TW202146435A - Compositions containing a pathogenic antigen and an immune stimulator - Google Patents

Abstract

Described are compositions comprising a pathogenic antigen and an immune stimulator for use in inducing an immune response to a pathogen. Also described are compositions comprising an immunostimulatory cytokine and a pathogenic antigen for use in treating cancer.

Description

Composition containing pathogenic antigen and immune stimulator

foreword

Coronaviruses, which can infect and cause disease in both mammals and birds, often cause mild respiratory infections such as the common cold. However, some strains of the coronavirus can be lethal. In December 2019, a new strain of coronavirus originally created by the World Health Organization (WHO) as 2019-nCoV and subsequently named SARS-CoV-2 after tracing the origin of the pneumonia outbreak in Wuhan, China discovered. SARS-CoV-2 was identified as a new betacoronavirus strain and capable of human-to-human transmission (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 included both 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. 2020 25 (3)). The World Health Organization has declared the SARS-CoV-2 outbreak a global pandemic.

Because novel coronaviruses and other emerging pathogens represent a global health threat, there is a need for improved vaccines that can be easily adapted to newly identified infectious pathogens.

Describe vaccines against pathogens. The described vaccine compositions can be used to vaccinate against pathogenic infections. Also described are methods of vaccinating an individual against infection using the described vaccine composition. A pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoa, parasites, and fungi. The 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, Polio virus, rubella virus, rubella virus virus, human papilloma virus, enterovirus, West Nile virus, Ebola virus, Zika virus, human immunodeficiency virus, lyssavirus, Rabies virus, yellow fever virus, Japanese encephalitis virus, hantavirus and coronavirus. Bacterial pathogens can be, but are not limited to: Corynebacterium diphtheriae , Haemophilus influenzae type b, Bordetella pertussis , Streptococcus pneumoniae , Streptococcus pneumoniae ( pneumococcus ), Clostridium tetani , Neisseria meningitidis , Salmonella typhi , Vibrio cholerae and Yersinia pestis . Parasitic pathogens can be, but are not limited to: Plasmodium spp. , P. falciparum , P. vivax , P. ovale , Plasmodium ( P. malariae ), Trypanosoma brucei (Trypanosoma brucei) and Leishmania ( Leishmania ). The described vaccine compositions combine at least one pathogen antigenic polypeptide with at least one immune stimulator. The immune stimulator can be an immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory interleukins can be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-[alpha], IFN-[beta], IFN-[gamma], TGF-[beta] and CXC 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 pathogenic antigen, and the encoded product comprises a genetic adjuvant-pathogenic antigen fusion polypeptide. A pathogenic antigen can be an isolated polypeptide or a nucleic acid encoding a pathogenic antigen. The immunostimulator can be an isolated polypeptide or a nucleic acid encoding an immunostimulator.

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

Nucleic acid-based vaccines are described. The nucleic acid-based vaccine compositions described can be used to vaccinate against infection. Also described are methods of vaccinating an individual against infection using the nucleic acid-based vaccine compositions described.

Describe the vaccine against the coronavirus. The coronavirus vaccine compositions described can be used to vaccinate against coronavirus infection. In some embodiments, the coronavirus vaccine compositions described can be used to vaccinate against coronavirus infection or viral replication in the lungs or nasal cavity. Methods of vaccinating an individual against coronavirus infection using the described vaccine compositions are also described. The coronavirus can be but not limited to betacoronavirus , A-series betacoronavirus (subgenus Embecovirus), B-series betacoronavirus (subgenus Sarbecovirus), C-series betacoronavirus (subgenus Sarbecovirus) Coronavirus (subgenus Merbecovirus ), D-line betacoronavirus (subgenus Nobecovirus), SARS-CoV, MERS-CoV, SARS-CoV-2 or related betacoronavirus Virus. The coronavirus vaccine compositions described combine at least one coronavirus antigenic polypeptide with at least one immune stimulator. The immune stimulator can be an immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory interleukins can be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-[alpha], IFN-[beta], IFN-[gamma], TGF-[beta] and CXC 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. A coronavirus antigenic polypeptide can be an isolated polypeptide or a nucleic acid encoding a coronavirus antigenic polypeptide. The immunostimulator can be an isolated polypeptide or a nucleic acid encoding an immunostimulator.

Describe the hybrid protein/nucleic acid coronavirus vaccine. The hybrid coronavirus vaccine compositions described can be used to vaccinate against coronavirus infection. Methods of vaccinating an individual against coronavirus infection using the described vaccine compositions are also described. The coronavirus can be but is not limited to betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), B betacoronavirus (subgenus Sabeicoronavirus), C betacoronavirus (subgenus Mobecoronavirus) genus), D-line betacoronavirus (subgenus Nobecoronavirus), SARS-CoV, MERS-CoV, SARS-CoV-2, or related betacoronaviruses. 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 immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory interleukins can be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-[alpha], IFN-[beta], IFN-[gamma], TGF-[beta] and CXC 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. Nucleic acids can be, but are not limited to, RNA, mRNA, DNA, plastids, or expression vectors.

Describes a nucleic acid-based vaccine for coronavirus. The described nucleic acid-based vaccine compositions can be used to vaccinate against coronavirus infection. Also described are methods of vaccinating individuals against coronavirus infection using the nucleic acid-based vaccine compositions described. The coronavirus can be but is not limited to betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), B betacoronavirus (subgenus Sabeicoronavirus), C betacoronavirus (subgenus Mobecoronavirus) genus), D-line betacoronavirus (subgenus Nobecoronavirus), SARS-CoV, MERS-CoV, SARS-CoV-2, or related betacoronaviruses. The coronavirus vaccine compositions described 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 immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory interleukins can be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-[alpha], IFN-[beta], IFN-[gamma], TGF-[beta] and CXC 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. Nucleic acids can be, but are not limited to, RNA, mRNA, DNA, plastids, or expression vectors.

The coronavirus antigenic polypeptide can be but is not limited to betacoronavirus antigenic polypeptide, A series betacoronavirus (Ebercoronavirus subgenus) antigenic polypeptide, B series betacoronavirus (Sabecoronavirus subgenus) antigenic polypeptide , C series betacoronavirus (Mobecoronavirus subgenus) antigenic polypeptide, D series betacoronavirus (Nobecoronavirus subgenus) antigenic polypeptide, SARS-CoV antigenic polypeptide, MERS-CoV antigenic polypeptide, SARS - CoV-2 antigenic polypeptide or related betacoronavirus antigenic polypeptide. The coronavirus antigenic polypeptide can be, but is not limited to, the 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, as well as in preclinical and clinical Middle East respiratory syndrome (MER)-CoV studies. The S protein antigenic polypeptide comprises a coronavirus spike polypeptide (that is, S glycoprotein or S protein) or an antigenic fragment thereof or a modified coronavirus spike polypeptide or an antigenic fragment thereof. The coronavirus spike protein or an antigenic fragment thereof may 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. The S protein antigenic polypeptide may comprise the extracellular domain of the S protein or an antigenic fragment thereof. In some embodiments, the S protein antigenic polypeptide may comprise amino acids 1-1208, 14-1208, 21-1208, 14- corresponding to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 33 Polypeptides of 305 (N-terminal domain) or 330-521 (receptor binding domain). The S protein antigenic polypeptide may include one or more additional components including, but not limited to, a secretion signal, a trimerization domain, or an Fc tag. The secretion signal (ie, signal sequence or signal peptide) can be a native secretion signal (ie, S protein secretion signal) or a heterologous secretion signal. The trimerization domain can be a native trimerization domain or a heterologous trimerization domain. The S protein antigenic polypeptide may also contain one or more mutations that disrupt the internal furin cleavage site and/or one or more mutations that stabilize the protein. Nucleic acids encoding S protein antigenic polypeptides can be codon-optimized.

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

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

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

In some embodiments, the 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, encoding a nucleic acid sequence having SEQ ID NO: 22 Nucleic acid sequences of polypeptides of amino acid sequence, nucleic acid sequences encoding polypeptides with at least 90% identity to the amino acid sequence of SEQ ID NO: 22, nucleic acid sequences comprising SEQ ID NO: 6, and SEQ ID NO: 6 A nucleic acid sequence having at least 90% identity, 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 with SEQ ID NO: 7.

In some embodiments, the nucleic acid encoding IL-12 and 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, encoding a nucleic acid sequence having SEQ ID NO: 25 The nucleic acid sequence of the polypeptide of the amino acid sequence of SEQ ID NO: 26, the nucleic acid sequence of the polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 26, the nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 8, and the nucleic acid sequence of SEQ ID NO: 8 NO: 8 is a nucleic acid sequence with at least 90% identity, a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 9, or a nucleic acid sequence encoding an amino acid sequence with at least 90% identity with SEQ ID NO: 9.

One or more isolated polypeptides or one or more nucleic acids encoding one or more polypeptides can be administered by injection. One or more isolated polypeptides and/or one or more nucleic acids encoding one or more polypeptides can be combined with one or more adjuvants, carriers, excipients, or combinations thereof. The one or more isolated polypeptides and/or one or more nucleic acids encoding the one or more polypeptides can be formulated for injection and for inducing an immune response. The one or more isolated polypeptides or one or more nucleic acids encoding the one or more polypeptides can be formulated for intramuscular injection, intradermal injection, intratumoral injection, or a combination thereof.

Nucleic acids encoding pathogenic antigens and/or immune stimulators 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). ), needle-free injection (with or without electroporation), microprojectile bombardment (eg, gene gun), hydrodynamic injection, magneto-fection, sono-poration (eg, ultrasound mediation) guided delivery), photo-poration, and hydro-poration. Nucleic acid sequences can be in nanoparticles, lipid nanoparticles, liposomes, lipoplexes, polymeric complexes, lipopolymeric complexes, or other non-viral particles or complexes.

Nucleic acids encoding pathogenic antigens and/or immune stimulators can be formulated for administration into the dermis (intradermal administration), into skeletal muscle (intramuscular administration), and/or into tumors ( intratumoral administration). In some embodiments, nucleic acids encoding pathogenic antigens and/or immune stimulators can be formulated for administration into the dermis by intradermal electroporation (ID-EP), by intramuscular electroporation (IM-EP) ) into muscle, into tumors by intratumoral electroporation (IT-EP), or a combination thereof. In some embodiments, nucleic acids encoding pathogenic antigens and/or immune stimulators can be formulated for injection by direct injection, needle-free injection, microprojectile bombardment, hydrodynamic injection, magnetotransfection, ultrasonic perforation, light The perforation or water perforation is administered into the dermis, muscle and/or tumor. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

The isolated pathogenic antigen or immunostimulatory peptide can be formulated for administration into the dermis (intradermal administration), into skeletal muscle (intramuscular administration), and/or into tumors (tumoral administration). Introduced and). The isolated pathogenic antigen or immunostimulatory peptide can be combined with an immunological adjuvant prior to administration. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

Describe the method for eliciting an immune response against the coronavirus. The coronavirus can be but is not limited to betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), B betacoronavirus (subgenus Sabeicoronavirus), C betacoronavirus (subgenus Mobecoronavirus) genus), D-line betacoronavirus (subgenus Nobecoronavirus), SARS-CoV, MERS-CoV, SARS-CoV-2, or related betacoronaviruses. In some embodiments, the methods comprise: administering to the individual 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 the second dose of the coronavirus antigenic polypeptide at a different site than the administration of the first effective dose of the coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide and the immune stimulator are simultaneously, within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 25 minutes, within about 30 minutes of each other , 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. The coronavirus antigenic polypeptides and immune stimulators can be administered intradermally, intramuscularly, intratumorally, or a combination thereof.

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

In some embodiments, the methods comprise: (a) administering to the individual a first effective dose of a coronavirus antigenic polypeptide and a first effective dose of an immune stimulator by intradermal administration, and (b) by intradermal administration Intramuscular Administration A second effective dose of a coronavirus antigenic polypeptide is administered to the individual. The coronavirus antigenic polypeptide can be, but is not limited to, the coronavirus spike protein or an antigenic fragment thereof. The immunostimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immunostimulator. In some embodiments, the first effective dose of the coronavirus antigenic polypeptide and the first effective dose of the immune stimulator are administered at the same site by intradermal administration. The coronavirus antigenic polypeptide and the immune stimulator can be administered separately to the dermis, or they can be combined prior to intradermal administration. The site of intradermal and intramuscular administration can be, but is not limited to, the shoulders, legs, or buttocks of an individual. The sites of intradermal and intramuscular administration can be very close (≤ 10 cm) or distant (> 10 cm).

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

Triggering an immune response against coronavirus can be used to: (a) elicit a cellular immune response against the coronavirus; (b) elicit a humoral immune response against the coronavirus; (c) elicit both cellular and humoral immune responses against the coronavirus; (d) Enhance the proliferation of cytotoxic T lymphocytes; (e) Trigger the production of anti-coronavirus antibodies; (f) trigger the production of neutralizing anti-coronavirus antibodies; (g) elicit the production of antibodies capable of preventing coronavirus infection; (h) reduce the likelihood or severity of coronavirus infection; (i) reduce the likelihood or severity or duration of COVID-19; (j) vaccinate patients against coronavirus; (k) elicit protective immunity against COVID-19 or severe COVID-19; (l) Prevention of at least one symptom of COVID-19 disease, and/or (m) Prevention of symptomatic COVID-19 disease.

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

In some embodiments, the method of eliciting an immune response against a coronavirus comprises: (a) administering to a subject by intradermal administration a first effective dose of a nucleic acid encoding a coronavirus antigenic polypeptide and a first effective dose of the encoding an immunostimulatory nucleic acid; (b) administering to the subject a second effective dose of a nucleic acid encoding a coronavirus antigenic polypeptide by intramuscular administration. The coronavirus antigenic polypeptide can be, but is not limited to, the coronavirus spike protein or an antigenic fragment thereof. The immunostimulator can be, but is not limited to, IL-12 or a combination of IL-12 and a second immunostimulator. 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 immunostimulator are administered at the same site by intradermal administration. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be administered to the dermis separately, or they can be combined prior to intradermal administration. The site of intradermal and intramuscular administration can be, but is not limited to, the shoulders, legs, or buttocks of an individual. The sites of intradermal and intramuscular administration can be very close (≤ 10 cm) or distant (> 10 cm). Intradermal administration can include intradermal electroporation (ID-EP). Intramuscular administration can include intramuscular electroporation (IM-EP). Intratumoral administration may comprise intratumoral electroporation (IM-EP).

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

The administration may comprise an initial administration (single administration) or an initial administration and one or more boost administrations (two or more administrations). The primary and booster administrations can be performed at the same site or at different sites in the subject, eg, in the same limb or different 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 performed in each round. The second round of injections (boost) can be performed 2 weeks to 12 months after the first round of injections (initial). 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 subsequent booster administrations may be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequent booster administrations can be about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks , about 11 weeks or about 12 weeks apart. In some embodiments, the additional boost is administered about 1-5 years after the initial or booster administration.

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

In some embodiments, the methods comprise a first (primary) intradermal administration of a coronavirus antigenic polypeptide and an immune stimulator. In some embodiments, the methods further comprise at least one booster intradermal administration of the coronavirus antigenic polypeptide and the immune stimulator, wherein the booster administration is administered after the primary administration. The booster administration can be 1, 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration (± 4 days each).

In some embodiments, the methods comprise a first (primary) 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 booster intradermal administration of a nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immune stimulator, wherein the booster administration is administered after the primary administration. The booster administration can be 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration (± 4 days each).

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

In some embodiments, the methods comprise a first (primary) intradermal administration of nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators and intramuscular administration of nucleic acids encoding coronavirus antigenic polypeptides. In some embodiments, the methods further comprise at least one boost intradermal administration of nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators and intramuscular administration of nucleic acids encoding coronavirus antigenic polypeptides, wherein the booster administration It is voted after the initial vote. The booster administration can be 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration (± 4 days each).

In some embodiments, the methods comprise a first (primary) intratumoral administration of a nucleic acid encoding an immunostimulator and, optionally, a nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the methods further comprise at least one boost intratumoral administration of a nucleic acid encoding an immune stimulator and, optionally, a nucleic acid encoding a coronavirus antigenic polypeptide, wherein the boost administration is administered after the primary administration and. The booster administration can be 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration (± 4 days each).

For intradermal administration, the coronavirus antigenic polypeptide and the immune stimulator can be administered at the same site or at different sites. For administration at different sites, the intradermal administration of the coronavirus antigenic polypeptide and the intradermal administration of the immune stimulator can be close to each other (≤ 10 cm), or far apart (> 10 cm). Distant sites include, but are not limited to, contralateral sites (eg, in the opposite shoulder or thigh).

The sites of intradermal and intramuscular administration can be close to each other (≤ 10 cm) or far apart (> 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 tethered to the contralateral site to the intramuscular site.

Intradermal administration can be performed prior to, concurrently with, or subsequent to intramuscular or intratumoral administration. In some embodiments, intradermal administration is performed within 60 minutes, within 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 of, before, or after intramuscular or intratumoral administration.

In some embodiments, the methods comprise a single round of ID-EP administration of nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators. In some embodiments, the methods comprise at least two rounds of ID-EP administration of nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators. The second round of ID-EP administration (enhancement) can be performed two weeks to 12 months after the first round of ID-EP administration (initial). 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 subsequent booster administrations may be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequent booster administrations can be about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks , about 11 weeks or about 12 weeks apart. In some embodiments, the additional boost is administered about 1-5 years after the initial or booster administration.

Nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators can be administered by ID-EP at the same site or at different sites. For ID-EP at different sites, the sites can be close to each other (≤ 10 cm), or far apart (> 10 cm) from each other. Distant sites include, but are not limited to, contralateral sites (eg, opposite shoulders or thighs). In some embodiments, the different sites are 1-5 cm or 2-3 cm apart.

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

In some embodiments, the methods comprise a single round of ID-EP administration of nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators and IM-ED administration of nucleic acids encoding coronavirus antigenic polypeptides. In some embodiments, the methods comprise at least two rounds of ID-EP administration of nucleic acids encoding coronavirus antigenic polypeptides and immunostimulators and IM-ED administration of nucleic acids encoding coronavirus antigenic polypeptides. Execute ID-EP and IM-EP investment in each round. The second round of ID-EP and IM-EP investment (enhancement) can be performed two weeks to 12 months after the first round of ID-EP and IM-EP investment (initial). 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 subsequent booster administrations may be performed at intervals of 2-12 weeks. In some embodiments, the first and subsequent booster administrations can be about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks , about 11 weeks or about 12 weeks apart. In some embodiments, the additional boost is administered about 1-5 years after the initial or booster administration.

The ID-EP and IM-EP sites can be close to each other (≤ 10 cm) or far apart (> 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 tied to the contralateral site of 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 tied in one shoulder and the IM-EP site is tied in the deltoid muscle of the opposite shoulder.

The ID-EP administration can be performed before, in parallel with, or after the IM-EP administration. In some embodiments, ID-EP administration is performed within 60 minutes, within 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, the ID-EP administration is performed prior to the IM-EP administration. In some embodiments, the ID-EP administration is performed after the IM-EP administration. In some embodiments, ID-EP administration is performed within 5-15 minutes of, before, or after IM-EP administration.

In some embodiments, the methods comprise at least one (cycle) administration of IT-EP of nucleic acid encoding an immunostimulator (IT-EP IL-12 therapy) and IT-EP of nucleic acid encoding a coronavirus antigenic polypeptide, ID-EP or IM-EP contribution. In some embodiments, the methods comprise at least two rounds (cycles) of IT-EP administration of nucleic acid encoding an immunostimulator and IT-EP, ID-EP or IM-EP administration of nucleic acid encoding a coronavirus antigenic polypeptide and, where the second round is cast after the first.

The IT-EP administration may be performed before, concurrently with, or subsequent to the ID-EP or IM-EP administration. In some embodiments, IT-EP administration is performed within 60 minutes, within 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 of, before, or after IT-EP or IM-EP administration.

In some embodiments, the cycle of IT-EP IL-12 therapy comprises Day 1 (± 2 days); Day 1 (± 2 days) and Day 5 (± 2 days); Day 1 of a 3 week cycle Days (± 2 days) and 8 (± 2 days); or Days 1 (± 2 days), 5 (± 2 days) and 8 (± 2 days) of nucleic acids encoding immunostimulators IT-EP investment. In some embodiments, IL-12 is administered on day 1 (± 2 days), day 5 (± 2 days), and day 8 (± 2 days) of each cycle, and the coronavirus antigenic polypeptide is Day 1 (± 2 days); Day 5 (± 2 days); Day 8 (± 2 days); Day 1 (± 2 days) and Day 5 (± 2 days); Day 1 (± 2 days) of each cycle Day 1 (± 2 days) and Day 8 (± 2 days); Day 1 (± 2 days), Day 5 (± 2 days) and Day 8 (± 2 days); or Day 5 (± 2 days) 2 days) and 8 days (± 2 days). In some embodiments, IL-12 is administered on day 1 (± 2 days) and 8 (± 2 days) of each cycle, and the coronavirus antigenic polypeptide is administered on day 1 (± 2 days) of each cycle 2 days), 8 days (± 2 days), or 1 day (± 2 days) and 8 days (± 2 days). In some embodiments, IL-12 is administered on day 1 (± 2 days) and 5 (± 2 days) of each cycle, and the coronavirus antigenic polypeptide is administered on day 1 (± 2 days) of each cycle 2 days), 5 days (± 2 days) or day 1 (± 2 days) and 5 days (± 2 days). In some embodiments, IL-12 is administered on day 1 (± 2 days), day 5 (± 2 days), and day 8 (± 2 days) of each cycle, and the coronavirus antigenic polypeptide is Administered on day 1 (± 2 days) of each cycle.

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

In some embodiments, the described vaccines are administered to cancer patients. In some embodiments, the coronavirus vaccination is by administration of the tumor to the individual in combination with tumor therapy, by intratumoral electroporation, in combination with nucleic acid encoding a coronavirus antigenic polypeptide and nucleic acid encoding an immunostimulatory interleukin . The coronavirus antigenic polypeptide can be, but is not limited to, the SARS-CoV-2 S protein or an antigenic fragment thereof. The immunostimulatory interleukin can be, but is not limited to, interleukin, IL-12, CXCL9, anti-CD3 half-BiTE, genetic adjuvants, or a combination thereof. The described vaccine, which combines intratumoral electroporation of coronavirus antigenic polypeptides (IT-EP) and IT-EP of immunostimulatory interferons such as IL-12, provides these patients with vaccination and resistance to coronavirus. cancer therapy. Immunocompromised patients, such as those with cancer, benefit from the ability to not only drive anti-tumor responses but also generate sustained immunity against SARS-CoV-2 by strengthening their immune systems to build defenses against viral infections such as COVID-19. vaccine.

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).

Any of the methods disclosed herein for vaccinating against a coronavirus can be modified to vaccinate against another pathogen by substituting an antigenic polypeptide of the pathogen of interest for a polypeptide of the coronavirus. Similarly, any of the coronavirus vaccine compositions described herein can be readily modified to prepare vaccine compositions against different pathogens by substituting the coronavirus polypeptides with antigenic polypeptides of the pathogen of interest.

