical vector (OncoSec) together a 60 bp Xhol and Notl linker excised from pSE280 vector (Invitrogen). The resulting plasmid was named as pUMVC3.SARS-CoV- 2-2_S-2P.foldon.tagoff. and is shown in FIG. 5. The plasmid encoding S protein is vialed at 1.67 mg/ml.

[421] The plasmid encoding IL-12 contains genes for the human IL-12 p35 and p40 subunits separated by an internal ribosomal entry site (IRES) and under control of a single cytomegalovirus (CMV) promoter. The plasmid encoding IL-12 is vialed at 3.33 mg/ml

[422] Vaccination with S protein and IL-12 comprises delivery of the genes via electroporation. IM-EP of S protein nucleic acid will provide expressed, diffusible S protein that can circulate and be presented to B cells to generate an anti-spike antibody response. ID- EP of S protein and IL-12 will provide for limited, local expression within the dermis, leading to antigen presentation in the draining lymph node that will result in the priming of an anti- viral immune response. Expression of IL-12 in the dermis will also polarize this local region towards a Thl-biased environment. This discrete modular deployment of antigen and immune- activating cytokine creates an immunogenic cellular microenvironment in the dermis with IL- 12 supporting innate effector cells, as well as presentation of the membrane bound (membrane anchored) or secreted (soluble) (S) glycoprotein and subsequent priming of a cellular anti-viral T cell response. Since this local cellular priming does not preclude the priming of humoral immunity in other secondary lymphoid tissues, the vaccine is expected to produce both cellular and humoral anti-SARS-CoV-2 immunity.

[423] Immunological Monitoring is performed as described in Example 4.

Example 6. Vaccination with coronavirus S protein in combination with IL-12.

[424] Combination of S protein with IL-12 induces production of neutralizing antibodies and increases cellular immunity (T cell response) following vaccination with a coronavirus antigenic polypeptide ( e.g. , nucleic acid encoding SARS-CoV-2 S protein) plus IL-12 (e.g, nucleic acid encoding IL-12). Human subjects are immunized with the combinations listed in

Table 2.

Table 2. Vaccines combining SARS-CoV-2 S protein with IL-12.

[425] For intradermal and intramuscular administration, the injection(s) can be in a single location or in two or more locations. For injection in two or more locations, the injections can be adjacent to each other or in separate locations. For example, a subject can receive 500 μL in two adjacent injections of 250 μL each. Similarly, a subject can receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be sufficiently close such that the electroporation applicator electrodes are able to span both injection sites. For administration of both S protein and IL-12 to dermis or muscle, the nucleic acids encoding the S protein and IL-12 can be injected separately at the same site, separately to distinct sites, or combined prior to injection. For administration of both S protein and IL-12 to tumor, the nucleic acids encoding the S protein and IL-12 can be injected separately into the tumor or combined prior to injection.

[426] In some embodiments, the plasmid(s) encoding the coronavirus antigen and/or the immune stimulator is(are) formulated in phosphate buffered saline (PBS) for injection followed by in vivo electroporation. [427] In some embodiments, the electroporative pulse is provided by an IGEA

Cliniporator® with an IGEA Applicator. The IGEA Applicator model can be, but is not limited to, a Model L-30-ST applicator containing an 8 needle array having 2 rows of 4 needles in each row, wherein the distance between the two rows of needles 4.7 mm and the distance between the needles in each row is 3.2 mm. The applicator electrodes are inserted to an appropriate depth for intradermal electroporation, intramuscular electroporation, or intratumoral electroporation such that the electrodes span the plasmid injection site. In some embodiments, electroporation comprises 2-10 (e.g., eight) pulses at a field strength of 400 V/cm and pulse width of 10 ms at 300 millisecond intervals.

[428] Each vaccination consists of two steps: injection, intramuscular, or intratumoral followed by administration of at least one electroporation pulse. Electroporation (EP) is co- localized at the site of each injection, and is performed as soon as reasonably possible, directly following injection. For intradermal administration, injection and administration of the electroporation pulse can be, for example, in the thigh over the vastus lateralis muscle or in the buttocks over the dorsogluteal muscle. Other locations, such as the shoulder are also suitable.