Expression vectors encoding immunostimulatory interferons, pathogenic antigens, or both interleukins and pathogenic antigens for use in the treatment of cancer are described. Also described are methods of treating tumors, including cancers and metastatic cancers, using the described expression vectors. When delivered to an individual by intratumoral electroporation (IT-EP), optionally combined with intradermal electroporation (ID-EP) or intramuscular electroporation (IM-EP), the described expression vector results in an encoded protein expression, resulting in T cell recruitment and antitumor activity. Such expression vectors and methods can be used to regress tumors (ie, reduce tumor volume), regress one or more tumors in an individual, soften tumors, prolong survival, prolong tumor-free survival, and/or enhance immune responses against tumors. In some embodiments, the described methods also produce a distal effect, ie, regression of one or more untreated tumors. In some embodiments, regression includes solid tumor debulking.

Described are methods of treating cancer comprising administering to an individual by intratumoral electroporation (IT-EP) a therapeutically effective amount of an expression vector encoding an immunostimulatory interleukin and a therapeutically effective amount of an expression vector encoding a pathogenic antigen Compositions. The expression vector encoding the immunostimulatory interleukin and the expression vector encoding the pathogenic antigen may be present in the same plastid or vector or in different plastids or vectors. For plastids or vectors encoding both the immunostimulatory interleukin and the pathogenic antigen, such as in a polycistronic plastid or vector, the sequences encoding the immunostimulatory interleukin and the pathogenic antigen are operably linked to A single promoter and linked via an IRES or 2A translational modification element; or sequences encoding immunostimulatory interkines and pathogenic antigens are operably linked to separate promoters. The composition is injected into the tumor, tumor microenvironment, and/or tumor margin tissue, and electroporation therapy is administered to the tumor, tumor microenvironment, and/or tumor margin tissue. Electroporation therapy can be administered by any suitable electroporation system known in the art. In some embodiments, electroporation is performed 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, electroporation is combined with electrochemical impedance spectroscopy (EIS). The individual can be a mammal. The mammal can be, but is not limited to, human, canine, feline, or equine.

Described are methods of treating 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 interleukin, and by intradermal electroporation (IT-EP) ID-EP) or intramuscular electroporation (IM-EP) to administer expression vectors encoding pathogenic antigens to individuals. Compositions containing expression vectors encoding immunostimulatory interkines are injected into tumors, tumor microenvironments, and/or tumor margin tissues, and electroporation therapy is administered to tumors, tumor microenvironments, and/or tumor margin tissues. Compositions containing expression vectors encoding pathogenic antigens are injected into the dermis or skeletal muscle, and electroporation therapy is administered to the dermis or skeletal muscle at the injection site. Electroporation therapy can be administered by any suitable electroporation system known in the art. In some embodiments, electroporation is performed 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, electroporation is combined with electrochemical impedance spectroscopy (EIS). The individual can be a mammal. The mammal can be, but is not limited to, human, canine, feline, or equine.

The immunostimulatory interleukin can be, but is not limited to, IL-12 or IL-15. In some embodiments, the immunostimulatory interferon comprises IL-12. IL-12 is a heterodimeric interleukin with both IL-12A (p35) subunits and IL-12B (p40) subunits. The encoded IL-12 may 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 by a polycistronic expression vector from a single promoter and linked via an IRES or 2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, the polycistronic expression vector comprises the IL12 p35 coding sequence and the IL-12 p40 coding sequence and the pathogenic antigen coding sequence, each of which is separated by an IRES or 2A element. In some embodiments, the 2A element is a P2A element. The expression vector encoding the immunostimulatory interferon can be delivered before, after or concurrently with one or more of the pathogenic antigens.

A pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoa, parasites, and fungi. The 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, Polio virus, German measles virus, Rotavirus, Human Papilloma virus, Enterovirus, West Nile virus, Ebola virus, Zika virus, Human immunodeficiency virus, Lyssa virus, 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 not limited to, betacoronavirus antigen, A-series betacoronavirus (Ebercoronavirus subgenus) antigen, B-series betacoronavirus (Sabecoronavirus subgenus) antigen, C-series betacoronavirus (Mobeycoronavirus) antigen. Coronavirus subgenus) antigen, D-line betacoronavirus (Nobecoronavirus subgenus) antigen, SARS-CoV antigen, MERS-CoV antigen or SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen is a spike protein antigen. Bacterial pathogens may be, but are not limited to, Bacillus diphtheriae, Haemophilus influenzae type b, Bordetella pertussis, Streptococcus pneumoniae, Neisseria pneumococcus, Clostridium tetani, Neisseria meningitidis, Salmonella typhi, Vibrio cholerae and Yarrowia. Parasitic pathogens can be, but are not limited to, Plasmodium, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium vivax, Trypanosoma brucei, and Leishmania. In some embodiments, the pathogenic antigen is a viral antigen. A pathogenic antigen can be any antigen that induces an immune response against a pathogen.

In some embodiments, the pathogenic antigen is the SARS-CoV-2 spike protein antigen and the immunostimulatory interleukin is IL-12.

In some embodiments, the methods further comprise the 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, the administration of one or more immune checkpoint inhibitors.

In some embodiments, the methods comprise: administering to the individual by intratumoral electroporation a first effective dose of a expression vector encoding a pathogenic antigen and a first effective dose of an expression vector encoding an immunostimulatory interleukin. The pathogenic antigen can be a viral antigen. The viral antigen may be a coronavirus antigenic polypeptide. The coronavirus antigenic polypeptide can be, but is not limited to, the coronavirus spike protein or an antigenic fragment thereof. The immunostimulatory interferon can be, but is not limited to, IL-12 or a combination of IL-12 and a second immunostimulator. In some embodiments, immunostimulatory interferons (eg, IL-12) and pathogenic antigens (eg, coronavirus S protein polypeptides) are encoded on polycistronic plastids.

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

Described are methods of treating a tumor in an individual comprising: administering to the individual at least one treatment cycle, 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). -EP IL-12 therapy), 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 therapy or IM-EP PA therapy) . In some embodiments, the cycle is a four-week, five-week 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 days 1, 2, 3, 4, 5 or 6 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, the IT-EP IL-12 treatment is administered on Day 1, Day 5±2, and Day 8±2 of each cycle. ID-EP PA treatment or IM-EP PA treatment can be administered on days 1, 2, 3, 4, 5 or 6 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 Day 1, Day 5±2, and Day 8±2 of each cycle. The cycle is repeated as often as necessary to treat the individual. In some embodiments, the cycle further comprises the administration of additional therapeutic agents. The additional therapeutic agent can be, but is not limited to, immune checkpoint therapy. In some embodiments, the immune checkpoint therapy is administered to the individual on day 1, day 2, or day 3 of the cycle.

In some embodiments, the individual is treated with more 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 One of the cycle treatment. Any of the above cycles may be repeated in subsequent cycles. Subsequent cycles may be continuous cycles or alternating cycles. Alternating cycles may have one or more intervening cycles of no therapy or alternative therapy (eg, immune checkpoint therapy). For example, any of the described expression vehicles can be on day 1, day 5±2, day 8±2 of alternating cycles (eg, cycles 1, 3, 5, etc., as desired) and one or more of Day 15±2, and the replacement therapy can be administered, for example, on Day 1±2 of consecutive cycles (eg, cycles 1, 2, 3, 4, 5, etc., as needed) , 2 days ± 2 days or 3 days ± 2 days administration.

Cycles of immunostimulatory interleukin plus pathogenic antigen therapy can be administered in the presence or absence of immune checkpoint inhibitor therapy. In other words, a composition comprising a therapeutically effective amount of one or more of the described expression vectors encoding immunostimulatory interleukins (eg, IL-12) and/or pathogenic antigens can be administered to an individual, and in odd-numbered Immune checkpoint inhibitor therapy is administered on cycles 1, 3, etc.; and immune checkpoint inhibitor therapy is administered on even numbered cycles (cycles 2, 4, etc.).

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

Such expression vectors and methods can be used to treat individuals with tumors predicted to be refractory to or unresponsive to one or more anticancer therapies. In some embodiments, the individual has low tumor-infiltrating lymphocytes, a low fraction of cytotoxic lymphocytes, or exhausted T cells. In some embodiments, the individual has progressed on one or more prior cancer therapies.

These expression vectors and methods can be used to treat individuals who have been previously infected with a pathogen, individuals who have previously had a disease or condition associated with infection with the pathogen, individuals who have been previously exposed to the pathogen, or individuals who have 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.

Cross-references to related applications

This application claims US Provisional Application No. 63/142,250, filed January 27, 2021, US Provisional Application No. 63/142,229, filed January 27, 2021, and US Provisional Application No. 63/142,229, filed March 29, 2020 The interests of Application No. 63/001,353, US Provisional Application No. 62/994,375, filed March 25, 2020, and US Provisional Application No. 62/985,192, filed March 4, 2020, in which Each of these is incorporated herein by reference. sequence listing

The size of the sequence listing written in document 555179_SeqListing_ST25.txt is 96 kilobytes, which was created on February 16, 2021 and is hereby incorporated by reference. 1. Definition:

A "vaccine" is one or more substances or one or more compositions used to stimulate an immune response, such as antibody production, and provide immunity against disease without inducing disease. Vaccines are often prepared from the causative agent of the disease or the product of the pathogen, such as a polypeptide or a nucleic acid encoding the polypeptide. When administered to an individual, the vaccine induces or stimulates an immune response. Vaccines can make an individual resistant to or immune to a particular disease or infection. Vaccines can also reduce the severity of an infection or reduce the duration of an infection.

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

"Nucleic acid" includes RNA and DNA. RNA and DNA include, but are not limited to, cDNA, genomic DNA, plastid DNA, condensed nucleic acids, nucleic acids formulated with cationic lipids, nucleic acids formulated with peptides or cationic polymers, RNA and mRNA. Nucleic acids also include modified RNA or DNA. Nucleic acids including plastids can be produced on a large scale and/or in high yields. Nucleic acids can be further manufactured using cGMP manufacturing. Nucleic acids can be formulated as safe and effective nucleic acids for injection into mammalian subjects.

A "homologous" sequence (eg, a nucleic acid sequence or an amino acid sequence) refers to a sequence that is identical or substantially similar to a known reference sequence such that, for example, it is at least 75%, at least 80% similar to the known reference sequence , at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. Sequence identity can be determined by using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics suite of software version 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., using preset gap parameters, or by checking and maximizing Sequences are aligned for optimal alignment (ie, yielding the highest percent sequence similarity over a comparison window). The percent sequence identity is calculated by comparing the two optimally aligned sequences over a comparison window, determining the number of positions where identical residues occur in the two sequences to obtain the number of matched positions, comparing the matched The number of positions is divided by the total number of matched and unmatched positions that do not count gaps in the comparison window (ie, the window size), and the result is multiplied by 100 to obtain the percent sequence identity. Unless otherwise indicated, the comparison window between two sequences is bounded by the entire length of the shorter of the two sequences.

"Expression vector" refers to a nucleic acid (eg, RNA or DNA) encoding one or more expression products (eg, peptides (ie, polypeptides or proteins)). Expression vectors can be, but are not limited to, viruses, attenuated viruses, plastids, linear DNA molecules, or mRNA. Expression vectors are capable of expressing one or more polypeptides in cells such as mammalian cells. The expression vector may contain one or more sequences required to express the encoded expression product. Various sequences can be incorporated into expression vectors to alter the presentation of the coding sequences. The expression vector may comprise one or more of the following: 5' untranslated region (5' UTR), enhancer, promoter, intron, 3' untranslated region (3' UTR), terminator and operably PolyA signal linked to DNA coding sequence. Any of the described nucleic acids encoding coronavirus antigenic polypeptides and/or immunostimulators may be part of an expression vector designed to express the coronavirus antigenic polypeptides or immunostimulators in cells.

The term "plastid" refers to a nucleic acid (eg, an expression vector) that includes at least one sequence encoding a polypeptide capable of being expressed in a mammalian cell. A plastid can be a closed circular DNA molecule. Various sequences can be incorporated into plastids to alter the expression of coding sequences in cells or to aid in plastid replication. Sequences that affect transcription, messenger RNA (mRNA) stability, RNA processing or translation efficiency can be used. Such sequences include, but are not limited to, 5' untranslated regions (5' UTRs), promoters, introns, and 3' untranslated regions (3' UTRs). In some embodiments, plastids can be transferred into bacteria such as E. coli.

A "promoter" is a DNA regulatory region capable of binding RNA polymerase in a cell (eg, directly or through other proteins or substances that bind a promoter) and initiate transcription of a coding sequence. A promoter may contain one or more additional regions or elements that affect the rate of transcription initiation, including but not limited to enhancers. The 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 not limited to CMV promoter, Igκ promoter, mPGK, SV40 promoter, β-actin promoter (such as but not limited to human or chicken β-actin promoter), α-actin promoter, SRα 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), S. sarcoma virus (rous sarcoma virus, RSV) promoter and EF1α promoter. A CMV promoter can be, but is not limited to, a CMV immediate early promoter, a human CMV promoter, a mouse CNV promoter, and a monkey CMV promoter. The promoter can also be a hybrid promoter. Hybrid promoters include, but are not limited to, the CAG promoter.

A "translational modification element" enables the translation of two or more genes from a single transcript. Translational modification elements include an internal ribosome entry site (IRES), which allows initiation of translation from internal regions of the mRNA, and a picornavirus-derived 2A peptide that enables ribosomes to skip the peptide at the C-terminus of the element key synthesis. Incorporation of translational regulatory elements results in the co-expression of two or more polypeptides from a single polycistronic mRNA. 2A regulons include, but are not limited to, P2A, T2A, E2A, or F2A. The 2A regulon contains a PG/P cleavage site.

"Operably linked" or "operably linked" refers to the juxtaposition of two or more components (eg, a promoter and another sequence element) such that both components function normally and allow The likelihood that at least one of the components may mediate an effect exerted on at least one of the other components. For example, a promoter is 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 regulators. Operably linked can include such sequences contiguous to each other or acting in trans (eg, regulatory sequences can act at a distance to control transcription of the coding sequence).

A "secretory signal" (also known as a 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. The signal peptide is usually located at the N-terminus of the polypeptide and removed by a signal peptidase. The secretion signal can be a native secretion signal or a heterologous secretion signal. The encoded signal peptide is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide.

A "trimerization domain" is an amino acid sequence that facilitates, aids or stabilizes the assembly of polypeptides in trimers. Assembly can occur via association with other trimerization domains. When attached to a polypeptide, the trimerization domain can be used to form artificial trimers. The trimerization domain can use a coiled-coil motif. The term is also used to refer to nucleic acid sequences encoding such amino acid sequences. Trimerization domains include, but are not limited to, the basic leucine zipper domain, the GCN4pl trimeric heptapeptide repeat region, its GCN4 modifier domain that allows trimeric coiled-coil formation, and the T4 fibrin C-terminal domain. The T4 fibrin trimerization domain (also known as the "folder") contains amino acid sequences that trimerize in nature. In some embodiments, the T4 fibrin trimerization domain is linked to a coronavirus S protein antigenic polypeptide. T4 fibrin trimerization domains include, but are not limited to, peptides comprising GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 29), GSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 30), or MQALQEAGYIPEAP-RDGQAYVRKDGEWVLLSTFLSPA (SEQ ID NO: 31). In some embodiments, a microfibrin trimerization domain comprising ADIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQ-YGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQEAGYIPEAPRDGQAY-VRKDGEWVLLSTFLSPALVPRGSHHHHHHSAWSHPQFEK (SEQ ID NO: 46) is linked to a coronavirus S protein antigenic polypeptide.

"Fc domain" refers to the "fragment crystallizable" region of an immunoglobulin heavy chain. 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 the IgG antibody isotype, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody. The Fc domain can be, but is not limited to, a human Fc domain. The Fc domain can be, but is not limited to, an IgG Fc domain, an IgG1 Fc domain, an IgG4 Fc domain. Compared to the native IgGl Fc domain, the Fc domain may contain one of more modifications (such as amino acid substitutions, deletions or insertions) that reduce binding affinity to Fc receptors and/or reduce effector function.

A "heterologous" sequence is a sequence that is not normally present in a cell, gene body, or gene in the context of the gene for which the sequence is currently found. A heterologous sequence can be a sequence derived from a gene or species different from the reference gene or species. Heterologous sequences can be from homologous genes in different species, from different genes in the same species, or from different genes in different species. For example, a regulatory sequence can be heterologous because it is linked to a different coding sequence than the native regulatory sequence.

An "immunostimulatory interleukin" includes an interleukin that mediates or enhances an immune response against foreign antigens including viral, bacterial or tumor antigens. Immunostimulatory interleukins may include, but are not limited to, TNFα, IL-1, IL-7, IL-10, IL-12, IL-12 p35, IL-12 p40, IL-15, IL-15Rα, IL-21IL -23, IL-27, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-5, IL-7, IL-9, IL-21, TGFβ and CXCL9.

The term "electroporation" refers to the use of electroporation pulses to facilitate entry of biomolecules such as plastids, nucleic acids or drugs into cells.

"Reversible electroporation" is the use of electrical pulse waves below the electric field threshold of the target cell to reversibly or temporarily penetrate into the cell membrane of molecules that are normally impermeable to the cell membrane. Because the electrical pulses are below the electrical threshold of the cells, the cells can be repaired and not killed by the electrical pulses. Reversible electroporation can be used to deliver macromolecules such as nucleic acids into cells without killing the cells. Reversible electroporation is a method of applying electrical pulse waves to aid in the absorption of macromolecules such as nucleic acids into cells. Unless otherwise indicated, references herein to electroporation include reversible electroporation.

"Draining lymph nodes" are lymph nodes that filter lymph from a specific area or organ. In the case of nucleic acid-based vaccines, the draining lymph nodes are downstream of the treatment area (eg, the site of intradermal or intramuscular administration).

"Immune checkpoint" molecules are any of a group of immune cell surface receptors/ligands that induce T cell dysfunction or apoptosis. These immunosuppressive targets reduce excessive immune responses and ensure self-tolerance. Tumor cells exploit the inhibitory effect of these checkpoint molecules. Immune checkpoint target molecules include but are not limited to cytotoxic T lymphocyte antigen-4 (CTLA-4), planned death 1 (PD-1), planned death ligand 1 (PD-L1), lymphocyte activation gene -3 (LAG-3), T cell immunoglobulin mucin-3 (TIM3), killer cell immunoglobulin-like receptor (MR), B-lymphocyte and T-lymphocyte attenuation factor (BTLA), adenosine A2a receptor (A2aR) and herpes virus entry mediator (HVEM). "Immune checkpoint inhibitors" include molecules that prevent immunosuppression by blocking the action 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. Immune checkpoint inhibitors can be, but are not limited to, PD-1 and/or PD-L1 antagonists. The PD-1 and/or PD-L1 antagonist can be, but is not limited to, an anti-PD-1 or anti-PD-L1 antibody. Anti-PD-1/PD-L1 antibodies include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

The term "cancer" includes a variety of diseases that are generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. Examples of cancers 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 sarcoma.

A "treatment-refractory cancer" is a cancer that has not responded or has not responded to at least one prior medical treatment. In some embodiments, with respect to treatment, treatment refractory indicates insufficient response to treatment or lack of partial or complete response to treatment. For example, a patient may be considered refractory to treatment (eg, checkpoint inhibitor therapy, such as PD-1 or PD-L1 inhibitor therapy) if the patient does not show at least a partial response after receiving at least 2 doses of therapy .

"Tumor microenvironment" refers to the environment surrounding a tumor and includes non-malignant vascular and stromal tissues that aid tumor growth and/or survival, such as by supplying the tumor with oxygen, growth factors, and nutrients, or by inhibiting immune responses against the tumor. The tumor microenvironment includes the cellular environment in which the tumor resides, including peripheral blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix.

"Tumor margin" or "marginal tissue" is the visually normal tissue in close proximity to or surrounding the tumor. Typically, marginal tissue is visually normal tissue within 0.1-2 cm of the tissue. When a tumor is surgically removed, tissue bordering the tumor is often removed. II. Vaccine composition

Described are vaccine compositions suitable for inducing an immune response against a pathogen in an individual, the composition comprising a pathogenic antigen and at least one immune stimulator. A pathogenic antigen can be an isolated polypeptide or a nucleic acid encoding a pathogenic antigenic polypeptide. The immunostimulator can be an isolated polypeptide or a nucleic acid encoding an immunostimulator. Nucleic acids can be, but are not limited to, RNA, mRNA, DNA, plastids, or expression vectors.

In some embodiments, the pathogen is a coronavirus. The coronavirus vaccine compositions described can be used to vaccinate against coronavirus infection. The coronavirus can be but is not limited to betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), B betacoronavirus (subgenus Sabeicoronavirus), C betacoronavirus (subgenus Mobecoronavirus) genus), D-line betacoronavirus (subgenus Nobecoronavirus), SARS-CoV, MERS-CoV, SARS-CoV-2, or related betacoronaviruses. The coronavirus vaccine compositions described combine at least one coronavirus antigenic polypeptide with one or more immune stimulators. The immune stimulator can be an immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. The immunostimulatory interleukins can be selected from the group consisting of: IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-[alpha], IFN-[beta], IFN-[gamma], TGF-[beta] and CXC 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 described vaccine compositions comprise nucleic acid-based vaccines. Nucleic acid-based vaccine compositions are advantageous for several reasons. Nucleic acids (eg, plastids or expression vectors) can rapidly adapt (within weeks or even days) to new mutations generated in the virus. The described nucleic acid-based vaccine induces humoral immunity while also stimulating a Th1-biased T cell response. Most vaccine approaches rely on neutralizing antibodies (humoral immunity). However, analysis of the most recently known SARS-CoV relative to SARS-CoV-2 has shown that the CD4 effector interleukin is an important vaccine component and that neutrophil hyperabundance associated with Th2-biased responses may limit vaccines effect. Coordination of humoral responses with Th1-biased T cell responses can provide an improved immune protective response.

Pathogenic antigens and/or immune stimulators can be administered to an individual by administering one or more nucleic acids encoding the pathogenic antigens and/or immune stimulators. 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), needle-free injection ( with or without reversible electroporation), microprojectile bombardment (eg, gene gun), hydrodynamic injection, magneto-fection, sono-poration (eg, ultrasound-mediated delivery), Photo-poration and hydro-poration. Nucleic acid sequences can be in nanoparticles, liposomes, lipid complexes, polymeric complexes, lipid polymeric complexes, or other non-viral particles or complexes. In some embodiments, the immunogenic composition further comprises one or more electroporation applicators.