[429] Immune response is monitored as described in example 4.

Example 7. Electroporation (EP) of SARS-CoV-2 Spike plasmid and IL-12 plasmid generate anti-spike immune response.

[430] Experiments were conducted on two different mice strains (Balb/C or C57bl/6. In brief, mice received priming dose (“EP Prime”) and boost dose (“EP boost”) 14 days apart. 10 days post-EP boost (Day 24), serum and splenocytes were collected for end point analysis.

[431] Mice were treated on day 0 (prime) and day 14 (boost) with:

(a) no treatment

(b) 50 μg plasmid encoding spike protein and 100 μg plasmid encoding IL-12 by IM-ED;

(c) 50 μg plasmid encoding spike protein by IM-ED and 50 μg plasmid encoding spike protein plus 100 μg plasmid encoding IL-12 by ID-EP or

(d) 50 μg plasmid encoding spike protein by IM-ED and 125 μg plasmid encoding spike protein by ID-EP

[432] Samples were taken on day 24 to assay for spike protein or IL-12 protein production by ELISA.

[433] On day 24, splenocytes were collected and cultured with spike peptides for 1-3 days. Sample were taken after 24 hours and assayed for cytokine production by ELISA.

[434] On day 24, serum was collected and assayed for SARS-CoV-2-specific IgG2a, IgGl and ACE binding inhibition by ELISA. [435] Combined electroporation of pIL12 and spike plasmid administered through intradermal or intramuscular treatments induced anti-spike IgGl antibodies and anti-spike IgG2a antibodies in both Balb/C and C57bl/6 mice (FIGs. 9-10). In addition, antibodies were able to inhibit binding of spike to ACE2 in vitro in a pseudo-neutralizing assay (inhibition of binding of a SARS-CoV-2 pseudovirus to ACE2) (FIG. 11).

Example 8. Spike protein in combination with IL-12.

[436] pIL-12 was administrated with spike plasmid via different routes of administration (intradermal, ID; intramuscular, IM). Prime and boost doses were 21 days apart. Experiments were conducted in two mice strains (Balb/C and C57bl/6). Serum samples were collected at different time points (D31, 42, 92) for anti-spike antibodies measurements (ELISA).

[437] Mice were treated on day 0 (prime) and day 21 (boost) with:

(a) 50 μg plasmid encoding spike protein by IM-EP and 50 μg plasmid encoding spike protein by ID-EP;

(b) 50 μg plasmid encoding spike protein by IM-EP and 50 μg plasmid encoding spike protein plus 100 μg plasmid encoding IL-12 by ID-EP;

(c) 50 μg plasmid encoding spike protein plus 100 μg plasmid encoding IL-12 by IM-EP and 50 μg plasmid encoding spike protein by ID-EP;

(d) 50 μg plasmid encoding spike protein plus 100 μg plasmid encoding IL-12 by IM-EP and control plasmid by ID-EP;

(e) control plasmid by IM-EP and 50 μg plasmid encoding spike protein plus 100 μg plasmid encoding IL-12 by ID-EP;

(f) control plasmid by IM-EP and control plasmid by ID-EP; or

(g) no treatment control

[438] On days 31, 42, and 92, serum was collected and assayed for SARS-CoV-2-specific IgG2a, IgGl and ACE binding inhibition by ELISA.

[439] In Day 31 samples, spike plasmid EP through concurrent ID and IM injections demonstrated the best anti-spike immune response among the test groups. Addition of pIL-12 to spike plasmid ID-EP at 100 μg/ml IL-12 plasmid does level did not enhance the immune response (FIG. 12A-B, 13A-B). Administration of 100 μg IL-12 by IM-EP decreased efficacy. Similar results were noted in serums from Day 42 and Day 92. While spike protein administration by ID-EP and IM-EM yielded the most neutralizing antibodies at day 92, each of the vaccination schedules yielded detectable levels of neutralizing antibodies (FIG. 14A-B). Example 9. Comparison of pILl 2-IRES and pIL12-P2A.