Nucleic acid-based vaccines represent an affordable safe and effective alternative to conventional vaccines. Nucleic acid-based vaccines are significantly faster and easier to manufacture than most other vaccine platforms. Additionally, nucleic acids are significantly safer to dispose of than live/attenuated virus 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). Plastid DNA vaccines also have room temperature stability and long shelf life. A) Pathogen antigenic polypeptides

A pathogen can be a microorganism that causes a disease or condition. Pathogenic microorganisms include, but are not limited to, viruses, bacteria, protozoa, parasites, and fungi. The 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, Polio virus, German measles virus, Rotavirus, Human Papilloma virus, Enterovirus, West Nile virus, Ebola virus, Zika virus, Human immunodeficiency virus, Lyssa virus, Rabies virus, Yellow fever virus, Japanese encephalitis virus, Hantavirus and coronavirus. Bacterial pathogens may be, but are not limited to, Bacillus diphtheriae, Haemophilus influenzae type b, Bordetella pertussis, Streptococcus pneumoniae, Neisseria pneumococcus, Clostridium tetani, Neisseria meningitidis, Salmonella typhi, Vibrio cholerae and Yarrowia. Parasitic pathogens can be, but are not limited to, Plasmodium, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium vivax, Trypanosoma brucei, and Leishmania.

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

The coronavirus antigenic polypeptide can be but is not limited to betacoronavirus antigenic polypeptide, A series betacoronavirus (Ebercoronavirus subgenus) antigenic polypeptide, B series betacoronavirus (Sabecoronavirus subgenus) antigenic polypeptide , C series betacoronavirus (Mobecoronavirus subgenus) antigenic polypeptide, D series betacoronavirus (Nobecoronavirus subgenus) antigenic polypeptide, SARS-CoV antigenic polypeptide, MERS-CoV antigenic polypeptide, SARS - CoV-2 antigenic polypeptide or related betacoronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide comprises a coronavirus spike glycoprotein (also known as S glycoprotein or S protein) or an antigenic fragment thereof. The coronavirus spike polypeptide or its antigenic fragment can be but not limited to betacoronavirus spike protein or its antigenic fragment, A series betacoronavirus (Ebecoronavirus subgenus) spike protein or its antigenic fragment, B series betacoronavirus Virus (Sabecoronavirus subgenus) spike protein or its antigenic fragment, C series betacoronavirus (Mobecoronavirus subgenus) spike protein or its antigenic fragment, D series betacoronavirus (Nobecoronavirus subgenus) ) spike protein or antigenic fragment thereof, SARS-CoV spike protein or antigenic fragment thereof, MERS-CoV spike protein or antigenic fragment thereof, SARS-CoV-2 spike protein or antigenic fragment thereof, or related betacoronavirus spike protein or antigenic fragment thereof.

The coronavirus spike glycoprotein (S protein) facilitates the interaction with host cells. SARS-CoV and SARS-CoV-2 spike glycoprotein (S protein) facilitates interaction with host cells by binding to the ACE2 receptor (Figure 1A). The spike glycoprotein contains a large extracellular domain followed by a transmembrane sequence and a short intracellular tail with a predicted molecular weight of ~141 kDa (Figure IB). The S1 subunit of the S-glycoprotein contains a receptor binding domain (RBD) that recognizes the host cell receptor for SARS-CoV-2, namely angiotensin-converting enzyme 2 (ACE2). The S2 subunit contains a fusion peptide, two heptapeptide repeats, and a transmembrane domain, all of which are required to mediate fusion of the virus to the host cell membrane by undergoing large conformational rearrangements. The S1 and S2 subunits trimerize to form large prefusion spinous bodies.

Genealogical analysis has revealed that among all virus strains from the family Coronaviridae, SARS-CoV-2 shares 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 SARS-CoV-2 strains together with two bat-derived SARS-like strains form distinct clades of coronaviruses. Therefore, the related immune mechanisms and related antigens of SARS-CoV have been applied to the SARS-CoV-2 coronavirus vaccine. The coronavirus S glycoprotein has demonstrated immunogenicity, thereby triggering 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 “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).

Conserved gene sequences encoding the S glycoprotein were identified by comparing multiple SARS-CoV-2 isolates (Lu et al. 2020). The S-glycoprotein gene sequence has limited synonymous mutations between virus strains, which may help limit immune evasion variants.

The S protein antigenic polypeptide may contain any combination of one or more of the following: one or more mutations that disrupt the internal furin cleavage site, one or more mutations that stabilize the protein, native or heterologous secretion signals, Native or heterologous transmembrane domains, native or heterologous trimerization domains, Fc tags, and reading frames codon-optimized for human expression (Figures 7-8). Additionally, the S protein antigenic polypeptide may optionally contain a HRV3C protease cleavage site and/or one or more affinity (epitope) tags.

Mutations in the S protein that disrupt the internal furin cleavage site can be, but are not limited to, RRAR substitutions with GSAS corresponding to amino acid positions 664-667 of SEQ ID NO: 1. In some embodiments, the S protein antigenic polypeptide contains a native furin cleavage site. In some embodiments, the S protein antigenic polypeptide contains a transmembrane domain and a native furin cleavage site.

In some embodiments, the S protein antigenic polypeptide contains one or more mutations that stabilize the protein. Mutations that stabilize the S protein can include mutations that stabilize the spike protein in the prefusion conformation. Stabilizing the prefusion conformation increases the immunogenicity of the spike protein. Stabilizing the prefusion conformation can also increase the expression of the spike protein by the nucleic acid expressing the spike protein. Mutations that stabilize the spike protein in the prefusion conformation can be, but are not limited to, amino acid substitutions with proline (2P) at the apex of the central helix and heptapeptide repeat 1 (corresponding to SEQ ID NO: 1 of the amino acids K ₉₈₆ and V ₉₈₇ ). The 2P mutation has been shown to stabilize the spike protein in the prefusion conformation in various betacoronaviruses including MERS-CoV, SARS-CoV and HCoV-HKU1.

In some embodiments, the S protein antigenic polypeptide contains a secretion signal. The secretion signal can be a native secretion signal or a heterologous secretion signal. The heterologous secretion signal can be, but is not limited to, a tyrosine protein phosphatase alpha secretion signal or an Ig-κ secretion signal. In some embodiments, native secretion signals are 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.

S protein antigenic polypeptides may or may not contain native S protein transmembrane and/or intracellular domain tails. The S protein antigenic polypeptide may contain a heterologous transmembrane domain.

The trimerization domain assists in the trimerization of the extracellular domain of the spike protein. The heterotrimerization domain can be, but is not limited to, a C-terminal phage T4 fibrin trimerization motif (folder domain). In some embodiments, the S protein antigenic polypeptide contains a native or heterologous trimerization domain. In some embodiments, the S protein antigenic polypeptide does not contain a native/heterologous trimerization domain.

In some embodiments, the nucleic acid encoding a coronavirus antigenic polypeptide comprises a mammalian codon-optimized nucleic acid encoding a microfibrin foldon sequence (amino acids 1-112 of SEQ ID NO: 46). The foldon sequence may further contain a C-terminal thrombin cleavage site, a 6xHis tag, and a Strep-tag II (ADIVLNDLPFVDGPPAEGQSRISWI-KNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQE-AGYIPEAPRDGQAYVRKDGEWVLLSTFLSPALVPRGSHHHHHSAWSHPQFEK (SEQ D NO: 46)).

The 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 6×His tag or an 8×His tag).

In some embodiments, the SARS-CoV-2 spike protein is encoded on a nucleic acid, as described by Wrapp et al. ("Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation." Science 2020 367(6483) :1260-1263). This SARS-CoV-2 antigenic polypeptide contains residues 1 to 1208 of the ectodomain/extracellular domain of the SARS-CoV-2 S protein and is C-terminal using a stabilization strategy proven to be effective against other betacoronavirus S proteins S2 fusion mechanism having two stabilizing proline mutation (e.g. belonging to SEQ ID NO: 1 and corresponds to residues MERS S proteins yl V1060 and L1061 and the K ₉₈₆ P V ₉₈₇ P) (Pallesen et al, "Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen.” Proc. Natl. Acad. Sci. USA 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). _Proline mutations (KV 986-987 PP, ie 2P) help stabilize the prototypical prefusion morphology in the polypeptide (ie, prefusion stabilizing mutations). In some embodiments, the nucleic acid encoding the S protein antigenic polypeptide comprises a secretion signal that causes secretion of the expressed polypeptide.

In some embodiments, the nucleic acid encoding a coronavirus antigenic polypeptide comprises DNA. DNA can be single-stranded or double-stranded, linear or circular, relaxed or supercoiled. DNA can be an expression vector or a plastid. In some embodiments, the nucleic acid encoding a coronavirus antigenic polypeptide comprises RNA. RNA can be mRNA.

Nucleic acid-based vaccines can be rapidly adapted to new mutations in targeted viruses such as SARS-CoV-2. Nucleic acid-based vaccines can adapt to new mutations in the viral genome within weeks or even days. For novel coronavirus strains or mutants of existing strains, the nucleic acid sequence encoding the S protein is sequenced using methods known in the art. Subsequently, a nucleic acid encoding a novel S protein antigenic polypeptide corresponding to a nucleic acid encoding an S protein antigenic polypeptide described herein is prepared and combined with a nucleic acid encoding an immune stimulator to form a vaccine composition to elicit a vaccine against the novel coronavirus Immune response to virus strain or mutation. B) Immune stimulants

The coronavirus antigenic polypeptide is co-administered with one or more immune stimulants. The immune stimulator can be an immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE. One or more immune stimuli support innate effector cells, presentation of coronavirus antigenic polypeptides, and activation of cellular antiviral T cell responses. The immunostimulator can be an isolated polypeptide or a nucleic acid encoding an immunostimulator. In some embodiments, the nucleic acid encoding the immunostimulator comprises DNA. DNA can be single-stranded or double-stranded, linear or circular, relaxed or supercoiled. DNA can be an expression vector or a plastid. In some embodiments, the nucleic acid encoding the immunostimulator comprises RNA. RNA can be mRNA.

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

In some embodiments, the immune stimulant comprises an immune stimulatory interleukin. Immunostimulatory interleukins include, but are not limited to, IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15/receptor alpha, IL-21, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TGF-β and CXCL9. C) IL-12

In some embodiments, the immunostimulatory interferon comprises IL-12. IL-12 is a pro-inflammatory interleukin known to effectively support NK (natural killer) cell and macrophage function and also polarize Th1-biased T cell responses. IL-12 has been reported to be increased in both cellular and humoral immune responses. Interleukin 12 (IL-12) plays a role in the induction of Th1-biased responses, coordinating both the innate and adaptive immune systems. IL-12 can also enhance antigenicity by activating antigen presenting cells and aid in humoral responses by promoting isotype switching towards more functional antibodies.

IL-12 is a heterodimeric interleukin with both IL-12A (p35) subunits and IL-12B (p40) subunits. The nucleic acid encoding IL-12 may comprise a nucleic acid sequence encoding an IL-12 p40-IL-12 p35 fusion protein (IL-12 p70), a nucleic acid encoding an IL-12 p35-IL-12 p40 fusion protein (IL-12 p70) Sequence or IL-12 p35 subunit and IL-12 p40 subunit. The nucleic acid sequences encoding the IL-12 p35 and IL-12 p40 subunits can be on contiguous nucleic acid sequences separated by translational modifying elements, allowing both subunits to be expressed from a single promoter or single mRNA. The translational modification element can be an internal ribosome entry site (IRES) element or a ribosome hopping regulator. Ribosome skipping regulators can be, but are not limited to, 2A elements (also known as 2A peptides or 2A self-cleaving peptides). 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 by polycistronic expression vectors from a single promoter and separated by IRES or 2A elements. D) CXCL9

In some embodiments, the immunostimulatory interferon comprises CXCL9. The C-X-C motif chemokine ligand 9 (CXCL9) is a small interleukin belonging to the CXC chemokine family. CXCL9 is also known as interferon-gamma-induced monoglobin (MIG). CXCL9 is a T cell chemoattractant and aids in the chemotactic recruitment of tumor-infiltrating lymphocytes (TILs). E) Flt3L

In some embodiments, the immunostimulatory interferon comprises a genetic adjuvant. A "genetic adjuvant" is a polypeptide that can be encoded by a nucleic acid and that acts as an adjuvant, enhancing an antigen-specific immune response compared to an 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 known as 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.

Flt3L is an immunoregulatory growth factor that supports dendritic cell differentiation and maturation, both of which are involved in T cell activation. Clinical data suggest that Flt3L enhances both humoral and T cell responses to antigens (Bhardwaj, 2016). Plasmids encoding the IL-12 interferon and the Flt3L immunomodulator have been shown to enhance immune responses induced by DNA-vaccine against the model antigen OVA (SIINFEKL epitope) (Figures 2A-C).

In some embodiments, the genetic adjuvant is linked to a pathogen antigenic polypeptide, 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.

In some embodiments, the immunogenicity of pathogen antigens is enhanced by co-delivery with IL-12 and/or Flt3 ligands. The pathogen antigen can be expressed by the same plastid as the IL-12 and/or Flt3 ligand or a different plastid than the IL-12 and/or Flt3 ligand. F) Anti-CD3 Half-BiTE

In some embodiments, the immunostimulatory interleukin comprises an anti-CD3 half-BiTE. Anti-CD3 half-BiTEs comprise anti-CD3 single-chain variable fragments (scFv) fused to a transmembrane domain (TM). scFvs comprise fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins linked to short linker peptides. Anti-CD3 scFvs can be recognized by phage display. Anti-CD3 scFvs can also be generated by subpopulating VH and VL with known anti-CD3 antibodies, such as from fusionomas. Known anti-CD3 antibodies have been described, for example, 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 the anti-CD3 scFv can be humanized. A humanized antibody (or antibody fragment or domain) is an antibody from a non-human species whose protein sequence has been modified to increase its similarity to antibody variants that occur naturally in humans. In some embodiments, humanized antibodies can be made by inserting the relevant complementarity determining regions (CDRs, also known as hypervariable regions (HVRs)) of anti-CD3 antibodies into the human VH and VL domain backbones.

An anti-CD3 scFv can be formed by linking the C-terminus of the VH chain to the N-terminus of the VL. Alternatively, the C-terminus of VL can be linked to the N-terminus of VH. The peptide linker can be from about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is rich in glycine. The 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 known as [(Gly) ₄ Ser] ₃ , (G ₄ S) ₃ or G4S (×3)). In some embodiments, the scFv peptide linker consists of G4S (x3).

The transmembrane domain (TM) comprises a polypeptide capable of inserting into the biological lipid bilayer (membrane) and anchoring the anti-CD3 half-BiTE to the membrane. TMs are known in the art and generally consist primarily of non-polar 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, the 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-VLTIISLIILIMLWQKKPR (SEQ ID NO: 35), AVGQDTQEVIVVPHSLPFKVVVISAILA-LVV LTIISLIILIMLWQKKPR (SEQ ID NO: 36), VVISAILALVVLTVISLIILI (PDGFRβ) (SEQ ID NO: 36) ID NO: 37), VVISAILALVVLTIISLIILI (PDGFRβ) (SEQ ID NO: 38), AAVLVLLVI-VIISLIVLVVIW (PDGFRα) (SEQ ID NO: 39) and AAVLVLLVIVIVSLIVLVVIW (PDGFRα) (SEQ ID NO: 40). In some embodiments, TM domain selected from the group comprising a nucleic acid sequence encoding the group of: gtgggccaggacacgcaggaggtcatcgtggtgc-cacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggt-gctcaccatcatctcccttatcatcctcatcatgct-ttggcagaagaagccacgt (SEQ ID NO: 41), gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgc-cctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaa-gccacgt (SEQ ID NO: 42), tggtgatct cagccatcctggccctggtggtgctcaccatcatctcccttatcatcctcatc (PDGFRβ) (SEQ ID NO: 43), gtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatca tcctcatc (PDGFRβ) (SEQ ID NO: 44), gctgcagtcctggtgctgttggtg attgtgatcatctcacttattgtcctggttgtcatt-tggaa (PDGFRα) (SEQ ID NO: 45).

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

In some embodiments, the one or more immune stimulators comprise IL-12 and any of CXCL9, Flt3L, or anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulators comprise IL-12, CXCL9, and anti-CD3 half-BiTEs. In some embodiments, the one or more immune stimulators comprise CXCL9 and an anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulators comprise IL-12 and CXCL9. In some embodiments, the one or more immune stimulators comprise IL-12 and an anti-CD3 half-BiTE. In some embodiments, the one or more immune stimulators comprise IL-12 and Flt3L. Flt3L can be linked to pathogen antigenic polypeptides. The pathogenic antigen linked to Flt3L can be any of the SARS-CoV-2 antigenic polypeptides described herein. G) Polycistronic expression vector

The nucleic acid encoding at least one immune stimulator can be present on a single plastid or on multiple plastids. Similarly, the nucleic acid encoding the pathogenic antigen may be present on a plastid containing one or more nucleic acid sequences encoding 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 encoding more than one polypeptide may contain polycistronic expression vectors.

In some embodiments, the nucleic acid encoding IL-12 also encodes a Flt3 ligand, a Flt3L-pathogenic antigen fusion polypeptide, CXCL9, an anti-CD3 half-BiTE, or a pathogen antigenic polypeptide. In some embodiments, IL-12 and Flt3L, Flt3L-pathogenic antigen fusion polypeptide, CXCL9, anti-CD3 half-BiTE, or pathogenic antigen are expressed by a polycistronic expression vector from a single promoter. In some embodiments, the nucleic acid encoding CXCL9 also encodes an anti-CD3 half-BiTE. CXCL9 and anti-CD3 half-BiTEs can be expressed by polycistronic expression vectors from a single promoter. Coding sequences in polycistronic expression vectors can be spaced apart by IRES or 2A translational modification elements. In some embodiments, the translational modification element comprises an IRES element. In some embodiments, the translational modification element comprises a 2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

The expression vector or plastid may contain a polycistronic expression vector. Polycistronic expression vectors express two or more separate proteins from the same mRNA and contain one or more translational modification elements. In some embodiments, an expression vector encoding IL-12 expresses two or three polypeptides expressed from a single promoter, wherein one or more translational modification elements enable the expression of two or three polypeptides from a single mRNA. In some embodiments, the expression vector ^{comprising: (a) PAT 1 -B,} (b) PBT 1 -A, (c) PBT 1 -B ', (d) PAT 1 -BT 2 -B' or (e) PBT ¹ -B'-T ² -A wherein P is a promoter, A encodes pathogenic antigenic polypeptide, Flt3L, Flt3L-pathogenic antigen fusion polypeptide, CXCL9 or anti-CD3 half-BiTE, and B and B' encode IL-12 or IL -12 subunit, and T ¹ and T ² are both translational modification element. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

In some embodiments, T ¹ and T ² are independently internal ribosome entry site (IRES) element or ribosome jumping regulator. Ribosome skipping regulators can be, but are not limited to, 2A elements (also known as 2A peptides or 2A self-cleaving peptides). The 2A element can be, but is not limited to, a P2A (SEQ ID NO: 28), T2A, E2A or F2A element. H) Nucleic acid formulations

Nucleic acids encoding any of the described pathogen antigenic polypeptides and/or immune stimulators can be formulated for in vivo administration. In some embodiments, the nucleic acid is formulated for in vivo administration by electroporation. Nucleic acids can be made using methods known in the art including, but not limited to, forming overlapping gene block fragments. Fragments can be spliced 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.

The mRNA can be in vitro transcribed mRNA. In vitro transcribed mRNA can be produced from a DNA template using RNA polymerase. The polymerase can be, but is not limited to, T7, T3 or Sp6 phage RNA polymerase. The DNA template can be supercoiled plastid, loose loop DNA or linear DNA template. The transcribed mRNA may contain one or more of the following: 5' UTR, 3' UTR, 5' cap, polyA tail, pseudouridine modified nucleotides, and 1-methyl pseudouridine modified core Glycosides. In some embodiments, the mRNA is self-amplifying mRNA. Self-amplifying RNA encodes both the antigen or immune stimulator of interest and the RNA-dependent RNA polymerase that enables RNA replication.

In some embodiments, a DNA template encoding a coronavirus antigenic polypeptide is flanked by 5' and 3' untranslated regions and a poly-A tail. The 5' UTR may be, but is not limited to, 5'-GGGA AAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUA-UAAGAGCCACC-3' (SEQ ID NO: 47). The 3' UTR may be, but is not limited to, 5'-UGAUAAUAGGCUGGAGCCCUGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC-CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAA-GUCUGA-3' (SEQ ID NO: 48).

In some embodiments, the donor methyl S-adenosylmethionine (SAM) is added to the methylated capped RNA. I) Polypeptide Formulations

In some embodiments, a pathogenic antigen or immune stimulator can be produced by polypeptide biosynthesis in a host cell. For biosynthesis in host cells, nucleic acids encoding pathogenic antigens or immune stimulators are produced and transformed or transfected into host cells. In some embodiments, the host cell is an industrially scalable microbial or eukaryotic culture cell. In some embodiments, the pathogenic antigen or immune stimulator is isolated and/or purified. Polypeptides can be isolated and purified from synthesis reaction mixtures by means of peptide purifications known in the art. For example, polypeptides can be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, fractionation or convective distribution. III. Formulations

The described vaccine compositions can be formulated for intradermal, intramuscular, and/or intratumoral administration to an individual. Intradermal administration includes injection into the dermis or delivery into the dermis. Intramuscular administration includes injection into skeletal muscle or delivery to skeletal muscle. Intratumoral administration includes injection into a tumor or delivery to a tumor. Intradermal, intramuscular, and intratumoral administration can be performed using devices known in the art for administration to the dermis, skeletal muscle, or tumors, including, but not limited to, conventional syringes and needles.

Nucleic acids encoding pathogen antigenic polypeptides and immune stimulators can be formulated for administration into the dermis (intradermal administration), into skeletal muscle (intramuscular administration), or into tumors (intratumoral administration) ).

The nucleic acid can be administered as naked nucleic acid, or the nucleic acid can be combined with protamine, cationic nanoemulsions, dendrimer nanoparticles, protamine liposomes, liposomes, cationic polymers, polysaccharide particles, polyliposomes, A combination of cationic lipid nanoparticles, cationic lipid cholesterol particles or cationic lipid cholesterol PEG nanoparticles. In some embodiments, electroporation is used to enhance the delivery of nucleic acids to cells.

In some embodiments, nucleic acids encoding pathogen antigenic polypeptides and/or immune stimulators are encapsulated in liposomes such as lipid nanoparticles (LNPs). LNP formation can be performed using methods available in the art including, but not limited to, ethanol drop nanoprecipitation. In some embodiments, the LNP contains 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and PEG-lipid; or ionizable cationic lipid, phospholipid, cholesterol and PEG - Lipids. In some embodiments, the LNP contains lipids in a 50:10:38.5:1.5 molar ratio (ionizable lipid:DSPC:cholesterol:PEG-lipid or ionizable cationic lipid, phospholipid, cholesterol, and PEG-lipid) , DSPC, cholesterol and PEG-lipid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the size of the LNPs is between 80-100 nm.