[440] ID-EP vaccination using a combination of a nucleic acid encoding IL12-P2A (plasmid encoding IL-12 p35-P2A-IL-12 p40) and a plasmid encoding Spike protein or a combination of a plasmid encoding IL12-IRES (plasmid encoding IL-12 p35-P2A-IL-12 p40) and a plasmid encoding spike protein were compared to determine if increased IL-12 expression from the IL12-P2A expression vector would enhanced anti-spike immune response. We have previously demonstrated that IL12-P2A consistently induces higher expression levels of IL-12 when compared with IL 12-IRES.

[441] Mice were vaccinated according to the following schedule, with a prime injection at day 0 and a boost injection at day 21.

(a) 50 μg spike IM-EP and 50 μg spike ID-EP

(b) 50 μg spike ID-EP

(c) 50 μg spike + 100 μg IL12-IRES ID-EP

(d) 50 μg spike + 100 μg IL12-P2A ID-EP

(e) empty plasmid ID-EP

[442] Serum was collected on days 31 and 42 and analyzed for SAR-CoV-2-specific IGgl and IgG2a antibodies and for neutralizing antibodies. Animal vaccination using the IL12-P2A vector had reduced the anti-spike immune response when compared to animals vaccinated with IL12-IRES vector. Thus, the results indicated that higher levels of IL-12 administered by ID- EP can depress immune response to a co-administered antigen (FIG. 15A-B). Nevertheless, the combination of spike and IL-12 resulted in induction of an immune response compared to empty vector control. Based on these observations, lower doses of the IL-12 plasmid were analyzed.

Example 10. Analyses of lower dose pIL-12 and spatial co-localization.

[443] Mice were vaccinated as described above using lower doses of IL 12-IRES expression vector. Two lower doses of pIL12 (50 μg or 10 μg) were co-administered with spike plasmid to immunize C57bl/6 mice. Some mice were injected with a solution containing both the pIL- 12 + spike plasmids (mix). Other mice were administered separate injections of IL-12 and spike plasmids. For separate injections, IL12 and spike plasmid injections were administrated at adjacent locations (~2 mm apart at injection sites) and each site was separated electroporated.

[444] Mice were vaccinated according to the following schedule, with a prime injection at day 0 and a boost injection at day 21.

(a) 50 μg spike IM-EP and 50 μg spike ID-EP (b) 50 μg spike ID-EP

(c) 50 μg spike ID-EP and 50 μg IL12-IRES ID-EP

(d) 50 μg spike ID-EP and 10 μg IL12-P2A ID-EP

(e) 50 μg spike + 10 μg IL12-P2A (mix) ID-EP

(f) empty plasmid ID-EP

[445] Serum was collected on days 30, 44, and 61 and analyzed for SAR-CoV-2-specific IGgl and IgG2a antibodies and for neutralizing antibodies. Intradermal co-injection with 50 μg of spike plasmid and 10 μg of pIL12 (“Spike(50)/IL12(10), mixed ID”) demonstrated superior immune response compared to 50 μg of spike plasmid ID-EP alone indicating the efficacy of IL-12 in enhancing immune response to the spike protein (FIG. 16A-B, 17B-B, 18A-B). Immunization of spike plasmid through IM-EP and ID-EP was used as positive control standard. The positive control standard treatment group received twice the dose of spike plasmid, 50 μg via each of IM and ID routes, compared to the other treatment groups, resulting in more spike protein expression overall.

Example 11. Dose- or schedule-dependent effects of pIL12.

[446] Mice were vaccinated as described above using different doses of IL 12-IRES expression vector administered in different schedules. Two lower doses of pIL12 (10 μg or 1 μg) were co-administered with spike plasmid to immunize C57bl/6 mice.

[447] Mice were vaccinated according to the following schedule, with a prime injection at day 0 and a boost injection at day 21 (except as noted).