In some embodiments, nucleic acids encoding pathogen antigenic polypeptides and immune stimulators can be formulated for intradermal electroporation (ID-EP), intramuscular electroporation (IM-EP), and/or intratumoral electroporation ( IT-EP) was administered into the dermis. Detection in serum of secreted protein following electroporation of nucleic acid encoding the model protein is shown in FIG. 3 . In some embodiments, nucleic acids encoding coronavirus antigenic polypeptides and immune stimulators can be formulated for injection by direct injection, needle-free injection, microprojectile bombardment, hydrodynamic injection, magnetic transfection, ultrasonic perforation, photoporation Or water perforated administration into the dermis, muscle or tumor. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

Intradermal electroporation (ID-EP) involves injecting one or more nucleic acids into the dermis and administering at least one electroporation pulse to the dermis at the injection site. The one or more nucleic acids can be injected prior to or substantially concurrently with the electroporation pulse administration. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

Intramuscular electroporation (IM-EP) involves injecting one or more nucleic acids into skeletal muscle and administering at least one electroporation pulse to the skeletal muscle at the injection site. The one or more nucleic acids can be injected prior to or substantially concurrently with the electroporation pulse administration. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

Intratumoral electroporation (IT-EP) involves injecting one or more nucleic acids into a tumor and administering at least one electroporation pulse to the tumor. The one or more nucleic acids can be injected prior to or substantially concurrently with the electroporation pulse administration. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

For administration of the isolated pathogen antigenic polypeptide or the isolated immunostimulatory polypeptide, the isolated polypeptide can be combined with a vaccine adjuvant. In some embodiments, the isolated pathogen antigenic polypeptide or the isolated immunostimulatory polypeptide is administered with a vaccine adjuvant. In some embodiments, the polypeptide is combined with an adjuvant prior to injection. The adjuvant can be, but is not limited to, alum, the Sigma adjuvant system, or Freund's adjuvant. The alum can be, but is not limited to, alum hydrogels. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

Any of the described vaccine compositions may contain 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 the nucleic acid encoding the pathogenic antigen or immune stimulator one or more of them. In some embodiments, the pathogenic antigen is a coronavirus antigenic polypeptide.

Pharmaceutically acceptable excipients ("excipients") are intentionally included in drug delivery systems other than active pharmaceutical ingredients (APIs, therapeutic products; such as nucleic acids encoding coronavirus antigenic polypeptides or immunostimulators) substances in. The excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. Excipients can be used to a) aid in processing the drug delivery system during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API, c) aid in product identification, and/or d) during storage or use Any other attributes that enhance the overall security, effectiveness of the API delivery during the period. Pharmaceutically acceptable excipients may or may not be inert substances.

Excipients include, but are not limited to: transfection enhancers, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colors, delivery enhancers, delivery polymers, polydextrose, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, slip agents, humectants, lubricants, oils, polymers, preservatives, saline, Salts, solvents, sugars, suspending agents, sustained-release bases, thickening agents, tonicity agents, vehicles, water-repellent and wetting agents. Transfection enhancers include, but are not limited to, lipids, cationic lipids, lipids, polycations, cell penetrating peptides, and combinations thereof.

Vaccine compositions may contain other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (eg, antihistamines, diphenhydramine, etc.). "Pharmacologically effective amount", "therapeutically effective amount" or simply "effective dose" as used herein refers to the amount of the described nucleic acid that produces the desired pharmacological, therapeutic or prophylactic result.

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, normal saline and phosphate buffered saline (PBS). IV. Set

Any of the described isolated polypeptides, nucleic acids, and/or vaccine compositions comprising the isolated polypeptides and/or nucleic acids disclosed herein can be packaged or included in a kit, container, pack, or dispenser. Any of the described isolated polypeptides, nucleic acids and/or vaccine compositions comprising isolated polypeptides, nucleic acids can be packaged in prefilled syringes or vials. The isolated polypeptide, nucleic acid, and/or vaccine composition may be provided as a lyophilized powder or it may be provided as a solution. A kit can include reagents for performing the methods disclosed herein. A kit may also include a composition, tool, or instrument disclosed herein, such as, but not limited to, an electroporation applicator. In some embodiments, a kit includes one or more of the described nucleic acid and/or nucleic acid-containing vaccine composition and an electroporation device or applicator. In some embodiments, a kit includes one or more of the described nucleic acid, one or more electroporation applicators, a syringe, and an injection needle. Nucleic acids encoding coronavirus antigenic polypeptides (such as SARS-CoV-2 spike protein and antigenic fragments thereof) and nucleic acids encoding immune stimulants (such as IL-12) may be provided in separate containers (vials, etc.) or they may be Combination in a single container. The isolated coronavirus antigenic polypeptide and the isolated immune stimulator can be provided in separate containers (vials, etc.) or they can be combined in a single container.

In some embodiments, the vaccine composition comprises a first container containing nucleic acid encoding a coronavirus antigenic polypeptide and a second container containing nucleic acid encoding an immune stimulator. In some embodiments, the vaccine composition comprises a first container comprising a coronavirus antigenic polypeptide and a second container comprising a nucleic acid encoding an immunostimulator. In some embodiments, the vaccine composition comprises a first container comprising a coronavirus antigenic polypeptide and a second container comprising an immunostimulatory polypeptide. In some embodiments, the vaccine composition comprises a first container comprising nucleic acid encoding a coronavirus antigenic polypeptide and a second container comprising an immunostimulatory polypeptide.

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

In some embodiments, the vaccine composition comprises a first container comprising nucleic acid encoding a coronavirus antigenic polypeptide and a nucleic acid encoding an immunostimulator and a second container comprising nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the vaccine composition comprises a first container comprising a coronavirus antigenic polypeptide and an immunostimulatory polypeptide and a second container comprising a coronavirus antigenic polypeptide.

The container may contain sufficient polypeptide or nucleic acid to provide a single effective dose or multiple effective doses. The polypeptide or nucleic acid encoding one or more coronavirus antigenic polypeptides and immunostimulators can be any of the described coronavirus antigenic polypeptides, immunostimulators, or combinations thereof.

In some embodiments, the kit further contains one or more of the following: instructions for use or a notice in the form of a regulation by a governmental agency governing the manufacture, use, or sale of the product. V. Vaccination

Prophylactic antiviral vaccines typically rely on neutralizing antibodies by triggering a humoral immune response. This antibody-directed immunity is typically generated by attenuated or subunit vaccines in a Th2 polarized environment. Th1-type interferons tend to generate pro-inflammatory responses responsible for killing intracellular pathogens. Excessive Th2 responses may inhibit or counteract Th1-type responses. Th1-type immune responses are elicited by co-administration of immune stimuli. Th1-type immune responses can limit the clinical pathology and associated immune subsets that are sometimes associated with Th2-specific interferons. Initiating a Th1-polarized immune response can help drive cellular T-cell responses, which can be critical for isotype switching and induction of antiviral effector interleukins. In some embodiments, the vaccine compositions and methods described can elicit both a Th2-biased response (humoral immunity) and a Th1-biased T cell response.

IL-12 plays a role in the induction of Th1-biased responses, coordinating both the innate and adaptive immune systems. In addition to triggering cellular immunity, IL-12 can also enhance antigenicity by activating antigen presenting cells. IL-12 may also aid humoral responses by promoting isotype switching towards more functional antibodies. With the inclusion of an immune stimulator such as IL-12, the described vaccine compositions drive a coordinated immune response capable of utilizing the innate, adaptive humoral and adaptive cellular repertoires.

The location and cellular architecture of the tissue used to deliver the nucleic acid-based vaccine can modulate the effectiveness and/or type of immune response generated by the vaccine. In some cases, intramuscular administration of non-viral nucleic acid vaccines has been shown to efficiently generate 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 of muscle tissue such as accessibility, large area and extensive vascularization enable persistent expression and efficient circulation of expressed proteins (Brown PA et al. 2008). Skin-targeted electroporation-mediated delivery 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). The skin contains antigen presenting cells (APCs) such as Langerhans cells that present antigens and activate cellular (T cell mediated) antiviral responses and skin resident dendritic cells (Hirao LA et al. 2011; and Tobin D "Biochemistry of human skin--our brain on the outside." Chem Soc Rev, 2006 35(1):52-67).

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

Local administration or presentation of coronavirus antigenic polypeptides and one or more immune stimulators can induce localized Th1-biased responses and cellular antiviral T cell responses. Since local cellular activation does not prevent activation of humoral immunity in secondary lymphoid tissues such as draining lymph nodes, the described vaccination method is able to generate both cellular and humoral anti-coronavirus immunity.

In some embodiments, the vaccine compositions and methods described can utilize the delivery of coronavirus antigenic polypeptides and immune stimuli to spatially distinct sites, such as the dermis and skeletal muscle. Discrete modules of one or more antigens and immune stimuli are deployed in the dermis to create an immunogenic cellular microenvironment where IL-12 readily supports innate effector cells and antigen presentation such as SARS-CoV-2 S antigenic polypeptides , and subsequently activated cellular antiviral T cell responses.

In some embodiments, the described vaccines induce coordinated immune responses involving humoral and cellular immune response pathways. In some embodiments, the described vaccines induce humoral and cellular immune responses.

In some embodiments, the described vaccines induce a humoral immune response. In some embodiments, the described vaccines mediate the production of neutralizing antibodies. In some embodiments, the neutralizing antibody prevents pneumovirus replication.

In some embodiments, the described vaccines induce cellular immune responses. Cellular immune responses can include both innate (NK) and adaptive (CD4 ⁺ T cells) groups and lead to the production of Th1-directed cytokines such as IFN-γ and TNF-α (Chen J et al. "The Immunobiology of SARS*"). Annu Rev Immunol , 2007 25:443-472; and Li CK et al. 2008).

In some embodiments, a nucleic acid encoding a coronavirus antigenic polypeptide and one or more nucleic acid sequences encoding at least one immune stimulator are delivered to an individual by ID-EP, IM-EP, and/or IT-EP.

In some embodiments, a nucleic acid encoding a coronavirus antigenic polypeptide and one or more nucleic acid sequences encoding at least one immune stimulator are delivered to patients with at least one immune stimulator by IT-EP, ID-EP, IM-EP, or a combination thereof An individual with a tumor.

In some embodiments, intramuscular electroporation (IM-EP) administration of nucleic acid comprises injecting a solution containing nucleic acid into skeletal muscle tissue and administering at least one electroporation pulse at the injection site. The muscles can be, but are not limited to, deltoid, leg muscles, or muscles in the buttocks.

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 nucleic acid is injected. In some embodiments, 0.25±0.15 mg of nucleic acid is injected. In some embodiments, 0.5±0.4 mg of nucleic acid is injected. In some embodiments, 0.8±0.5 mg of nucleic acid is injected. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector.

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 one or more injections can be in a single location or in two or more locations. For injections in two or more locations, the injections can be adjacent to each other or in separate locations. For example, an individual may receive 500 μL in two adjacent injections of 250 μL each. Similarly, an individual may receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be close enough to enable the electroporation applicator electrode to span the two injection sites.

In some embodiments, the nucleic acid is injected into skeletal muscle at a depth of 0.5 to 1.5 cm or greater. 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.

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

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 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 nucleic acid is injected. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector.

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 μL. The one or more injections can be in a single location or in two or more locations. For injections in two or more locations, the injections can be adjacent to each other or in separate locations. For example, an individual may receive 500 μL in two adjacent injections of 250 μL each. Similarly, an individual may receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be close enough to enable the electroporation applicator electrode to span the two injection sites.

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 active tissue layer, the granular layer.

In some embodiments, intratumoral electroporation (ID-EP) administration of nucleic acid comprises injecting a solution containing nucleic acid into the tumor and electroporating the injection site. Tumors can be, but are not limited to, solid tumors, skin tumors, subcutaneous tumors, or visceral tumors.

In some embodiments, 0.01-3 mg of 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 nucleic acid are injected. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector.

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 volumes 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 is injected into the tumor at a concentration of 0.5-1.0 mg/ml in a volume corresponding to 25% ± 10% of the calculated volume of the tumor. Injection into a tumor may include injection into marginal tissue surrounding the tumor. Injecting into the tumor may include injecting the tumor as uniformly as possible and, as appropriate, the peripheral tissue surrounding the tumor (ie, dispersing the injection throughout the tumor and optionally throughout the peripheral tissue surrounding the tumor).

For each of ID-EP, IM-EP, and IT-EP, the electrodes of the EP applicator are inserted so that the electrodes span one or more injection sites. The electrodes are usually placed at about the same depth as the injection. In some embodiments, the electrodes of the EP applicator are set to the same depth as the injection ±0.3 cm. In some embodiments, the electrodes of the EP applicator are set to the same depth as the injection ±0.2 cm. In some embodiments, the electrodes of the EP applicator are set to the same depth as the injection ±0.1 cm. The electrodes of the EP applicator were inserted so that electroporation pulses were delivered to the cells at the injection site.

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

In some embodiments, one or more pulse waves 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, one or more pulse waves are administered at field strengths of 400±250 V/cm, 400±100 V/cm, 400±75 V/cm, or 400±50 V/cm. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 administrations of the pulse wave. In some embodiments, the pulse at a field strength of about 400 V/cm is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times Wave. In some embodiments, the 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 ms. In some embodiments, the 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 milliseconds and a field strength of about 400 V/cm.

The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator may be present in the same solution or in different solutions. If present in separate solutions, the nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be injected concurrently or sequentially. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be combined prior to injection.

The isolated coronavirus antigenic polypeptide and the isolated immunostimulatory polypeptide may be present in the same solution or in different solutions. If present in separate solutions, the isolated coronavirus antigenic polypeptide and the isolated immunostimulatory polypeptide can be injected concurrently or sequentially. The isolated coronavirus antigenic polypeptide and the isolated immunostimulatory polypeptide can be combined prior to injection.

The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be co-injected into the dermis, muscle and/or tumor. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be combined prior to injection into the dermis, muscle and/or tumor. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be present on the same vector (eg, plastid or RNA). When present on the same vector, the nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding the immunostimulator can be expressed by different promoters or by a single promoter, such as in a polycistronic vector.

Intradermal administration includes, but is not limited to, injection into the dermis (eg, the dermis of the shoulder, thigh, or buttocks). In some embodiments, the intradermal administration comprises ID-EP.

Intramuscular administration includes, but is not limited to, injection into a muscle (eg, shoulder muscle (eg, deltoid), leg muscle (eg, anterolateral thigh muscle, vastus lateralis, or gastrocnemius), or gluteal muscle (eg, dorsal gluteal muscle)) . In some embodiments, the intramuscular administration comprises IM-EP.

Intratumoral administration includes, but is not limited to, injection into tumors. In some embodiments, the intratumoral administration comprises IT-EP.

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

In some embodiments, vaccination occurs in two steps comprising: (a) administering the first dose by intradermal administration and administering the second dose by intramuscular administration; (b) The first dose is administered by intradermal administration and the second dose is administered by intratumoral administration; or (c) the first dose is administered by intramuscular administration and is administered by intratumoral administration administering a second dose; 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 coronavirus Combinations of antigenic polypeptides and immunostimulators. Intradermal administration can be performed before, after, or concurrently with intramuscular administration. Intradermal administration can be performed before, after, or concurrently with intratumoral administration. Intramuscular administration can be performed before, after, 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.

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

The coronavirus antigenic polypeptide can be any of the coronavirus antigenic polypeptides described herein that include nucleic acid encoding a coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigenic polypeptide comprises a SARS-CoV-2 spike polypeptide or an antigenic fragment thereof. The immunostimulator can be any of the immunostimulators described herein that include a nucleic acid encoding the immunostimulator. In some embodiments, the second dose comprises nucleic acid encoding IL-12. In some embodiments, the second dose comprises nucleic acid encoding IL-12 and Flt3L, CXCL9, or an anti-CD3 half-BiTE.

A single vaccination may be administered to an individual or a primary vaccination and one or more booster vaccinations may be administered to the individual. In some embodiments, the primary and booster vaccinations can be administered to the individual in the same location (eg, in the same limb) or in different locations (eg, different limbs) on the individual.

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

In some embodiments, the first and subsequent booster administrations may be performed at 2-6 week intervals. 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

In some embodiments, the vaccines described 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 CD8+ T cell proliferation and/or response Memory T cell proliferation and/or response, balanced Th1/Th2 antibody isotype response, S-specific IgG2a and IgG1 response, prevention of upper airway coronavirus infection, prevention of lower airway coronavirus infection, protective immunity against coronavirus infection , to prevent symptomatic COVID-19 disease, to prevent at least one symptom of COVID-19 disease, to reduce the severity or duration of one or more symptoms of COVID-19 disease, and to prevent severe COVID-19 disease.

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 cells following administration of a single dose Proliferation and/or response, induction of balanced Th1/Th2 antibody isotype responses, induction of S-specific IgG2a and IgG1 responses, prevention of upper airway coronavirus infection, prevention of lower airway coronavirus infection, induction of protective immunity against coronavirus infection, Prevent symptomatic COVID-19 disease, prevent at least one symptom of COVID-19 disease, reduce the severity or duration of one or more symptoms of COVID-19 disease, or prevent severe COVID-19 disease.

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 following administration of an initial administration and at least one booster administration Response, increase memory T cell proliferation and/or response, induce balanced Th1/Th2 antibody isotype response, induce S-specific IgG2a and IgG1 response, prevent upper airway coronavirus infection, prevent lower airway coronavirus infection, induce anti-coronavirus Protective immunity against infection, prevention of symptomatic COVID-19 disease, prevention of at least one symptom of COVID-19 disease, reduction in the severity or duration of one or more symptoms of COVID-19 disease, or prevention of severe COVID-19 disease.

In some embodiments, administration of the described vaccines prevents coronavirus infection in the lungs and/or nose. In some embodiments, administration of the described vaccines results in reduced viral replication in the lungs and nose following challenge. In some embodiments, administration of the described vaccine results in reduced viral replication in the turbinate following challenge. VII. Methods of treating cancer

Pathogenic antigens and immune stimulators can be administered to an individual by administering one or more nucleic acids encoding the pathogenic antigens and immune stimulators. In some embodiments, the immunostimulator is an immunostimulatory interleukin. In some embodiments, the immunostimulatory interleukin is IL-12. In some embodiments, a nucleic acid encoding an immune stimulator is administered to a tumor, and a nucleic acid encoding a pathogenic antigen is administered to the tumor, dermis, or skeletal muscle.

Described are methods for treating a tumor in an individual comprising administering to the individual an effective amount of an expression vector encoding an immunostimulatory interleukin (eg, IL-12) and an effective amount of an expression vector encoding a pathogenic antigen. Expression vector encoding immunostimulatory interleukin 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 tumor margin tissue ( IT-EP therapy) to be administered to an individual. An expression vector encoding a pathogenic antigen is administered to an individual by (a) injecting the expression vector into the tumor, tumor microenvironment and/or tumor marginal tissue and administering electroporation therapy to the tumor, tumor microenvironment and /or tumor margin tissue (IT-EP treatment), (b) inject the expression vector into the dermis and administer electroporation therapy to the dermis at the injection site (ID-EP treatment), or (c) inject the expression vector into the dermis Electroporation therapy is administered to the muscle in skeletal muscle and at the injection site (IM-EP treatment). The expression vector encoding the immunostimulatory interferon can be administered prior to, concurrently with, or subsequent to administration of the expression vector encoding the pathogenic antigen. To administer both the expression vector encoding the immunostimulatory interleukin and the expression vector encoding the pathogenic antigen to the tumor, the expression vectors may be combined prior to injection, injected separately, or the expression vectors may be present on a single plastid , RNA or viral vectors.

Treated tumors can be skin tumors, subcutaneous tumors, or visceral tumors. Tumors can be cancerous or noncancerous. Tumors can be, but are not limited to, solid tumors, superficial lesions, non-superficial lesions, lesions within 15 cm of the body surface, or visceral lesions. In some embodiments, the described methods and expression vectors can be used to treat primary tumors as well as distal (ie, untreated) tumors and metastases. In some embodiments, the described compositions and methods provide tumor size reduction or tumor growth inhibition, cancer cell growth inhibition, metastasis inhibition or reduction, metastatic cancer development reduction or inhibition, and/or cancer recurrence in individuals with cancer reduce. Tumors are not limited to a particular type of tumor or cancer.

IT-EP Pathogenic Antigen (PA) therapy or treatment comprises injecting an effective dose of a described expression vector encoding a pathogenic antigen into the tumor, tumor microenvironment and/or tumor marginal tissue and administering at least one electroporation pulse to the tumor . Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

IT-EP S protein antigen therapy or treatment comprises injecting an effective dose of the described expression vector encoding the SARS-CoV-2 S protein antigen into the tumor, tumor microenvironment and/or tumor margin tissue and administering at least one electroporation to the tumor pulse wave. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

ID-EP pathogenic antigen (PA) therapy or treatment comprises injecting into the dermis an effective dose of the described expression vector encoding the pathogenic antigen and administering at least one electroporation pulse wave to the dermis at the injection site. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

ID-EP S protein antigen therapy or treatment comprises injecting into the dermis an effective dose of the described expression vector encoding the S protein antigen and administering at least one electroporation pulse wave to the dermis at the injection site. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

IM-EP pathogenic antigen (PA) therapy or treatment involves injecting an effective dose of a described expression vector encoding a pathogenic antigen into skeletal muscle and administering at least one electroporation pulse wave to the skeletal muscle at the injection site. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

ID-EP S protein antigen therapy or treatment comprises injecting into skeletal muscle an effective dose of the described expression vector encoding the S protein antigen and administering at least one electroporation pulse wave to the skeletal muscle at the injection site. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

IT-EP immunostimulatory interleukin therapy or treatment involves injecting an effective dose of an expression vector encoding an immunostimulatory interleukin into the tumor, tumor microenvironment, and/or tumor marginal tissue and administering at least one electroporation pulse to the tumor . Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

IT-EP IL-12 therapy or treatment comprises injecting an effective dose of an IL-12-encoding expression vector into the tumor, tumor microenvironment and/or tumor marginal tissue and administering at least one electroporation pulse to the tumor. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

IT-EP immunostimulatory interleukin-pathogenic antigen therapy or treatment comprises injecting into the tumor, tumor microenvironment and/or tumor marginal tissue an effective dose of an expression vector encoding an immunostimulatory interleukin and an effective dose of an encoded pathogenic agent expression vector for the antigen and at least one electroporation pulse is administered to the tumor. Electroporation pulse waves can be performed using any known electroporation device suitable for use in mammalian subjects.

The described expression vectors and methods are contemplated for use in individuals suffering from cancer. The tumor treated by the method of this embodiment can be any one of the following: non-invasive tumor, invasive tumor, superficial tumor, papillary tumor, flat tumor, metastatic tumor, localized tumor, monocentric tumor, multicentric tumor Tumors, low-grade tumors, and high-grade tumors. These growths may manifest themselves as any of the following: lesions, polyps, neoplasms (eg, papillary urothelial neoplasia), papillomas, malignancies, tumors (eg, Klatskin tumor, Hilar tumor, non-invasive papillary urothelial tumor, germ cell tumor, Ewing's tumor, Askin's tumor, primitive neuroectodermal tumor, Leydig cell tumor , Wilms' tumor, Sertoli cell tumor), sarcoma, carcinoma (eg, squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma , hepatocellular carcinoma, invasive papillary urothelial carcinoma, squamous urothelial carcinoma), lump, or any other type of cancerous or noncancerous growth. These expression vectors and methods can be used to treat advanced, metastatic, or treatment-refractory cancers.

The term "cancer" includes a variety of diseases that are generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. Cancers can be, but are not limited to, solid cancers, sarcomas, carcinomas, and lymphomas. Cancer may also be but not limited to pancreatic cancer, skin cancer, brain cancer, liver cancer, gallbladder cancer, stomach cancer, lymph node cancer, breast cancer, lung cancer, head and neck cancer, throat cancer, pharyngeal cancer, lip cancer, throat cancer, heart cancer, kidney cancer cancer, muscle cancer, colon cancer, prostate cancer, thymus cancer, testicular cancer, uterine cancer, ovarian cancer, skin cancer and subcutaneous cancer. 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" refers to malignant as well as non-malignant cell populations that often appear to be morphologically and genotypically distinct from surrounding tissue. In some embodiments, the described methods can be used to treat humans. In some embodiments, the described methods can be used to treat non-human animals or mammals. Non-human mammals can be, but are not limited to, mice, rats, rabbits, dogs, cats, pigs, cows, sheep, and horses.

The expression vectors and methods described herein are contemplated for use in, for example, adrenal cortical cancer, anal cancer, cholangiocarcinoma (eg, hilar cholangiocarcinoma, distal cholangiocarcinoma, intrahepatic cholangiocarcinoma), bladder cancer, benign and cancerous bone cancers (eg, osteoma, osteoid osteoma, osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma , lymphoma, multiple myeloma), brain and central nervous system cancers (e.g., meningioma, astrocytoma, oligodendritic glioma, ependymoma, glioma, medulloblastoma, neural gangliocytoma, schwannoma, blastoma, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, gynecomastia), cardia Castleman disease (eg, giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (eg, endometrial adenocarcinoma, adenoacanthoma, papillary serous gonadal carcinoma, clear cell), esophagus, gallbladder, (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoids (eg, choriocarcinoma, destructive chorioadenoma), Hodgkin's disease, Non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (eg, renal cell carcinoma), larynx and hypopharyngeal cancer, liver cancer (eg, hemangioma, hepatic adenoma, focal nodular hyperplasia) , hepatocellular carcinoma), lung cancer (such as small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and sinus cancer (such as sensitive neuroblastoma, midline granuloma), nasopharyngeal carcinoma, Neuroblastoma, oral and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary gland cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, polymorphism rhabdomyosarcoma), salivary gland cancer, skin cancer, both melanoma and non-melanoma skin cancer), gastric cancer, testicular cancer (e.g. seminoma, non-seminoma germ cell carcinoma), thymic cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal carcinoma, vulvar carcinoma and uterine carcinoma (eg uterine leiomyosarcoma).

The described methods can be used to induce one or more of the following: aggravate the tumor, induce T cell infiltration into the tumor or the tumor microenvironment (increase the number of tumor infiltrating lymphocytes (TIL)), enhance the systemic T cell response , induce T cell activation, induce tumor-specific T cell activation, enhance T cell response, enhance antigen-specific T cell response, increase T cell proliferation, increase antigen-specific T cell proliferation, enhance multi-line T cell response, increase tumor or NK and/or NKT cells in the tumor microenvironment, enhancing immune responses against treated and/or untreated tumors, reducing T cell exhaustion, increasing lymphocytes in one or more treated or untreated tumors, and Monocyte surface markers, increasing intratumoral levels of INFγ-regulated genes in one or more treated or untreated tumors, increasing proliferative effector memory T cells in the blood of an individual, increasing short-lived effects in the blood of an individual cells, increasing expression of genes present in activated natural killer cells in cancerous tumors, increasing expression of genes in cancerous tumors that play a role in antigen presentation, increasing T cell survival and T cell mediated cytotoxicity in cancerous tumors Gene expression at play, induction of treated and/or untreated tumor regression, induction of treated and/or untreated tumor debulking, and improved response to secondary therapies such as, but not limited to, immune checkpoint inhibitor therapy reaction. In some embodiments, cancer treatment includes one or more of: enhancing an immune response to a tumor, tumor regression, metastatic tumor regression, prolonging individual survival, and increasing tumor-free survival.

In some embodiments, ID-EP or IT-EP pathogenic antigen therapy enhances IL-12 effects, resulting in increased efficient trafficking of tumor-specific lymphocytes, increased tumor regression, decreased tumor volume, and/or survival or tumor-free Survival is prolonged. A) Treatment regimen/cycle

The described cancer therapies can be administered at various time intervals depending on factors such as tumor nature, individual condition, molecular size and chemical characteristics, and molecular half-life.

In some embodiments, and tumor IT-EP IL-12-pathogenic antigen therapy is administered on days 1 (± 2 days), 5 (± 2 days), and 8 (± 2 days) of a cycle . In some embodiments, tumor IT-EP IL-12-pathogenic antigen therapy is administered on days 1 (± 2 days) and 5 (± 2 days) of a cycle. In some embodiments, tumor IT-EP IL-12-pathogenic antigen therapy is administered on days 1 (± 2 days) and 8 (± 2 days) of a cycle.

A treatment cycle may contain 1-6 IT-EP treatments. In some embodiments, the treatment cycle comprises 1, 2 or 3 IT-EP treatments. Cycles can range from about 1 week to about 6 weeks. In some embodiments, the cycle is 2 weeks. In some embodiments, the cycle is 3 weeks. In some embodiments, the cycle is 4 weeks. In some embodiments, the cycle is 5 weeks. In some embodiments, the cycle is 6 weeks.

In some embodiments, the cycle comprises 1-3 IT-EP treatments. Treatment can occur on Day 1 (± 2 days), Day 5 (± 2 days), and/or Day 8 (± 2 days) (i.e., Day 0 (± 2 days), Day 4 (± 2 days) day) and/or day 7 (± 2 days)). Each treatment may comprise one or more of the following: 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.

In some embodiments, the treatment may be administered every cycle or every other cycle. Cycles can be repeated such that 2 or more cycles are administered to an individual. Repeated cycles may be administered continuously, alternating with one or more different treatment cycles, or operating in parallel with one or more different treatment cycles. Any of the treatments described above can be combined with other cancer therapies. For example, the IT-EP cycle can be combined with checkpoint inhibitor therapy. B) Combination therapy

In some embodiments, the method of treatment includes combination therapy. Combination therapy comprises a combination of therapeutic molecules or treatments. Therapeutic treatments include, but are not limited to, electrical pulse waves (ie, electroporation), radiation, antibody therapy, checkpoint inhibitor therapy, and chemotherapy. Therapeutic electroporation can be combined or administered with one or more additional therapeutic treatments. The one or more additional therapeutic agents can be delivered by systemic delivery, intratumoral injection, intratumoral injection with electroporation, and/or radiation. One or more additional therapeutic agents can be administered prior to, concurrently with, or subsequent to IT-EP IL12-pathogenic antigen therapy. VIII. Electroporation (EP):

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 are 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, 2012 23(3), 157-68). Electroporation (EP) is a technique of applying electrical pulse waves to temporarily penetrate cell membranes, thereby facilitating the absorption of macromolecules such as nucleic acids into cells. 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 greatly 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 , 2010 28 (4):1056-61).

The Apollo C-System (ACS) is a medical electroporation device system consisting of three main components: an electroporation generator that generates electrical pulse waves, a sterile applicator containing a 2-needle array, and a proximal connection to the electroporation generator The applicator cable assembly is connected at the distal end to the applicator. The APOLLO-C generator delivers electrical pulses with specific voltages and durations. The generator delivers controlled electroporation (EP) pulses in a square-wave pulse mode, creating a transmembrane potential for electroporation to occur. The applicator electroporation parameters are pre-programmed into the applicator cable assembly via a memory chip that determines the applied pulse width, pulse interval, pulse number, and applied potential for each electroporation sequence . The ACS applicator is a sterile (gamma-irradiated), single-piece assembly consisting of a plastic molded assembly with two stainless steel Trocar needles and a reusable cable assembly with a proximal connector attached to the APOLLO-C generator. Disposable device for secondary use. The reusable cable assembly contains a memory chip that stores the following specific pulse parameters: applied pulse width, pulse interval, pulse number, and applied potential for each electroporation therapy. Stainless steel Trocar needles are spatially 0.5 cm apart. The needle length is 1.5 cm, and the needle insertion depth can be adjusted by the user to be between 0 cm and 1.5 cm.

Additional electroporation devices suitable for use with the described compounds, compositions and methods include, but are not limited to, electroporation devices described in US Pat. Nos. 7,245,963, 5,439,440, 6,055,453, 6,009,347, Nos. 9020605 and 9037230 and US Patent Publication Nos. 2005/0052630, 2019/0117964 and Patent Application PCT/US2019/030437 and US Patent Application No. 16/269,022.

In some embodiments, electroporation therapy involves the administration of one or more voltage pulses. The properties of the electric field to be generated are determined by the properties of the tissue, the size of the selected tissue and its location. The voltage pulses that can be delivered to the tumor can range from about 100 V/cm to about 1500 V/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 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 electricity 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.

The pulse duration of the electroporation pulse may be 10 microseconds to 1 second. In some embodiments, the pulse duration is from about 10 microseconds to about 100 milliseconds (ms). In some embodiments, the pulse duration is 100 microseconds, 1 ms, 10 ms, or 100 ms. The time interval between sets of pulse waves can be any desired time, such as one second. The waveform, electric field strength, and pulse duration are also dependent on the cell type and the type of molecules that enter the cell via electroporation.

The waveform electrical signal provided by the pulse generator may be an exponentially decaying pulse, square pulse, unipolar vibratory pulse train, bipolar vibratory pulse train, or a combination of any of these forms. Square wave electroporation systems deliver controlled electrical pulses that rapidly rise to a set voltage, remain at that level for a set period of time (pulse length), and then rapidly drop to zero.

1 to 100 pulses can 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, 6 x 0.1 millisecond pulses are administered. In some embodiments, 6 pulses are administered. In some embodiments, 6 x 0.1 millisecond pulses at 1300-1500 V/cm are administered. In some embodiments, 8 pulses are administered. In some embodiments, 8 x 10 millisecond pulses are administered. In some embodiments, 8 x 10 millisecond pulses at 300-500 V/cm are administered.

In some embodiments, the EP applicator is inserted such that the electrode spans the nucleic acid injection site.

The electroporation device may comprise a single needle electrode, a pair of needle electrodes, or multiple needle electrodes or an array of needle electrodes. In some embodiments, the electroporation device comprises a hypodermic needle or equivalent device. In some embodiments, an electroporation device may include an electrodynamic device ("EKD device") capable of generating a series of programmable constant current pulse patterns between electrodes in an array based on user control and pulse parameter input . IX. List of Examples

1. A method of eliciting an immune response against a pathogen in an individual, comprising: administering to the individual an effective dose of a pathogenic antigen and an effective dose of an immune stimulator, wherein the immune stimulator comprises an immune stimulatory interleukin, a gene Adjuvant or anti-CD3 half-BiTE.

2. The method of embodiment 1, wherein the pathogen is a coronavirus selected from the group consisting of betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), and B betacoronavirus (Sabecoronavirus). virus subgenus), C series betacoronavirus (Mobecoronavirus subgenus), D series betacoronavirus (Nobecoronavirus subgenus), SARS-CoV, MERS-CoV and SARS-CoV-2.

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.

4. The method of embodiment 3, wherein the coronavirus spike protein or its antigenic fragment comprises the extracellular domain of the coronavirus spike protein or its antigenic fragment.

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

6. The method of embodiment 5, wherein the SARS-CoV-2 spike protein or antigenic fragment thereof comprises amino acids 1-1208, 14 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 -1208 or 21-1208 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.

7. The method of any one of embodiments 3 to 6, wherein the coronavirus spike protein or its antigenic fragment comprises a modified coronavirus spike protein or an antigenic fragment thereof.

8. The method of embodiment 7, wherein the modified coronavirus spike protein or its antigenic fragment comprises one or more of the following: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain and Affinity label.

9. The method of embodiment 7 or 8, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises one or more mutations that destroy the internal peptidase cleavage site and/or one or more that make the fusion prefabricated. Mutations in protein stabilization in shape.

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 .

11. The method of any one of embodiments 1-10, wherein administering the effective dose of the pathogenic antigen to the individual comprises administering to the individual an isolated pathogen antigenic polypeptide, optionally, wherein the pathogen Antigens include coronavirus antigenic polypeptides.

12. The method of any one of embodiments 1-10, wherein administering the effective dose of the pathogenic antigen to the individual comprises administering to the individual a nucleic acid encoding the pathogenic antigen and/or administering the individual to the individual. An effective dose of the immunostimulator comprises administering to the individual a nucleic acid encoding the immunostimulator, optionally wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

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

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

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

16. The method of embodiment 12 or 15, wherein the nucleic acid comprises a plastid or mRNA.

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, encoding an IL-12 p35-IL-12 p40 fusion The nucleic acid sequence of the protein or the nucleic acid sequence encoding the IL-12 p35 subunit and the IL-12 p40 subunit separated by an internal ribosome entry site (IRES) element or a 2A peptide skipping motif.

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

19. The method of embodiment 17, wherein the nucleic acid encoding IL-12 further encodes a Fms-like tyrosine kinase 3 ligand (Flt3L), a CXC motif chemotactic interleukin ligand 9 (CXCL9) or anti-CD3 Half BiTE.

20. The method of 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 with the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 22; (c) the 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% identity with the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 21.

21. The method of 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) the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8; or (d) A nucleic acid sequence having at least 90% identity with the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.

22. The method of any one of embodiments 15-21, wherein administering to the individual the effective dose of the coronavirus antigenic polypeptide and the effective dose of the immune stimulator comprises: (a) administering to the individual by intradermal administration a first effective dose of the nucleic acid encoding the coronavirus spike protein or an antigenic fragment thereof and a first effective dose of the nucleic acid encoding IL-12; or (b) administering to the individual by intradermal administration a first effective dose of the nucleic acid encoding IL-12 and, optionally, administering a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof and a second effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof is administered to the individual by intramuscular administration.

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 middle.

24. The method of embodiment 23, 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 administered intradermally combination.

25. The method of any one of embodiments 15-21, wherein administering to the individual the effective dose of the coronavirus antigenic polypeptide and the effective dose of the immune stimulator comprises: (a) administering to the individual a first effective dose of the nucleic acid encoding the coronavirus spike protein or an antigenic fragment thereof and a first effective dose of the nucleic acid encoding IL-12 by intratumoral administration; (b) administering to the individual a first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof by intradermal or intramuscular administration and administering the first effective dose to the individual by intratumoral administration An effective dose of the nucleic acid encoding IL-12; or (c) administering to the individual by intratumoral administration 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 and by intradermally or intramuscularly administering to the individual a second first effective dose of the nucleic acid encoding the coronavirus spike protein or the antigenic fragment thereof; wherein the individual has a cancerous tumor.

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

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 tumor Internal administration involves intratumoral electroporation (IT-EP).

28. The method of embodiment 27, wherein ID-EP, IM-EP and IT-EP comprise the administration of at least one voltage pulse with a field strength of about 100-1500 V/cm.

29. The method of embodiment 28, wherein ID-EP, IM-EP, and IT-EP comprise the administration of at least one voltage pulse with a field strength of about 400 V/cm and a duration of about 10 milliseconds.

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

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 immune response and a humoral immune response.

32. The method of any one of embodiments 1-31, wherein the immune response comprises one or more of the following: 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 Th1/Th2 antibody isotype response, S-specific IgG2a and IgG1 response, prevention of upper airway coronavirus infection, prevention of lower airway coronavirus infection, Protective immunity against coronavirus infection, preventing symptomatic COVID-19 disease, preventing at least one symptom of COVID-19 disease, reducing the severity or duration of one or more symptoms of COVID-19 disease, and preventing severe COVID-19 disease 19 Diseases.

33. A method of eliciting an immune response against the SARS-CoV-2 virus in an individual, comprising: (a) administering to the individual by ID-EP a first effective dose of a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12; (b) administering to the subject a first effective dose of a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof by IM-EP and administering to the subject a first effective dose of the coding by ID-EP Nucleic acid of IL-12; or (c) administering to the individual by ID-EP a first effective dose of a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12; and by IM - EP administers to the individual a second effective dose of a nucleic acid encoding a SARS-CoV-2 spike protein or an antigenic fragment thereof.

34. A method of eliciting an immune response against the SARS-CoV-2 virus in an individual with cancer, comprising: (a) administering to the individual by IT-EP a first effective dose of a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12; (b) administering to the individual 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 administering the first effective dose to the individual by IT-EP an effective dose of a nucleic acid encoding IL-12; or (c) administer to the individual by IT-EP a first effective dose of a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof and a first effective dose of a nucleic acid encoding IL-12 and by ID- The EP or IM-EP administers to the individual a second effective dose of a nucleic acid encoding the SARS-CoV-2 spike protein or an antigenic fragment thereof.

35. A vaccine for generating an immune response against a pathogen in an individual, the vaccine comprising: an effective dose of a pathogenic antigen or 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 immune stimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE or a combination thereof.

36. The vaccine of embodiment 35, wherein the pathogen is a coronavirus selected from the group consisting of betacoronavirus, A betacoronavirus (subgenus Ebecoronavirus), and B betacoronavirus (Sabecoronavirus). virus subgenus), C series betacoronavirus (Mobecoronavirus subgenus), D series betacoronavirus (Nobecoronavirus subgenus), SARS-CoV, MERS-CoV, SARS-CoV-2.

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.

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

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

40. The vaccine of embodiment 39, wherein the SARS-CoV-2 spike protein or antigenic fragment thereof comprises amino acids 1-1208, 14 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 -1208 or 21-1208 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.

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

42. The vaccine of embodiment 41, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises one or more of the following: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain, and Affinity label.

43. The vaccine of embodiment 41 or embodiment 42, wherein the modified coronavirus spike protein or the antigenic fragment thereof comprises one or more mutations that disrupt the internal peptidase cleavage site and/or one or more fusions Mutation of protein stabilization in pre-configuration.

44. The vaccine of embodiment 43, 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.

45. As the vaccine of any one of embodiments 35 to 43, the coronavirus antigenic polypeptide comprises an isolated coronavirus antigenic polypeptide.

46. As the vaccine of any one of embodiments 35 to 43, the coronavirus antigenic polypeptide comprises a nucleic acid encoding the coronavirus antigenic polypeptide.

47. The vaccine of any one of embodiments 35-46, the immune stimulator comprising an isolated immune stimulator polypeptide.

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

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

50. The vaccine of embodiment 46 or 49, wherein the nucleic acid comprises plastid or mRNA.

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

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 nucleic acid sequences encoding IL-12 p35 subunits and IL-12 p40 subunits separated by an internal ribosome entry site (IRES) element or a 2A peptide skipping motif.

53. The vaccine of embodiment 52, wherein the nucleic acid encoding IL-12 further encodes a Fms-like tyrosine kinase 3 ligand (Flt3L), a CXC motif chemokine ligand 9 (CXCL9) or anti-CD3 Half BiTE.

54. The vaccine of 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 with the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 22; (c) the 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% identity with the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 21.

55. The vaccine of 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) the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8; or (d) A nucleic acid sequence having at least 90% identity with the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.

56. The vaccine of any one of embodiments 35 to 54, wherein the pathogenic antigen or the nucleic acid encoding the pathogenic antigen and the immunostimulator or the nucleic acid encoding the immunostimulator are formulated for intradermal, intramuscular use. Intratumoral and/or intratumoral administration, where appropriate, wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

57. The vaccine of any one of embodiments 35 to 56, wherein the pathogenic antigen or the nucleic acid encoding the pathogenic antigen is combined with the immunostimulator or the nucleic acid encoding the immunostimulator, as the case may be, wherein The pathogenic antigen comprises a coronavirus antigenic polypeptide.

58. The vaccine of any one of embodiments 35 to 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 The nucleic acid of the stimulator is formulated for ID-EP, IM-EP and/or IT-EP, as appropriate, wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

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.

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

61. The vaccine of any one of embodiments 35 to 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 immunostimulator 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 immunostimulator 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 immunostimulator formulated for administration by ID-EP and formulated for use by a second effective dose of the nucleic acid encoding the coronavirus antigenic polypeptide administered 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 coronavirus formulated for administration by IM-EP Nucleic acids of viral antigenic polypeptides; (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 formulated for administration by IT-EP a dose of the nucleic acid encoding the immunostimulator; (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 immunostimulator formulated for administration by IT-EP and formulated for use by A second effective dose of the coronavirus antigenic polypeptide administered by ID-EP or IM-EP.

62. The vaccine of any one of embodiments 35 to 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 immunostimulator or the nucleic acid encoding the immunostimulator are in solution in separate containers or as a lyophilized powder; (b) The effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide and the effective dose of the immunostimulator or the nucleic acid encoding the immunostimulator are in solution in a container or Provided together 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 in the first Provide together in the form of solution or lyophilized powder in a container, and the second effective dose of the coronavirus antigenic polypeptide or the nucleic acid encoding the coronavirus antigenic polypeptide is in solution form or in the second container. Supplied as lyophilized powder.

63. The vaccine of any one of embodiments 35 to 62, wherein the effective dose of the pathogenic antigen or the nucleic acid encoding the pathogenic antigen and the effective dose of the immunostimulator or the nucleic acid encoding the immunostimulator Provided independently with one or more pharmaceutically acceptable carriers and/or excipients.

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

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

66. A method of reducing the likelihood or severity of infection by a pathogen in a patient, comprising the steps of: (a) administering to the patient a first immunogenicity comprising the vaccine of any one of embodiments 35-64 composition; (b) waiting 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 Examples 35-63.

67. A method of eliciting an immune response against a pathogen in an individual diagnosed with cancer, comprising: administering to the individual by intratumoral electroporation an effective amount of a nucleic acid encoding a pathogenic antigen and an effective amount of an immunostimulatory encoding nucleic acid of a substance, wherein the immune stimulator comprises an immunostimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE, optionally, wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide.

68. The method of embodiment 67, wherein the coronavirus antigenic polypeptide comprises SARS-CoV-2 spike protein or an antigenic fragment thereof and the immunostimulator comprises IL-12.

69. An expression vector comprising: a first nucleotide sequence encoding an immunostimulatory interleukin and a second nucleotide sequence encoding a pathogenic antigen, wherein the first nucleotide sequence and the second nucleoside The acid sequence is operably linked to a translational modification element.

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

71. The expression vector of embodiment 70, wherein the promoter is selected from the group consisting of: CMV promoter, mPGK, SV40 promoter, β-actin promoter, SRα promoter, herpes thymidine kinase promoter, Herpes simplex virus (HSV) promoter, mouse breast tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), Rous sarcoma virus (RSV) promoter and EF1α Promoter.