(a) positive control, spike IM-EP and spike ID-EP

(b) spike ID-EP

(c) spike ID-EP + 10 μg IL12 ID-EP (IL12 at prime only)

(d) spike ID-EP + 1 μg IL12 ID-EP (IL12 at prime only)

(e) spike ID-EP + 10 μg IL12 ID-EP

(f) spike ID-EP + 1 μg IL12 ID-EP

(g) spike ID-EP + 10 μg IL12 ID-EP (IL12 at boost only)

(h) spike ID-EP + 1 μg IL12, ID-EP (IL12 at boost only)

(i) empty vector negative control

[448] Use of 10 μg or 1 μg of IL-12 plasmid increased anti-Spike IgGl and neutralization antibody response at days 31 and 42 compared to spike plasmid administered by ID-EP (FIG. 19A-C, 20). The increase occurred for IL-12 administration at prime and boost, prime only, or boost only. Use of 10 μg or 1 μg of IL-12 plasmid also increased CD19 ⁺ B cells, CD3 ⁺ CD4 ⁺ T cells, CD3 ⁺ CD8 ⁺ T cells, effector memory (CD3 ⁺ CD4 ⁺ CD127 ⁺ CD62L-) T cells (T _em cells), and CD3 ⁺ CD4 ⁺ CD127-CD62L- T Cells (Thymic epithelial cells (T _ec ) cells) and at day 90 compared to spike plasmid administered by ID-EP. Use of 10 μg or 1 μg of IL-12 plasmid, administered at prime and boost, also increased Memory B cells at day 90 when compared to spike plasmid administered by ID-EP (FIG. 21 A-D) or spike plasmid administered by both IM- EP and ID-EP. The results indicate that adding IL-12 to the pathogenic antigen may induce immune memory response and longer-lasting immunity.

Example 12. Dose analysis of spike plasmid

[449] Multiple dose levels of spike plasmid was administered to mice with or without pIL12. pIL12 was either mixed with spike plasmid to be co-administered or administered separately. In two independent experiments with two distinct administration approaches, anti- spike response levels were not significantly different based on spike plasmid doses (FIG. 22A- B, 23A-B). As in the experiments shown above, IL-12 plasmid administered at 10 μg induced stronger immune response to the pathogenic antigen than IL-12 plasmid administered at 50 μg. Example 13. Vaccination of subjects having cancer, intratumoral electroporation (IT-EP) of nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immunostimulatory cytokine.

[450] Mice were anesthetized and subcutaneously injected with tumor cells into the right and/or left flank. Tumor growth was monitored by digital caliper measurements until tumors developed. Tumors were then treated as shown in Table 3. Tumor volumes and survival were monitored. In addition, sample were collected to determine immune response to SARS-CoV- 2. PBMC immune profiling and IgG and ACE2 inhibit assays (see example 4) were performed on days 15 and 25.

Table 3. Vaccination of cancer subjects.

[451] Analysis of peripheral blood B cells: Intratumoral electroporation of IL-12 in combination with S protein induced a high percentage of B cells in peripheral blood (FIG. 24).

[452] Analysis of peripheral blood memory B cells: Intratumoral electroporation of IL-12 combined with S protein induced a high percentage of memory B cells in peripheral blood (FIG. 25).

[453] Analysis of tumor draining lymph node B cells subset: Intratumoral electroporation of IL-12 combined with S protein induced a high percentage of germinal center B cells and class switched B cells (where antibodies are produced) in tumor draining lymph nodes (FIGs. 26-27).

[454] Analysis of tumor draining lymph node T cells subset: Intratumoral electroporation of IL-12 combined with S protein induces high percentage of CD4 ⁺ and CD8 ⁺ T cells in tumor draining lymph nodes (FIGs. 28-29).

[455] Analysis of anti-Spike IgGl and IgG2a: Serum was diluted 1:500. Intratumoral electroporation of S protein with or without IL-12 yielded high levels of anti-spike IgGl. One treatment (prime, day 15) or two treatments ( prime + boost, day 25) yielded similar anti-spike IgG levels (FIG. 30, upper panel). Animals vaccinated with IT-EP IL-12 plus S protein had significantly increased production of anti-spike IgG2a antibodies (FIG. 30, lower panel).