72. The expression vector of any one of embodiments 69-71, wherein the immunostimulatory interleukin comprises interleukin.

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

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 translational modification element.

75. The expression vector of any one of embodiments 69 to 74, wherein the pathogenic antigen comprises a viral antigen.

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

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

78. The expression vector of any one of embodiments 69 to 77, wherein the expression vector comprises a formula represented by: PAT ¹ -BT ² -B' or PT ¹ -BT ² -B'-A wherein P is the activation child, A encoding immunogenic disease antigen, T ¹ and T ² for the translational modifications element, B encoding IL-12 p35, and B 'encoding IL-12 p40.

Example 79. The expression vector of embodiment 78, 2A of the peptide group wherein T ¹ T ² and selected from the group consisting encoding of: P2A peptide, T2A peptide, E2A peptides and peptide F2A.

80. A plastid for expressing an immunostimulatory interleukin and a pathogenic antigen, comprising the expression vector of any one of embodiments 69-79.

81. The expression vector of any one of embodiments 69 to 79 or the plastid of embodiment 80 for use in the treatment of cancer in a subject.

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

83. A method of treating an individual with a tumor, comprising injecting an effective dose of an expression vector encoding an immunostimulatory interleukin into the tumor, the tumor microenvironment, and/or tumor marginal tissue, and injecting the tumor, the tumor microenvironment, and /or the tumor margin tissue is administered electroporation therapy, 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 tumor margin tissue; (b) injecting an effective dose of an expression vector encoding a pathogenic antigen into the dermis and administering electroporation therapy to the dermis at the injection site; or (c) injecting an effective dose of an expression vector encoding a pathogenic antigen into a skeletal muscle and administering electroporation therapy to the muscle at the injection site, Thereby reducing tumor size or inhibiting tumor growth, inhibiting cancer cell growth, inhibiting or reducing metastasis, reducing or inhibiting the development of metastatic cancer, and/or reducing cancer recurrence in the individual.

84. The method of embodiment 83, wherein the expression vector encoding an immunostimulatory interleukin and the expression vector encoding the pathogenic antigen are operably linked to a translational modification element.

85. The method of embodiment 84, wherein the expression vector encoding an immunostimulatory interleukin and the expression vector encoding the pathogenic antigen are operably linked to a promoter.

86. The method of embodiment 85, wherein the promoter is selected from the group consisting of: CMV promoter, mPGK, SV40 promoter, β-actin promoter, SRα promoter, herpes thymidine kinase promoter, simplex Herpes virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), Rous sarcoma virus (RSV) promoter and EF1α promoter.

87. The method of any one of embodiments 83-86, wherein the immunostimulatory interleukin comprises interleukin.

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

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

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

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

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

93. The method of any one of embodiments 83 to 92, wherein the expression vector comprises a formula represented by: PAT ¹ -BT ² -B' or PT ¹ -BT ² -B'-A wherein P is the promoter , A encoding immunogenic disease antigen, T ¹ and T ² for the translational modifications element, B encoding IL-12 p35, and B 'encoding IL-12 p40.

2A peptide of the group of 93 in Example 94. The method of embodiment, wherein T ¹ and T ² is selected from the group consisting of the coding: P2A peptide, peptide T2A, and the E2A peptide F2A peptide.

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

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

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

98. The method of any one of embodiments 95 to 97, wherein the field strength of the at least one voltage pulse is from about 200 V/cm to about 1500 V/cm.

99. The method of any one of embodiments 83 to 98, wherein the first nucleic acid and the second nucleic acid are injected into the tumor into the tumor on days 1 ± 2 days, days 5 ± 2 days, and /or Administer electroporation therapy on day 8 ± 2 days.

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

101. The method of any one of embodiments 83-100, further comprising administering to the individual at least one additional therapeutic agent.

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

103. The method of any one of embodiments 83 to 102, wherein the method comprises injecting the expression vector encoding the immunostimulatory interleukin and the expression vector encoding a pathogenic antigen into the tumor, tumor microenvironment and/or or tumor margin tissue, and electroporation therapy is administered to the tumor, the tumor microenvironment, and/or the tumor margin tissue.

104. The method of any one of embodiments 83 to 102, wherein the method comprises injecting the expression vector encoding the immunostimulatory interleukin into the tumor, tumor microenvironment, and/or tumor marginal tissue, and injecting the expression vector into the tumor, tumor microenvironment, and/or tumor marginal tissue. Electroporation therapy is administered to the tumor, tumor microenvironment, and/or the tumor marginal tissue, and the expression vector encoding the pathogenic antigen is injected into the dermis and the electroporation therapy is administered to the dermis at the injection site.

105. The method of any one of embodiments 83 to 102, wherein the method comprises injecting the expression vector encoding the immunostimulatory interleukin into the tumor, tumor microenvironment, and/or tumor marginal tissue, and injecting the expression vector into the tumor, tumor microenvironment, and/or tumor marginal tissue. Electroporation therapy is administered to the tumor, tumor microenvironment, and/or the tumor margin tissue, and the expression vector encoding the pathogenic antigen is injected into skeletal muscle and the electroporation therapy is administered to the skeletal muscle at the injection site.

106. The method of any one of embodiments 83-105, wherein the method further comprises administering to the individual an immune checkpoint inhibitor. EXAMPLES Example 1 The sequence alignment

Evaluation of approximately one hundred DNA sequences from SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) cases in the United States (Global Initiative for Sharing All Influenza Data (GISAID)). All sequences of the "S" gene encoding spike glycoprotein were aligned with reference gene ID 43740568 (NC_045512.2 bases 21563-25384). These multiple sequence alignments of ~100 gene sequences did not create alignment gaps (ie, no insertions or deletions or major structural changes). The aligned DNA sequences are translated into amino acids and evaluated for percent identity to the reference protein sequence. All positions of the spike glycoprotein are 100% identical to the reference sequence, amino acids H49 (99%), F157 (99%), G181 (99%), V483 (96%), D614 (97%) and Except H655 (99%). Notably, V483 is in the receptor binding domain (RBD) of angiotensin-converting enzyme 2 (ACE2). V483 is not a contact residue and exists as an alanine substitution with low conservation among homologous coronavirus strains. Example 2. Phase I trial of SARS-CoV-2 spike protein (S protein ) and pIL-12 nucleic acid vaccine in human subjects

Describes a Phase 1 study to evaluate the safety and profile of the S protein and 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. Individuals are given a primary vaccination, and booster vaccinations are given 3-4 weeks after the primary vaccination. Subjects were divided into age groups 18-50 and >50 years old. Table 1. Vaccine combining SARS-CoV-2 S protein with IL-12 and healthy individuals aged 18-50 and 50 years old. Day 1 and Day 30 (Initial and Enhanced) group age Peptide throw in Dosage (mg) Volume (μL) 1A 18-50 S protein S protein ID-EP IM-EP 0.25 0.5 50 100 1B 18-50 S protein/IL-12 S protein ID-EP IM-EP 0.25/0.5 0.5 100 100 2A > 50 S protein S protein ID-EP IM-EP 0.25 0.5 50 100 2B > 50 S protein/IL-12 S protein ID-EP IM-EP 0.25/0.5 0.5 100 100

pIL-12 (TAVO; pIL-12): Plasmids 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) was performed. The mass system was supplied at a concentration of 3.3 mg/mL in a fill volume of 0.225 mL.

SARS-CoV-2 spike protein (S protein): plastids encoding the 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 mass system was supplied in a fill volume of 0.7 mL at a concentration of 1.67 mg/mL.

Electroporation can be performed using, for example, the Apollo C-System (ACS) electroporation equipment or the IGEA Cliniporator electroporation system.

Electroporation (EP) was co-localized at each injection site. Electroporation can be administered concurrently with or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes of plastid injection. In some embodiments, electroporation (EP) comprises 6 to 8 10 millisecond pulses at a field strength of 400 V/cm.

Administration: Prior to plastid injection, a local anesthetic such as 1% lidocaine can be injected around the planned injection site to obtain local anesthesia. Alternative local anesthetics may be used as clinically indicated and considered clinically appropriate. Additionally, analgesics, anxiolytics, or conscious sedation may be administered to the patient. Ice packs that cool an area and render it unconscious are also acceptable.

In some embodiments, a suitable site is selected that readily allows intramuscular (IM) delivery to a depth of 0.5 to 1.5 cm (such as but limited to the shoulder) and adjacent intradermal (ID) delivery 2-3 cm from the IM injection site . In some embodiments, the ID-EP site may be more than 10 cm from the IM-EP site. In some embodiments, the ID-EP site is tied to the contralateral site of the IM-EP. In some embodiments, vaccination occurs in two steps: intradermal injection + EP (eg, right shoulder); followed by intramuscular injection + EP to the matched contralateral site (eg, left shoulder deltoid). In some embodiments, the ID-EP is administered before the IM-EP.

Intramuscular administration (eg, IM-EP) can be performed concurrently with intradermal administration (eg, ID-EP), before intradermal administration, or after 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.

Intradermal administration can be performed 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 within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm of the site of intramuscular administration.

Vaccines can be administered to individuals in an outpatient setting. Example 3. Intramuscular, intradermal and intratumoral push-out and electroporation of nucleic acids encoding coronavirus S protein antigenic polypeptides and IL-12 . A. Encoding coronavirus S protein antigenic polypeptides, IL12 or coronavirus S protein antigenicity Intramuscular Electroporation (IM-EP) Administration of Nucleic Acids in Combination of Polypeptide and IL12.

IM-EP administration of the nucleic acid encoding the 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 Electroporate the injection site.

In some embodiments, 0.1-3 mg of nucleic acid encoding 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 nucleic acid encoding coronavirus S protein antigenic polypeptide and/or IL-12. In some embodiments, 0.1 mg, 0.1 ± 0.01 mg, or 0.1 ± 0.05 mg of 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 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 coronavirus S protein antigenic polypeptide and/or IL-12 nucleic acid 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 coronavirus S protein antigenic polypeptide and/or IL-12 nucleic acid is injected. The coronavirus S protein antigenic polypeptide can be, but is not limited to, the SARS-CoV-2 spike protein or an antigenic fragment thereof. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector. The amount indicated above may be the amount of each of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12, or the nucleic acid encoding the coronavirus S protein antigenic polypeptide and encoding IL-12. 12 The amount of nucleic acid taken together (whether on separate vectors or the same vector).

In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is divided into 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 volume. 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 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 divided into Injection at a depth of 1.5 ± 0.3 cm, 1.5 ± 0.4 cm or 1.5 ± 0.5 cm. The EP applicator was set to the same depth as the injection ± 0.1 cm. Insert the EP applicator so that the electrode spans the nucleic acid injection site. In some embodiments, pulses at a field strength (E+) of 400 ± 100 V/cm and a pulse width of about 1-20 ms are administered 1-10 times at intervals of about 1 second. In some embodiments, pulses at a field strength (E+) of 400 V/cm and a pulse width of about 10 ms are administered eight times at 1 second intervals. 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 a coronavirus S protein antigenic polypeptide and IL12.

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

In some embodiments, 0.1-3 mg of nucleic acid encoding 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 are injected , 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 nucleic acid encoding coronavirus S protein antigenic polypeptide and/or IL-12. In some embodiments, 0.1 mg, 0.1 ± 0.01 mg, or 0.1 ± 0.05 mg of 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 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 nucleic acid encoding 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 nucleic acid encoding 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, the SARS-CoV-2 spike protein or an antigenic fragment thereof. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector. The amount indicated above may be the amount of each of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12, or the nucleic acid encoding the coronavirus S protein antigenic polypeptide and encoding IL-12. 12 The amount of nucleic acid taken together (whether on separate vectors or the same vector).

In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is divided into 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, 650, 700, 750, 800, 900, or 1000 μL in volume. 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 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 was set to the same depth as the injection ± 0.1 cm. Insert the EP applicator so that the electrode spans the nucleic acid injection site. In some embodiments, pulses at a field strength (E+) of 400 ± 100 V/cm and a pulse width of about 1-20 ms are administered 1-10 times at intervals of about 1 second. In some embodiments, pulses at a field strength (E+) of 400 V/cm and a pulse width of about 10 ms are administered eight times at 1 second intervals. C. Intratumoral electroporation (ID-EP) administration of nucleic acid encoding a coronavirus S protein antigenic polypeptide, IL12, or a combination of a coronavirus S protein antigenic polypeptide and IL12.

IT-EP administration of nucleic acids encoding coronavirus S protein antigenic polypeptides and/or IL-12 comprises injecting into the tumor one or more solutions containing nucleic acids encoding coronavirus S protein antigenic polypeptides and/or IL-12 and Electroporate the injection site.

In some embodiments, 0.1-3 mg of nucleic acid encoding 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 are injected , 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 nucleic acid encoding coronavirus S protein antigenic polypeptide and/or IL12. In some embodiments, 0.1 mg, 0.1 ± 0.01 mg, or 0.1 ± 0.05 mg of 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 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 coronavirus S protein antigenic polypeptide and/or IL-12 nucleic acid 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 coronavirus S protein antigenic polypeptide and/or IL-12 nucleic acid is injected. The coronavirus S protein antigenic polypeptide can be, but is not limited to, the SARS-CoV-2 spike protein or an antigenic fragment thereof. Nucleic acids can be DNA or RNA. Nucleic acids can be expression vectors or plastids. In some embodiments, the nucleic acid is a non-viral vector. The amount indicated above may be the amount of each of the nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12, or the nucleic acid encoding the coronavirus S protein antigenic polypeptide and encoding IL-12. 12 The amount of nucleic acid taken together (whether on separate vectors or the same vector).

In some embodiments, the nucleic acid is injected in a volume of 20-1000 μL. In some embodiments, the nucleic acid is divided into 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 volume. 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 of the tumor volume. 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 is injected into the tumor at a concentration of 0.5-1.0 mg/ml in a volume corresponding to 25% ± 10%, 25% ± 5%, 25% ± 2.5% of the calculated volume of the tumor. Injection into a tumor may include injection into marginal tissue surrounding the tumor. Injecting into the tumor may include injecting the tumor as uniformly as possible and, as appropriate, the peripheral tissue surrounding the tumor (ie, dispersing the injection throughout the tumor and optionally throughout the peripheral tissue surrounding the tumor).

The nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12 may exist in the same solution or in different solutions. If present in separate solutions, the nucleic acid encoding the coronavirus S protein antigenic polypeptide and the nucleic acid encoding IL-12 may 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. The nucleic acid encoding the coronavirus antigenic polypeptide and the nucleic acid encoding IL-12 can be present on the same vector (eg, plastid or RNA). When present on the same vector, the nucleic acid encoding a coronavirus antigenic polypeptide and the nucleic acid encoding IL-12 can be expressed by different promoters or by a single promoter, such as in a polycistronic vector.

In some embodiments, the ID-EP site may be 0.5 to 10 cm from the 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 from the IM-EP site , 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. 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 from the IM-EP site , 2.5 ± 1 cm. In some embodiments, the ID-EP site is 2-3 cm from the IM-EP site. Example 4. Immune Response Analysis .

Immunological monitoring . Can be performed on one or more of Day 1, Day 2, Day 3, Day 15, Day 30, Day 31, Day 32, Day 45, Day 60, and Day 90 of the study Blood sampling for plastid expression and immune response measurement. The samples were analyzed for antibodies to the vaccine and cellular immune responses. Flow cytometry was performed with a panel of markers including CD4, CD8, NK, B cells and monocyte/DC panels. These markers include CD4 T-learning, 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, CD158e1, KIRc, CD158b, CD11b, CD15, CD33, CD54, CD80, CD83, CD86, PD-L1, PD-L2. Sorted immune cell subsets were processed for RNA/DNA extraction and genome-wide CpG methylation by target capture DNA library preparation and Illumina sequencing.

Gene expression analysis of immune response : Ki-67, CD38, HLA-DR, CD40, ICOS, Granzyme B, PD-1, TIM-3, LAG-3, 4-1BB, CTLA by gene expression or protein expression analysis -4, one or more of OX40R, NKG2A, CD94, NKp46, NKG2D, NKp30, CD158e1, KIRc, CD158b, CD11b, CD15, CD33, CD54, CD80, CD83, CD86, PD-L1 and PD-L2 for analysis .

Neutralization analysis . Antibody neutralization of SARS-CoV-2 was assessed using a pseudo-patterned lentivirus expressing the SARS-CoV-2 S protein and luciferase. The ability of serum from a vaccinated individual to prevent infection of susceptible target cells is indicative of the induction of an anti-SARS-CoV-2 immune response in the individual. The readout is the ability of sera from vaccinated individuals to prevent infection of susceptible target cells. Alternatively, the ability of serum to block the binding of hACE2R to spinous bodies was performed.

Cytokinin analysis . Serum cytokinin profiling was assessed using the Quanterix Simoa platform. Analysis of systemic IL-12 (p70) and including IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A and up to 35 additional cytokines of TGF-β.

T cell responses . SARS-CoV-2 S protein-specific cellular immune responses were measured by interleukin release assays for type 1, type 2 and IL-17 interleukin responses using interleukin bead arrays. IFN-γ positivity was assessed by IFN-γ ELISPOT analysis using unfractionated PBMC and peptide pools of epitopes deemed to be dominant epitopes of HLA expressed by a particular individual. Peptide pools containing 15-mer peptides overlapping by 11 amino acids were also investigated. The ELISPOT response to the holoprotein was predicted to be ^{dominated by the CD4+} T cell response. It is further expected that responses to overlapping peptides include both CD4 ⁺ T cell responses and CD8 ⁺ T cell responses. Alternatively, ^{fractionated PBMCs were tested for CD4+} T cell response and CD8 ⁺ T cell response by intracellular intercellular staining (ICS).

B cell response . Antibody responses to SARS-CoV-2 spike-like S glycoprotein were assessed using micro-affinity proteosome (MAP) bead technology. Bead arrays containing SARS-CoV-2 protein were used to assess IgM and IgG isotype responses developed following vaccination. Humoral responses were monitored by B cell ELISPOT, assessing IgA, IgM and IgG responses of PBMCs to COVID-19 S spinous bodies to measure B cell responses.

Coordinated humoral / cellular responses . Single cell RNA sequencing is performed on one or more of day 0, day 15, day 30, day 45, day 60, and day 90. Paired αβ TCRseq and tandem BCRseq using the Archer Immunoverse platform allow tracking of T and B cell clones. Evaluation of epitope recognition of the SARS-CoV-2 spike protein by dominant clones. Sustained neutralizing Ab responses are predicted to result from coordinated CD4, CD8 and B cell responses against the SARS-CoV-2 spinous body (S) glycoprotein.

Whole genome sequencing . Whole-genome sequencing was performed to assess polymorphism in immune response-related genes and to provide HLA haplotypes. This information was evaluated for its relationship to the magnitude of the immune response to vaccination. Correlations with the depth and magnitude of response to vaccination were also analyzed.

Immune response to variants . In addition to analyzing immune responses against viruses with specific antigenic polypeptides used in vaccines, the above analysis can also be used to analyze immune responses against pathogen variants. For example, in the case of SARS-CoV-2, analysis can also be performed for the United Kingdom (UK) variant (referred to as 20I/501Y.V1, VOC 202012/01 or B.1.1.7), the South African variant (referred to as 20H/501Y.V2 or B.1.351), the Brazilian variant (referred to as P.1 or B11248) and the California variant (referred to as CAL.20C or 20C/S:452R;/B1429). Induction of an immune response against the variant indicates that the vaccine is effective against the variant. Phase I study of Example 5. inoculated encoding SARS-CoV-2 spinous glycoprotein (S protein) and an immune response in an individual nucleic acid of the IL-12 health.

This study will evaluate the safety and antiviral immunostimulatory efficacy of combined electroporation of plastids encoding both the coronavirus spike protein (S protein) and IL-12. The effects of IL-12 vaccination other than S protein on neutralizing antibody production, induction of cellular immunity, induction of Th1 response and prevention of SARS-CoV-2 infection were analyzed.

Recent reports suggest that patients who have died from COVID-19 have depleted T cells. Since IL-12 supports T cell responses and reduces T cell exhaustion, it is expected that individuals receiving the S protein along with IL-12 will develop more sustained T cell responses against SARS-CoV-2. Sustained T cell responses would support higher titers of anti-spike neutralizing antibodies. Vaccination is expected to prevent exposure to SARS-CoV-2 and to provide cross-protection against other coronaviruses.

The phenomenon of antibody-dependent enhancement (ADE, in which a non-neutralizing antiviral protein assists the entry of the virus into host cells) has been described 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)) were reported . Wang et al. experimentally showed that anti-SARS-CoV spike protein monoclonal antibodies cause ADE (Wang, SF et al. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochemical and biophysical research communications 451, 208-214 (2014)), however multiple strains, undiluted antisera neutralized SARS-CoV infection. Inclusion of IL-12 in the vaccine platform studied prevents ADE, thereby acting as ^{an adjuvant for triggering both effector CD8+} T cell responses and complementary CD4 ⁺ T cell responses, which can enhance humoral responses.

All individuals in this protocol will receive plastids encoding the S protein via ID-EP and/or IM-EP. Half of the individuals will also receive plastids encoding IL-12 via ID-EP. Subjects are administered an initial round of S protein and IL-12 on day 1 (prime vaccination) and a second round of S protein and IL-12 (boost) approximately 3-4 weeks later. Various doses of both the S protein-encoding nucleic acid and the IL-12-encoding nucleic acid optimized the immune response.

The plastid encoding the S protein encodes the spike protein from SARS-CoV-2 and is driven by the cytomegalovirus (CMV) promoter or the chicken beta actin promoter. The S protein has been stabilized by the addition of two prolines and reported). The S protein is the prefusion spike ectodomain 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), has a "Furin cleavage site (residues 682-685), C-terminal T4 fibrin trimerization motif, HRV3C protease cleavage site, TwinStrep tag and 8 × His tag" GSAS" substituted gene encoding residues 1-1208 of SARS-CoV-2 S (GenBank: MN908947) was synthesized and cloned into the mammalian expression vector pαH. The plastid map of SARS-CoV-2 S-2P_desfurin_F3CH2S is in Figure 4. The SARS-CoV-2 S-2P_desfurin_F3CH2S plasmid system encoding the SARS-CoV-2 S protein was obtained from the Vaccine Research Center NIAID (21MAR2020). The His8 and StrepII tags were removed by restriction enzyme digestion with BamHI and XhoI and replacement of the plastid from SARS-CoV-2 S-2P_desfurin_F3CH2S with the 122 bp PCR product encoding the fold-only subdomain. SARS-CoV-2 S-2P_desfurin_F3CH2S plastid was used as template for PCR. The resulting plastid was named SARS-CoV-2 S-2P_desfurin_foldon. The CMV promoter, chimeric intron, and S-2P foldon fragments encoding the prefusion hybrid S-2P ECD and Foldon fusion proteins were excised by restriction digestion with SpeI and XhoI and cloned into Spel and NotI digested IL-12 p35_p40 pUMVC3 clinical vector (OncoSec) and 60 bp XhoI and NotI linkers excised from pSE280 vector (Invitrogen). The resulting plastid was named pUMVC3.SARS-CoV-2-2_S-2P.foldon.de-tagged.and is shown in FIG. 5 . The plastids encoding the S protein were vials at 1.67 mg/ml.

Plasmids encoding IL-12 contain genes for human IL-12 p35 and p40 subunits separated by an internal ribosome entry site (IRES) and under the control of a single cytomegalovirus (CMV) promoter. Plastids encoding IL-12 were vial at 3.33 mg/ml.

Vaccination with S protein and IL-12 included gene delivery via electroporation. IM-EP of S protein nucleic acid will provide an expressed diffusible S protein that can be recycled and presented to B cells to generate anti-spike antibody responses. The ID-EP of the S protein and IL-12 will provide a restricted local presentation in the dermis, leading to antigen presentation in the draining lymph nodes, leading to activation of the antiviral immune response. The expression of IL-12 in the dermis will also polarize this local area towards a Th1-biased environment. This discrete module deployment of antigens and immune-activating interleukins creates an immunogenic cellular microenvironment in the dermis in which IL-12 supports innate effector cells and one or more membrane-bound (membrane-anchored) or secreted (maybe lysis) presentation of glycoproteins and subsequent activation of cellular antiviral T cell responses. Since this local cellular activation does not prevent the activation of humoral immunity in other secondary lymphoid tissues, the vaccine is expected to generate both cellular and humoral immunity against SARS-CoV-2.

Immunomonitoring was performed as described in Example 4. Example 6. Inoculation of coronavirus S protein and IL-12.

Following vaccination with a coronavirus antigenic polypeptide (eg, nucleic acid encoding SARS-CoV-2 S protein) plus IL-12 (eg, nucleic acid encoding IL-12), the combination of S protein and IL-12 induces neutralizing antibody production and Increases cellular immunity (T cell response). Human subjects were immunized with the combinations listed in Table 2. Table 2. Vaccines combining SARS-CoV-2 S protein and IL-12. treat Exemplary Dose (mg) Exemplary volumes (μL) Day 1 ± 3 days Day 30 ± 7 days ID-EP S protein (control) 0.8 ± 0.15 mg 500 + + ID-EP S protein ID-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + ID-EP S protein IM-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + ID-EP S protein IT-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IM-EP S protein (control) 0.8 ± 0.15 mg 500 + + IM-EP S protein ID-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IM-EP S protein IM-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IM-EP S protein IT-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IT-EP S protein (control) 0.8 ± 0.15 mg 500 + + IT-EP S protein ID-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IT-EP S protein IM-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + IT-EP S protein IT-EP pIL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 500 250 + + + + ID-EP S protein IT-EP S protein IT-EP IL-12 0.8 ± 0.15 mg 0.8 ± 0.15 mg 0.8 ± 0.15 mg 250-500 250-500 250 + + + + + +

For intradermal and intramuscular administration, the one or more injections can be in a single site or in two or more sites. For injections in two or more locations, the injections can be adjacent to each other or in separate locations. For example, an individual may receive 500 μL in two adjacent injections of 250 μL each. Similarly, an individual may receive 750 μL in two adjacent injections of 375 μL each. For two adjacent injections, the injections can be close enough to enable the electroporation applicator electrode to span the two injection sites. To administer both the S protein and IL-12 to the dermis or muscle, the nucleic acids encoding the S protein and IL-12 can be injected separately at the same site, injected separately to different sites, or combined prior to injection. To administer both the S protein and IL-12 to a tumor, nucleic acids encoding the S protein and IL-12 can be injected into the tumor separately or combined prior to injection.

In some embodiments, one or more plastids encoding coronavirus antigens and/or immunostimulators are formulated in phosphate buffered saline (PBS) for injection followed by in vivo electroporation.

In some embodiments, the electroporation pulse is provided by an IGEA Cliniporator® with an IGEA applicator. The IGEA applicator model may be, but is not limited to, a model L-30-ST applicator containing an 8-pin array with 2 rows of 4 needles in each row, wherein the distance between the needles in the two rows is 4.7 mm and each The distance between the needles in the column is 3.2 mm. The applicator electrode is inserted to the appropriate depth for intradermal electroporation, intramuscular electroporation, or intratumoral electroporation so that the electrode spans the plastid injection site. In some embodiments, the electroporation comprises 2-10 (eg, eight) pulses at a field strength of 400 V/cm and a pulse width of 10 ms separated by 300 ms.

Each vaccination consists of two steps: intramuscular or intratumoral injection, followed by administration of at least one electroporation pulse. Electroporation (EP) was co-localized at each injection site and performed directly after injection as quickly as reasonably possible. For intradermal administration, injection and administration of electroporation pulse waves can be performed, for example, in the thigh over the vastus lateralis muscle or in the buttocks over the dorsal gluteus muscle. Other locations such as the shoulders are also suitable.

Immune responses were monitored as described in Example 4. Example 7. Electroporation (EP) of SARS-CoV-2 spiny plastids and IL-12 plastids generates anti-echinoid immune responses .

Experiments were performed on two different mouse strains (Balb/C or C57bl/6). Briefly, mice received an initial dose ("prime EP") and a booster dose ("boost EP") 14 days apart. Ten days after the EP boost (day 24), serum and splenocytes were collected for endpoint analysis.

Mice were treated on day 0 (prime) and day 14 (boost): (a) No treatment (b) 50 μg plastids encoding spike protein and 100 μg plastids encoding IL-12 using IM-ED; (c) 50 μg plastids encoding spiculin using IM-ED and 50 μg plastids encoding spiking protein using ID-EP plus 100 μg plastids encoding IL-12, or (d) 50 μg of plastids encoding spike protein with IM-ED and 125 μg of plastids encoding spike protein with ID-EP.

Samples were taken on day 24 for analysis by ELISA for spike protein or IL-12 protein production.

On day 24, splenocytes were harvested and cultured with echinoid for 1-3 days. Samples were collected after 24 hours and analyzed by ELISA for interferon production.

On day 24, sera were collected and analyzed by ELISA for SARS-CoV-2 specific IgG2a, IgG1 and ACE binding inhibition.

Electroporation of a combination of pIL12 and acanthoplasts administered via intradermal or intramuscular treatment induced anti-spike IgGl and anti-spike IgG2a antibodies in Balb/C and C57bl/6 mice (Figures 9-10). In addition, the antibody was able to inhibit the binding of spinosad to ACE2 in vitro (inhibition of the binding of SARS-CoV-2 pseudovirus to ACE2) in a pseudo-neutralization assay (Figure 11). Example 8. Spike protein and IL-12.

pIL-12 and acanthoplasts were administered via different routes of administration (intradermal, ID; intramuscular, IM). The initial dose and booster dose were separated by 21 days. Experiments were performed in two mouse strains (Balb/C and C57bl/6). Serum samples were collected at different time points (D31, 42, 92) for anti-spike antibody assay (ELISA).

Mice were treated with the following on day 0 (prime) and day 21 (boost): (a) 50 μg of plastids encoding spiculin using IM-EP and 50 μg of plastids encoding spiculin using ID-EP; (b) 50 μg of IM-EP-encoding plastids and 50 μg of ID-EP-encoding plastids plus 100 μg of IL-12-encoding plastids; (c) 50 μg of IM-EP-encoding plastids plus 100 μg of IL-12-encoding plastids and 50 μg of ID-EP-encoding plastids; (d) 50 μg plastids encoding spiky protein using IM-EP plus 100 μg plastids encoding IL-12 and control plastids using ID-EP; (e) control plastids using IM-EP and 50 μg plastids encoding spiculin plus 100 μg plastids encoding IL-12 using ID-EP; (f) a control plastid using IM-EP and a control plastid using ID-EP; or (g) No treatment control.

On days 31, 42 and 92, sera were collected and analyzed by ELISA for SARS-CoV-2 specific IgG2a, IgG1 and ACE binding inhibition.

In the day 31 samples, spiny plastid EP via concurrent ID and IM injections demonstrated the best anti-echinoid immune response among the test groups. Addition of pIL-12 to ID-EP in spinous plastids at the 100 μg/ml IL-12 plastid dose level did not enhance the immune response (Figures 12A-B, 13A-B). Administration of 100 μg of IL-12 by IM-EP reduced efficacy. Similar results were noted in day 42 and day 92 serum. While Spikein administration by ID-EP and IM-EM produced mostly neutralizing antibodies on day 92, each over the vaccination time course produced detectable amounts of neutralizing antibodies (Figure 14A-B) . Example 9. Comparison of pIL12-IRES and pIL12-P2A .

Comparison of the use of nucleic acids encoding IL12-P2A (plastids encoding IL-12p35-P2A-IL-12p40) in combination with plastids encoding spiky protein or plastids encoding IL12-IRES (encoding IL-12p35- ID-EP vaccination in combination with plastids of P2A-IL-12 p40) and plastids encoding spiculin to determine whether increased IL-12 expression by the IL12-P2A expression vector would enhance anti-spike body immune response. We have previously shown that IL12-P2A induces consistently higher levels of IL-12 expression when compared to IL12-IRES.

Mice were vaccinated according to the following schedule, with the primary injection on day 0 and the booster injection on day 21. (a) 50 μg Spinosad IM-EP and 50 μg Spinosad ID-EP (b) 50 μg Spinosad ID-EP (c) 50 μg Spinosad + 100 μg IL12-IRES ID-EP (d) 50 μg Spinosad + 100 μg IL12-P2A ID-EP (e) Empty plastid ID-EP

Serum was collected on days 31 and 42 and analyzed for SAR-CoV-2 specific IGg1 and IgG2a antibodies and for neutralizing antibodies. Vaccination of animals with the IL12-P2A vector has reduced anti-echinoid immune responses when compared to animals vaccinated with the IL12-IRES vector. Thus, the results indicated that higher levels of IL-12 administered by ID-EP inhibited the immune response to the co-administered antigen (FIG. 15A-B). However, the combination of spinous bodies and IL-12 resulted in induction of an immune response compared to the empty vehicle control. Based on these observations, lower doses of IL-12 plastids were analyzed. Example 10. Analysis of lower doses of pIL-12 and spatial colocalization .

Mice were vaccinated with lower doses of the IL12-IRES expression vector as described above. Two lower doses of pIL12 (50 μg or 10 μg) were co-administered with acanthoplasts to immunize C57bl/6 mice. Some mice were injected with a solution containing pIL-12 + acanthoplasts (mixture). Separate injections of IL-12 and acanthoplasts were administered to the other mice. For individual injections, IL12 and spiny plastid injections were administered at adjacent sites (~2 mm apart at the injection sites), and the sites were electroporated to separate.

Mice were vaccinated according to the following schedule, with the primary injection on day 0 and the booster injection on day 21. (a) 50 μg Spinosad IM-EP and 50 μg Spinosad ID-EP (b) 50 μg Spinosad ID-EP (c) 50 μg Spinosad ID-EP and 50 μg IL12-IRES ID-EP (d) 50 μg Spinosad ID-EP and 10 μg IL12-P2A ID-EP (e) 50 μg Spinosad + 10 μg IL12-P2A (mixture) ID-EP (f) Empty plastid ID-EP

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 of 50 μg acanthoplasts and 10 μg pIL12 (“Echinoid(50)/IL12(10), Mixed_ID”) showed superiority compared to 50 μg acanthoid ID-EP alone Immune response, indicating the efficacy of IL-12 in enhancing the immune response to spike protein (FIGS. 16A-B, 17B-B, 18A-B). Immunization of acanthoplasts via IM-EP and ID-EP was used as a positive control standard. Compared to the other treatment groups, the positive control standard treatment group receiving twice the dose of spiny plastids, 50 μg via each of the IM and ID routes, caused more spinous protein expression overall. Examples of 11. pIL12 dose or time course dependent effect.

Mice were vaccinated as described above with different doses of the IL12-IRES expression vector administered over different time courses. Two lower doses of pIL12 (10 μg or 1 μg) were co-administered with acanthoplasts to immunize C57bl/6 mice.

Mice were vaccinated according to the following schedule, wherein the primary injection was on day 0 and the booster injection was on day 21 (unless otherwise indicated). (a) Positive control, spinous body IM-EP and spinous body ID-EP (b) Spinous body ID-EP (c) Spinosad ID-EP + 10 μg IL12 ID-EP (IL12 only at primary) (d) Spinosad ID-EP + 1 μg IL12 ID-EP (IL12 only at primary) (e) Spinosad ID-EP + 10 μg IL12 ID-EP (f) Spinosad ID-EP + 1 μg IL12 ID-EP (g) Spinosad ID-EP + 10 μg IL12 ID-EP (IL12 only when boosted) (h) Spinosad ID-EP + 1 μg IL12, ID-EP (IL12 only when boosted) (i) Empty vector negative control

Administration of 10 μg or 1 μg of IL-12 plastids enhanced anti-spiky IgG1 and neutralizing antibody responses on days 31 and 42 compared to administration of spiculoids by ID-EP (Figure 19A- C. 20). For IL-12 administration, potentiation occurred at prime and boost, prime only, or boost only. Administration of 10 μg or 1 μg of IL-12 plastids 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 (T _ec ) cells). When compared to spinoplasts administered by ID-EP (FIG. 21A-D) or by both IM-EP and ID-EP, administration at primary and booster The use of 10 μg or 1 μg of IL-12 plastids also increased memory B cells at day 90. The results indicate that addition of IL-12 to pathogenic antigens induces an immune memory response and longer lasting immunity. Example 12 Dose analysis of plastid spinous

Multiple dose levels of spiny plastids were administered to mice with or without administration of pIL12. pIL12 is mixed with the spiny plastids to be co-administered or administered separately. In two independent experiments utilizing two different routes of administration, there was no significant difference in the level of anti-acanthosome response based on acanthoplast dose (FIGS. 22A-B, 23A-B). As in the experiments shown above, administration of 10 μg of IL-12 plastids induced a stronger immune response against pathogen antigens compared to 50 μg of IL-12 plastids. Example 13. Vaccination of individuals with cancer, intratumoral electroporation ( IT-EP ) of nucleic acids encoding coronavirus antigenic polypeptides and nucleic acids encoding immunostimulatory interleukins .

Mice were anesthetized and tumor cells were injected subcutaneously into their right and/or left flanks. Tumor growth was monitored by digital caliper measurements until tumor appearance. Subsequently, tumors were treated as indicated in Table 3. Tumor volume and survival were monitored. Additionally, samples were collected to determine the immune response against SARS-CoV-2. PBMC immunoprofiling and IgG and ACE2 inhibition assays were performed on days 15 and 25 (see Example 4). Table 3. Vaccination of individuals with cancer. group Day 0 Day 15 1 IT-EP 10 μg IL-12 IT-EP 10 μg IL-12 2 IT-EP 10 μg IL-12 ID-EP 115 μg S protein IT-EP 10 μg IL-12 right flank ID-EP 115 μg S protein right flank 3 IT-EP 10 μg IL-12 + 115 μg S protein IT-EP 10 μg IL-12 + 115 μg S protein 4 IT-EP 115 μg S protein IT-EP 115 μg S protein 5 IT-EP 10 μg EV ID-EP 115 μg S protein IT-EP 10 μg EV ID-EP 115 μg S protein 6 IT-EP 10 μg EV IT-EP 10 μg EV 7 ID-EP 115 μg S protein + 1 μg IL-12 ID-EP 115 μg S protein + 1 μg IL-12 8 untreated

Analysis of peripheral blood B cells: Intratumoral electroporation of IL-12 and S protein induces a high percentage of B cells in peripheral blood.

Analysis of peripheral blood memory B cells: Intratumoral electroporation of IL-12 and S protein induces a high percentage of memory B cells in peripheral blood.

Analysis of tumor-draining lymph node B cell subsets: Intratumoral electroporation of IL-12 and S protein induces a high percentage of germinal center B cells and class-switched B cells in tumor-draining lymph nodes in which antibodies have been produced.

Analysis of T cell subsets in tumor draining lymph nodes: Intratumoral electroporation of IL-12 and S protein induces high percentages of CD4 ⁺ and CD8 ⁺ T cells in tumor draining lymph nodes.

Analysis of anti-spiky IgG1 and IgG2a: 1:500 dilution of serum. Intratumoral electroporation of the S protein in the presence or absence of IL-12 produced high levels of anti-spiky IgGl. One treatment (prime, day 15) or two treatments (prime+boost, day 25) produced similar levels of anti-spiky IgG. Animals vaccinated with IT-EP IL-12 plus the S protein had significantly increased production of anti-spiky IgG2a antibodies.

Pseudo-neutralization assay: Intratumoral electroporation of the S protein in the presence or absence of IL-12 produces a high level of inhibition of spinous body binding to ACE2. An increase in neutralizing antibody levels was observed following booster injections. Animals vaccinated with IL-12 + echinoids showed higher increases in neutralizing antibodies after booster vaccination.

Conclusion: Intratumoral electroporation of the combination of IL-12 and S protein produces 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 in tumor-draining lymph nodes cells and CD8 ⁺ T cells. IT-EP in combination of IL-12 and S protein also produced high levels of anti-spiky IgGl and IgG2a including neutralizing antibodies in serum. A strong antiviral response was observed, indicating that IT-EP, a coronavirus antigenic polypeptide and an immunostimulatory interleukin, effectively induced an immune response against the coronavirus. Example 14. Tumor regression in mice treated with IL-12 and SARS-CoV-2 S protein antigen.

Mice were implanted with tumor cells. Cells were injected subcutaneously into the right and/or left flanks of anesthetized mice. By digital caliper measurement of tumor growth was monitored until the average tumor volume of ~ 100 mm ³ of reach.

Tumors were treated with IT-EP control vehicle, IT-EP IL-12 or IT-EP IL12-2 + SARS-CoV-2 S protein antigen on days 1, 5 and 8. Tumor volume and survival were monitored. If the total tumor burden contralateral primary tumor and the tumor reached 2000 mm ^3, the mice were euthanized. Example 15. Intratumoral expression of pathogenic antigens and IL-12 enhances immune responses against tumors .

Tumor cells were implanted into mice on day -7. The tumor was implanted in the right flank. A single tumor in each mouse was treated on days 0 and 15 with the following: (a) untreated, (b) IT-EP control vector, (c) IT-EP 10 μg IL-12 plastids, (d) IT-EP 115 μg SARS-CoV-2 S protein antigenic plastid, (e) IT-EP 10 μg IL12 plastid + 115 μg SARS-CoV-2 S protein antigenic plastid, (f) IT-EP 10 μg IL-12 plastid + ID-EP 115 μg SARS-CoV-2 S protein antigenic plastid, (g) ID-EP 1 μg IL-12 plastid + 115 μg SARS-CoV-2 S protein antigenic plastid, (h) IT-EP 10 μg control plastid + ID-EP 115 μg SARS-CoV-2 S protein antigenic plastid. For the ID-EP SARS-CoV-2 S protein, the plasmid encoding the SARS-CoV-2 S protein was administered in the same flank as the tumor.

Animals treated with: (a) no treatment; (b) IT-EP control vehicle; (g) ID-EP IL-12 plastids + SARS-CoV- 2 S protein; (h) IT-EP control plastid + ID-EP SARS-CoV-2 S protein; or (d) IT-EP SARS-CoV-2 S protein. Animals treated with (c) IT-EP IL-w12 exhibited substantially reduced tumor growth by 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 activity by day 23 Tumor growth is shown. As expected, intratumoral IL-12 treatment resulted in decreased tumor growth. Combining intratumoral IL-12 therapy with intradermal or intratumoral pathogen antigen therapy enhanced the therapeutic efficacy of IL-12 therapy (Figures 24-25).

Intratumoral electroporation of the combination of pIL12 and pathogenic antigenic plastids enhanced antitumor responses compared to IL12 plastids alone. On day 25 after the first treatment, 85% of mice treated with pIL12 + SARS-CoV-2 S protein were tumor free compared to 60% of mice treated with IL12 alone (Figure 26). Example 16. Intratumoral expression of pathogenic antigens and IL-12 enhances immune responses against tumors .

Tumor cells were implanted into mice on day -7. Tumors were implanted in the right and left flanks. Single tumor lines in each mouse were treated with IT on days 1, 5±2 and 8±2, 1 and 5±2, or 1 and 8±2 -EP Control Vehicle, 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 plastid treatment. Subsequently, tumor and immune responses in primary (treated) tumors and secondary (untreated) tumors were 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.

In some mice, tumors and splenocytes were harvested 2 days after the last EP (ie, day 10) for NanoString and flow-based analysis. Alternatively, tumor volume was measured three times a week for regression/survival studies. Changes in gene expression in electroporated CT26 lesions were assessed by NanoString nCounter® technology. Intratumoral expression of pathogenic antigens was confirmed using ELISA in tumor lysates from tumor-bearing mice 48 hours after electroporation.

Volcano plots showing p-values and log2 fold changes for various immune response genes were generated in mice.

Flow cytometry analysis was used to analyze splenocytes in treated mice. Antigen-specific AH1+ CD8+ T cells were measured via tetramer assay (Immudex). The fold increase in number of AH1+ CD8+ T cells compared to the empty vector control was determined. Example 17. Intratumoral expression of pathogenic antigens and IL-12 enhances immune responses against tumors .

Tumor cells were implanted into mice on day -7. Tumors were implanted in the right and left flanks. A single tumor line in each mouse was treated on day 1 with IT-EP control vehicle, IT-EP 10 μg IL-12 plastids, IT-EP 10 μg IL12 plastids + 115 μg SARS-CoV-2 S protein antigen body or IT-EP 10 μg IL-12 plastid + ID-EP 115 μg SARS-CoV-2 S protein antigen plastid treatment. In some embodiments, each tumor was again treated with IT-EP control vehicle or IT-EP 10 μg IL-12 plastids on day 5±2. In some embodiments, each tumor was again treated with IT-EP control vehicle or IT-EP 10 μg IL-12 plastid on day 8±2. In some embodiments, each tumor is again treated with IT-EP control vehicle or IT-EP 10 μg IL-12 plastid on days 5±2 and 8±2 days. Subsequently, tumor and immune responses in primary (treated) tumors and secondary (untreated) tumors were monitored.

In some embodiments, the above 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.

In some mice, tumors and splenocytes were harvested 2 days after the last EP (ie, day 10) for NanoString and flow-based analysis. Alternatively, tumor volume was measured three times a week for regression/survival studies. Changes in gene expression in electroporated CT26 lesions were assessed by NanoString nCounter® technology. Intratumoral expression of pathogenic antigens was confirmed using ELISA in tumor lysates from tumor-bearing mice 48 hours after electroporation.

Volcano plots showing p-values and log2 fold changes for various immune response genes were generated in mice.

Flow cytometry analysis was used to analyze splenocytes in treated mice. Antigen-specific AH1+ CD8+ T cells were measured via tetramer assay (Immudex). The fold increase in number of AH1+ CD8+ T cells compared to the empty vector control was determined.

It should be understood that the 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 may be made without departing from the scope and spirit of the invention.

Figure 1A-B. (A) A coronavirus model showing the membrane presentation of the coronavirus spike glycoprotein. (B) Schematic of the coronavirus spike protein domain (SS = signal sequence (secretion signal), ectodomain, TM = transmembrane domain, ICD - intracellular domain).

Figures 2A-C. (A) Schematic representations of nucleic acids encoding IL-12 and Flt3L fusion proteins and antigens. (B) Flow cytometry plot showing 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 depicting the percentage of CD8+ cells (n=5) following administration of PIIM-OVA.

Figure 3. Schematic depicting differential system performance of secreted human model proteins 7 days after EP.

Figure 4. Plastid map of SARS-CoV-2 S-2P_desfurin_F3CH2S.

Figure 5. Plasmid map of pUMVC3.SARS-CoV-2-2_S-2P.foldon.de-tagged.

Figure 6. Schematic representation of nucleic acid constructs encoding coronavirus antigenic polypeptides (eg, CoV antigens), IL-12, CXCL9, anti-CD3 half-BiTE, Flt3L, and combinations thereof (T1, T2 = translational 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).

Figure 7. Schematic representation of nucleic acid constructs encoding coronavirus antigenic polypeptides (trimer = trimerization domain, SS = secretion signal, • = cleavage site mutation, * = stabilizing mutation).

Figure 8. Schematic representation of nucleic acid constructs encoding coronavirus antigenic polypeptides (ECD=extracellular domain, trimer=trimerization domain, SS=secretory signal).

Figure 9. Shows the comparison of Balb/C (A) or C57bl/6 (B) mice after IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 Graph of anti-S protein IgGl antibody levels in peripheral blood in control mice.

Figure 10. Shows the comparison of Balb/C (A) or C57bl/6 (B) mice after IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 Graph of anti-S protein IgG2a antibody levels in peripheral blood in control mice.

Figure 11. Shows the comparison of Balb/C (A) or C57bl/6 (B) mice after IM-EP S protein and ID-EP S protein plus IL-12 or IM-EP S protein plus IL-12 Graph of the presence of anti-S protein neutralizing antibody levels in peripheral blood in control mice.

Figure 12A. Graph depicting serial dilution analysis of anti-S protein IgGl (upper panel) and IgG2a (lower panel) at day 31 in mice vaccinated with various combinations of echinoplasts and IL-12 (legend shown in Figure 12A. 12B).

Figure 12B. Graph depicting day 31 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 13A. Graph depicting serial dilution analysis of anti-S protein IgGl (upper panel) and IgG2a (lower panel) at day 42 in mice vaccinated with various combinations of echinoplasts and IL-12 (legend shown in Figure 13A. 13B).

Figure 13B. Graph depicting day 42 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 14A. A graph depicting the presence of anti-S protein IgGl levels in serum at day 92 in mice vaccinated with various combinations of echinoplasts and IL-12 (legend is shown in Figure 14B).

Figure 14B. Graph depicting day 92 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 15A. Graph showing serial dilution analysis of anti-S protein IgG2a (upper panel) and IgG1 (lower panel) at day 31 in mice vaccinated with various combinations of echinoplasts and IL-12 (legend shown in Figure 15A. 15B).

Figure 15B. Graph depicting day 31 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 16A. Graph depicting anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody content in day 31 sera of mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 16B. Graph depicting day 31 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 17A. Graph depicting anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody content in sera at day 42 in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 17B. Graph depicting day 42 neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 18A. Graph depicting anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody content in day 61 sera in mice vaccinated with the indicated combinations of plastid protein and IL-12.

Figure 18B. Graph depicting day 61 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 19A. Graph showing anti-S protein IgGl antibody levels in serum at day 31 in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 19B. Graph depicting anti-S protein IgG2a antibody levels in sera at day 31 in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 19C. Graph depicting day 32 anti-S protein neutralizing antibody analysis in mice vaccinated with various combinations of echinoplasts and IL-12.

Figure 20. Depicts serial dilution assays of anti-S protein IgG1 (top panel) and IgG2a (middle panel) or anti-S protein neutralizing antibody assay at day 42 in mice vaccinated with various combinations of echinoplasts and IL-12 (bottom image).

^{Figure 21A. Graph depicting day 90 (CD19+} ) B cell content in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

^{Figure 21B. Graph depicting day 90 (CD3 +} CD4 ⁺ ) T cell content in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

^{Figure 21C. Graph depicting day 90 (CD3 +} CD8 ⁺ ) T cell content in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 21D. Graph showing memory B cell content at day 90 in mice vaccinated with the indicated combinations of echinoplasts and IL-12.

Figure 22A. Graph showing anti-S protein IgGl (upper panel) and IgG2a (lower panel) antibody content at day 31 in mice vaccinated with various amounts of echinoplasts and IL-12.

Figure 22B. Graph depicting day 31 anti-S protein neutralizing antibody analysis in mice vaccinated with various amounts of echinoplasts and IL-12.

Figure 23A. Graph depicting anti-S protein IgGl (upper panel) or IgG2a (lower panel) antibody content in day 31 serum in mice vaccinated with various amounts of echinoplasts and IL-12.

Figure 23B. Graph depicting day 31 anti-S protein neutralizing antibody analysis in mice vaccinated with various amounts of echinoplasts and IL-12.

Figure 24. Graph depicting tumor growth in mice treated with IT-EP IL-12 and IT-EP or ID-EP SARS-CoV-2 spike protein or in control mice.

Figure 25. Graph depicting tumor growth in mice treated with IT-EP IL-12 and IT-EP or ID-EP SARS-CoV-2 spike protein or in control mice.

Figure 26. Graph depicting the percentage of tumor-free mice in mice treated with IT-EP IL-12 and IT-EP or ID-EP SARS-CoV-2 spike protein or control mice.

Claims (34)

A use of a pathogenic antigen and an immunostimulatory agent for the manufacture of a medicament for eliciting an immune response against a pathogen in an individual, wherein the immunostimulatory agent comprises an immunostimulatory interleukin, a genetic adjuvant or an anti-CD3 half-BiTE. The use of pathogenic antigens and immune stimulators as claimed in claim 1, wherein the pathogenic antigens are selected from the group consisting of: bacterial antigenic polypeptides, viral antigenic polypeptides, protozoal antigenic polypeptides, parasite antigenic polypeptides, fungi Antigenic polypeptides, betacoronavirus antigenic polypeptides, A series betacoronavirus (subgenus Embecovirus) antigenic polypeptides, B series betacoronavirus (subgenus Sarbecovirus) antigens β-coronavirus C series (subgenus Merbecovirus ) antigenic polypeptide, D series βcoronavirus (subgenus Nobecovirus) antigenic polypeptide, SARS-CoV antigenicity Polypeptide, MERS-CoV antigenic polypeptide, SARS-CoV-2 antigenic polypeptide, coronavirus spike protein or its antigenic fragment, SARS-CoV-2 spike protein or its antigenic fragment, among the corresponding SEQ ID NO: 1 The amino acids at amino acid positions 986 and 987 comprise a proline-substituted SARS-CoV-2 spike protein or an antigenic fragment thereof, comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 Polypeptide of amino acid 1-1208, 14-1208 or 21-1208 or amino acid 1 to 1208, 14-1208 or 21-1208 with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 Polypeptides with at least 90% identity. The use of a pathogenic antigen and an immune stimulator according to claim 2, wherein the pathogenic antigen comprises one or more of the following: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain, an affinity tag, a One or more mutations that disrupt the internal peptidase cleavage site and one or more mutations that stabilize the protein in the prefusion conformation. The use of the pathogenic antigen and immunostimulator according to claim 1, wherein the pathogenic antigen is an isolated pathogenic antigen or a nucleic acid encoding the pathogenic antigen, and the immunostimulator is a nucleic acid encoding the immunostimulator. The use of a pathogenic antigen and an immune stimulator according to claim 4, wherein the nucleic acid encoding the immune stimulator comprises: a nucleic acid encoding IL-12, a nucleic acid sequence encoding an IL-12 p40-IL-12 p35 fusion protein, a nucleic acid encoding an IL-12 p40-IL-12 p35 fusion protein, Nucleic acid sequences of IL-12 p35-IL-12 p40 fusion proteins, encoding IL-12 p35 subunits and IL-12 p40 subunits separated by internal ribosome entry site (IRES) elements or 2A peptide skipping motifs The nucleic acid sequence, SEQ ID NO: 3 or SEQ ID NO: 4. The use of pathogenic antigens and immune stimulators as claimed in claim 4, wherein: (a) the nucleic acid encoding the pathogenic antigen and the nucleic acid encoding IL-12 are formulated for intradermal administration; (b) The nucleic acid encoding IL-12 and, optionally, a first effective dose of the nucleic acid encoding the pathogenic antigen are formulated for intradermal administration; and a second effective dose of the nucleic acid encoding the pathogenic antigen The nucleic acid is formulated for intramuscular administration; (c) the nucleic acid encoding the pathogenic antigen and a first effective dose of the nucleic acid encoding IL-12 are formulated for intratumoral administration; (d) a first effective dose of the nucleic acid encoding the pathogenic antigen is formulated for intradermal or intramuscular administration, and a first effective dose of the nucleic acid encoding IL-12 is formulated for intratumoral administration; or (e) a first effective dose of the nucleic acid encoding the pathogenic antigen and a first effective dose of the nucleic acid encoding IL-12 are formulated for intratumoral administration, and a second first effective dose of the nucleic acid encoding the pathogenic antigen The nucleic acid of the antigen is formulated for intradermal or intramuscular administration. The use of pathogenic antigens and immune stimulators as claimed in claim 6, wherein the first effective dose of the nucleic acid encoding the pathogenic antigen and the first effective dose of step (a), (b), (c) or (d) An effective dose of the nucleic acid encoding IL-12 is formulated for injection into the same site, optionally, wherein the first effective dose of the nucleic acid encoding the pathogenic antigen is the first effective dose of the nucleic acid encoding the pathogenic antigen Nucleic acids encoding IL-12 were combined prior to administration. The use of a pathogenic antigen and an immune stimulator as claimed in claim 6, wherein the intradermal administration comprises intradermal electroporation (ID-EP), the intramuscular administration comprises intramuscular electroporation (IM-EP), and the Intratumoral administration includes intratumoral electroporation (IT-EP), where the ID-EP, IM-EP and IT-EP comprise at least one voltage pulse with a field strength of about 100-1500 V/cm, as appropriate or at least one application of a voltage pulse having a field strength of about 400 V/cm and a duration of about 10 milliseconds. The use of a pathogenic antigen and an immune stimulator as claimed in any one of claims 1 to 8, wherein the pathogenic antigen and the immune stimulator are formulated for administration to an individual at a first time and a second time, wherein the The second time is 14 days to 6 months after the first time, as the case may be, wherein the pathogenic antigen comprises a coronavirus antigenic polypeptide. The use of pathogenic antigens and immune stimulators as claimed in claim 1, wherein the immune response comprises one or more of the following: cellular immune response, humoral immune response, both cellular and humoral immune responses, 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 Th1/Th2 antibody isotype response, antigen-specific IgG2a and IgG1 response, protective immunity against the pathogen, prevention of symptomatic disease associated with the pathogen, prevention of at least one symptom associated with infection with the pathogen, reduced severity of one or more symptoms associated with infection with the pathogen or reduction in duration and prevention of severe disease associated with infection with this pathogen. Use of a pathogenic antigen and an immunostimulator for the manufacture of a medicament for eliciting an immune response against a SARS-CoV-2 virus in an individual, the pathogenic antigen and the immunostimulator being characterized by: (a) The pathogenic antigen is formulated for administration by ID-EP to a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof, and the immune stimulator is formulated for administration by ID-EP EP administered a nucleic acid encoding IL-12; (b) The pathogenic antigen is formulated for administration by IM-EP of a nucleic acid encoding the SARS-CoV-2 spike protein or an antigenic fragment thereof, and the immune stimulator is formulated for administration by ID- EP administered a nucleic acid encoding IL-12; or (c) The pathogenic antigen is formulated for administration by ID-EP and IM-EP to a nucleic acid encoding SARS-CoV-2 spike protein or an antigenic fragment thereof, and the immune stimulator is formulated for administration by ID-EP administered a nucleic acid encoding IL-12. Use of a pathogenic antigen and an immune stimulator for the manufacture of a medicament for eliciting an immune response against the SARS-CoV-2 virus in an individual suffering from cancer, the pathogenic antigen and the immune stimulator being characterized by: (a) The pathogenic antigen is formulated for administration by IT-EP of a nucleic acid encoding the SARS-CoV-2 spike protein or an antigenic fragment thereof, and the immune stimulator is formulated for administration by IT-EP EP administered a nucleic acid encoding IL-12; (b) The pathogenic antigen is a nucleic acid encoding the SARS-CoV-2 spike protein or an antigenic fragment thereof formulated to be administered by ID-EP or IM-EP, and the immune stimulator is formulated for administration by IT-EP administers a nucleic acid encoding IL-12; or (c) The pathogenic antigen is a nucleic acid or antigenic fragment thereof that is formulated for administration by IT-EP and ID-EP or IM-EP, encoding the SARS-CoV-2 spike protein, and the immune stimulator is IL-12-encoding nucleic acid is formulated for administration by IT-EP. A vaccine for generating an immune response against a pathogen in an individual, the vaccine comprising: an effective dose of a pathogenic antigen or nucleic acid encoding the pathogenic antigen and an effective dose of an immunostimulator or a nucleic acid encoding the immunostimulator, wherein The immunostimulator comprises an immunostimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE or a combination thereof. The vaccine of claim 13, wherein the pathogen is a coronavirus selected from the group consisting of bacterial antigenic polypeptides, viral antigenic polypeptides, protozoal antigenic polypeptides, parasite antigenic polypeptides, fungal antigenic polypeptides, beta coronavirus Virus antigenic polypeptides, A series betacoronavirus (Ebercoronavirus subgenus) antigenic polypeptide, B series betacoronavirus (Sabecoronavirus subgenus) antigenic polypeptide, C series betacoronavirus (Mobecoronavirus subgenus) genus) antigenic polypeptide, D series betacoronavirus (Nobe coronavirus subgenus) antigenic polypeptide, SARS-CoV antigenic polypeptide, MERS-CoV antigenic polypeptide, SARS-CoV-2 antigenic polypeptide, coronavirus spike protein or antigenic fragment thereof, SARS-CoV-2 spike protein or antigenic fragment thereof, SARS-CoV-containing proline substitutions at amino acids corresponding to amino acid positions 986 and 987 of SEQ ID NO: 1 2 Spike protein or an antigenic fragment thereof, a polypeptide comprising amino acid 1-1208, 14-1208 or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 or a polypeptide with SEQ ID NO: 1. A polypeptide having at least 90% identity between amino acids 1 to 1208, 14-1208 or 21-1208 of SEQ ID NO: 2 or SEQ ID NO: 33. The vaccine of claim 14, wherein the pathogenic antigen comprises one or more of the following: a heterologous secretion signal, a heterologous trimerization domain, a heterologous transmembrane domain, an affinity tag, one or more disrupting internal peptidase cleavage Mutations at sites and one or more mutations that stabilize the protein in the prefusion conformation. The vaccine of any one of claims 13 to 15, wherein the pathogenic antigen and the immune stimulator are formulated for intradermal, intramuscular and/or intratumoral administration. The vaccine of claim 16, wherein the vaccine comprises the nucleic acid encoding the pathogenic antigen and the nucleic acid encoding the immune stimulator. The vaccine of claim 17, wherein the nucleic acid encoding the immunostimulator comprises: a nucleic acid encoding IL-12, a nucleic acid sequence encoding an IL-12 p40-IL-12 p35 fusion protein, a nucleic acid encoding IL-12 p35-IL-12 Nucleic acid sequence of p40 fusion protein, nucleic acid sequence encoding IL-12 p35 subunit and IL-12 p40 subunit separated by an internal ribosome entry site (IRES) element or a 2A peptide skipping motif, SEQ ID NO: 3 or SEQ ID NO:4. The vaccine of claim 17, 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. The vaccine of claim 13, wherein the pathogenic antigen or the nucleic acid encoding the pathogenic antigen is combined with the immune stimulator or the nucleic acid encoding the immune stimulator. The vaccine of claim 17, 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, optionally, wherein the The first expression vector and the second expression vector are encoded on a single plasmid. The vaccine of any one of claims 13 to 21, wherein the vaccine comprises (a) a first effective dose of the nucleic acid encoding the pathogenic antigen 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 pathogenic antigen 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 pathogenic antigen and a first effective dose of the nucleic acid encoding the immunostimulator formulated for administration by ID-EP and formulated for administration by IM - EP administration of a second effective dose of the nucleic acid encoding the pathogenic antigen; (d) a first effective dose of the nucleic acid encoding the immunostimulator formulated for administration by ID-EP and a first effective dose of the nucleic acid encoding the pathogen formulated for administration by IM-EP Nucleic acids of sexual antigens; (e) a first effective dose of the nucleic acid encoding the pathogenic antigen formulated for administration by ID-EP or IM-EP and a first effective dose formulated for administration by IT-EP the nucleic acid encoding the immunostimulator; (f) a first effective dose of the nucleic acid encoding the pathogenic antigen and a first effective dose of the nucleic acid encoding the immunostimulator formulated for administration by IT-EP and formulated for administration by ID - EP or IM-EP administration of a second effective dose of the coronavirus antigenic polypeptide. The vaccine of claim 13, 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 associated with one or more drugs Scientifically acceptable carriers and/or excipients are provided, and as appropriate, as solutions or lyophilized powders. Use of a nucleic acid encoding a pathogenic antigen and a nucleic acid encoding an immunostimulator for the manufacture of a medicament for eliciting an immune response against a pathogen in an individual diagnosed with cancer, wherein the nucleic acid encoding the pathogenic antigen has been Formulated for administration to the individual by IT-EP, ID-EP or IM-EP, and the nucleic acid encoding the immunostimulator is formulated for administration to the individual by IT-EP, wherein the immunostimulatory The agent comprises an immunostimulatory interleukin, a genetic adjuvant, or an anti-CD3 half-BiTE, where the pathogenic antigen comprises a coronavirus antigenic polypeptide, where the pathogenic antigen comprises SARS-CoV- 2 Spike protein or an antigenic fragment thereof, and the immune stimulator comprises IL-12. An expression vector comprising: a first nucleotide sequence encoding an immunostimulatory interleukin and a second nucleotide sequence encoding a pathogenic antigen, wherein the first nucleotide sequence and the second nucleotide sequence operably linked to a translational modification element, where appropriate, wherein The expression vector of claim 25, wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to a promoter, optionally, wherein the promoter is selected from the group consisting of: CMV promoter promoter, mPGK, SV40 promoter, β-actin promoter, SRα 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 (rous sarcoma virus, RSV) promoter and EF1α promoter. The expression vector of claim 25, wherein the immunostimulatory interferon comprises IL-12, and optionally, wherein the nucleotide sequence encoding IL-12 comprises IL-12 p35 operably linked to a translational modification element Coding sequence and IL-12 p40 coding sequence. The expression vector of claim 25, wherein the pathogenic antigen comprises bacterial antigenic polypeptide, virus antigenic polypeptide, protozoal antigenic polypeptide, parasite antigenic polypeptide, fungal antigenic polypeptide, betacoronavirus antigenic polypeptide, A-strain antigenic polypeptide Betacoronavirus (Ebercoronavirus subgenus) antigenic polypeptide, B series betacoronavirus (Sabecoronavirus subgenus) antigenic polypeptide, C series betacoronavirus (Mobecoronavirus subgenus) antigenic polypeptide, D Betacoronavirus (Nobecoronavirus subgenus) antigenic polypeptide, SARS-CoV antigenic polypeptide, MERS-CoV antigenic polypeptide, SARS-CoV-2 antigenic polypeptide, coronavirus spike protein or its antigenic fragment, SARS-CoV antigenic polypeptide - CoV-2 spike protein or antigenic fragment thereof, SARS-CoV-2 spike protein or antigen thereof comprising proline substitutions at amino acids corresponding to amino acid positions 986 and 987 of SEQ ID NO: 1 Fragment, polypeptide comprising amino acid 1-1208, 14-1208 or 21-1208 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 33 or with SEQ ID NO: 1, SEQ ID NO: 2 Or a polypeptide having at least 90% identity to amino acids 1 to 1208, 14-1208 or 21-1208 of SEQ ID NO: 33. The expression vector of any one of claims 25 to 28, wherein the expression vector comprises a formula represented by: PAT ¹ -BT ² -B' or PT ¹ -BT ² -B'-A wherein P is the promoter , A encoding immunogenic disease antigen, T ¹ and T ² for the translational modifications element, B encoding IL-12 p35, and in terms of B 'p40, optionally encode 12 IL-, wherein T ¹ and T ² is an internal ribosome entry Site or 2A peptide selected from the group consisting of P2A peptide, T2A peptide, E2A peptide and F2A peptide. The expression vector of claim 25 for use in the treatment of cancer in a subject. A use of an expression vector encoding an immunostimulatory interleukin and an expression vector encoding a pathogenic antigen for the manufacture of a medicament for treating an individual suffering from a tumor, the expression vector encoding the immunostimulatory interleukin having been formulated for injection into the tumor, tumor microenvironment, and/or tumor margin tissue, wherein electroporation therapy is performed on the tumor, tumor microenvironment, and/or tumor margin tissue, and (a) The expression vector encoding the pathogenic antigen is formulated for injection into the tumor, tumor microenvironment and/or tumor margin tissue, wherein the tumor, tumor microenvironment and/or tumor margin tissue are electroporated therapy; (b) the expression vector encoding the pathogenic antigen is formulated for injection into the dermis, wherein electroporation therapy is applied to the dermis at the injection site; or (c) the expression vector encoding the pathogenic antigen is formulated for injection into skeletal muscle, wherein electroporation therapy is applied to the muscle at the injection site, wherein treating the individual comprises reducing tumor size or inhibiting tumor growth, inhibiting cancer cell growth, inhibiting or reducing metastasis, reducing or inhibiting the development of metastatic cancer, and/or reducing cancer recurrence in the individual. The use of an expression vector encoding an immunostimulatory interleukin and an expression vector encoding a pathogenic antigen according to claim 31, wherein the immunostimulatory interleukin comprises IL-12. The use of an expression vector encoding an immunostimulatory interleukin and an expression vector encoding a pathogenic antigen of claim 31, wherein the electroporation therapy comprises administering at least one voltage pulse for a duration of about 100 microseconds to about 1 millisecond wave, optionally, wherein the field strength of the at least one voltage pulse is about 200 V/cm to about 1500 V/cm, and optionally, wherein the at least one voltage pulse comprises 1-10 voltage pulses . The use of an expression vector encoding an immunostimulatory interleukin and an expression vector encoding a pathogenic antigen of any one of claims 31 to 33, further comprising administering an immune checkpoint inhibitor to the individual.

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