[456] Pseudo-neutralizing Assay: Intratumoral electroporation of S protein with or without IL-12 yielded high levels of inhibition of binding of spike to ACE2. Increased levels of neutralizing antibodies were observed following the boost injection. Animals vaccinated with IL-12 + spike showed a greater increase in neutralizing antibodies following boost vaccination.

[457] Conclusions: Intratumoral electroporation of IL-12 combined with S protein yielded high levels of B cells and memory B cells in peripheral blood, and high levels of germinal center B cells, class switched B cells, CD4 ⁺ T cells, and CD8 ⁺ T cells in tumor draining lymph nodes. IT-EP of IL-12 combined S protein also yielded high level of anti-spike IgGl and IgG2a, including neutralizing antibodies, in serum. A strong anti-viral response was observed, indicating that IT-EP of a coronavirus antigenic polypeptide and an immunostimulatory cytokine are effective in inducing an immune response to a coronavirus.

Example 14. Tumor regression in mice treated with IL-12 and SARS-CoV-2 S protein antigen.

[458] Mice were implanted with tumor cells. Anesthetized mice were injected with cells subcutaneously into the right and/or left flank. Tumor growth was monitored by digital caliper measurements until average tumor volume of -100 mm ³ was reached.

[459] Tumors were treated on days 1, 5, and 8 with IT-EP control vector, IT-EP IL-12, or IT-EP IL12-2 + SARS-CoV-2 S protein antigen. Tumor volumes and survival were monitored. Mice were euthanized if the total tumor burden of the primary and contralateral reached 2000 mm ³ .

Example 15. Intratumoral expression of pathogenic antigen, in combination with IL-12, enhances immune response to tumors.

[460] Tumor cells were implanted in mice on day -7. Tumors were implanted in the right flank. A single tumor in each mouse was treated on days 0 and 15 with

(a) Untreated,

(b) IT-EP control vector,

(c) IT-EP 10 μg IL-12 plasmid,

(d) IT-EP 115 μg SARS-CoV-2 S protein antigen plasmid,

(e) IT-EP 10 μg IL12 plasmid + 115 μg SARS-CoV-2 S protein antigen plasmid,

(f) IT-EP 10 μg IL-12 plasmid + ID-EP 115 μg SARS-CoV-2 S protein antigen plasmid,

(g) ID-EP 1 μg IL-12 plasmid + 115 μg SARS-CoV-2 S protein antigen plasmid,

(h) IT-EP 10 μg control plasmid + ID-EP 115 μg SARS-CoV-2 S protein antigen plasmid.

For ID-EP SARS-CoV-2 S protein, plasmid encoding SARS-CoV-2 S protein was administered in the same flank as the tumor.

[461] Animals treated with (a) no treatment; (b) IT-EP control vector; (g) ID-EP IL-12 plasmid + SARS-CoV-2 S protein; (h) IT-EP control plasmid + ID-EP SARS-CoV-2 S protein; or (d) IT-EP SARS-CoV-2 S protein exhibited substantial tumor growth by day 15 after the first injection. Animals treating with (c) IT-EP IL-wl2 exhibited substantially decreased tumor growth out to day 23. Animals treated with (e) IT-EP IL12 + SARS-CoV-2 S protein antigen, or (f) IT-EP IL-12 + ID-EP SARS-CoV-2 S protein antigen showed little to no tumor growth out to day 23. As expected intratumoral IL-12 treatment resulted in decreased tumor growth. Combining intratumoral IL-12 treatment with intradermal or intratumoral pathogenic antigen treatment enhanced the therapeutic efficacy of the IL-12 treatment (FIG. 31-32)

[462] Intratumoral electroporation of pIL12 combined with pathogenic antigen plasmid increased the anti -tumor response compared with IL12 plasmid alone. On day 25 post first treatment, 85% of the mice treated with pIL12 + SARS-CoV-2 S protein were tumor free compared with 60% of mice treated with IL12 alone (FIG. 33).

Example 16. Intratumoral expression of pathogenic antigen, in combination with IL-12, enhances immune response to tumors.

[463] Tumor cells are implanted in mice on day -7. Tumors are implanted in both the right and left flank. A single tumor in each mouse is treated on days 1, 5 ±2 days, and 8±2 days, days 1 and 5±2 days, or days 1 and 8 ±2 days with IT-EP control vector, IT-EP 10 μg IL-12 plasmid, IT-EP 10 μg IL12 plasmid + 115 μg SARS-CoV-2 S protein antigen plasmid, or IT-EP 10 μg IL-12 plasmid + ID-EP 115 μg SARS-CoV-2 S protein antigen plasmid. Tumor and immune response in the primary (treated) and secondary (untreated) tumors are then monitored. In some embodiments, the treatment (cycle) is repeated every three to six weeks. In some embodiments, the cycle is repeated every three weeks. In some embodiments, the cycle is repeated every four weeks. In some embodiments, the cycle is repeated every six weeks.

[464] In some mice, tumor and splenocytes are harvested 2 days after last EP ( i.e ., Day 10) for NanoString and flow based analysis. Alternatively, tumor volumes are measured three times a week for regression/survival studies. Gene expression changes in electroporated CT26 lesions are assessed by NanoString nCounter® technology. Intratumoral expression of pathogenic antigen is confirmed using ELISA 48hrs post-electroporation in tumor lysates from mice bearing tumors.

[465] Volcano plots displaying p-values and log2 fold change for various immune responsive genes are generated in mice.

[466] Flow cytometric analysis is used to analyze splenocytes in treated mice. Antigen specific AH1+ CD8+ T cells are measured via tetramer analysis (Immudex). The fold increase in the number of AH1+ CD8+ T cells compared to empty vector control is determined.

Example 17. Intratumoral expression of pathogenic antigen, in combination with IL-12, enhances immune response to tumors.

[467] Tumor cells are implanted in mice on day -7. Tumors are implanted in both the right and left flank. A single tumor in each mouse is treated on days 1 with IT-EP control vector, IT- EP 10 μg IL-12 plasmid, IT-EP 10 μg IL12 plasmid + 115 μg SARS-CoV-2 S protein antigen plasmid, or IT-EP 10 μg IL-12 plasmid + ID-EP 115 μg SARS-CoV-2 S protein antigen plasmid. In some embodiments, each tumor is treated again on day 5±2 days with IT-EP control vector or IT-EP 10 μg IL-12 plasmid. In some embodiments, each tumor is treated again on day 8 ±2 days with IT-EP control vector or IT-EP 10 μg IL-12 plasmid. In some embodiments, each tumor is treated again on days 5±2 days and 8±2 days with IT-EP control vector or IT-EP 10 μg IL-12 plasmid. Tumor and immune response in the primary (treated) and secondary (untreated) tumors are then monitored.

[468] In some embodiments, the treatment (cycle) above is repeated every three to six weeks. In some embodiments, the cycle is repeated every three weeks. In some embodiments, the cycle is repeated every four weeks. In some embodiments, the cycle is repeated every six weeks.

[469] In some mice, tumor and splenocytes are harvested 2 days after last EP (z.e., Day 10) for NanoString and flow based analysis. Alternatively, tumor volumes are measured three times a week for regression/survival studies. Gene expression changes in electroporated CT26 lesions are assessed by NanoString nCounter® technology. Intratumoral expression of pathogenic antigen is confirmed using ELISA 48hrs post-electroporation in tumor lysates from mice bearing tumors.

[470] Volcano plots displaying p-values and log2 fold change for various immune responsive genes are generated in mice. [471] Flow cytometric analysis is used to analyze splenocytes in treated mice. Antigen specific AH1+ CD8+ T cells are measured via tetramer analysis (Immudex). The fold increase in the number of AH1+ CD8+ T cells compared to empty vector control is determined.

[472] It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention.